progesterone an anti-convulsant
TRANSCRIPT
Progesterone as an Anti-convulsant
BY
Afshin Shahzamani
A thesis submitted to the Department of Pharmacology
in conformity with the requirements for
the degree of Masters of Science
University of Toronto
Toronto, Ontario, Canada
July, 2001
8 Copyright Afshin Shahzamani, 2001
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Acknowledgements
There are many people that I'd like to thank for helping me in this long journey.
First, 1 would like to thank Dr. Bumham. His love for life is contagious. He opens doors
when others close them. All dong, despite cornpeting responsibilities, he has been
helpful.
1 would like to thank the following "lab-relates people who have, in many ways, heiped
me complete this project. Some of these individuals have helped more than professional
duty requires; some have become friends.
Ted Brown Antonio Mendonca Jerome Cheng Brian Scott Sergei Likhodi Nancy Mingo Janet Eric Law Elizabeth Ng David Lai John Misek Heather Edwards Emmanuel Ho Sue Ambrosini
1 would like to thank Alla Vilner. She cheers me up, always.
1 would like to thank Paula Thiessen who never "doodles all-day-long" when fiiends
need her.
1 would like to th& David Peiroff who studies Gravity - we c m sleep a sound sleep,
some things are still as they should be.
1 would like to thank my aunt's entire family for having open hearts.
1 would like to thank my sister and her farnily, who gave me a second chance.
1 would like to thank my parents, Ali and Azar, and my brother, Ramin, who have always
- without equivocation - believed in my innate abilities and my integrity.
Most of all, 1 would like to hank my wife, Andrea Griggs, who has already been there for
me and with me, in the tough times, and in the good times. She gives meaning to what is
othenvise absurd, and 1 could not have done anything meaningfui without her.
III
Progesterone as an Anti-convulsant
Master of Science, 200 1
Afshin S hahzamani
Department of Pharmacology
University of Toronto
Abstract
Progesterone (Pd) has been successfully used as an anticonvulsant. Its use,
however, has been limited to patients whose seinires are exacerbated by varying
hormonal States in the menstrual cycle. Its anticonvulsant mechanism of action is
unclear. Therefore, the following studies were designed to increase the understanding of
the mechanism of action of P4 and promote its broader use in a clinical setting: 1) Effect
of P, on Amygdala-kindled seisure 2) Effeci of chronic low doses of P, in various seizure
rnodels 3) Eflect of gender and age on the anticonvu~sant activity of P4 4 ) The role of
GABA and the classic intracelizdar receptor in mediating PI S anticonvulsant action.
These studies indicate that P4 is an etrective anticonvulsant in males and infants. Clinical
trials in non-catamenial adults and children would be warranted. Its mechanism of
action may involve the newly discovered cell-surface P4 receptor and rnay provide the
possibility of new targets for future drug development.
% - . . .
1 INTRODUCTION
1.1 Epilcpsy 1.1.1 Definitions
1.2 Types of Seizures 1.2.1 Comrnon Seizure Types
1.3 Therapy for Epikpsy 1.3.1 Antiepileptic D ~ g s (AED's) 1.3.2 Prognosis for Seizure Control
1.4 Steroid Sex Hormones and Epilepsy 1.4.1 Catamenial Seizurcs 1.4.2 Steroid Sex Hormones: General Background 1.4.3 Progesterone and Estrogen as Ncurosteroids 1.4.4 Synthetic and Metabolic Pathways 1.4.5 Comrnercially Available Progesterone, MPA and Ganaxolone 1.4.6 Sex Steroids and the Brain: Reccptors 1.4.7 Somc Other Notable Neuro-active Steroids
1.5 Animal Seizure Models 1 .S. 1 Electroconvulsivc Shock Seizures: Maximal (MES) and Threshold (ECS) 1.5.2 Pentylenetetrazol: Maximal (MMT) and Threshold (MET) 1-53 Kindling
1.6 Fast studies of Progestin EfCects on Seizures 1.6.1 Clinical Studies 1.6.2 Animal Studies
1.7 Objectives
2 GENERAC METHODS
2.1 Animals
2.2 Drugs
2.3 Procedures for the Kindling Experiments 2.3.1 Implantation of Electrodes 2.3.2 Kindling Procedure 2.3.3 Detemination of AAer-discharge Threshold 2.3.4 Determination of Stability 2.3.5 Procedurc for Drug Testing 2.3.6 Verification of Electrode Placement 2.3.7 Procedure for Ovariectomy and Gonadectomy
2.4 Procedurc for Vaginal Smears
2.5 Proccdurc for the MET Test
2.6 Proccdure for t hc MMT Tcsl
2.7 Procedure for the ECS Test
2.8 Proccdure for the MES Test
2.9 Procedu rc for Pen tylenetetrazol Infusion
2.10 Procedure for Tests of Sedation and Ataxia
2.1 1 Statistical Analysis
3.1 Rationale Experiment l a Experimcnt l b Experiment Ic Experiment Id
3.2 Methods Su bjects Kindling and Threshold Tcsting Dose-Response Testing Seimre Scoring Data Analysis
3.3 Results Sedation Experiment la: Progesterone Dose-Response: Route of Administeration and Vehicle Expriment 1 b: Progesterone Dose-Response: Estrogen Priming Experiment I c: Progesterone Dose-Response: tntact subjects Experiment Id: Progesterone dose-response, male rats, hormone replacement
3.4 Summary 75
4 ANTICONVULSANT EFFECTS OF CHRONIC LOW-DOSE PROGESTERONE AND MPA IN SIX SElZURE MODELS 78
4. I Rationale 78
4.2 Methods Subjects Drug Administration Testing and Seizure Scoring Data Analysis
4.3 Results Effects on the Estrous Cycle
4.4 Summary 92
5 THE ANTICONVULSANT EFFECTS OF PROGESTERONE IN ADULT AND 15-DAY-OLD MALE AND FEMALE RATS IN THE MMT AND THE MES SEIZURE MOOECS 94
5.1 Rationalc 94
5.2 Mcthods Dnig Preparation and Administration Testing and Seizure Scoring Data Analysis
5.3 Results 5.3.1 Expcriment 3a: Adult Female Subjects 5.3.2 Experiment 3b: Adult Male Subjects 5 -3.3 Experiment 3c: immature Female Subjccts 5.3.4 Experiment 3d: Imrnahm Male Subjects
5.4 Summiry 107
6 MECHANISTIC STUDIES OF THE ANTICONVULSANT EFFECTS OF PROGESTERONE 309
6.1 Rationale 109
6.2 Methods S u bjects Drugs and Dnig Administration Testing and Seizure Sconng Data Analysis
6.3 Results 6.3.1 Experiment 4a: lndomethacin 6.3.2 Experimcnt 4b: RU486
6.3 Summary 113
1. DISCUSSION 118
7.1 The Kindl ing Data: Experiment 1 118
7.2 The "Chronic, Low-Dose" Data: Experiment 2 120
7.3 The Influence ofgender and Age: Studies at 15-Minute Injcction/rcst Interval 121
7.4 Studies OC Mechanism: Experiment 4 124
7.5 How Does Progcstcronc Stop Seizures? : The Role of Allopregnanolone 125
7.6 How Does Progesterone Stop Seizures? : Non-CABkMediatcd Actions 1 ) Non-Specific Effects 2) Binding to the lntracellular Receptor 3) Other Metabolites 4) Binding to the Membrane Receptor
7.7 How Does Progesteronc Stop Seizures? Th reshold Riscs 129
7.8 How Does Progcsteronc Stop Seizurcs: Clinical Actions 1 ) Contraceptive effects 2) Anti-estrogen Effects
7.9 Clinical Relevance o f the Prcsent Study 132
7.1 O Future Studies 133
APPENDIX 2: NEUROSTEROIDS - THREE IMPORTANT METABOUC ENZYMES 142
A2.1 Aromatasc 142
A2.2 5-alpha reductase 144
2 . 3-alpha hydroxystcroid dchydrogenase (3-alpha HSD) 147
APPENDIX 3: THE ROLE OF SEX HORMONES IN SOME IMPORTANT ASPECTS OF NEURONAL PUiSTlClTV 150
A3.1 Somc Behavioral Changes During the Fcrtility Cycle 150
AL2 Neuronal Plasticity and the Fertility Cycle: Cellular Changes 152
A3.3 Neuronal Plasticity and the Fcrtility cycle: Somc Molccular Changes A3.3.1 GABA A3.3.2 Glutamate A3.3.3 Connexin A3.3.4 C- OS A3.3.5 BDNF
List of Figures
Figure 1
Figure 2a
Figure 2b
Figure 2c
Figure 3
Figure 4
Figure Sa
Figure 5b
Figure 6
Figure 7
Figure 8
Figure 9
Figure 10
Figure 1 1
Figure 12
Metabolic Pathways of Sex Hormones
Systemic Plasma Hormone Concentrations During the Menstnial Cycle in the Human
Systemic Plasma Hormone Concentrations During the 4-Day Estrous Cycle in the Rat
Feedback Control of the Hypothalamus and the Pituitary by Progesterone and Estrogen a Different Times of the Fertility Cycle
The Anti-convulsant Effects of Progesterone in the Kindling Model Progesterone Was Administered IP or SC
The Anti-convulsant Effects of Progesterone in the Kindling Model Progesterone Was Administered in Cyclodextrin and Corn Oil
The EfTect of Estrogen on Aftu-discharge Threshold
The Anti-convulsant Effects of Progesterone in the Kindling Model Subejects Were Estrogen-Primed
The Anti-convulsant Effects of Progesterone in the Kindling Model Intact Subjects Were Used
The Anti-convulsant Effects of Progesterone in the Kindling Model Subjects Were Gonadectomized and Hormone-Replaced Males
The Effect of Chronic Progesterone and MPA on the Genetalized Convulsive Threshold in Amygdala-Kindled Rats
The Effect of Chronic Progesterone and MPA on the Threshold for ECS Seizures
The Effect of Chmnic Progesterone and MPA on the Threshold for MES Seinues
The Effect of Chronic Progesterone and MPA on the Latencies to the Onset of MET Clonic Seizures
The Effect of Chronic Progesterone and MPA on the Latencies to the Onset of MMT Tonic (Forelimb Extension) Seizures
Figure 1 3 The Effect of Chronic Progesterone and MPA on the Threshold Dose 92 of Penty lenetetrazol Required to lnduce Clonic Seizures (FLC)
Figure 14 The Anti-convulsant Effects of Progesterone in Adult, Female Wistar 99 Rats
Figure 15 The Anti-convulsant Effects of Progesterone in Adult, Male Wistar 102 Rats
Figure 16 The Anti-convulsant Effects of Progesterone in 1 May-old, Female 104 Wistar Rats
Figure 17 The Anti-convulsant Effects of Progesterone in 1 May-old, Male 105 Wistar Rats
Figure 18 The Influence of Indomethacin (1 Smg/kg) on the Anticonvulsant 116 Effects of Progesterone (1 00mgkg)
Figure 19 The Influence of RU486 (20mg/kg) on the Anticonvulsant Effects of 1 17 Progesterone (1 OOmgkg)
List of Tables
Table t Brain Distribution of Sex Hormones and Related Enzymes 23
Table 2 Past Studies on the Anticonvulsant Effects of Progesterone 40
Table 3 The Anticonwlsant EDSOs of Progesterone for the Suppression of 77 Kindled Seizures
Table 4 The Anticonvulsant EDSOs of Progestemne for the Suppression of 107 MMT and MES Seizures
List of Abbreviations
Pl3 3-alpha HSD
ADT
ADT
AED
AED
aMPN
CA
CAmg
CAMP
clMPN
Cyclodextrin
Cyclodextrin
DHEA
ElB
ECS
EDa
EEG
ER
EUE
ERa
ERP
FEBP
FLC
FLE
FSH
GABA-A
micro-Amps
m icrogram
3-alpha hydroxy-steroid dehydrogenase
afterdischarge threshold
afterdischarge threshold
antiepileptic dnig
antiepileptic drugs
anterior medial preoptic nucleus
ammon's hom
central arnygdaloid nucleus
cyclic adenosine mono-phosphate
caudal parts of the lateral periphery of the preoptic nucleus
2 hydroxypropyl-pcyclodextrin
hydroxypropy 1-beta-cyclodextrin
Dihydroepiandrosterone
estradiol benzoate
threshold electroconvulsive shock seizure test
effective dose (50%)
electroencephalogram
estrogen receptor
estrogen receptor elements
estrogen receptor alpha
estrogen receptor beta
fetoheonatal estrogen binding protein
forelimb clonus
forelimb extension
Follicular stimulating hormone
gamma-aminobutyric acid
XII
GCT
GD
GDX
GnRH
GRIP I
HLE
HPOA
ICV
ILS
IP
kg
LH
mA
MES
MET
mg
MMT
MPA
mRNA
NDv
ovBNST
OVX
PdMAmg
Pa
PND
PO
PR
PRA
PRB
prBNST
R .
rMPN
SC
SMRT1
general ized convulsive threshold
gestational day
gonadectom ized
generalized releasing hormone
glucocorticoid receptor interacting protein 1
hindlimb extension
Hypothalamus
intracerbralventricular
lateral septal nucleus
intraperitoneal
k i lograrn
Lutenzing hormone
milli- Amps
maximal electroconvulsive shock seizure test
threshold pentylenetetrazol seizure test
milligrarn
maximal pentylenetetrazol seinire test
medroxy progesterone acetate
messenger RNA
neurotoxic dose (50%)
oval nucleus o f the bed nucleus o f the stria terminalis
ovariectom ized
posterodonal part o f the medial amygdaloid nucleus
Progcsterone
Post-natal day
oral
progesterone receptors
progesterone receptor A
progesterone receptor B
principle nucleus o f the bed nucleus of the stria terminalis
maximal response
rostoral portion o f the medial preoptic nucleus
subcutaneous
silencing mediator for retinoid and thyroid hormone receptors
SRCl
StPA
TERP- I
steroid receptor coactivator 1
stria1 part of the preoptic area
truncated estrogen receptor product- 1
XIV
Chapter 1
Introduction
1 INTRODUCTION
1.1 Epilepsy
1 . Definitions
The "epilepsies" are a group of neurological disorciers characterized by
spontaneous, recurrent seizures (Engel and Starkman, 1994). ''Seizures" are episodes of
excessive neuronal activity that can be identified by characteristic 'spike' or 'spike and
wave' abnormalities in electroencephalographs (EEGs) (Burnharn, 1 997).
Epilepsy is a very common disorder of the cenaal nervous system (CNS),
affecting up to 1% of the population (Berg et al., 1996; Hauser et al., 1991).
Seizures occur in epileptic patients due to the abnonnally low seizure thresholds
seen in these patients (Scher, 1997). Al1 brains have the circuitry necessary to produce
seizures, and h g s that biock inhibition or enhance excitation will induce a seizure in
anyone (Engel and Starkman, 1 994). In non-epileptic people, seizure thresholds are high,
however, and spontaneous seizures do not occur. In epileptic patients enhanced
excitation - or insufficient inhibition - leads to lowered thresholds and spontaneous
seizures (Engel, 1996).
1.2 Types of Seizures
1.2.1 Common Seizure Types
Seizures cm be classified as "generalized, involving the entire brain, or "partial"
involving only a part of the brain (Proposal for revised clùùcal and
electroencephalographic classification of epileptic seizwes. From the Commission on
Classification and Terminology of the International League Against Epilepsy ,198 1 ).
There are a number of types of generalized seinires. The two most commonly seen are
"tonic-clonic" and "absence seizures". "Tonic-clonic" seizures involve a loss of
consciousness with tonic-clonic convulsions. Constant "spiking" is seen in the EEG
(Swoboda and Drislane, 1 994). "Absence" seinires involve a loss of consciousness
without convulsions. A pattern of three per second "spike and wave" is seen in the EEG
(Mirsky et al., 1986).
There are only two categones of partial seizures: "simple" partial and "cornplex"
partial (So, 1995). "Simple" partial seinves involve abnormal sensations, emotions or
movements. They do not involve an impairment of consciousness. EEG "spiking" is
seen in neo-cortical or limbic regions (Devinsky et al., 1988). "Cornplex" partial seizures
are episodes of confused behavior, during which the patient is out of contact with the
environment. Consciousness is said to be "impaired". "Spiking" is unilateral or more
commonly bilateral (Gastaut, 1970).
Partial seinires of any sort may "generalize" to the whole brain, producing a loss
of consciousness and tonicîlonic convulsions. The generalized seizure is then tenned a
"secondarily" generalized seinire (So, 1 995).
1.3 Tberapy for Epilepsy
1.3.1 Antiepileptie Drugs (AED's)
Currently, pharmacological treatment is the first line of defense against seizures.
In most cases, it is the best option available (Aiken and Brown, 2000).
Traditional drugs (such as phenytoin, carbarnazepine and phenobarbital) stop
seizures by either acting as GABA-A receptor agonists or by blocking calcium or sodium
channels (Macdonald and McLean, 1986). These drugs are called "first generation"
AED's in contrast to the mwer drugs which have brrn introduced recentfy. The newer
drugs are called "second generation" AED's (Loscher, 1998).
A number of "Second generation" AED's have been introduced in the past
decade. In general, they have the same mechanisms of action as the old AED's (Loscher,
1998) (see Appendix 1 ). Perhaps for this reason, the new AED's have failed to provide
significantly greater control of dmg-resistant seizures than the first generation AED's
(Loscher, 1 998) - although they do have fewer side e ffects (Rogvi-Hansen and Gram,
1995). Clinical success with non-drug therapies (such as the ketogenic diet) has proved
that drug - resistant seizures can be suppressed, and has provided hope that dmgs
working through novel mechanisms could be effective against drug-resistant seizures.
A discussion of both the older and newer dmgs that are on the market in Canada is
provided in Appendix 1.
1.33 Prognosis for Seizure Control
The majority of patients (about 60%) suffering from epilepsy can control their
seinires adequately with current medications (Shorvon, 1996). Approximately 20% of
patients attain partial control, and about 20% completely resist drug control (Shorvon,
1996). Patients with hg-resistant seinire are said to have "intractable" epilepsy.
Anticonvulsants that act through novel mechmisms rnay be more effective against
intractable seizures, and may lead to fewer and less severe side effects. It is possible that
drugs related to steroid sex hormones might provide anticonvulsant compounds that work
by a novel mechanism.
1.4 Steroid Scx Hormones and Epikpsy
1.4.1 Catamenial Seizures
The possibility that sex hormones may affect seizure thresholds is suggested by
the phenomenon of catamenial epilepsy.
Seizure fkquency in many wornen is related to the phases of their fertility cycle-
and. thus, to the levels of progesterone and estrogen (Herzog, 1999). Seinires in these
patients are called "catamenial". The prevalence of catamenial seizures is unclear, but,
estimates suggest that they occur in ten to seventy percent of the female epileptic
population (Backstmm, 1976; Bauer et al., 1995). The majority of women suffenng fiom
catamenial epilepsy have seinves of the complex partial variety (Curnmings et al., 1995).
Until recently, it was thought that catamenial exacerbation was due simply to a
high ratio of estrogen to progesterone, estrogen king thought to be proconvulsant and
progesterone king though to be anticonvulsant (Backstrorn, 1976). Henog (Herzog et
al., 1997a), however, has recently proposed that there are three distinct patterns of
catamenial seizures with different hormonal profiles: 1 ) Pattern One is seen in women
whose seizure frequency increases around the time of ovulation. This correlates with a
surge in estrogen levels (Herzog et a!., 1997b). 2) Pattern Two is seen in women who
have increased seizures during the entire luteal (ps t ovulation) phase. During the luteal
phase, progesterone (and estrogen) levels are normally high. Women displaying this
pattern of seizure frequency. however, rnay have chronically elevated levels of estrogen
or may have deficient levels of progesterone (Bonuccelli et al., 1989; Murri and Galli,
1997; Narbone et al., 1990). 3) Pattern T h e is seen in women whose seinues get worse
during the perimenstd period. This occurs at the end of the luteal phase when the
levels of both estrogen and progesterone are are dropping. This pattern may correspond
to a period of "progesterone withdrawal" (Herzog et al., 1997b).
Relative to progesterone withdrawai, Smith's group has recently shown that
progesterone withdrawal enhances excitation by rendering GABA-A receptors less
insensitive to the effects of endogenous GABA (Smith et al., 1998~).
Further evidence that seizure incidence is affected by sex hormones cornes from
studies that show seinire onset, or an increase in seizure fiequency, at puberty (Herzog,
199 1). Other studies have shown that seizure fiequency is reduced, or that seimres lose
their catamenial pattem, d e r rnenopause (Harden et al., 1999).
The phenornenon of catamenial epilepsy suggests that estrogen is proconvulsant,
and that progesterone is anticonvulsant. If progesterone is anticonvulsant, conceivably it
could be used as a treatment for epilepsy. The focus of this thesis is an in-depth
pharmacological study of progesterone as an anticonvulsant.
1.4.2 Steroid Sex Hormones: General Background
The steroidal rnetabolic pathway is illustnited in Figure 1. The best-known
steroid sex hormones are progesterone and estrogen, which are secreted primarily fkom
the ovaries in females, and testosterone, which is secreted primarily fiom the testicles in
males. Estrogen and progesterone are not exclusively found in females, however, and
testosterone is not exclusively found in males. Low levels of testosterone are found in
females (McNatty, 198 1). Likewise, males produce low levels of progesterone and
estrogen (Bemti, 1 998).
Peptide hormones released from the hypothalamus and the pituitary control the
release of steroid sex hormones in the periphery. The hypothalamus releases the peptide
gonadotropin-releasing hormone (GnRH) (Reindollar et al., 1985). GnRH controls the
release of the peptides follicle-stimulating hormone (FSH) and luteinizing hormone (LH)
from the anterior pituitary. FSH and LH control spematogenesis in males, which
involves the production of testosterone (Franz, 1988). They also control the development
of follicular growth and the corpus luteurn in fernales, which results in the production of
progesterone and estrogen (Baird and McNeilly, 198 1).
Steroid sex hormones released in the periphery enter the brain and provide
positive and negative feedback to both the hypothalamus and the anterior pituitary
(McNeilly, 1988). Since they have a high partition coefficient, they easily cross the
b lood-b rain barrier .
- Metabolic Patliways of Sex Hormones
20a hydrocortisol
20a, 22 dihydrocholesterol
20,P d - l u (Pt- SU)
pregnenolone
Sa rcducuK MI
androsterone
P4SOcI 1 Q (Z Fm)
stenediol Ir i P450e11 P3(2 FP)
Sa nductasc
2 -5a DH corticosterone
P450,I 103 3a hydroxystcroid dehydrogtnasc
corticosterone
P450,I 1 AS corticosterone
: 3a hydroxystcroid dchydmgcnase 111111 1. aldosterone
Hormonal release is relatively constant in males, but it is cyclical in fernales.
Figure 2 demonstrates the variation in levels of progesterone and estrogen during the
female fertility cycle. The fertility cycle in humans is divided into the follicular phase,
ovulation, and the luteal phase. A primary follicle contains an ovum surrounded by
several layen of granulosa and theca cells, which produce estrogen (and some
progesterone) (Baird and McNeilly, 198 1). In the follicular phase, a primordial follicle
grows to maturity - mainly under the control of FSH (Baird and McNeilly, 198 1). Just
prior to mid cycle, increasing LH and FSH secretion cause rapid follicular growth and
begin to alter the function of the granulosa and theca cells fiom the production of
estrogen to the production of progesterone (Fr= 1988). This culminates in a surge in
LH and FSH release, which causes ovulation (Franz, 1988). Ovulation is the expulsion
of the ovum fiom the follicle and the conversion of the granulosa cells to lutein cells.
Together, these cells make up the corpus luteum. The conversion is mainly under the
control of LH (Cooke, 1988). AAer ovulation, the luteal phase begins. The corpus
luteum produces large quantities of progesterone and estrogen (Cooke, 1988). Negative
feedback fkorn progesterone ont0 the hypothalamas leads to Iow levels of LH and FSH.
The end of the luteal phase is marked by the degeneration of the corpus luteum, which
results in a sharp drop in the levels of progesterone and estrogen. The degradation of the
corpus luteum also causes menstration, and the start of a new ovarian cycle (Franz, 1988).
1.4.3 Progesterone and Estrogen as Neurosteroids
Progesterone and estrogen are gonadai hormones that act peripherally, and also
enter the brain to provide feedback. In addition, they are "neurosteroids", which means
that they are both produced and catabolized within the brain (McEwen, 1992). The
enzymes that synthesize and metabolize sex hormones are widespread in the brain, and
are found in many structures outside of those involved in sexual function.
1.4.4 Synthetie and Metabolie Pathways
The synthetic pathways involved in steroidogenesis are shown in figure 1. They
are similar in the body and the brain. Progesterone is derived from pregnenolone, which
is derived fiom cholesterol (figure 1). Progesterone is metabolized to allopregnanolone
(figure 1 ). Through another pathway , progesterone is also the precursor for testosterone
and estrogen. Aromatase converts testosterone to estrogen.
The enzymes involved in the catabolism of progesterone to allopregnanolone are
two Cyp450 enzymes: 5-al pha reductase and 3-alpha dehyàrogenase. Allopreganaolone
is a potent agonist and an allosteric modulator of the alpha subunit of the OABA-A
receptor (Maitra and Reynolds, 1998). Brain distribution and other pertinent details of
these enzymes are provided in Appendix 2.
In the periphery, it is primarily the gonads, which synthesize sex steroids.
Catabolism o c c m primarily in the liver (Feuer, 1983). In the brain, both glia and
neurons contribute to steroidogenesis (Zwain and Yen, 1999). Astrocytes are the most
active steroidogenic cells. They produce both progesterone and dehydroepiandrosterone
(DHEA). Oligodendrocytes predominantly produce pregnenolones. Neurons
predominantly produce estrogens (Zwain and Yen, 1999).
1.4.5 Commercirlly Available Progesterone, MPA and Ganaxolone
Progestins are synthetic compounds that mimic progesterone. Progesterone and a
number of progestins are cornmercially available and are used clinically to treat a variety
of conditions. Indications include contraception, hormone replacement therapy,
amenorrhea, dysfunctional uterine bleeding and endometrial hyperplasia (Apgar and
Greenberg, 2000).
Progestins are categorized according to the time of market introduction or
according to structural derivation. Ethynoldiol diacetate and norethindrone are examples
of "estranes". Norgestrel, levonorgestrel, desogestrel, gestodene, and norgestimate are
examples of "gonanes". Medroxyprogesterone acetate (MPA) is an example of a
"pregnane" (Apgar and Greenberg, 2000).
MPA and natural progesterone have been used successfully in clinical trials to
treat catarnenial seimes (Herzog, 1995;Mattson et al., 1984). MPA is available in oral
tablets (Provera@) and in injectable form (Depo-Provera@). Natural progesterone is
orally administered in micronized progesterone tablets (PrometriumQ) and as a vaginal
gel (CrinoneO) (Apgar and Greenberg, 2000).
In the present studies, n a d progesterone and MPA were used. An important
di fference between these is that natural progesterone is metabol ized to allopregnanolone,
which enhances GABAergic activity, whereas MPA is not.
Ganaxolone is a synthetic steroid whose structure mimics allopregnanolone, the
progesterone metabolite. Like allopregnanolone, ganaxolone binds to the steroid site of
the GABA-A receptor, and increases chloride flux (Carter et al., 1997). Like most
GABA-A-acting drugs, ganaxolone exacerbates absence seizures (Snead, 1998). In
clinical trials for epilepsy, ganaxolone has had only mild beneficial effects on intractable
seizures (Kemgan et al., 2000;Laxer et al., 2000). Allopregnanolone itself may be usefbl
in pnventing the withdrawat effects of progesterone in catarnenial seizures (Reddy and
Rogawski, 2000).
1.4.6 Sex Steroids and the Brain: Receptors
Many lines of evidence suggest that sex hormones affect brain excitability
(McEwen, 1999) and seizure thresholds (Woolley and Schwartzkroin, 1998). Most
studies have found that estrogen is excitatory (and proconvulsant) and that progesterone
is inhibitory (and anticonvulsant) (Woolley and Schwaiukroin, 1998).
While the main focus of this thesis is progesterone and its receptors, estrogen and
its receptors will be discussed as well, since the two hormones are intimately related.
Progesterone and estrogen both cause physiological changes in the brain at the molecular
and cellular levels (Smith et al., 1998a;Woolley and Schwartzkroin, 1998) and at the
behaviod level (Steiner, l992), and there is some evidence of functional antagonism
between the two hormones (Brann et al., 1988). There are also instances of synergism
between the two (McNicol, Jr. and Crews, 1979;Williams et al., 198 1). It is difficult to
discuss the effects of one hormone without delving into the effects of the other. It is
possible that some of the anticonvulsant effects of progesterone relate to its functional
antagonism of estrogen.
1.4.6.1 Estrogen Receptors
The best-studied estrogen receptors are the intracellular receptors, which mediate
gene transcription. There are two types of the "classic" (intercellular) estrogen receptors
(ER'S): alpha estrogen receptors (ERa), and beta estrogen receptors (ERP). The two
isofoms are highly homologous in al1 domains except in the amino terminal (Delaunay et
al., 2000). Both isofoms are capable of transcriptional activation, although ERP has
weaker activity (Delaunay et al., 2000). After binding estrogen, the ER'S homo- or hetro-
dimerize and bind to the estrogen receptor response elernents (ERE), enhancing
transcription (Delaunay et al., 2000).
Estrogen is involved in the regulation of a multitude of genes, including the
induction of progesterone receptors (Blankenstein et al., 1995), c-fos (Giannakopoulou et
al., 200 l), glanin (Howard et al., 1997), gonadotropin recepton (Xiong et al., 1994) and
oxytocin (Richard and Zingg, 1990). Interestingly, estrogen quickly down-regulates its
own receptors (Brown et al., 1996a).
In the rat brain, both ERa and ERP are widely distributed (table 1) (Shughe et
al., 1998). In the limbic structures, ERa and ERP mRNA are most abundant in the nuclei
of the arnygdala. The lateral nuclei, however, lack both isoforms, and the central nuclei
lack ERP. Pirifonn, entorhinal, and isocortex are weakly labeled for ERa, but are more
intensely labeled for ERP. The sme pattern is s e m in the hippocarnpus, with pater
concentration of receptors in the ventral region. It should be noted that Shughure's data
contrast with Orikasa's findings that ERa, but not ERB, are present in the hippocampus
(Orikasa et al., 2000). Orikasa, however, used intact rats, whereas Shughure used
ovariectomized rats. Within the hippocampus, McEwen's group has found that ER are
highly concentrated in the hilus and that they are found only in internewons (Weiland et
al., 1997a).
Gender and age do not seem to affect ER distribution (Orikasa et al., 2000).
Males and females have similar levels of ER'S (in males testosterone is metabolized into
estrogen, which then binds the ER'S) (Brown et al., 1996b). Sex differences in ER
distribution, however, are found in the hypothalamus (Brown et al., 1996b), with females
having higher levels than males. Raab et al. found that in the rnidbrain ERa is present in
both the pre and postnatal periods, whereas ERP is only expressed postnatally (Raab et
al., 1999). ERa and ERP are both found in the brains of infants and children, although
levels of stimulation are low until puberty.
Significant interspecies differences do exist in ER distribution (Osterlund et al.,
2000b). As compared to the rat, primates lack ERa mRNA in the media1 nuclei of the
arnygdala. Primates also lack ERa mRNA in the amygdoloidal areas related to fear
(central and basolateral nuclei). The highest expression of ER in the arnygdala of
primates (including humans) is in the amygdala-hippocarnpal area. the posterior cortex
nucleus, the accessory basal, and the periamygdala cortex. There are significant species
differences in the hypothalamus and cerebral cortex also. In the hypothalamus, ERa
mRNA levels are very high in the paraventricular and supraoptic regions of primates as
compared to rats. In rats, ERP is highly expressed in these regions. In the cerebral
cortex, there is no larninar pattern of ERa distribution in rats. In the human, there are
' George, Ojeda, 1982 Laubcr, 1994 ' Lephari, 1992 ' MacLusky, 1985 ' Sanghera, 1 99 1 ' Pelletier, 1994 ' ducna- rai ta, et al. ZOO0 : Shughrue et al. 1997
Shughnic et al. 1998 'O brikasa et al. 2000 " hagihara, et al. 1992 l2 $hughure, Lane, 1997 " kiland, Orikasa, 1 997
14 Compagnone, Mellon, 2000 l 5 Camacho-Arroyo et al. 1998 " Lauber et al. 1991 " Szabo et al. 2000 " Cammancho-Arroyo, 1 998 'O Shinoda, 1994 l9 Brown et al., 1987 " Lauber, Lichensteiger, 1994 " MacLusky et al. 1994
Lauber, Lichtensteiger, 1996 " Poletti, Martini, 1999 '' Cheng, 1994
Reâieiase ( HSD 1 ERa
+" u -
+a
+++" ++-ta
+++-ta
+++" -H-+~
0
a
+-+-kW
+"
++agir
++O
+" - a
O - +&
ERP
a - +'
+++a'1'
fa
++-++a
++-ka
+' ++lu
+tu*'L ++++"em
+' +"
- 6
-t+" uU
++@*IL
II - +++O
.t+r
Brain Region ER
Subtbalamic nucleus +" Habeaula
a Media1 (Habenula) - Lateral (Habenula) .tu
Hypothalamus +teo Preoptic area ++a*n
Lateral +&
Media1 ++++" Periventricular
Pefiventricular nucleus ++"*'o
Pqaventricular nucleus Su)mchiasmatic nucleus ttm S9raoptic nucleus +$-tu
Arpuate nucleus ++' Dqrsalmedial nucleus +' F e r a l hypothalamus +" Vqtralmedial nucleus tu Pr(mammil1ary nuclei ++O
Su~ramammillary nuclei +-ka
T u ~ r o m a m m illary nuc lei Mammillary nuclei Subcomissural organ
Pituitary Basil ganglia Cerebellum Medulla
Mem. ER
wu
PR
* ~ l . l b
++lb
+++"
-ulb
+t Io
++l
++I
A
PRA
+ I I , IU .~
+l itia.T3
+ I V
+IY
+fU ++l l
+++l '
utii
PRB
++li,m
++l
+Iy
Mem. Aromatase
+ l 0Lr)t4*1J
+l J4*4*14
low levels of ERa in al1 cortical areas, and layer five of the temporal cortex expresses
higher levels of ERa rnRNA. In the human hippocampus, both ERa and ERP are present
(with ERP being more prominent in the hippocampus than in any other region of the
brain) (Osterlund et al., 2000a). In the rat, however, only ERa is present in the
hippocampus (Orikasa et al., 2000). Osterlund notes that ER distribution in human brains
is in areas involved in emotion, memory and cognition (and also endocrine fiction of
course) and may reflect estrogen's role in these fùnctions. Furthemore, estrogen levels
are significantly different between primates (including the human) and rodents - primates
having much higher levels than rodents. Therefore, estrogens or anti-estrogens may have
differential effects on behavior in rats and humans.
Recently, cell-surface (membrane) receptors for estrogen have also been described
(Ramirez et al., 1996). These receptors do not have transcriptional activity and
presumably mediate changes in membrane excitability (Ramirez et al., 1996). The "fast"
or "non-genomic" effects of estrogen have k e n attributed to these membrane receptors
(Wong et al., 1996).
Functionally, at a molecular level. cell-surface estrogen recepton have been found
to: 1 ) activate the adenylate cyclase system (Morozova et al., 1 WO), 2) induce the
phosphorylation of the CAMP response element (Zhou et al., 1996), and 3) stimulate
nitnc oxide synthase activity by increasing intracellular calcium concentrations (Baldi et
al., 2000;Stefano et al., 2000). Their net effect on ion channels is to: 1) decrease L-type
calcium currents (Mermelstein et al., 1996), and 2) increase kainate-induced currents (Gu
et al., 1999). These effects are excitatory in nature (Joels, 1997). The relevance of these
recepton to the effects of estrogen on behavior, or brain excitability, is not well
understood yet.
1 A6.2 Progesterone Receptors
Like the ER'S, the "classic" progesterone receptors (PR) are intracellular. They
also corne in two fonns: progesterone receptor A (PR-A) and progesterone receptor B
(PR-B). PR-A is identical to PR-B except that PR-A is amino-terminal muicated
(Giangrande et al.. 1997). PR-A is missing the first 164 amino acids thai are present in
PR-B. The two isoforms are obtained fiom the same copy of the PR gene, but they have
distinct estrogen-inducible promoters (Giangrande and McDonnell, 1999).
PR-B is a transcriptional activator. PR-A, on the other hand, lùnctions as a
transcriptional repressor (Giangrande and McDonneII, 1999). PR-A inhibits the
transcriptional activity of al1 steroid hormone receptor genes. The opposing
transcriptional activities of the two isoforms are mediated through distinct signaling
pathways (Wen et al., 1994). This is due to differential cofactor binding. PR-A has a
higher affinity for the corepressor Silencing Mediator for Retinoid and Thyroid hormone
receptors (SMRT). PR-B has a higher afinity for the coactivators Glucocorticoid
Receptor Interacting Protein 1 (GRIPI) and Steroid Receptor Coactivator 1 (SRC 1)
(Giangrande et al., 2000).
PR-A and PR-B are estrogen-inducible, but they also engage in 'cross-talk' by
affecting ER transcriptional activity (Katzenellenbogen, 2000). PR-A is a trans-dominant
repressor of the transcriptional activity of other sex and stress hormones. This effect
involves a noncornpetitive interaction through distinct cellular targets or distinct contact
sites within the same target (Wen et al., 1994). The repressor effects of PR-A, including
its anti-estrogenic effects, can be induced by progesterone receptor antagonists such as
RU486 (mifepristone) (McDonnell and Goldman, 1994) or ZK98299 (onapristone)
(Vegeto et al., 1993). Hence it appears that these cornpounds have agonist activity with
PR-A.
PR'S are involved in the regulation of many genes. These include the GnRH
(Kepa et ai., 1996) and FSH genes (OIConner et al., 1997), as well as the genes related to
ER'S.
Similar to the ER, PR-A and PR-B have a pattern of brain distribution that
extends far beyond the hypothaiamic nuclei involved in sexual function. Within the
isocortex, layers two and four express high levels of PR. The cortical nucleus of the
amygdala also expresses high levels of PR (Hagihara et al., 1992) (note: Hagihara did not
distinguish between receptor sub-types). Both PR-A and PR-B are equally expressed in
tne hippocampus (Guerra-Araiza et al., 2000). Within the hippocampus, the pyramidal
layers of CA 1 and CA3 fields express high levels of PR (Hagihara et al., 1992). The
normal f ic t ion of these widespread receptors is not clear. They may, however, play a
role in progesterone's anticonvulsant effects.
PR levels change during the estrous cycle. This is probably due to the estrogen-
inducible nature of the PR. Therefore, the question arises as to whether estrogen
synergizes progesterone's anticonvulsant effects or whether progestemne antagonizes
estrogen's proconvulsant e ffect? The interaction between estmgen and progesterone may
be important in progesterone is actions as an anticonvulsant. It is important to note that
both PR-A and PR43 expression can be estrogen sensitive or insensitive (Camacho-
Arroyo et al., 1998b). This sensitivity is determined by the region of the brain and not
necessarily (but possibly) by the receptor isiform. The PR levels Vary during the estrous
cycle in the hypothalamus a d the frontal cortex, but the hippocampal receptors are not
affected (Guerra-Araiza et al., 2000). In the hypothalamus, both isoforms are induced by
estrogen and down-regulated by progesterone(Camacho-Arroyo et al., 1998a). Estrogen
treatrnent does not affect PR mRNA in the amygdala of either male or female rats
(Lauber et al., 1991).
PR'S are found in the brains of both females and males. There are some
male/female differences in the distribution and levels, however. These differences are
most evident in regions of the brain related to sexual fùnction such as the hypothalamus
(Khisti et al., 1998). There is no significant gender-related difference in estrogen-induced
PR in cortical regions.
PR's are also found in pre-pubertal brains. In the cortex, the intracellular PR is
detectable at post-natal day one. PR levels rapidly increase, starting on day seven, and
reach maximum levels (higher than adult) at day ten. Thereafter, PR expression drops
gradually to adult levels. A sirnilar early pattern of expression is seen in the
hypothalamic preoptic area, except that levels remain constant after day fourteen (Gee et
al., 1988). The ontogenic development of PR's is sirnilar in both sexes (with the
exception of particular nuclei mentioned above).
Recently, cell-surface PR's have been discovered. These are distinct fiom the
"classic" intracellular PR's. Little is known as yet about the function of these ce11 surface
receptors, although the "fast" effects of progesterone have been attributed to them (Joels,
1997). These include inhibition of purkinje neurons (Smith, 199 1) and stimulation of
sperm functions (Calogero et al., 2000). Recently, Momson's group has s h o w that
membrane PR'S stimulate tyrosine kinase activity, which results in phospholipase C
activation (Momson et al., 2000). In sperm, progesterone enhances calcium currents
through its membrane receptor (Foresta et al., 1993). M i l e these effects should enhance
excitability, membrane PR's are curiously associated with decrease excitability in
neurons (Joels, 1997).
1.4.7 Some Other Notable Neuro-active Steroids
Progesterone, allopregnanolone and estrogen are not the only neuro-active
steroids involved in the normal and abnormal h c t i o n of the brain. Stress hormones,
androgens and pregnenalone cm also alter neuronal excitability (Rupprecht and Holsboer,
1 999).
Administration of stress hormones (Le., corticosterone) generally increases
seinire thresholds in both animals (Stitt and Kinnard, 1968) and in humans (Aird 195 1).
Paradoxically, stress can trigger seizws in some epileptic patients (Frucht et al., 2000;
Spector et al., 2000).
Testosterone appears to be proconvulsant (Rosse et al.. 1 990). This may be due to
the conversion of testosterone to estrogen (Edwards et al., 1999a).
Pregnenolone sulfate is proconvulsant and dec~ases seinire thresholds (Kokate et
al., 1999; Maione et al., 1992; Reddy and Kulkarni, 1998). Pregnenolone does this in two
ways. Fint, pregnenolone antagonizes GAB A-A receptoa (Majewska et al., 1 988;
Majewska and Schwartz, 1987; Mienville and Vicini, t 989). Second, it allosterically
potentiates NMDA receptors (Wu et al., 1991).
1.5 Animal Seizure Models
The fact that sex hormones affect excitability in many regions of the brain - and
that some of the effects are inhibitory - suggests that sex hormones might be used
pharmacologically to control seizures. The focus of the present thesis was the
pharmacological antagonism of seizures with progesterone. The work was done using
animal seizure models. It was done to supplement past clinical studies, and to provide a
basis for fbture clinical work.
Animal seizure models are used in drug development because unproven potential
AED's cannot be tested legally or ethically on epileptic patients. These models should
reliably produce seizure activity. They should also be easy to use and have clinical
relevance (Fisher, 1 989).
A number of animal models are used to study seizures and the epilepsies (Fisher,
1989). Most cornmonly, acute "seizure" models are used. These involve electrical brain
stimulation or the systemic administration of various chernical convulsants. In these
models, seinws do not occur spontaneously. They occur only when the experimenter
appks the epiteptogenic stimuius (Fisher, 1989).
Some investigators have argued that these acute preparations do not fùlly modei
clinical epilepsy since the seizures are not spontaneous (Avanzini, 1995). A number of
chronic "epilepsy" models - where the seizures occur spontaneously - do exist (Fisher,
1989). Some of these are genetic models, whereas others involve post-status
"spontaneous recurring seizures" (SRS) (Niuinen et al., 2000).
While these spontaneous models have some attractive features, they have never
been validated pharmacologically. To test the anticonvulsant properties of new
compounds, models with known and valid phannacological profiles are necessary.
Therefore, acute, validated models have been used in the present studies. These models
are discussed below.
1.5.1 Electroconvulsive Sbock Seizures: Maximal (MES) and Tb reshold
@CS)
The maximal electroconvulsive shock seinw (MES) model is the standard animal
model for human tonic-clonic seizures (Woodbury, 1972). High-intensity electrical
stimulation is passed through the subject's brain using comeal or pinneal electrodes.
Usually, mice or rats are used. Standard current parameters are 60 Hz sine-wave current
for 0.2 seconds at 50 mA in mice or 150 mA in rats (Swinyard, 1972). The seizures
begin with a brief period of tonic flexion, followed by forelimb and hind limb tonic
extension. Anticonvuhnts that suppress the tonic hindtimb extension in rodents - such
as phenytoin and phenobarbital - also suppress tonic-clonic seizures in man (Woodbury,
1972).
Lower-intensity electrical currents - applied in the same manner - produce
seizures that are exclusively clonic (Woodbury, 1972). These submaximal seizures are
called "threshold" electroconvulsive shock seizures (ECS). Anticonvulsants that suppress
forelimb clonus in the "threshold" ECS mode! - such as ethosuximide and valproic acid -
also suppress absence seizures in man (Woodbury, 1972).
In administering ECS or MES, investigators should be aware of species, gender
and age differences. With regard to species, the threshold for ECS and MES is higher in
rats than in mice (Swinyard, 1972).
With regard to gender, females tend to have lower seinire thresholds than males
(Kokka et al., 1992; Sackeim et al., 1987). The ECS threshold for female rats is 16 rnA,
for instance, and for males it is 19 rnA (Kokka et al., 1992).
With regard to age, developmental variaiions are particularly dramatic (London
and Buterbaugh, 1978). Immediately afler birth, rats have remarkably high ECS and
MES seizure thresholds (over 40 mA for the clonic threshold, over 160 rnA for the tonic
threshold). The threshold drops rapidly to base-line levels over the first three weeks of
li fe. At day 2 1, the threshold for clonic seizures is approximately 1 5 mA and for tonic
seizures, appximateiy 30 mA. Thereafter, thRsholds rise slightly into adul thood
(London and Buterbaugh, 1978).
1.5.2 Pentylenetetrazol: Maximal (MMT) and Threshold (MET)
Pentylenetetrazol (also called "Metrazol" and "PTZ") is a chemical convulsant,
which antagonizes GABA-A receptoa non-selectively (Pellmar and Wilson, 1977).
A subcutaneous injection of a high dose of pentylenetetarazol(8Smg/kg in rat)
produces forelirnb and hindlimb tonic fiexion and extension in rats. This is the "maximal
metrazol seizure" model (MMT) (Fisher, 1989). Like the tonic seizures induced by
electrical currents, MMT seizures mimic tonic-clonic seinves in man (Woodbury, 1972).
A lower subcutaneous injection of 7Omgkg in rats (8Smgkg in mice) of
pentylenetetrazol produces clonic seizures. This is the "threshold metrazol seinin"
model (MET) (Stone, 1972). Like the c h i c seizures produced by electrical stimulation,
MET seizures mimic absence seizures in man (FerrendeIli et al., 1989).
There are also species, gender and age differences in the MMT and MET models.
With regard to species, rats require slightly lower doses of the convulsant than mice to
produce clonic or tonic seinires (Fisher, 1989).
With regard to gender, females have higher seizure thresholds than males (Kokka
et al., 1992). Twenty-h~o mgkg of tail-vein infused pmtyienetetmot produces clonic
and tonic seizures in female rats. The sarne response is seen with 17mgkg in male rats
(Kokka et al., 1992).
With regard to age, two conflicting alterations occur. Infant rats have very high
seizure thresholds that drop over the fint three weeks of life. After that they rise again.
Curiously, although the thresholds are high in infants, the latency to the onset of seizures
is much shorter in infants than in adults (Velisek et al., 1992).
1.5.3 Kindling
Kindling is a technique for producing focal seizures with secondary generalization
(Goddard et al., 1969;Racine, 1972). in the kindling model, an electrode is pemanently
implanted in the brain of in an animal subject (oflen a rat). The subjects are then
stimulated at intervals (often daily) with a low level of electrical stimulation. At first,
only a localized ("focal") seizwe occurs. With repeated stimulations, however, the
seizure activity spreads to other sites (or "generalizes"), as measured by the
electroencephalograph (EEG). Eventually, afier motor structures have become involved
convulsions occur (Racine, 1972).
When the kindling electrode is implanted in limbic structures, such as the
amygdala or the hippocarnpus, the kindling preparation models complex partial s e i m s
with secondary generalization (Albright and Bumham, 1980). Albright and Bumharn
showed that focal limbic discharge in kindled subjects has a pharmacological profile
similar to human complex partial seizure (Albright and Bumham, 1980). They found that
anticonvulsants successfully suppressed the generalized component of seizures, but were
iinable to raise seizure thresholds in the focus at clinically relevant doses (Albright and
Burnharn, 1980; Albright, 1 983). In humans, anticonvulsants also suppress secondaril y
generalized seizures, but fail to suppress lim bic focal (corn plex partial) activity . Hence.
the kindling mode1 is a pharmacologically valid mode1 of human complex partial
seizwes.
Species, gender, and age also affect kindling. Al1 species that have k e n tested
(including primates) kindle (Wada et al., 1978), (Wada, 1981). Seinire thresholds and
the rate of kindling, however, Vary with the species. Higher animals, including primates.
tend to kindle more slowly (Wada, 198 1).
With regard to gender, differences in kindling rate or seizure threshold between
males and females have not been studied. There are, however, reports on the effect of the
sex hormones on kindling thresholds and kindling rates. With regard to estrogen.
Edwards et al. found that it lowered seizure threshold and increased the kindling rate
(Edwards et al., 1999b). Buterbaugh also saw a similar increase in kindling rate with
estrogen (Buterbaugh, 1987). WahnschafTe, however, did not observe variations in
seizure threshold across the estrous cycle (WahnschafTe and Loscher, 1992). With regard
to progesterom, Edwards et al. (1999) have found that it raises the seizure threshold and
decreases the kindling rate.
With regard to age, Moshe (1998) has shown that rat pups kindle at a faster rate
and to higher seizure stages than adult subjects. Kindling threshold in rat pups has not
been reported. Recently, however, Edwards et al. (unpublished data) have show that rat
pups (fourteen days old) have remarkably high seizure thresholds.
1.6 Past studies of Progestin Effects on Seizures
1.6.1 Clinical Studies
Clinicians have used progestin therapy to control intractable catamenial seizures
for more than half a century. Table 2a presents a sumrnary of the case studies and open
trails that have used progesterone or medtoxyprogesterone acetate, a synthetic progestin.
Positive results have been reported in al1 studies. Although the earlier studies were only
case reports, Herzog (1988, 1995) and Matson's (1984) studies were actual clinical trials.
Matson used medroxyprogesterone acetate (MPA), whereas Herzog used natural
progesterone. Signifiant and similar anticonvulsant effects were shown in both studies.
Despite this success, progestins have not found widespread use even in the
treatment of catamenial epilepsy. Moreover, no clinician has yet ventured to try them on
the non-catamenial epileptic population. Part of the reason for this failure may be that no
"blind" (single or double) clinical studies have as yet been performed to evaluate the
anticonvulsant effects of progesterone. Equally lacking are convincing animal studies to
support the clinical observations.
1.6.2 Animal Studies
A number of past animal studies have investigated the anticonwlsant effects of
progesterone. Unfonunately, many of these have used unreasonably high (sedating)
doses. When lower (therapeutic) doses have been used, the data have been inconsistent.
Table 2b and table 2c provide a summary of these studies. Table 2b summarizes studies
involving high doses of progesterone (over 30 mgkg) and table 2c surnrnarizes studies
involving low doses (under 5 mgkg).
The high dose data (Table 2b) are not contradictory. High doses of progesterone
are clearly anticonvulsant (Table 2b). These anticonvulsant effects cm be seen in the
MET, the MES and the kindling models. The anticonvulsant doses used in these studies,
however, are highly sedative (Table 2b), and they produce blood levels that are well
above physiological levels (Finn and Gee, 1994). These studies do not resemble the
clinical trials in which women have received relatively low doses of progesterone and
progestins (Table 2a), which produce plasma levels within the physiological range. In
clinical trials, patients have not suffered from excessive sedation (Table 2a).
The low dose data (Table 2c) an less cornpletc and consistent. In the kindling
model, for instance, Holmes and Weber (1 984) found that progesterone retarded quick
kindling in infants, but not in adult male rats. Edwards et al. (1999) reported that
progesterone retards kindling in adult female rats. Also in this model, Mohammad et al.
( 1998) found that progesterone doses below 75 rng/kg are not anticonvulsant in male rats.
Edwards et al. (1 999), however, found significant anticonvulsant activity at 5 mgkg in
female rats. These data seem to suggest that progesterone, at low doses, works in female
rats, but not in male rats.
Wooley and Tirnaras (1 962), testing electroshock threshold, found that low dose
progesterone partially countend the small threshold drops caused by estrogen in
ovariectomized adult female rats. Stin and Kinnard (1 968), however, found that low dose
progesterone in female rats was not protective in the MES model. These data might
suggest that progesterone works against threshold, but not maximal seinues. Once again,
no direct cornparisons have been made.
In the threshold pentylenetetrazol model, dose-response studies have s h o w
anticonvulsant effects of progesterone at high doses only . Kokate ( 1 998) did these
studies in adult male mice, and Craig (1 966) did them in both male and female mice.
In kainic acid seimres, Nicoletti et al. (1985) found anticonvulsant effects using
low doses of medroxyprogesterone acetate. In this model, Frye and Bayon (1 999) have
also found anticonwlsant effects using low doses of progesterone. These investigaton,
however, have used latency measures. Changes in latency generally imply small changes
in seizure thresholds.
To summarize, at low doses of progesterone - which resembte the doses used in
clinicai studies - the findings are incomplete and inconsistent.
Table 2a: Clinical studies of progesterone (P,) as anticonvulsant
Patients
R.H., et al., (1 984)
Zimmennan, A.W., et al., ( 1973)
case report
' adult woman Open dinical trial 14 adult women
Case report adult woman
Epilepsy
8ackstrdm, T., et al., (1 984)
Herzog, A., ( 1 986)
Herzog, A., (1 995)
Herzog, A., ( 1 999)
Generalized Seinires (catarnenial)
Open clinical study 7 adult women 22-4 3 Y cars old Open clinical trial 8 adult women 1 6-4 1 Y =an old Open clinicat trial 25 adult women 18-40 years old Open clinical trial 15 adult women (3 year follow-
Primidone 250mg, Phenobarbital 30mg & 1. Povera' ( 1 0,20,50
mg/day 2. Depo-Provera
(250,250,150 mg12 wcek intervals)
1
Generalized 1 90mg Phmobarbital ton ic-clonic seizures (catamenial) 13 complex partial, 1 absence al1 intractable
& Progesterone (0.35mglday)
Prior medication & Provera (in gpatients, IOmg, q2=4d, PO) Depo-Provera (in 6 patients, 120- 1 SOmg, IM)
Complex partial Prior medication with one distinct focus, sclected based on greater than 1 epileptic discharge per 5 minutes of EEG
& P4 (IV in alcohol & Ringers Glucose solution, 0.5-3 mg bolus + 4- 12 mghr infusion, achieved luteal phase levels, 72nmoVL )
paroxy smal 50-400 mg natural P, q2d of temporal during times of high seinire origin, catamenial)
Complex partial (with secondary generalization, 13/25) (catamenial)
Complex partial (catarnenial)
frequency (achieved luteal phase levels, 5-25ng/mL)
200 mg natural P, lozenges q2d during times of high seizure fiequency (achieved luteal phase levels, S- 25ng/m L)
200 mg natural P4 lozenges q2d during times of high seizure fiequency (achieved luteal phase levels, 5- 25nglmL)
1. No effect; 50 mgkg showcd slight efficacy
2. Complete cessation of seizures for 4 months
Adverse cffects: amenonhea
Complete seizure control over 7 months Adverse effects: axnenorrhea 39% reduction in seizure fiequency , (3 patients withdrew fkom swdy Adverse effects: arncnorrtiea, spotting Significant reduction in hzquency of epileptic discharges (4/7 patients)
68% reduction in seizure frequency Adverse effects: transient t iredness, depression (4/8 patients)
54% reduction in seizure frequcncy Adverse effects: asthenia, depression (U25 patients)
62% reduction in seizure fiequency
Table 2b: Anixnai studies involving high-dose progesterone (P,)
Selye, H., ( 1 942)
Spiegel, E., & Wycis, H., ( 1 945)
Craig, CR., (1 966)
Craig, C.R., 8t Deason, J.R., (1 968)
Selye, H., (1 970)
Mohammad, S., et al. ( 1998)
Kokate, T.G., et al., ( 1 998)
Subjects
White rats Young 8
White rats e Sw iss- Webster m ice 20-30g 8 & 9
Swiss- Webster m ice 20-30s 8&Q Holtzma n rats lOOg 9 White cats 260- 700g (3 NIH Swiss mice 25-50 g 6
Seizure Mode1
Pentylenetetrazol
E lectroshock seizure threshold
1 . Penty lenetet m o l 85 mgkg* SC
2. Maximal electroshock seizure, 50 mA (0.2 sec)
Picrotox in 3.5mgkg SC
Kindling (amygdala) 5 consecutive stage 5s
1. Pentylenetet raz01 85 mgkg* SC
2. Maximal electroshock seizure 0.2s, 60Hq 50mA
P, dose-response: (0- 10oomgn<g)
P, (chronic, 100mg/kg, q2d)
P, dose-responsc: (0,10,30,60,75,90mg/kg)
1. P, dose-response: (50-200mg/kg)
2. P, dose-response: ( 1 00-400mg/kg)
Results
T Seizure threshold Adverse effects: anesthesia
1. o Seizure threshold 2. T Adverse effects: hypnosis, anesthesia T Seizure threshold 1 . EDw=200mg/kg 2. ED,e300mg/kg Adverse effects: acute neuro-toxicity, N Ds0=720mg/kg
7 Seinire threshold EDm=200mg/kg Adverse effects: acute neuro-toxicity, NDso=720mg/kg ? Seizure threshold Adverse effects: catatoxic
? Seizure threshold only @ 75 m g / k Adverse effects: mi Id sedat ion @3 Omg/kg, severe sedation @90mg/kg 1. T Seizure threshold
EDm=94mg/kg 2. ? ED,o=250mg/kg Adverse effects: sedation
Table 2 C : Animal studies invoiving low-dose ptogesterone (P#j
Woolley, D., & Timaras, P., ( 1962)
Stitt, S.L., & Kinnard, W.J., ( 1968)
Holmes, G.L., & Weber, D.A., ( 1984)
Nicoletti, F., et al., (1985)
Landgren, S., et al.
Frye, C., & Bayon, L., ( 1999)
Edwards, H., et al. ( 1 999)
Subjects
Long Evans Rats lmmatur e & mature 9 (OVW
Wistar Rats 125- 150 g 9
Sprague -Daw ley raïs Immatur e T (15 & 30 days) & Adult $ (36-70 day s) Wistar rats 200g 8&9
Cat 9 ( O W Long Evans rats Young Adult (aprox. 60 days) 9 W X ) Wistar rats 200- 225g 3
Seizure Model
Electroshock seizure threshold ( Woodbury paradigm, 1952)
Electroshock seizure threshold ( Woodbury paradigm, 1952)
Kindling (am ygdala) Kindled houriy and daily
Kainic acid 1 5 SC
Penicillin focus (cerebral cortex)
1. Kainic acid, 32 mg/kg, SC
2. Kindling (Perforant path, for 1 hr, 0.1 ms, 20 Ht, I2.W)
Kindling (am ygda ta) 15 consecutive stage 5s
1. Chronic E,B (40,l OOpglkglday)
2. Chronic P4 ( Smg/kg/day) 3. Chronic &B+P,
1. P4 (acute, 70mgkg) 2. P4 (chronic, Smgkg) 3. MPA (chronic, lOmg/kg) 4. MPA+E, (chronic,
1 Om!#kg)
P4 (5,25 mgkg, hourly & daily)
MPA (chronic, 2.5 rng/kg)
1. E,B+P4 & withdrawal; (GB: lOpg/kg@Ohr, P,: O.Smg/kg @44hrs, kianic acid @48hrs)
2. Silastic implants (cholesterol, E&B+P,, intermittent E$+P4)
Results
1. $4 Seizure threshold 2. 7' 3. 4- Adverse effects:
arnenorrhea in intact adultÇ?
I . t, Seizure threshold 2. t, 3. O 4. 4 Adverse effects: amenorrhea 3 Kindling rate in immature rats * in mature rats Adverse effects: sedation @2Smg/kg in immature r
9, 4 Severity &++latency 0. &t,
& Spontaneous interictal spikes
1. 4 Seizure duration 2. 5.
? Seizure threshold
1.7 Objectives
1. Past expenments from this laboratory have suggested that progesterone is
an effective anticonvulsant at a low dose (5mg/kg) in the kindling model of complex
partial epilepsy (Edwards et al., 1999b). Adult female rats were used and only a single
dose of progesterone was tested. The first objective of these snidies was to perfocm a full
pharmacological study of progesterone in the kindling model. Dose-response studies
were done. These data are described in Experiment 1.
2. The second objective of these studies was to determine whether low dose
progesterone was an effective anticonvulsant in other seizure models, especially when
applied chronically.
In addition, MPA was tested. MPA has pharmacokinetic advantages over
progesterone, and it is not converted to allopregnanolom (Sturm et al ., 199 1). Thercfore,
it was hoped that MPA would provide valuable information about the mechanism of the
anticonwlsant action of progesterone. Thesa data are reported in Experiment 2.
3. The third objective of these studies was to perfom dose and time-response
studies in both immature rats and in adult males. This was to determine whether
progesterone could be used in chilcihoocLepilepsies, or in adult men. These chta are
reported in Experiment 3.
4. The forth objective of these studies was to determine whether
progesterone's anticonvulsant effects were mediated via binding to progesterone
receptors (intracellular or cell surface), or whether they mlated to progesterone's
neuroactive metabo l ite, al lopregnanolone. Progesterone' s anticonvulsant effects were
assessed in the presence of RU486 - which blocks the "classic" progesterone receptors -
and in the presence of indomethach - which blocks progesterone's conversion to
allopregnanolone. These data are reported in Experiment 4.
Chapter 2:
General Methods
2 General Methods
Wistar rats (Charles River, Canada) served as subjects. The age and ser of the
subjects varied and is specified for each expriment. Subjects were housed in transparent,
plastic cages (24x24~45 cm) in a vivarium maintained at 18OC on a 12-hour light/dark
cycle (lights on at 7:00 8.m.). Adult subjects were housed individually, while immature
subjects were housed with their dams. All subjects were given fiee access to food (Purina
Rat Cho*) and water. On test days, subjects were transported to the test room in their
home cages and were allowed to acclimatize for 30 minutes prior to experimentation.
2.2 Drugs
Medroxyprogesterone acetate and indomethacin were obtained from a local
phmacy . Al1 other dmgs including pmgesterone, estrogm, testostezone, and beta-
hydroxy cyclodextrin were obtained fiom Sigma-Aldrich.
2.3 Procedures for the Kindling Expcriments
2.3.1 Implantation of Electrodes
Two weeks (minimum) after amval in the animal facility, subjects were
anesthetized with sodium pentobarbital (Somnitol@, 40mgkg for female subjects,
6Omg/kg for male subjects, IP) and their heads were fixed in a stereotaxic instrument.
Heads were sterilized with 70% alcohol, and an iodine solution. A saggitd incision (1.5
cm) was then made in the scalp, and the superficial muscles were separated from the skull
with Q-tipsm. The skull was washed with normal saline solution, and bregma was
located. Three holes were drilled (each 5 mm fiom bregma) in three quadrants of the skull
and jeweler's screws were inserted to act as anchors for the implant. A hole was then
d d e d at the implant site and a bipolar electrode was lowered into the right amygdala
using the following coordinates: 1 mm caudal fiom bregma, 4.8 mm lateral from the
saggital suture, 8.5 mm ventral from the dura, The incisor bar was set at +5 mm . The
electrode was cemented in place with a mixture of cranioplastic powder and cranioplastic
liquid (Cranioplastics One Inc., Roanoke, VA).
Etectrodes consisted of two pot yunide@-insulated, stainless wi res (0.25 mm
diameter) (MS303f1, Plastic One, Roanoke, Virginia). Subjects were allowed 9 days for
post-surgical recovery, and then daily handling was begun.
2.3.2 Kindling Procedure
Fourteen days afier surgery, kindling was initiated. Kindling was prefonned in a
plastic stimulation cage (width: 40cm, length: 50cm, height: 30cm). Daily stimulations
were administered between 12:OO and 6:00 PM. The stimulus consisted of a 1s train of
lms, 6OHz biphasic (positive - and negative - going), square-wave pulses, 500 UA (peak
to peak), generated by Grass Model S88 stimulator coupled to two Grass PSIU6 constant-
current stimulus-isolation units (Grass Instruments Co., Quincy, MA). Convulsive
responses were scored according to Racine's (1 972) 5-stage scale of motor seizures: stage
1) mouth clonus; stage 2) head nodding; stage 3) forelimb clonus; stage 4) rearing; stage
5) loss of postural control. Electroencephalographic (EEG) activity was recorded for the
duration of the afier-discharge, using a Grass Model 7D polygraph. Subjects were
kindled until 30 stage 5 seizures had been evoked.
2.3.3 Determination of After-discbarge Threshold
AAer-discharge threshold (ADT) was deterrnined using slight variations of the
asceding series technique (Pinel et ai., 1976) or half-split technique (Racine, 1972). The
ascending series technique was used in Experiment 1. The half-split technique was used
in Expenment 2. ADT was detemined before kindling began and also after the subjects
had had 5 stage 5 seinires.
The ascending series technique of threshold measurement involves the initial
administration of a sub-threshold electrical stimulus followed by subsequent, step-wise
increases in the electrical stimulus separated by a short "inter-stimulus interval". In the
present study, ADT determination began with a stimulus of 20 UA (peak to peak). The
stimulus intensity was then increased in 20 UA steps up to 320 UA; then the intensity was
increased in 40 UA steps. A 1 -minute inter~al was allowed between stimuli. ADT was
defined as the minimum stimulus intensity needed to provoke an after-discharge of at
least 4 seconds in non-kindled subjects or a generalized seinire in well-kindled subjects.
The half-split technique is also called the "up and dom" method (Kokaia et al.,
1994). The electrical current-interval between a stimulus that produces seizures and one
that doesn't is halved to obtain a new current-interval. This procedure is continued until
the threshold is deterrnined within an established rnargin of error. Long inter-stimulus
intervals are used. In the present shidy, the ADT was detemined over several days. On
the first day, a current of 100 UA was used. Depending on whether this current induced a
seizure (after-discharge of at least 4 seconds in non-kindled subjects or a generalized
seizure in well-kindled subjects), the current intensity was halved or doubled on the
second day. This process was continued until the interval between the lowest effective
stimulus and the highest ineffmtive stimulus was 20 UA or less. ADT was defined as the
halfway point within this interval.
2.3.4 Determination of Stability
ABer the ADT was determined, seizure stability at ADT plus 40% was assessed.
Subjects were stimulated daily at a curent intensity equal to ADT plus 40%. 'Stability'
was defined as 5 consecutive stage 4 or 5 seizures. If subjects were not stable, the
stimulus intensity for kindling was increased by an additionai 40%.
2.3.5 Procedure for Drug Testing
The procedure for drug testing is specified for each expriment in the specific
methods section.
2-3.6 Verification of Electrode Placement
Once kindling experiments were cornpiete, subjects were anesthetized with
sodium pentobarbital (SomnitolQ, 80mgkg) and perhsed. After loss of consciousness,
the site of the implantation was lesioncd by a cwcnt of 8mA, lasting 30 seconds. The
thoracic cavity was then opened via a medial incision, exposing the heart. A cut was
made in the right auricle and a cannula (gauge 16) was inserted into the lefi ventride.
Perfùsion began with 1 OOmL of normal saline. The perfusion rate was 1 SmLlminute.
Perfusion was completed with lOOmL of 4% formaldehyde solution (9g NaCI, 108g 37%
formaldehyde, in 1 L of distilled water). Brains were then extracted and stored in 20%
sucrose solution containing 4% fomaldehyde. Forty-eight hours later, the brain was
withdrawn From the fixative and stored until sectioning at -80°C in a sealed plastic
container.
At the time of sectioning, the brains were attached to a specimen disc using
ernbedding medium (Tissue Tek@, Sakura Finetechnicd Co., Ltd., Tokyo). They were
then transferred to a cryostat (Jung CM3000, Leica Inc., Canada), maintained at -2S°C,
and allowed to equilibrate for 20 minutes. Sections (30um) were cut coronally. Every
fifth section in the area of the electrode tip was thaw-mounted on poly-L-lysine coated
glass slides and allowed to air dry.
Dried sections were stained 6 t h cresyl violet. Slides were immersed for 3
minutes in each of each of the following media: 100% ethanol, 95% ethanol, 70%
ethanol, distilled water, 0.5% cresyl violet, distilled water, and 70% ethanol. Slides were
then incubated for 2 minutes in 95% ethanol containing 0.1% acetic acid, and then twice
in 100% alcohol. Finally, slides were incubated twice for 5 minutes each in xylene. The
slides were then covered using a cover slip and Permount@ (Fisher Chemicals).
The electrode tract was examined with a magnification-projection system
(Research Analysis System mode1 42 125 1, Amersham, MI). Only subjects with verified
electrode placements in the targeted region were used in subsequent data analyses.
2.3.7 Procedure for Ovariectomy and Gonadectomy
Ovariectorny and gonadectomy were performed on some of the subjects in the
kindling experiments.
Twenty-four hours after the 10th stage 5 seinues, female subjects were
ovariectomized. Subjects were anesthetized with 4% halothane and then anesthesia was
rnaintained using 1.5% halothane. Bilateral flank incisions (1 cm) were made. The
uterine horn attached to the ovary and the accompanying fat were pulled âhrough the
incision. The ovary and the accompanying fat and vessels were tied (5-0 silk suture) and
the oviduct severed and removed with a single cut. The uterine hom was returned to the
peritoneal cavity. The muscle and skin incision were closed with one and two sutures,
respectively. Subjects were allowed a 14-day postsperative recovery period before
funher procedures.
Male subjects were gonadectomized at the time of electrode implantation. Under
general anesthesia, a single, saggital incision (1 cm) was made in the scrotal sac. The
testes and epididiymes was pulled through the incision. Both tissues were severed and
removed by a single cut, following suturing of the tissue above the cut. The muscle and
skin incision were closed with one and two sutures respectively. Subjects were allowed
1 Cday post-operative recovery period before fùrther procedures.
2.4 f rocedure for Vaginal Smears
In some experiments, daily vaginal smears were performed (between 10:OO h and
10:30 h) to characterize each day of the 4-day estrous cycle in the intact female rat
(Freeman. 1996). Smears were obtained by placing a blunted, plastic 20 pl Eppendorf tip
on the vaginal opening and flushing the orifice twice with 10 pl of 0.9% saline. Tips
were rinsed thoroughly with 70% ethanol d e r each use. The saline flush was displayed
on a clean microscope slide and observed at 20X magnification. Phases of the estrous
cycle were classified according to the following criteria: 1) diestrus - smears were
characterized by a heterogeneous mixture of polymorphic leukocytes and epithelial cells;
2) proestrus - smears had predominantly large round nucleated epithelial cells; 3) estrus -
smears consisted primarily of comified epitheliurn with few or no nuclei visible, and 4)
metestrus - smears with exclusively leukocytes in high density.
The threshoid pentylenetetrazol test (MET) was administered according to a
modification of the protocol of Kra11 et al. (Wl a al. 1978). This test was used in
Experirnent 2 on adult female Wistar rats. Subjects were injected with 70mgkg of
pentylenetetrazol (SC) in the rough of the neck. Pentylenetetrazol was dissolved in
normal saline to a concentration of 19 mgkg. The volume of injection was 4mLkg.
Following injection, the subjects were placed in a test chamber and observed for 30
minutes. Seinires were scored as "present" or "absent". The latency to the onset of
seinires was also recorded. Seizure absence was defined as the absence of forelimb
clonus (FLC).
2.6 Procedure for the MMT Test
The maximal pentylenetetrazol test (MMT) was administered according to a
modification to the protocol of Desmedt et al. (Desmedt et al.. 1976). This test was used
in Experiments 2,3, and 4. Adult subjects were in injected with 85mg/kg of
pentylenetetrazol (SC) in the rough of the neck. Pentylenetetmzol(2lmg/mL) was
dissolved in normal saline. The volume of injection was 4mUkg. Rat pups were injected
with 150mgkg of pentylenetetrazol (SC) in the rough of the neck. Pentylenetetrazol
(1 5 rng/mL) was dissolved in normal saline. The volume of injection was 1 OmLkg.
Following injection, the subjects were placed in a test charnber and observed for
30minutes. The seizures were scored as "present" or "absent". The latency to the onset
of seizures was also recorded. Seizure absence was defined as the absence forelirnb
extension (FLE). Fotelimb extension was measure as opposed to hind-limb extension
(HLE), because subjects did not produce hind-limb extensions reliably in this model.
2.7 Procedure for the ECS Test
The threshold electroconvulsive shock test (ECS) was administered according to a
modification to the protocol of Woodbury (1972). This test was used in Expriment 2 on
adult female Wistar rats. The ECS stimulus was administered via corneal electrodes.
Electrodes were wetted with normal saline prior to application to the comea. The
following parameten were used for stimulation: pulse codiguration - sine wave, pulse
fiequency-60 Hz, train duration-0.2 seconds, pulse intensity - variable (1 0-50 mA).
Variable pulse intensity was used because the threshold for ECS seinires was being
measured. To determine ECS threshold, the half split method (as described previously)
was used. The seinires were scored as "present" or "absent". Seinire absence was
defined as the absence of forelimb clonus.
2.8 Procedure for the MES Test
The maximal electroconvulsive shock test (MES) was administered according to a
modification to the protocol of Krall et al. (Kra11 et al. 1978). This test was used in
Experiments 3 and 4. The ECS stimutus was administercd via corneal etectrodes.
Electmdes were wetted with normal saline prior to application to the comea. The
following stimulus parameters were used: pulse configuration-sine wave, pulse fiequency
- 60 Hz, train duration-0.2 seconds, pulse intensity - 150 mA. Seizures wete scored as
"prcsent" or "absent". Seizwe absence was defined as the absence of hindlimb extension
(HLE).
In Experiment 2 the threshold for MES was detemined in adult female Wistar
nits. The same procedure was followed as in the standard MES test except that variable
pulse intensity (1 0-1 00mA) was used. To detemine this threshold, the half split method,
as described previously, was used.
2.9 Procedure for Pentylenetetrazol Infusion
The pentylenetetrazol infusion test allows accurate threshold measurements of the
clonic or tonic seizures resulting fiom the administration of pentylenetettazol. This test
was used in Experiment 2 in adult female Wistar rats. It was administered according to
the protocol of Thavendiranathan et al. (2000). Pentylenetetrazol was dissolved in
normal saline to a concentration of IOmgImL. The mixture was administered through the
tail vein at an injection rate of 1 mL/minute. A Sage Instrument synnge purnp (mode1
35 1) was used for administration. The infusion was stopped when tonic seizures were
produced. The time to the onset of tonic seizures was recorded.
2.10 Procedure for Tests of Sedation and Ataxia
In al1 experiments, subjects were challenged with the righting-reflex test and
Loscher's ataxia rating scale immediately pnor to testing. The righting reflex test
involves rolling the subject ont0 its back and observing whether it is able to rotate 180
degrees ont0 its limbs.
Loscher's test of ataxia involves simple observation, and uses the following 6-
stage catagones of behavioral impairments:
1. Slight ataxia tottering of hind limbs
2. More pronounced ataxia dragging of hind limbs
3. Further increase of ataxia more pronounced dragging of hind limbs
4. Marked ataxia animal occasionally loses balance during forward
locomotion
animal fiequently loses balance during forward
locomotion
animal, despite attempts, is unable to move fonvard
5. Very marked ataxia
6. Extreme ataxia
2.1 1 Statistical Analysis
Time-response and dose-respome cuves were plotted for both the clonic and
tonic seinues. Quantal curves were constructed by ploning the percentage of animais at
each time or dose that were protected against the clonic or tonic seizures. Whenever
possible, dose-response curves were fitted with sigrnoidal curves and using linear
regression, and ED& were calculated fiom the fitted curves.
Interval-ratio data (e.g., latencies) were expressed as mean standard error of the
mean (SEM). When two groups were king compared and the data were normally
distributed, a student t-test or a paired t-test was used. When the data were not normally
distributed, the Mann-Whitney Rank Sum test was used. When more than two groups
were being compared and the data were normally distributed, analysis of variance (one-
way ANOVA) was used. Provided that the overall analysis of variance was significant,
post-hoc cornparison used Duncan's Multiple Range test. When the data were not
normally distributed, the Kniskal-Wallis one-way analysis on ranks was done. A cntical
significance level of P < 0.05 was used for al1 tests.
Chapter 3
Anticonvulsant Effects of Progesterone
in Amygdala-kindled Rats
3 Anticonvulsant Effects of Progesterone in Amygdala-kindled Rats
3.1 Rationale
In small, open clinical triais, progesterone has proven to be an anticonvulsant.
Women with catamenial seizures, who are refiactory to other treatments, consistently
respond to progestemne (see table 2a). These patients generally suffer fiom intractable
complex partial seizures. Complex partial epilepsy is the most common adult f o m of
epilepsy, and it is often intractable (see Introduction).
The kindling preparation provides an animal mode1 in which to study the effects
of progesterone on complex partial seizures (Introduction). Past studies on the
anticonvulsant efTects of progestemne in the kindling mode], however, have produced
conflicting results. Edwards et al. (1 999) found that 5 mgkg of progesterone abolished
amygdala-kindled seinires in 60% of adult female Wistar rats. Mohammad et al. (1998),
however, f o n d that in male Wistar amygdala-kindled rats, doses bel ow 75 mgkg were
ineffective at blocking generalized kindled seizures. Edwards did not perform dose-
response studies, whereas Mohammad did. These data seem to suggest that progesterone
may have low-dose anticonvulsant effects in femaies but not males.
Expenment 1 was designed to clarify the effects of progesterone on kindled
seizures. Dose-response studies with progesterone were perfonned in amygdala-kindled
female and male Wistar rats. ED,'s were determined, and the influences of endogenous
and exogenous hormones on the response were explored. It was originally hypothesized
that progesterone would be anticonvulsant at low doses (5 mgkg), at least in fernales.
In an attempt to replicate the procedure of Edwards et al., adult Wistar rats were
used, and progesterone was injected 2 hours before seizures were triggered. It was
assurned that the responses would be genomic and rnediated by the classic progesterone
receptor. Genomic responses take some time to develop.
When initial trials failed to show low-dose progesterone effects (data not shown),
a number of subsequent trials were performed, varying different expenmental parameters
(The same ovariectomized subjects were used in Experiments la and 1 b).
Experiment la
In Experiment la, parameters related to administration were manipulated. To
assess the possible importance of first-pass effects, the route of delivery was varied. The
drug was administered IP (first p a s effects) or SC (no first pass cffkct). To assess the
possible effect of the vehicle, the drug vehicle was varied. The drug was administered
either in beta-hydroxy cyclodextrin or corn oil (Edwards et al. had used oil as their drug
vehicle).
Experiment 1 b
In Experiment 1 b, the possible role of estrogen was investigated. The effect of
"estrogen priming" on after-discharge threshold (ADT) was determineci. Subjects were
then "estrogen-primed" before tests of the anticonvulsant effects of progesterone.
Experiment le
In Experiment 1 c, to determine effect of endogenous hormones (including
estrogen) a new group of intact (not ovariectomized) subjects were used. This allowed
the evaluation of anticonvulsant effects of progesterone within a normal hormonal milieu
(Edwards et al. used intact subjects). (Ail other kindling experiments used castrated
subjects to prevent the confounding effect of endogenous hormones when testing
progesterone.)
Experiment Id
In Experiment 1 d, castrated male subjects, with and without hormone
replacement, were used to provide a cornparison with the fernale subjects in Experirnents
la, l b and fc.
3.2 Methods
Subjects
The same subjects were used in Experiment l a and 1 b. They were 33 right
amygdala-implanted, ovariectomized female Wistar rats. In Experiment 1 c, the new
subjects were 24 right amygdala-implanted, intact (not ovariectomized) female Wistar
rats. In Expenment Id, the subjects were 36 right amygdala-implanted, gonadectomized
and hormone-nplaced, male Wistar rats. Hormone replacement consisted of SC
implantation of Silastic@ capsules containhg cholesterol, estrogen or testosterone.
Subjects were housed as described in the General Methods.
Kindling and Threshold Testing
The subjects (in al1 experiments) were kindled daily to a critenon of 30 stage 5
seizures (procedure as in General Methods). Their thresholds were measured using the
ascending series rnethod as described in the General Methods. Thresholds were checked
for stability as described in the Generai Methods.
Dose-Response Testing
Four days fottowing the last kindling stimulus (stability testing), progesterone
dose-response studies were initiated. In al1 studies, the following doses of progesterone
were used: 4,8, 1 6 ,3 2, and 64 mgkg. For all expenments (except l a, when corn oil was
used as vehicle), progesterone was dissolved in beta-hydroxy cyclodextrin to a
concentration of 32mgfmL for the highest dose, followed by seriai dilution for lower
doses. It was adrninistered in a volume of 2mL/kg. In part of Experiment 1 b, the drug
(progesterone) was prepared in a similar m a w r as above, except that corn oit was used
as the vehicle.
In the first part of Experiment la, the subjects were randomized and injected SC
or IP with different doses (4,8, 16,32,64 mgkg) of progesterone. Two hours after drug
injection, the anticonvulsant action of progesterone was tested by administering a
kindling stimulus equivalent to ADT+40%. The same subjects were tested repeatedly
with different doses. A four-day interval was allowed between tests. It permits complete
clearance of progesterone.
In the second part of Experiment la, the same subjects (ovariectomized fernales)
were randomized and injected SC with different doses of progesterone dissolved in corn
oil or beta-hydroxy cyclodextrin. Seizure testing was the same as described above.
In the first part of Experiment lb, the same subjects (ovariectomized females)
were "estrogen primed" with a SC injection of I Ougkg estradiol benzoate 48 hours prior
to ADT testing. Estradiot was dissolved in corn oit, and injected in a volume of ZmL/kg.
ADTs were detemined (ascending series method as described in General Methods)
before and after "estrogen priming".
In the second part of Experiment lb, a progesterone dose-response study was
then perfonned in "estrogen primeà" subjects (ovariectomized females). Forty-six hours
after estrogen priming, they were randomized and injected SC with different doses of
progesterone. Seizure testing was identical to Experiment la, except that a newly found
ADT + 40% was used. It was based on the ADT measured in the presence of estradiol.
In Experiment le, new subjects (intact females) were randomized and injected
SC with different doses of progesterone. S e i m testing was identical to Experiment la.
I t should be noted that the intact subjects were not cycling and exhibited persistent
estrous. The kindling stimulus cause female rats to cycling anest and the subjects
perpetually appear to be in the proestrous/estrous phase. (Edwards et al., 1999).
In Experiment Id, adult mate subjects were (gonadectomized and hormone-
replaced males) with cholesterol (control), estrogen or testosterone. They were then
randomized and injected SC with different doses of progesterone. Seinire testing was
identical to Experiment la.
Seizure Scoring
Two compomnts of the kindled seizure were scored: the generalized convulsion
and the focal component. Each component was marked as "present" or "absent".
Generalized seizures were defined as stage 4 and 5 seizures, as catagorized by Racine's
classification (General Methods). The generalized se iwe was scored as absent if seinire
stages 4 and 5 were suppressed. The focal seizure was defined as 3 or more seconds of
"spiking" in the EEG record. This component was scored as absent, if a normal EEG
followed the stimulation. These definitions are consistent with Albright's scoring
technique (Albright and Bumharn, 1980).
Data Analysis
Dose-response curves were plotted for progesterone's effects on both the
generalized and focal components of the seizures. Quantal curves were constmcted by
plotting the percentage of animals that were protected at each dose. ED,o's and Rmax's
were calculated. A paired Student t-test was used to compare ADT's in Experiment 1 b.
3.3 Results
Sedation
Sedation was generally not apparent for doses of 32rngkg or less. A dose of
64mgkg in the SC, cyclodextrin groups fiom experiments 1 a and 1 b (data are combined),
however. caused significant sedation, including loss of the righting reflex in 36 % of the
subjects (N=14). Ataxia was measured on the Loscher scale (General Methods) and
found to be 4.9 +/- 0.4 in these groups. This was significantly different from control
subjects (P<O.OOl) using the paired t-test.
Experiment la: Progesterone Dose-Response: Route of Administeration and
Vehicle
Figure 3 shows the progesterone (injected IP or SC) dose-response curve for the
suppression of generalized and focal seizures in well-kindled, ovariectomized, female
Wistar rats. For generalized seizures in both the IP and the SC treated groups, the ED,
was 33 mgkg and the maximal response (100%) occurred at 64mgkg. The route of
administration did not matter. SC administration was therefore used in al1 subsequent
studies.
Dose mglkg
4 8 16 32 64
Dose mgkg
Fiaure 3: The Anti-convulsant Effects of Proaesterone in the Kindlina Model Proclesterone Was Administered IP or SC
The anticonvulsant effects of progesterone against generalized kindled convulsions (closed circles) or focal seizures (open circles) Each point represents data from at least 7 subjects. Subjects were adult, ovariectomized female Wistar rats. Figure 3a: progesterone was administered I P. Figure 3b: progesteorne was administered SC.
No substantial suppression of focal seizure activity was seen at any of the doses
tested in either group. No low-dose anticonvulsant effects were seen.
Figure 4 shows the progesterone (dissolved in cyclodextrin or corn oil) dose-
response curve for the suppression of generalized and focal seinires in well-kindled,
ovariectomized, fernale Wistar rats. For generalized seinires in the beta-hydroxy
cyclodextrin treated group, the ED,, was 33 mgkg and the maximal response (100%)
occuned at 64mgkg. There was no substantial suppression of generalized seizures in the
corn oil treated group. Cyclodextrin was, therefore, used in al1 subsequent studies.
No substantial suppression of focal seizure activity was seen at any of the doses
tested in either group. No low dose progesterone effects were seen.
Experiment 1 b: Progesterone Dose-Response: Estrogen Priming
Figure 5a shows the effect of "estrogen-priming" on the ADT as measured by the
axending series method (General Methods). The same subjects were used as in
Experiment la. The ADT was significantly reduced by 23% (P < 0.00 1, using t-test).
Thus, estrogen priming lowea seinire thresholds at the seimre focus. In the subsequent
4 8 16 32 64
Dose mgkg
l Progesterone , corn oil
Dose mgkg
Fiaure 4: The Anticonvulsant Effects of Frocresterone in the Kindlina Model Prociesterone Was Administered in Cvclodextrin or Corn Oil
The anticonvulsant effects of progesterone against generalized kindled convulsions (closed circles) or focal seizures (open circles) Each point represents data from at least 7 subjects. Subjects were adult, ovariectomized female Wistar rats. Figure 4a: progesterone was administered in cyclodextrin. Figure 3b: progesteorne was administered in corn oil.
The Effect of Estrogen on Afterdischarge Threshold (ADT)
control "estrogen-prirned"
Fiaure 5a: The Effect of Estroaen on After-discharae Threshold
The proconvulsant effects of estrogen on after-discharge threshold. Data are represented as mean +!- standard error. Each group contains 33 subjects; C) indicates a signifiant difference from the control group using a paireâ t-test (Pc0.001).
Progesterone, "estrogen-primed"
4 8 16 32 64
Dose mgkg
Ficlure Sb: The Anticonvukant - Effects of Proaesterone in the Kindlina Model Subiects Were Estroaen-Primed
The anticonvulsant effects of progesteione against generalized kindled convulsions (closed circles) or focal seizures (open circles). Each point represents data from at least 5 subjects. Subjects were adult, ovariectomized (estrogen-primed) female Wistar rats.
dose-response study (Figure Sb), estrogen primed subjects were tested at their
newiy measured ADTYs+40%.
Figure Sb shows the progesterone dose-response curve for the suppression of
generalized and focal seizures in well-kindled, ovariectomized, "estrogen-primed" female
Wistar rats. For generalized seizures in the "estrogen primed" subjects. the ED,, was
40mg/kg and the maximal response (80%) occurred at 64mgkg. No low-dose
anticonvulsant effects were seen. Therefore, estrogen does not synergize progesterone's
anticonvulsant effect in the kindling model.
No substantial suppression of focal seinire activity was seen at any of the doses
tested.
As a positive control for the testing method, a dose-response of phenobarbital was
prefonned (data not shown). The ED,. against generalized s e i m s was 12.5 mg/kg.
Experiment le: Progesterone Dose-Respoose: Intact subjects
Figure 6 shows the progesterone dose-response curve for the suppression of
generalized and focal seizures in well-kindled, intact, female Wistar rats (These subjects
were not the sarne as those used in previous studies). For generalized seizures in the
intact subjects, the ED, was 30mglkg and the maximal response (100%) occurred at
Progesterone, "intact"
4 8 16 32 64
Dose mglkg
Fiaure 6: Anticonvulsant - - Effects . of Proaesterone in the Kindlina Model Intact Subiects Were Used
The anticonvulsant effects of progesterone against generalized kindled convulsions (closed circles) or focal seizures (open circles) Each point represents data from at least 5 subjects. Subjects were adult, intact (not ovariedomized) fernale Wstar rats.
64mgkg. No low-dose anticonvulsant effects were seen. Therefore, normal levels of
endogenous hormones do not alter progesterone's anticonvulsant effect in the kindling
model.
No suppression of focal seinire activity was seen at any of the doses tested.
Experiment Id: Progesterone dose-response, male rats, hormone
replacement
Figure 7 shows the progesterone dose-response curves for the suppression
of generalized and focal seizures in gonadectomized, cholesterol-replaced, estrogen-
replaced and testosterone-replaced, well-kindled, male Wistar rats. For generalized
seizures, the ED, was 35mg/kg in the cholesterol-replace subjects, 37mgkg in the
estrogen-replaced subjects, and IO 1 mgkg in the testosterone-replaced subjects. The
maximal response (90%) occurred at 64mglkg in the cholesterol-replace subjects, and
a b in the estrogen-replaced subjects. The maximal response (60%) occurred at 98mg/kg
in the testosterone-replaced subjects. No low-dose anticonvulsant effects were seen.
Hence, the anticonvulsant effects of progesterone in gonadectomized and cholesterol-
replaced or estrogen-replaced rats are similar to the response in femaie rats. Testosterone
in male rats, however, makes them less responsive to the anticonvulsant effects of
progesterone.
4 8 16 32 64
Oose rngiûg
I
o . /a -v
4 8 16 32 64
Dose mghg
Progesterone, testosterone-replaced
100.
cn 8 0 .
2 a .Y u 60- Y> C 3 O r 40 .r 3
* 2 0 .
0
Figure 7: The Anticonvulsant Effect of Progesterone in the Kindlina Model Subjects Were Gonadectomized and Hormone-Replaced Males
Progesterone, estrogen-replaœd
1 I b 1 b
The anticonvulsant effects of progesterone against generalized kindled convulsions (closed circles) or focal seizures (open circles) Each point represents data from at least 7 subjects. Subjects were adult , ovariectornized female Wistar rats. Figure 7a: progesterone was administered in cholesterol-replace subjects. Figure 7b: progesteorne was administered in estrogen-replaced su bjects. Figure 7c: progesteorne was administered in testosterone-replaced subjects.
75
No substantial suppression of focal seinire activity was seen at any of the doses
tested in any of the groups.
3.4 Summary
Progesterone was an effective anticonvulsant in female rats, but at doses that were
higher than expected. ED,s were usualiy in the range of 30-40mglkg (summarized in
Table 3). Manipulations of the route of drug delivery, the dnig vehicle, and of exogenous
(estrogen priming) and endogenous (intact subjects) hormonal States did not irnprove
progesterone's anticonvulsant response. Estrogen prirning, however, lowered seizure
thresholds, thus supporting the theory that estrogen i s an important cause of catarnenial
seizures of the complex partial vmiety.
Castrated males (cholesterol or estrogen replaced) had ED,s similar to the
fernales. Testosterone-replaced castrated males, however, had high EDs,,s, suggesting that
testosterone blocks the anticonvuf sant effects of progesterone.
The original hypothesis that progesterone would have anticonvulsant effects at
low doses was not supported.
Tabfe 3
The anticonvulsant EDSO's of progesterone for the suppression of Amygdala-generalized convulsions
Route of Administeration
-
Vehicle
- -
C yclodextnn
C yclodextrin
Corn oil
Cyclodextrin
Cyclodextrin
Cyclodextrin
Cyclodextrin
Cyclodextrin
Sex
Female
Female
Female
Female
Female
Male
Male
Male
Hormonal Parameters
Ovariectomized
Ovariectomized
Ovariectomized
Ovariectomized / Estrogen-replaced
Intact
Gonadectomized / Cholesterol-replaced
Gonadectomized / Estrogen-replaced
Gonadectomized / Testosterone-replaced
Amygdala EDSO (mg/kg)
Chapter 4:
Anticonvulsant Effects of Chronic Low-Dose
Progesterone and MPA in Six Seizure Models
4 Anticonvulsant Effects of Chronic Low-Dose Progutvone and MPAim Six
Seizu r e Models
4.1 Rationale
The results of Experiment 1 indicated that progesterone is not anticonvulsant at
low doses in amygdala-kindled rats. These findings are consistent with sirnilar studies by
Mohammad's group who also found suppression of kindled convulsion ai high doses
(Moharnrnad et al., 1998b).
The fact that only high doses - which caused sedation - were effective suggests a
GABAergic mechanism of action for progesterone's anticonvulsant effects. These
findings. however, are not consistent with the clinical data, which indicate that low doses
of both progesterone and MPA (which is not metabolized into ailopregnanolone) are
effective anticonvulsants.
One important difference between Experiment 1 study and the clinical studies is
that we administered progesterone acutely, whereas epileptic patients use progesterone or
MPA chronically. It seemed possible that low doses of progesterone or MPA might be
anticonvulsant in rats if they were administered chronically (for 1 week).
The effects of chronicaily administered progesterone and MPA were therefore
assessed in several different seizure models. In each model, seinire latency or threshold
- rather than seinire absence or presence - was measured, so that even srnall changes
would be seen. The tests included amygdala kindling, ECS-threshold, MES-threshold,
MET, MMT, and pentylenetetrazol-infusion. It was hypothesized that low doses of
progestins, chronically administered, would have anticonvulsant effects in these tests.
4.2 Methods
Subjects
In al1 experiments. intact fernale Wistar rats were used as subjects. Subjects were
60 days old upon arriva1 from the breeding f m s . They were allowed one week for
acclimatization and daily handling before any treatments or tests were performed.
The subjects' stage in the estrous cycle was monitored by daily vaginal smears, as
described in the General Methods.
Dmg Administration
In al1 experiments, subjects were randomized into three groups. The first group
(the control subjects) was injected with normal saline. The second group was injected
with MPA (1 Smgkg, 0.2mL, IM, vehicle: saline). This dose of MPA reliably stopped
the fertility cycle in subjects, indicating that it was effective. The third group was treated
with X inch Silastic@ capsules containing progesterone. The Silastic0 capsules were
implanted subcutaneously in the mff of the neck under light halothane-induced
anesthesia. This dose of progesterone has produced luteal phase levels of progesterone in
ovariectomized rats (Edwards et al., 1999).
Afier one week of dnig treatment seinire testing was initiated. Control subjects
were tested during proestrous. Drug-treated subjects who stopped cycling were tested
once every four days (in studies where repeated testing was required). Othenvise,
subjects were tested during proestrous.
Testing and Seùure Sconng
Experiment 2a: Kindling Before dmg administration, subjects were amygdala-kindled to 5 stage 5 seizures,
using procedures described in the General Methods. Progesterone or MPA were then
administered for 1 week. On the 8'h day, the threshold for amygdala-kindled generalized
convulsions was determined by the half-split method, as described previously. The
lowest current that produced generalized seizures (stage 4 or 5) as defined by Racine's
classification was considered the "thresh~ld'~.
Experiment 2b: ECS Threshold ECS threshold was measured by the half-split method, as described in the General
Methods section. The lowest current required to produce forelimb clonus was considered
the threshold.
Experiment 2c: MES Threshold
MES threshold was measured by the half-split method, as described in the
General Methods section. The lowest current required to produce hind-limb tonus was
considered the "threshold".
Experiment 2d: MET The MET test was administered as described in the General Methods section.
Subjects were scored for presence or absence of clonic seizures within 30 minutes after
injection, and also for the delay to onset of the seizures (latency).
Experiment 2e: MMT The MMT test was administered as described in the General Methods section.
Subjects were scored for presence or absence of tonic (forelimb extension) seinws
within 30 minutes afier injection, and also for the delay to onset of the seizures (latency).
Experiment 2E Pentylenetetrazol Infusion
Pentylenetetrazol was administered via the tail vein, as described in the General
Methods section. The dose of pentylenetetrazol required to cause forelimb clonous was
calculated and was considered to be "threshoid".
Data Analysis
In al1 experiments, the data were andyzed using one-way analysis of variance
(ANOVA) or - when they were not norrnally distributed - the Kniskal-Wallis one-way
analysis on ranks.
4.3 Rcsults
Effects on the Estrous Cyck
The estrous cycle was arrested in 84% of subjects that were treated with MPA
(N=50). Vaginal smears from subjects that had stopped cycling displayed characteristics
of cells from diestrous or metestrous. With progestemne, only 28% of the subjects
stopped cycling (N=43).
Experiment 2a: Kindling As indicated by Figure 8, the mean thresholds (+/- SE) for generalized
amygdala-kindled convulsions (GCT) for saline, progesterone and MPA-treated subjects
were 63 f 2 1,68 f 32, and 83 + 53 PA, respectively. Analysis of variance revealed no
significant difference among these means (P=O.94 1). These data indicate that chronic
administration of progestins does not significantly alter the threshold for amygdala-
Amygdala-Kindled Convulsive Threshold
control progesterone MPA
Fiaure 8: The Effect of Chronic Proclesterone and MPA on the Generalized Convulsive Threshold in Amvadala-Kindled Rats.
The control, progesterone, and MPA groups contain 13, 8, and 6 subjects respectively. The data are ptesented as mean +1- standard error. No significant difference was found using Kruskal-Wallis one-way analysis on ranks (P=O.Q4l).
kindled convulsions in rats. There was, however, a bbtrend" towards
anticonvulsant activity with both progesterone and, to a greater extent, the MPA
treatments.
Experiment 2b: ECS Tbreshold As indicated by Figure 9, the mean ECS thresholds (+/O SE) for saline,
progesterone and MPA-treated subjects were 25 f 3,25 f 2, and 27 f 3 rnA, respectively.
Analysis of variance revealed no significant difference among these means (P=0.140).
These data indicate that chronic administration of progestins does not significantly alter
the ECS threshold in rats. There was, however, a slight "trend" towards anticonvulsant
activity with the MPA treatrnent.
Experiment 2c: MES Threshold As indicated by Figure 10, the mean MES thresholds (+/- SE) for saline,
progesterone and MPA-treated subjects were 40 f 6,42 + 6, and 42 t 5 mA, respectively.
Analysis of variance revealed no significant difference among these means (P=0.7 18).
These data indicate that chronic administration of progestins does not significantly alter
the MES threshold in rats. There was, however, a slight "trend" towards anticonvulsant
activity with both the progesterone and the MPA treatrnents.
ECS Convulsive Threshold
control progesterone MPA
Fiaure 9: The Effect of Chronic Progesterone and MPA on the Threshold for ECS Seizures.
Each group contains at least 13 subjects. The data are presented as mean +/- standard error. No significant difference was found using the Kruskal-Wallis one-way analysis on ranks (P=O. 140).
MES Convulsive Threshold
control progesterone MPA
Figure 10: The Effect of Chronic Prooesterone and MPA on the Threshold for MES Seizures.
Each group contains at least 13 subjects. The data are presented as mean +Io standard error. No significant difference was found using the Kruskal-Wallis one-way analysis on ranks (P=O.718).
Experiment 2d: MET Al1 test subjects had a clonic seizure within 30 minutes of pentylenetetrazol
injection.
As indicated by Figure 1 1, the mean latencies (+/- SE) to onset of forelimb clonus
following penty lenetetrazol injection in saline, progesterone and MPA-treated subjects
were 457 k 1 33,590 I 423, and 87 1f 465 seconds, respectively. Kruskal-Wallis one-
way analysis on ranks revealed no significant difference among these means (P=0.082),
although the data were approaching significance. These data indicate that chronic
administration of progestins does not significantly alter response to the MET stimulus in
rats. There was a "trend", however, for progesterone and, to a greater extent, for MPA to
increase latency. Using the Mann-Whitney rank sum test to compare only the saline and
MPA groups, the MPA group would have differed significantly fiom the saline group
(P=O.O 18).
Experiment 2e: MMT Eight out of 9 test subjects in each group had a tonic seizwe (forelimb extension)
within 30 minutes of pentylenetetrazol administration. There was no noteworthy
difference in seizure occurrence between two groups.
Latency to MET Clonic Seizures
control progesterone MPA
Fiaure 11: The Effect of Chronic Prwesterone and MPA on tatencies ta the Onset of MET Clonic Seizures.
Each group contains at least 8 subjects. The data are presented as mean +/- standard error. No significant differenco was found using the Kruskal-Wallis one-way analysis on ranks (Pz0.082).
Latency to MMT Tonic Seizures
control progesterone MPA
Ficiure 12: The Effect of Chronic Proclesterone and MPA on Latencies to the Onset of MMT Tonic (Forelirnb Extension) Seizu res .
Each group contains at least 8 subjects. The data are presented as mean +/- standard error. No significant difference was found using the Kruskal-Wallis one-way analysis on ranks (P-0.254).
As indicated by Figure 12, the mean latencies (+/- SE) to onset of forelimb
extension following pentylenetetrazol injection in saline, progesterone and MPA-treated
subjects were 547 f 1 11,678 f 374, and 774 f 323 seconds, respectively. Kniskal-
Wallis one-way analysis on ranks revealed no significant difletence among these means
(P-0,254). These data indicate that chronic administration of progestins does not
significantly alter response to the MMT stimulus in rats. There was, however, a "trend"
for progesterone and, to a greater extent, MPA to increase latency.
Experiment 2f: Peatylenetetrazol Infusion As indicated by Figure 13, the mean doses (+/- SE) that produced clonic seizures
in the pentylenetetrazol inhision test for saline, progesterone and MPA-treated subjects
were 52 i 17,48 f 20, and 56 f 21 mgkg, respectively. Andysis of variance revealed no
significant difference among these means (P=0.666). These data indicate that chronic
administration of progestins does not significantly alter the convulsant pentylenetetrazol
infusion dose in rats. There was, however, a "trend" towards anticonvulsant activity with
MPA treatment.
Tonic Threshold: Pentylenetetrazol Infusion
control progesterone MPA
Fiaure 13: The Effect of Chronic Proaesterone and MPA on the Threshold Dose of Pentylenetetrazol Reauired to lnduce Clonic Seizures (FLC).
The control. progesterone, and MPA groups contain 22, 12. and 13 subjects, respectively. The data are presented as mean +/- standard error. No signifiant difference was found using the Kruskal-Wallis one-way analysis on ranks (P=0.666)
4.4 Summary
Chronically administered progesterone and, to a greater degree, MPA displayed
trends towards anticonvulsant activity in several tests. In no case, however, was the
anticonvulsant activity of progesterone and MPA statistically significant. Our hypothesis
that low doses of progestins, chronically administered, would have significant
anticonvulsant effects was not supported.
Chapter 5
The Anticonvulsant Effects of Progesterone in
Adult and 15-day-old Male and Female rats in the
MMT and the MES Seizure Models
5 The Anticonvubant Effects of Progesteroae in Adult and 15-day-old Male and
Female rats in the MMT and the MES Seizure Models
5.1 Rationale
Experiment 1, the initial dose-response study, was conducted with a 2-hour
injectiodtest interval. This interval was the interval used by E d w d s et al. (1999), and
was based on the assurnption that pmgesterone's effects were genomic effects, mediated
by the "classic" intracellular receptors. Genomic effects take some time to appear.
Recently, Edwards et ai. (submitted) have performed fiuther experiments, which
showed anticonvulsant effects of a single dose of progesterone using a 15-minute
injectiodtest interval. In Experiment 3, therefore, the time-response of progesterone was
studied. These studies established that 15 minutes is an optimal injectiodtest interval.
Subsequently, dose-response studies were perfomed using a 1 5-minute injectiodtest
interval.
The time-response studies were performed in the MMT model. The dose-
response studies were performed in both the MMT and the MES rnodels. The MES
model was used because it is a standard model of tonic-clonic seizures. The MMT model
was used because it allows the evaluation of both tonic and the clonic responses
simultaneously.
In experiment 3, studies were done in both female and male adults rats, and in
male and female 1 S-day-old rat pups. The following experiments were performed:
Experiment 3a: the anticonvulsant efTects of progesterone in female rats
Experiment 3b: the anticonvulsant effects of progesterone in male rats
Experiment 3c: the anticonvulsant effects of progestemne in female rat pups
Experiment 3d: the anticonvulsant effects of progesterone in male rat pups
These experiments were designed to determine the phannacology of progesterone
in tonic-clonic seinire models, and also to detemine whether progesterone is as effective
in stopping seinires in males and infants as it is in females. It was hoped that these
studies would provide a basis for using progesterone in clinical trials in a broader
epileptic population (children and men as well as women). It was hypothesized that
progesterone would be as effective in infants and adult males as it is in adult females.
5.2 Methods
Subjects
In experiments 3a and 3b, intact young adult female or male Wistar rats were used as
subjects. Subjects were 60 days old upon arriva1 fiom the breeding f m . Subjects were
allowed one week for acclimatization and daily handling before any treatments or tests
were performed.
In experiment 3c and 3d, 1 S-day-old, Wistar male and femaie rats were used as
subjects. Subjects were 8 days old upon arriva1 fiom the breeding farm. They arrived as
"made up" Mers of 12 with their dam.
Drug Preparation and Administration
In d l of the experiments, progesterone was prepareù with beta-hydroxy
cyclodextnn and administered SC in the rough of the neck.
In experiments 3a and 3b, progesterone (various doses) was injected in a volume
of 4mLkg. Pentylenetetrazol(8Smgkg) was prepand in normal saline and administered
in a different part of the rough of the neck in a volume of 4.2mLIkg.
In experiments 3c and 3d, progesterone (various doses) was injected in a volume
of 1 OrnLkg. Pentylenetetrazol(1 SOmg/kg) was prepared in normal saline and
administered in a different part of the rough of the neck in a volume of 10 xnL/kg.
Testing and Seizure Scoring
In the MMT test, pentylenetetrazol was administered at 85mg/kg in experiments
3a and 3 b and at 1 50mgkg in experiment 3c and 3d as described above. S ubjects were
observed for 30 minutes, and scored as described in the General Methods.
MES was administered according to the standard procedures that were described
in the General Methods.
In the time-response studies, a dose of 6Omgkg of progesterone was used, plus
the following injectiodtest intervals: 5 minutes, 15 minutes, 1 hour, 2 hours, 6 hours and
24 hours.
Data Analysis
Time-response and dose-response curves were plotted for both the tonic and
clonic elements of the seizures. Quantal curves were constructed by plotting the
percentage of animals at each time or dose that were protected against the clonic or tonic
seizures. Dose-response curves were fitted with linear regression, and ED,'s and
maximal responses were calculated.
5.3 Results
5.3.1 Experimeat 3.: Adutt Female Subjects
Figure 14a shows the time-response curve in adult females for the suppression of
tonic and clonic se imes in the MMT mode1 &er a single dose of progestemne
(6OmgIkg). Both tonic (forelimb extension) and clonic components of the seizure were
considerably suppressed 15 minutes after treatment. Protection against the c h i c
component began to disappear after 1 hour. Thereafter, protection against both
Figure 14a,b,c: Anticonvulusant effects of Progesterone in Adult, Female Wistar Rats
The anticonvulsant effects of progesterone on suppression of forelimb clonus (m), forelimb extension (O), and, in figure 14c. hind-limb extension (w). Each point represents data fiom at least 5 subjects. Figure 14a: time-response study following a 60mgkg injection of progesterone in the MMT seinire test (8Smgkg of pentylenetetrazol, SC). Figure 14b: dose-response study in the MMT s e i m test. Progesterone was administered 15 minutes prior to the seinw test. Figure 14c: dose-response study in the MES seinire test (1 X h A of current delivered via comeal electrodes). Progesterone was administered 15 minutes prior to the seizure test.
time
Progesterone Dose-Response, MM 2--
dose (mglkg) 1 I 1 I I I I
1 5 1 O 20 40 80 160
Progesterone Dose-Response, MES
1 dose (mglkg)
Firiure 14a.b.c: adult fernales
components declined. Curiously. there was major protection against both components of
the seizure only 5 minutes &er SC progesterone injection.
Based on the tirne-response study, dose-response tests were carried out using a
15-minute injectiodtest interval. Figure 14b shows the dose-response c w e for the
suppression of MMT-induced tonic and clonic seizures in adult females. The ED,s for
tonic (forelirnb extension) and clonic seinws were 14 and 19 mgkg, respectively.
Maximal responses (1 00% protection) were observed at 80 and 160 mgkg for tonic and
clonic seizures, respectively .
Figure 14c shows the dose-response curve for the suppression of MES-induced
tonic and clonic seimes in adult fernales. The ED, for both tonic (hind-limb extension)
and clonic seizuies was >320 mgkg. Maximal response for tonus (40% protection) was
observed at 320rng/kg.
5.33 Experiment 3b: Adult Male Subjects
Figure 15a shows the time-response curve in adult males for the suppression of
tonic and clonic seizures in the MMT mode1 after a single dose of progesterone
(6Omgkg). 60th tonic (forelimb extension) and clonic components of the seizures were
drastically suppressed 15 minutes after treatment. Protection against both components
began to disappear after 2 h o u . At 5 minutes, there was complete protection against the
tonic seizure, but only partial protection against the clonic seinire.
Based on the time-response study, and also for consistency with the experiments
on female rats, dose-response tests were carried out using a 15-minute injectiodtest
interval. Figure 1 Sb shows the dose-response curve for the suppression of MMT-induced
tonic and clonic seinires. The ED,s for tonic (forelimb extension) and clonic seizures
were 23 and 35 mgkg respectively. Maximal responses (1 00% protection) were
observed at 80 rnglkg for both tonic and clonic seizws.
Figure 1 Sc shows the dose-response curve for the suppression of MES-induced
tonic and clonic seizures. The ED,*s for tonic (hind-limb extension) and clonic seizures
were 220 and >320mg/kg respectively. The maximal response for tonic hind-limb
extension (1 00%) was reached at 320mgkg.
5.3.3 Experiment 3c: Immature Female Subjects
Figure 16a shows the tirne-response curve in immature fernales for the
suppression of tonic and clonic seizures in the MMT mode1 after a single dose of
progesterone (6Omgkg). Both the tonic (forelimb extension) and clonic components of
the seizure were considerably suppressed 15 minutes &er treatment. Protection against
Figure 1 Sa,b,c: Anticonvulusant effects of Progesterone in Adult, Male Wistar Rats
The anticonvulsant effects of progesterow on suppression of forelimb clonus (O), forelimb extension (O), and, in figure 15c, hind-limb extension ( Each point represents data fiorn at least 5 subjects. Figure 1 Sa: time-response study following a 6Omg/kg injection of progesterone in the MMT seizure test (85mgkg of pentylenetetrazol, SC). Figure 15 b: dose-response study in the MMT seinire test. Progesterone was administered 15 minutes prior to the seinire test. Figure 1 Sc: dose-response study in the MES s e i w test (150rnA of current delivered via comeal electrodes). Progesterone was administered 15 minutes prior to the seizure test.
Progesterone Time-Response, MMT
time 1 , r 1 I
Sm 1Sm lhr 2hr 6hr
dose (mglkg) 1 1 I I 1 1 r
1 5 10 20 40 80 1 60
O
Q n 1
n m
n w O a
Dose (mglkg)
Ficiure 1Sa.b.c: adult males
both components began to disappear after 2 hours. Even at 5 minutes, there was
major protection against both the clonic and the tonic components of the seinires.
Based on the the-response study, dose-response tests were carried out using a
15-minute injectiodtest interval. Figure 16b shows the dose-mponse curve for the
suppression of MMT-induced tonic and clonic seizures. The EDs0s for tonic (forelimb
extension) and clonic seizures were 15 and 28 mg/kg respectively. Maximal responses
(1 00% protection) were observed at 80 and 160 mgkg for tonic and clonic seizures,
respectively .
Figure 16c shows the dose-response c w e for the suppression of MES-induced
tonic and clonic seizures. The ED,,s for tonic (hind-limb extension) and clonic seizures
were 23 and 1 6Omgkg respective1 y. Maximal responses (1 00% protection) were
observed at 320 mgkg for both tonic and clonic seizures.
53.4 Experiment 3d: Immature Male Subjects
Figure 17a shows the time-response curve in immature males for the suppression
of tonic and clonic seizures in the MMT mode1 after a single dose of progesterone
(6Omglkg). Both tonic (forelimb extension) and c h i c components of the seinires were
considerably suppressed 15 minutes after treatment. Protection against the tonic and
clonic components began to disappear aRer 2 hours and 1 hour, respectively. Even at 5
Figure 16a,b,c: Anticonvulusant effects of Progesterone in 1 5-Day-Old, Female Wistar Rats
The anticonwlsant effects of progesterone on suppression of forelimb clonus (e), forelimb extension (O), and, in figure 16c, hind-limb extension ( Each point represents data fiom at least 8 subjects. Figure 1 6a: time-response study following a 6Omg/kg injection of progesterone in the MMT seizure test (1 SOmgkg of pentylenetetrazol, SC). Figure 16b: dose-response study in the MMT seizure test. Progesterone was administered 15 minutes prior to the seinire test. Figure 16c: dose-response study in the MES seizure test (1 5OmA of current delivered via comeal electrodes). Progesterone was adrninistered 15 minutes prior to the s e i m test.
& i5m lhr P hr 6hr
Progesterone Dose-Response, MMT
dose (mglkg)
Progesterone Dose-Response, MES I
- Q a dose (rnglkg)
I 1 I 1 . Fiqure 16aabac: 15-day-old female rat pups
Figure 1 7a,b,c: Anticonvulusant efTects of Propesterone in 1 5-Daysld, Male Wistar Rats
The anticonvulsant effects of progesterone on suppression of forelimb clonus (*), forelimb extension (O), and, in figure 17c, hind-limb extension ( D . Each point represents data fiom at least 8 subjects. Figure 17a: time-response study following a 6Omgkg injection of progesterone in the MMT seizure test (1 50mgkg of pentylenetetnwl, SC). Figure 17b: dose-response sîudy in the MMT seizure test. Progesterone was administered IS minutes prior to the seizure test. Figure 17c: dose-response study in the MES seizure test (1 SOmA of current delivered via corneal electrodes). Progesterone was administered 15 minutes pior to the seiaire test.
Progesterone Time-Response. MMT 100 -
l
80 -
60 -
40 - O
time -20 1 , m 1 l 1
5m 15m 1hr 2hr 6hr 24hr
1 Progesterone Dose-Response, MMT 100 O 13
I dose (mglkg) I I r I I
1 5 1 O 20 40 80 160
1 Prqlestemne Dose-Response, MES 7 0 0 v 7
1 Dose (mglkg)
E r iiLE FE]
Figure 17a.b.c: 1 5-day-old male rat pups
minutes there was important protection against the tonic seizures, and partial
protection against the clonic seizures.
Based on the time-response study, dose-response tests were canied out using a
15-minute injectiodtest interval. Figure 17b shows the dose-response curve for the
suppression of MMT-induced tonic and clonic seizures. The E D g for tonic and clonic
seizures were 12 and 85 mgkg, respcctively. Maximal responses (1 00% protection) were
observed at 80 and 160 mgkg for tonic and clonic seizures, respectively.
Figure 17c shows the dose-response curve for the suppression of MES-induced
tonic and clonic seinires. The ED,,s for tonic (hind-limb extension) and clonic seizures
were 8 and 160 mgkg, respectively. Maximal responses (100% protection) were
observed at 20 and 320 mgkg for tonic and clonic seinws, respectively.
Table 4 The anticonvulsant EDSO's of progesterone for the suppression of MMT / MES seizures
*Tonic seizures were defined as forelimb extension in the MMT model, and hind-limb extension in the MES model.
Seizure Test
M T
MMT
MMT
MMT
MES
MES
MES
MES
Adult
Adult
PUP
PUP
Adult
Adult
PUP
PUP
Gender
Female
Male
Female
Male
Female
Male
Female
Male
- --
EDSO Owkg) (Tonic*)
14
23
15
12
>320
220
23
8
The results of Expriment 3 are s m a r i z e d in Table 4. Progesterone is an
anticonvulsant in both female and male rats. Progesterone is an anticonvulsant in both
female and male rat pups. The ED,,s in the MMT model are fat lower than in the MES
model.
The original hypothesis was supported that progesterone is anticonvulsant in
males and in infants. These data are novel and warrant M e r study in clinical trials.
In addition, the very rapid onsets of progesterone's anticonwlsant actions
cal1 into question the previous assumptions about the genomic nature of progesterone's
anticonvulsant effects. Preliminary studies of progesterone's mechanism of action were
perfomed in Experiment 4.
Chapter 6:
Mechanistic Studies of the Anticonvulsant E ffects
of Progesterone
6 Mechanistic Sîudies of the Anticonvulsant ERects of Progesterone
6.1 Rationale
It seems probable that the anticonvulsant effects of progesterone are mediated by
one of three different mechanisms: 1) by its active metabolite, allopregnanolone; 2) by
binding to its "classic" (intracellular) receptors; or 3) by binding to the newly discovered
ceIl surface receptors: Expriment 4 was designed as an initial investigation of these
mechanisms.
Most investigators believe that allopregnanolone, progesterone's GABA-A-acting
metabolite, is responsible for its anticonvulsant effects (General Introduction). We could
not test this hypothesis by directly antagonizing GABA-A receptors, because al1 known
GABA-A antagonists are proconvulsant. Therefore, we blocked the conversion of
progesterone to allopregnanolone by using indomethacin. Indomethacin binds to, and
blocks the action of, Sa-reôuctase, fi rst of the enzymes involvcd in the conversion of
progesterone to allopregnanolone.
The initial hypothesis of this thesis had been that progesterone worked through
the classic (intracellular) progesterone receptor. RU486 (Mefipristone), an antagonist of
the classic progesterone receptor, was used to test this hypothesis.
nie onset of progesterone's anticonvulsant effects in Experiment 3 seems too
rapid to be explained either by conversion to allopregnanolone or by binding to the
intracelldar (genomic) receptors. It was hypothesized. therefore, that progesterone's
"fast" anticonvulsant effects were mediated by binding to the ce11 surface receptor. The
expectation was that neither indomethacin nor RU486 would block progesterone's
anticonvulsant effects. The MMT mode1 was used in these tests.
6.2 Methoda
Subjects
Intact, young adult, female Wistar rats were used as subjects. Subjects were 60
days old upon arriva1 from the breeding f m . They were allowed one week for
acclimatization and daily handling before any treatrnents or tests were performed. After
one week, subjects were randomly sorted into the following eight treatment groups:
Experiment 4a (Indomethesin)
1) Vehicle
2) Vehicle
3) Indomethacin
4) Indomethacin
Expenment 4b (RU486)
5) Vehicle
6) Vehicle
+ Vehicle + MMT
+ P4 + MMT
+ Vehicle + MMT
+ P4 + MMT
+ Vehicle + MMT
+ P4 + MMT
+ Vehicle + MMT
+ P4 + MMT
Dmgs and Dmg Administration
Progesterone (1 OOmg/kg) was dissolved with beta-hydroxy cyclodextrin to a
concentration of 20mgkg. It was administered SC in the rough of the neck in a volume
of 4mLkg. This dose is above the ED,, (Experiment 3).
Afier 15 minutes, pentylenetetrazol(85mglkg) - prepared in normal saline at a
concentration of 2 1 mg/mL and injected in a volume of 4mLkg - was administered in a
different part of the rough of the neck in a volume of 4mLkg. This dose prodixes
maximal seizures.
Indomethacin (1 50 mgkg) was pnpared as a suspension, using normal saline, at a
concentration of 37mghL. It was administered IP in a volume of 4mLkg, fifieen
minutes before injecting progesterone. This dose of indomethacin is above doses used
previously to block 5-alpha reductase.
RU486 was prepared as a suspension, using cy do-h ydroxy cyclodex-
in a concentratration of SrnghL. It was administered IP, at a dose of 20 mg/Kg in a
volume of 4mL/kg, 15 minutes before injecting progesterone. This dose of RU486 is
well above doses used previously to block the progesterone receptor.
Testing and Seizun Scoring
Seven days after arrival fiom the breeding fam, testing w a initiated. Subjects
were preinjected with either RU486 or indomethacin. Fifteen minutes later, progesterone
was administered and, 15 minutes after that, pentylenetetrazol was administered.
Subjects were scored for the presence or absence of tonic seizures within 30 minutes after
injection.
Data Analysis
In both experiments, the Chi Square test was used to analyze the data.
6.3 Results
6.3.1 Experiment 4.: Indomethacin
Figure 1 8 shows the e ffect of pre-treatment with indomethach on the
anticonvulant effects of a single high dose of progesterone. These data show that: 1)
pentylenetetrazol was an effective convulsant agent (vehicle+vehicle group), 2)
progesterone at 100mgkg was an effective anticonvulsant (vehicle~progesterone group),
3) indomethacin did not interfere with the convulsant action of pentylenetetrazol
(indomethacin+vehicle group), and 4) indomethacin did not block the anticonvulsant
action of progesterone (indomethacin+progesterone group). These data are statistically
significant using the Chi Square test (P 5 0.001).
6.3.2 Experiment 4b: RU486
Figure 19 shows the effect of pre-treatment with RU486 on the anticonvulant
effects of a single high dose of progesterone. These data show that 1) pentylenetetrazol
was an effective convulsant agent (vehicle+vehicle group), 2) progesterone was an
effective anticonvulsant (vehicle+progesterone group), 3) RU486 did not interfere with
the convulsant action of pentylenetetrazol (RU486+vehicle group), and 4) RU486 did not
block the anticonwlsant action of progesterone (RU486+progesterone group). These
data are statistically significant using the Chi Square test (P 5 0.001)
6.3 Summay
These data suggest that the "rapid" anticonvulsant effects of progesterone are not
mediated by the classic progesterone receptor or by the GABA-A-binding metabolite of
progesterone, allopregnanolone. They suggest that progesterone's "rapid" anticonvulsant
effects are mediated by the newly discovered ce11 surface receptors.
Anticonvulsant effect of progesterone in the presence or absence of indomethacin
vehicle vehicle indomethacin indomethacin + + + +
vehicle progesterone vehicle progesterone
Fiaure 18: The Influence of lndomethacin (1 50 mglkri. IP) on
the Anticonvulsant Effects of Proaesterone (1 00 m a l k a
Seizures were triggered by pentylenetetrazol. Subjects
were young adul fernale rats. Each point represents
data from 10 rats. (*) indicates a significant difference
frorn the ''vehicle+vehicle" controfs. using the Chi
Square test (P<0.001).
Anticonvulsant effect of progesterone in the presenee or absence of RU486
vehicle vehicle RU486 RU486 + + + +
vehicle progesterone vehicle progesterone
Figure 19: The Influence of RU486 (20 mdks. IP) on the
Anticonvulsant Effects of Proaesterone 11 O0 malka
Seizures were triggered by pentylenetetrazol. Subjects
were young adult female rats. Each point represents
data from 10 rats. (*) indicates a significant difference
from the "vehicle+vehicle" controls, using the Chi Square
test (PeO.001)
Chapter 7:
Discussion
1. Discussion
7.1 The Kindling Data: Experiment 1
Experiment 1 was designed to test the anticonvulsant effects of progesterone in
dose-response studies involving the kindling (arnygdala) mode!. Since Experiment 1
attempted to replicate a single-dose study by Edwards et al. (1999), an injectiodtest
interval of 2 hours was used. The hypothesis was that progesterone would be
anticonvulsant at low doses. It was thought that the low-dose anticonwlsant effects
would be "genomic" effects, and that they might be seen in female, but not male, rats.
Anticonvulsant effects were observed only at high doses in Experiment 1 (ED,, >
30mgIkg). These findings are in agreement with Mohammad et al.'s (1998) findings.
Moharnmad et al. (1 998), working in amygdala-kindled male rats, found seizure
suppression with ED,,s of greater than 60mg/kg. The results of Experiment 1, however,
do not agree with the findings of Edwards et al. ( 1 999), since low dose effects were not
observed in either males or fernales. Edwards et al. (1999) found that a low dose of
progesterone (5mgkg) suppressed seizures in 60% of female subjects.
Since progesterone was administered two hours before testing, high
concentrations of allopregnanolone, but not of the parent compound (progesterone) would
have been present at the time of seizure testing. The data fiom Experiment 1 appear to
support the hypothesis that the anticonvulsant effects of progesterone are mediated by
allopregnanolone. They appear to refbte the original hypothesis that low doses of
progesterone, acting through genornic mechanisms, have anticonvulsant effects.
Although the animal data - fiom Experiment 1 and Mohammad's (1998) - appear
to show considerable seizure suppression only at high doses, the clinical data
(Introduction) indicate that progesterone can suppress seizures at low doses. Therefore,
low-dose effects were further investigated. Some features of the technique, which might
have "masked" the low-dose effect of progesterone, were considered, and appropriate
experiments were performed: 1) Phararncokinetic effects were ruled out by altering the
route of administration and the vehicle. Neither changing the route nor the vehicle
revealed a low dose effect. 2) The role of estrogen in synergizing progesterone's
anticonvulsant effect was explored by using estrogen-primed (in ovariectomized rats) and
intact (as opposed to ovariectomized) subjects. Since subsets of progesterone receptors
are "inducible" by estmgen (Introduction) and the initial preparation used ovariectomized
rats, it was thought that these "inducible" receptors might mediate a low-dose
anticonvulsant effect of progesterone. Neither estrogen "priming", however, nor the use
of intact subjects revealed a low dose anticonvulsant response to progesterone. 3) As a
positive control, dose-response studies were preformed with phenobarôital in
ovariectomized and estrogen "primed" subjects. These studies replicated Albnght and
Burnham's findings (1980) in male rats, and indicated that anticonvulsant effects could
be found in the paradigm used in the present study.
7.2 The 'Chronic, Low-Dose" Data: Experiment 2
In a M e r search for low-dose effects, Experiment 2 was designed to test the
anticonvulsant action of chronically adrninistered, low-dose progesterone and MPA. This
treatment regimen imitates clinical practice more closely than the acute treatments used in
Experiment 1. The hypothesis was that low-dose of progesterone and MPA adrninistered
chronically would be anticonvulsant. It was assumed again that the anticonvulsant effects
would be "genomic" effects.
Chronic (7 day), low-dose administration of progesterone (Silastic@ capsules
resulting in luteal phase levels) or MPA (1 Smgkg, IM), however, failed to show a
significant anticonvulsant action in a variety of seinire models. These findings are in
agreement with Holmes and Weber (1 984) who reported that npeated administration of
low doses of progesterone (Smgkg) had no effect on kindling parameters. These findings
are in disagreement with Edwards et al. (1 999) who reported that chronic low-dose
progesterone (Silastic@ capsules) retarded kindling rates and raised seizure thresholds. It
appears then that chronic, treatment with progesterone does not produce noteworthy
anticonvulsant effects at low doses. These data are in agreement with Stitt and Kinnard
(1 968) who found that chronic low-doses of progesterone (5mg/kg) and MPA (1 Omglkg)
did not alter MES seizure thresholds. These data are also in agreement with Woolley et
al. (1 962) who found that chronic progesterone (5mg/kg) only altered MES s e h e
thresholds by 5%. These data refùte the original hypothesis. It should be noted,
however, that trends were seen in several models. These represented the first suggestion
of low-dose effects that were seen.
7.3 The Influence of gender and Age: Studies at 15-Minute InjectiodTest
Intewal
Experiment 3 was undertaken &ter Edwards et al. (submitted) reported
anticonvulsant effects of progesterone in 15-day-old rat pups using a 15-minute
injectiodtest interval and a dose of 2Omgkg. These data suggested that Experiments 1
had failed to show low dose efiects because the injectiodtest interval had been too long,
and Experiment 2 had failed to show low dose eEects because the dose had been too low.
Time-response studies were therefore performed in adults and pups. These were
designed to rneasure the time of onset and offset of progesterone's anticonvulsant actions.
Anticonvulsant actions were seen at 5 minutes d e r SC injection. These peaked by 15
minutes, and declined after 2 hours.
Subsequent to the time-response studies, dose-response studies were performed in
adult and 15-day-old female and male rats in the MMT model. It was hypothesized that
progesterone would be an effective anticonwlsant in the female and male adults and in
15-day-old rat pups. This was found to be true in the MMT seinw test, where relatively
low dose effects were found (for tonic foreiimb exteasion), but not in the MES seizure
test, where only high dose effects were found, at least in adults.
Comparing the effects of progesterone in the different MMT groups, few
differences are seen. The ED,,s for tonic forelimb extension are similar in adult females
and 15-day-old rat pups (1 2- 14 mg/kg). They are somewhat higher in adult males
(3Omgkg) but still relatively low. These data are consistent with the kindling data
(Expriment 1). where male testosterone-replaced subjects had higher EDs0s than female
subjects. The MMT data are a new addition to the literature.
Comparing the effects of progesterone in the different MES groups, ED,s for
tonic hind-lirnb extension are much higher in adults (>320 mgkg in females. 240mg/kg
in males) than in 15-day-old subjects (24rnglkg in females, 7mg/kg in males). 11 is
interesting that progesterone is not a7 effective anticonvulsant against adult MES
seizures, but it is quite effective against infant MES seizures. This may reflect the
difference in seizure thresholds between adult and 15-day-old subjects - immature
subjects having much higher thresholds. The MES stimulus may be closer to threshold in
infants than adults. Consequently, a lower anticonwlsant dose of progesterone is
required to raise thresholds above the seizure threshold.
In the MES model. the data regarding the immature subjects are a new addition to
the literature. The data regarding the adult subjects are consistent with Craig (1966) and
Kokate et al. (1998), who found high EDs0s for progesterone in the MES model using
adult mice (300mgkg and 2SOmg/kg, respectively). These data are also consistent with
Stitt and Kirnard (1968), who found that a 70 mgkg of progesterone had no effect on
MES seizure threshold in adult female rats.
lt is unclear why progesterone was a more effective anticonvulsant in the MMT
seizure test in adults than in the MES seizure test. It is possible, however, that in the
MMT test the epileptogenic stimulus is closed to threshold than in the MES test. The
MES stimulus is 5 times threshold (Experiment 2), whereas the MMT stimulus is
possibly just above threshold, since some rats full to seize. This would suggest that
progesterone raises seizw thresholds by a small amunt.
The time-course studies provided some surprising findings, which suggest a
possible novel mechanism of action for progesterone. Progesterone was effective as early
as five minutes after administration. It must be remembered that MMT seizures have a
latency of 5 to 10 minutes, so it would be fair to Say that effects were seen at 10 to 15
minutes afier injection. Still, this is a very rapid response. These findings suggested that
progesteront's anticonvutsant action was not mediated by the gene transcription through
the classical PR. Genomic effects do not occur this fast. They M e r suggested that the
parent compound, and not the active metabolites, might mediate progesterone's
anticonvulsant effect. The time-course data led to Experiment 4.
l IT-
7.4 Studies of Mechanism: Experiment 4
Experiment 4 was designed to test whether. at an early time after injection (1 5
minutes), progesterone's metabolites were involved in the anticonvulsant action of
progesterone. It was hypothesized that they were not. Indomethacin was used to block
the Sa-reductase-mediated conversion of progesterone to metabolites such as
al lopregnanolone. Lt was hypothesized that progesterone would be an effective
anticonvulsant in the presence of indomethacin. This was found to be true. niese
findings are in disagreement with Kokate et al. (1 998), who found that finasteride -
another inhibitor of 5-alpha reductase - blocked the anticonvulsant effects of
progesterone. This conflict will be discussed below.
Experiment 4 was also designed to test whether genomic effects via the "classic"
receptors participated in progesterone's anticonvulsant action. RU486 (20mglkg) was
used to block the classical progesterone receptor. It was hypothesized that RU486 would
not block progesterone's anticonvulsant action. This was found to be true. This finding
is consistent with the previous findings of Mohammad et al. (1998), who also found that
RU486 does not block progesterone's anticonvulsant effects. It appears, then, that the
progesterone's anticonvulsant action does not involve the classic progesterone receptor.
This is consistent with the initial hypothesis.
7.5 How Does Progesterone Stop Seizum? : The Role of AUopregnanolone
Herzog has argued that the clinical anticonvulant actions of progesterone are
completely mediated by its GABA-modulating metabolite, allopregnanolone. A nurnber
of animal researchers agree with this point of view (eg. Mohammad et ai., Kokate et al.,
Landgren et al., and Frye et al.) Two facts, however, have always suggested that this is
not the case: 1) Matson's Clinical study (1985) found that MPA was effective in treating
catamenial seizures. MPA is not metabolized to any major degree to a GABA-A
receptor-binding compound like allopregnanolone. 2) Relatively low doses of
progesterone are used clinically. Herzog's progesterone treatment achieves normal luteal
phase levels of 5-25ng/mL (Herzog, 1986). This results in allopregnanolone
concentrations lower than those necessary to produce its anticonwlsant effects(Kokate et
al., 1994;Kokate et al., 1996).
Data fiom present experiments provide new and direct evidence that non-GABA-
mediated anticonvulsant effects exist for progesterone. In Experiment 4, blockade of the
conversion of progesterone to allopregnanolone had no effect on progesterone's
anticonvulsant actions. This implies that progesterone has major anticonvulsant effects
that are entirely independent of GABA.
These data are not in complete agreement with the past studies of Kokate et al.
(1 998). There are only iwo cornmercially available Sa-reductase inhibitors, finasteride
( P r o s c d ) and indomethacin (IndocidO). Finasteride has been used by other
investigators in the epilepsy field to determine the mechanism of action of progesterone.
Kokate et al. (1 998) used high doses of finasteride in conjunction with progesterone, and
found that it completely blocked progesterone's anticonvulsant actions. Hence, Kokate
concluded that progesterone's anticonvulsant action is entirely due to its metabolite,
allopregnanolone. Recent studies (Edwards et al., personal communication), however,
have shown that finasteride is itself a convulsant, and that high doses of finasteride cause
seizures in both mature and immature rats (unpublished data). Therefore, this excludes
the use of finasteride for mechanistic studies in seizure models. The finding, by Kokate,
that finasteride blocks the anticonvulsant effects of progesterone could be due to
finasteride's proconvulsant effects, rather than its inhibitory effects on Sa-reductase. It
seems unlikely, therefore, that allopregnanolone plays a major role either in the clinical,
or in the "fast" effects seen in the present study.
7.6 How Does Progesterone Stop Seizures? : Non-GABA-Mediated Actions
What are the possible non-GABA-modulating mechanisms of progesterone's
"fast" anticonvulsant action? Four possible mechanisrns may be considered: 1) non-
specific effects, 2) binding to the intracellular receptor, or 3) non-GABA-acting
metabolites, and 4) binding to the membrane receptor.
1) Non-Specilc Effects
Non-specific effects refer to alterations in neuronal excitability due to changes in
membrane fluidity, or membrane expansion. In other worâs, might progesterone be
acting as an anesthetic? If this were an important mechanisrn of progesterone's
anticonvulsant action, then it would be expected that cholesterol, which has a similar
chemical structure, would be an equally good anticonvulsant. Expriments on cholesterol
have not shown it to be an effective anticonvulsant at similar doses (Likhodi et al.,
unpublished data).
2) Binding to the Intracellular Receptor
The assumption that guided the original hypothesis of this thesis was that
progesterone was acting through the "classic" intracellular receptors. The rapid onset of
progesterone's anticonvulsant actions in Experiment 3 made this dubious, and the fact
that RU486 in Experiment 4 failed to block progesterone's anticonvulsant action seems to
rule it out. This indicates that the classic progesterone receptor is not involved in
progesterone's anticonvulsant actions.
3) Other Metabolites
As well as being metabolized to allopregnanolone, progesterone is also
metabolized to IO-alpha dihydroprogesterone and 1 1 -deoxycorticosterone (Figure 1).
There is no evidence that 20-alpha dehydroprogesterone has any anticonvulsant effects.
1 1 -deoxycorticosterone, however, is M e r broken d o m to GABA-A-binding
metabolites and does have GAB A-mediated anticonvulsant effects. Recently , Edwards et
al. (unpublished) have shown that 1 1 -deoxycorticosterone has sigaif~cant anticonvulsaat
properties even in the presence of finasteride. This contrasts with the anticonvulsant
effects of progesterone, which can be blocked by finasteride (Kokate et al., 1998). This
irnplies that progesterone's and 1 1 -deoxycorticosterone's non-GABA-mediated effects
are different. Therefore, it is highly unlikely that progesterone's anticonvulsarit effects
are mediated via l 1 -deoxycorticosterone.
4) Binding to the Membrane Receptor
The most likely candidate for the non-GABA-mediated "fast" anticonwlsant
effects of progesterone is the membrane progesterone receptor. The RU486 (Experiment
4) data support this mechanism. RU486 blocks the intracellular progesterone receptor but
has a very low affiinity for the membrane progesterone receptor.
The MPA data are also consistent with this hypothesis. MPA binds the
intracellular, but not the cell surface receptor.
Assuming that propesterone's anticonvulsant action is mediated by its membrane
receptor, what downstream mechanisms could be involved? This is not clear as yet. the
pi-imary known effect of the membrane progesterone receptor is to increase calcium
currents, which should be procovulsant (Introduction) in the brain. These findings,
however, are from sperm data. There is no evidence as yet, that the progesterone
membrane receptors enhance calcium currents in the brain.
There is currently a lack of comprehensive information on the
electrophysiological effects of progesterone membrane receptors in the brain. Further
research will be required. It seems probable. however, that progesterone's "fast"
anticonvulsant effects are mediated by binding to ce11 surface progesterone receptors.
7.7 How Does Progesterone Stop Seizures? Tbresbold Rises
What is the effect of progesterone on the "systems" level? The fact that
progesterone was effective against MMT seinws, but not against MES seizures (in
adults), suggests that progesterone raises s e i m thresholds, but that the threshold rises
are not large.
Previous studies have also shown that small changes in seime thresholds can
have clinical significance. The ketogenic diet, for instance, raises seizure thresholds
minutely in animal seizure models by only about 20% (Thavendiranathan et al., 2000),
yet it is an effective treatment for intractable childhood seizures. Smail rises in threshold
can be important if they occur in patients that resist the standard anticonvulsant drugs.
7.8 How Does Progesterone Stop Seizures: Clinical Actions
The present work has shown "fast" anticonvulsant effects of progesterone,
unrelated to aliogregnanolone and possibly mediated by membrane receptors. Do these
play a role in progesterone's clinical actions? While they could play an important role
(below), it seems unlikely that they can account for the effects seen clinically by Herzog
and others.
Clinical studies establish progesterone concentrations within the normal
physiological range. Even Smg/kg would produce supra-physiological concentrations,
and the doses used in Experiment 3 (EDso's were 10-1 5 mgfkg.) are certainly supra-
physiological.
Thus, while we have discovered new anticonvulsant actions of progesterone, they
are probably unrelated to the effects seen in clinical studies. Those effects probably relate
to progesterone's contraceptive and anti-estrogen effects.
1) Contraceptive effectg
The contraceptive-dated anticonvulsant effects of progesterone relate to its
ability to eliminate the seizure triggers in catamenial exacerbation - the seinire triggers
being estrogen and progesterone withdrawal. Progesterone does this by stopping the
production of endogenous hormones through negative feedback to the hypothalamus and
the anterior pituitary (Introduction). The progesterone intracellular receptor may be
involved in this process. This mechanism would require prolonged exposure to
progesterone.
2) Antkstrogen Effects
Progesterone's anti-estrogen effects may also be nlated to its actions on the
classic progesterone receptors (Introduction). This mechanism involves the ability of
PR-A to act as a transcriptional inhibitor of other hormone receptors, including the
estrogen receptors. Together, these mechanisms may explain progesterone's low dose
clinical effects. This hypothesis is tenned the bbcontraceptive" hypothesis.
The "contraceptive hypothesis" of the anticonvulsant actions of progesterone -
outlined above - would deal with dl three patterns of catamenial epilepsy: Pattern 1)
exacerbation at the time of ovulation, Pattern 2) exacerbation during the entire luteal
phase, and Pattem 3) exacerbation during the preimenstnial period. Progestins (used as
contraceptives) stop the production of sex hormones through negative feedback in the
hypothalamus and the ovaries. Hence, a patient who uses progestins as contraceptives: 1)
would not have an estrogen surge which is associated with ovulation, 2) would not have
elevated levcls of estrogen which are associated with the luteai phase, and 3) would not
have the progesterone withdtawal which is associated with the perimenseual period.
Bauer's clinical studies ( 1992) support the hypotheses that the anticonvulsive
properties of progestins are linked tc their contraceptive mechanisms of action. He used
a synthetic GnRH analogue to suppress the fertility cycle in intractable epileptic patients
with catamenial exacerbation. Using this treatment, he was able to reduce the freguency
or abolish seizures to an equal or greater degree as in the studies, which have used
progestins.
In summary, progesterone may stop seinws in three ways. At tmly low doses, in
the ch ic , it may have "contraceptive" effects. At very high doses, in animal studies, it
may have "slow" anticonvulsant effects related to its sedative metabolite,
allopregnanolone. In addition, as we have show in the present work, at m o d e m doses,
progesterone has "fast" anticonvulsant effects, possibly related to binding to the ce11
surface receptor.
The "fast effects" of progesterone constitute a novel finding. It may provide a
new target, and a new rnechanism of action, for anticonvulsant drug development.
7.9 Clinical Relevance of the Present Study
To date, only a srna11 number of female patients suffering fiom catarnenial
seinires have benefited From progesterone therapy. The present study found major
moderate-dose anticonvulsant actions of progesterone in adult male as well as female
subjects (Expriment 3). Similady, major anticonvulsant effects were seen in immature
subjects (Experiment 3). These "fast", moderate-dose effects were not associated with
sedation. These findings imply that progesterone might be used clinically in males,
infants and non-catarnenial females. A series of expanded clinical trials are in order.
7.10 Future Studies
Future studies must investigate the mechanisms of action of progesterone, and
also the nature of catamenial and intractable seizures:
1) To investigate the "fast" effects of progesterone, specific agonist and antagonists
against the membrane progesterone receptor should be tested. Currently, these
compounds exist, although they are not available commercially. This work should be
carried on in whole animals, as well as in slice preparations. An alternative to specific
agonists is BSA-conjugated progesterone. This cornpound does not cross ceIl membranes
and its activity is limited to membrane receptors. Since BSA-conjugated progesterone
also does not cross the blood-brain-bamer, it should be used in slices or administered
ICV via an implanted cannula in whole animals.
2) To show more conclusively that the "fast* effects of progesterone are not
mediated by its GABA-modulating metabolite, plasma levels of progesterone and
allopregnanolone should be measured. Hormone levels should be measured in a time
response study, using the same parameters as Experiment 3. Hormone levels should also
be measured in the presence or absence of indomethacin.
3) In the kindling experiments, dose-response studies were performed two hours
afler the administration of progesterone. These studies should be repeated at a test
interval of fifteen minutes. This would show whether the "fmt" effects are also present in
the kindling model, a model of complex partial seinires.
4) One of the anticonvulsant mechanisms of progestemne may be antiestrogenic
effect. This cm be tested easily and directly using antiestrogens such as roloxifene.
These should be tested as anticonvulsants.
5 ) The intractable nature of seizures with catamenial exacerbation may relate to the
role that estrogen plays in complex partial seizures. In Experiment 1, Estrogen treatment,
alone, lowered seizure thresholds at the (amygadala-kindled) seinve focus. This finding
that estrogen is proconvulsant is in agreement with al1 previously published data. The
fact that seizure thresholds drop at the seizure focus, however, explains why the
catamenial exacerbation is often associated with complex partial seizures. Estrogen
lowers seizure thresholds focally (in limbic structures) and also globally by a small
dcgm (less than 30°/0). While this wvould not be important for the treatment of
generaiized seizures, it has radical consequences for complex partial seizum. Albright
and Bumham (1980) showed that AED's raise seime thresholds globally, but not in
limbic structures. Hence, an endogenous substance (estrogen) that lowers limbic seizure
thresholds by as much as 30% could cause seinires despite the presence of therapeutic
doses of AED's. In fact, toxic doses of AED's would be required to return limbic seizure
thresholds back to normal.
The role that estrogen plays in cornplex partial seizures, therefore, can be studied
in the following series of experiments. First dose-response studies should be performed
on the effect of estrogen on after-discharge threshold in amygdala and cortical implanted
subjects (before and after kindling). Next. the effect of AED's and antiestrogens on
raising focal and generalized seizure thresholds in estrogen-pretreated, amygdala and
cortical kindled subjects should be studied. If there is a difEerence in response between
cortical and amygdala kindled subjects (as hypothesized), then the mechanism of action
should be studied in slice preparations, which compare the effect of estrogen (on current
fluxes for instance) on tissue fiom the cortex and the amygdala.
6 ) The finding that estrogen lowers seimre thresholds at the seizure focus also has
significance for the role that glia play in the development of cornplex-partial seizures.
There are no estrogen receptors in the neurons of the baselateral amygdala (Table 1) - the site of the seinire focus - in the normal rat. Garcia-Segura's group (1 999), however,
found that in response to injury activated glia express aromatase (Appendix 2). This
response is irrespective of the site of injury. Hence, it is IikeIy that at the site ofthe
seimre focus, estrogen is stimulating activated glia rather than neurons to lower the
seizure threshold.
This theory can be verified by studying the effect of activation and inactivation of
astrocytes on kindling thresholds. Alpha-arninoadipate, a compound that selectively
destroys astrocytes, can be used at the site of kindling focus to test this hypothesis.
Appendix 1:
AEDs
Appendix 1 : AED's
Pharmacological intervention in epilepsy began with the use of potassium
bromide by Locock in 1857(Friedlander, l986b).
In 1 9 1 2, Hauptman introduced phenobarbital as the first organic antiseizure
compound (cited by Woodbur~ and Fingl, 1975). Ticku and Olsen discovered that
phenobarbital binds to the barbiturate site on the gamma-aminobutyric acid (GABA)
receptor, and enhances GABA-mediated inhibitory pathways(Ticku and Olsen, 1978).
Phenobarbital is no longer used as a first line therapy due to its severe sedative side
effects. It is still used however, in poly-pharmacy and, in some cases, in the treatment of
tonic-clonic and partial seizures(Leman-Sagie and Leman, 1 999; Yukawa, 2000).
Meria and Putnarn introduced phenytoin in 1938(Glazko, 1986). The discovery
of phenytoin was the result of a conscious effort to find a phenobarbital-like dmg with
less sedative side effects. Many variants of the phenobarbital molecule were generated
and tested. Phenytoin was the compound discovered. Phenytoin also marked an
important advancement in the development of anticonvulsant drugs, since it was the first
marketed antiseinire drug to be developed using an animal mode1 of
epilepsy(Fned1ander 1 986a). Willow and Catterail later discovered that phenytoin exerts
i ts efiect by blocking voltage gated Na' channels(Willow and Catterall, 1982). Phenytoin
is used pnmarily in the treatment of tonic-clonic and partial seizures(Penicca, 1996).
Phenobarbital, and phenytoin are not effective in the treatment of absence
seinues(Murphy and Delanty, 2000). In 1945, Lemox found that trimethadione - a
variant of phenobarbital - was selectively effective against absence seizures. It was
ineffective against tonic-clonic or partial seimes. In 1963, it was found that
ethosuimide - another variant of Phenobarbital - was effective in the treatment of
absence seizures (Chen et al. 1963). Ethoswimide replaced trimethadione due to leu
severe side effects. Coulter has show that ethoswimide (and trimethidione) act through
inhibition of T-type voltage gated calcium channels in the thalamus (Coulter et al.,
1989;Davies, 1995). This finding was possible because of the work of Rowgoski who
discovered how neuronal firing in the thalamus is afFected by T-type calcium channels
(Suniki and Rogawski, 1989).
Benzodiazepines were introduced in the 1 970's. They include clonazepam,
clobazam, and diazepam. They are effective against a broad range of seizures including
partial. tonic-clonic and absence seimes(Kral1 et al., 1978). Mohler and Okada
discovered the benozodiazepine receptor, which was later found to be a site on the
GABA-A-related chlode chan.net (Mohler and Okada, 1977). Benzodiazepines increase
GABAergic inhibition by binding to the benzodiazepine site of the GABA-A
receptor(Doble, 1 999). B y tradition, clonazepam and cloba~un are used in the chronic
treatment of seizures, whereas diazepam is adrninistered i.v. to stop recurrent seizures
('status epilepticus').
Two important drugs introduced in the 1970's were carbarnazepine and viilproate.
Carbarnazepine, has a similar mechanism of action to phenytoin (Macdonald and Kelly,
1995), but it has fewer side effects than phenytoin. It has therefore, become more widely
used in the treatrnent of tonicslonic and partial seizures(Perucca, 1996).
Valproic acid was discovered accidentally when it was used as a solvent for
potential AED. It has a broad range of activity, including activity against partial, tonic-
clonic, and absence seinws(Perucca, 1996). This is possibly due to valproate's multiple
mechanisms of action. Valproic acid both increases GABAergic inhibition and inhibits
NMDA-mediated ce11 firing (Loscher, 1993).
A number of new AED's have entered clinical PR-Actice in the past decade.
These are called "second generation" AED's (Loscher, 1998). Vigabatrin and tiagabine,
enhance GABA-ergic inhibition. They are effective for the treatment of partial seinws
and tonic-clonic seinires. Vigabatrin increases the levels of the neurotransmitter GABA
by covalently binding to and inhibiting GABA transaminase, GABA's catalytic enzyme
(Graves and Leppik, 1993). Tiagabine hydrochloride increases synaptic GABA levels by
blocking the re-uptake mechanism of GABA (Bourgeois, 1998).
Gabapentin and topiramate are "second generation" AED's with multiple
mechanisms of action. They are effective for partial, tonic-chic and absence seinues.
Gabapentin has also been used as an add-on therapy in cases of intractable cornplex-
partial seizures (Marson et al., 2000). Gabapentin's mechanisms of action include raising
GAB A levels in the brain and decreasing glutamte levels (McLean. 1999). Lt is u n c k
which of gabapentine's mechanisms of action is primarily responsible for its
anticonvulsant effects. Topiramate blocks sodium channels (Shank et al., 2000).
Lamotrigine acts mainly through sodium charme1 blockade. It also inhibits
glutamate release (Coulter, 1997). It is effective against partial, tonic-clonic, and absence
seinws. It also protects against some instances of drop attacks, which are a part of the
Lemox-Gastaut syndrome (Perucca, 1999).
Appendix 2:
Neurosteroids - Tbree Important
Meta bolic Enzymes
Appendix 2: Neurosteroids - Tbree Important Metabolie Enzymes
A2.1 Aromatase
Aromatase converts testosterone into estrogen. Aromatase is normally found only
in the neurons of the limbic structures such as the amygdala, the hippocampus, and the
hypothalamus. After brain injury however, astrocytes begin to express aromatase
regardless of the location of the injury(Bre~er et al., ). This strongly supports the notion
that estrogen is involved in brain repair, and helps explain the positive effects of this
hormone in the treatment of neurodegenerative diseases such as Alzheimer's(0riowo et
al., 1980).
Aromatase distribution in the rat brain changes in three distinct, ontological,
phases(0riowo et al., 1980). Aromatase is expressed in moderate levels in the following
structures between GD16 and PND2 only: anterior mediai preoptic nucleus (aMPN), the
stria1 part of the preoptic area (SPA), and the rostoral portion of the medial preoptic
nucleus (MPN). In a second set of structures arornatase appears at GD 16, and reaches
maximum levels between GD 18 and PND2. Aromatase expression then declines
gradually to adult levels. Expression in these second set of structures forms a continuum
that extends from the caudal parts of the lateral periphery of the media1 preoptic nucleus
(clMPN) to the principal nucleus of the bed nucleus of the stria teminalis (PR-BNST)
and on M e r to the posterdorsal part of the medial amygdaloid nucleus @dMAmg). In
a third group of neurons aromatase appears only after PND 14. These newons are in the
lateral septal nucleus (iLS), the oval nucleus of the bed nucleus of the stria terminalis
(ovBNST), and the central amygdaloid nucleus (CAmg). These findings by Shinoda
( 1994) were in complete agreement with Lauber and Lichtestiger who also characterized
the brain distribution of aromatase simultaneously(Oriowo et al., 1980). Using a different
technique, Maclusky's group found that in addition to the high levels of ammatase in the
hypothalamus-preoptic area and the amygdala, there are lower levels in the hippocarnpus,
midbrain, and the cingulated cortex(0riowo et al., 1980).
The spatial as well as the temporal distribution of aromatase in the brain suggests
that it plays an in important role in the sexual differentiation of the brain. Ontologically,
masculinization of the brain, or lack of it, is determined by sex hormones. Mammalian
brains are ' female' by default. It is the presence of estrogen that "masculinizes" the
brain. This is possible because of aromatase(McEwen et al., 1997): the enzyme that
converts testosterone to estrogen. Aromatase distribution and expression however, is
very similar betweeti male and fernales(0riowo et al., 1 980). The only di fference seems
to be that males express higher Ievels o f the enzyme(lauber et al., 1997;Oriowo et al.,
1980). It is primmily the pre and postnatal surge in testosterone in males (and lack
thereof in fernales) that is converted to estrogen via arornatase and thereby masculinizes
the brain(Shinoda, 1994). Matemal estrogen does not effect the fetal brain because of the
expression of high concentrations of fetolneonatal estrogen binding protein (FEBP) that
keep fiee-estrogen levels very low. FEBP binds estrogens with high affinity, but it does
not sequester androgens(Komeyev et al., 1993).
Subcellular localization of aromatase by electron microscopy has revealed the
presence of this enzyme on the surface of vesicles in presynaptic buttons(Med1ock et al..
1991). This finding lends support to theones on the actions of estrogen at surface
receptors modulating membrane potentiais(Leyendecker et al., 1975).
Two studies have looked at ammatase expression and activity in brain samples
fiom human patients with epilepsy(0riowo et al.. 1980). Expression and activity of
aromatase in hman brains is consistent with data obtained fiom the rat. The authors of
these studies suggest that enhanced aromatase activity in epileptic patients may increase
their risk for having seizures. The present studies however, fail to prove this point since
proper controls are lacking.
A2.2 5-alpha reductase
5-alpha reductase metabolizes a number of hormones, including progesterone. 5-
alpha reductase is ubiquitously distributed in the brain in both neurons and
glia(Leyendecker et al., 1975 j. The enzyme activity however is not unifom in the brain.
Melcangi's group has show two seemingly contradictory results with respect to 5-alpha
reductase activity. niey showed that neurons have greater enzyme activity than
glia(Reddy and Kulkarni, 1998). They also showed that 5-alpha reductase activity is
greater in white matter structures than in the cerebral cortex(Kokate et al., 1999). They
fail to explain this discrepancy in their results, however, the latter finding has k e n
reproduced and investigated in subsequent experiments(Gee et al., 1988;Komeyev et al.,
1993;Medlock et al., 1992).
The finding that 5-alpha reductase activity is greater in white matter led
Melcangi's group to speculate that this enzyme is involved in the process of myelin
formation. They found support for this hypothesis by showing that 5-alpha reductase is
present in myelin sheaths, while axons are devoid of the enzyme(Med1ock et al., 1992).
They also showed that dihyroprogesterone, the 5-alpha reduced metabolite of
progesterone that binds to the progesterone receptor, induces gene expression of
peripheral myelin protein zero (PO)(Kokate et al., 1999). This metabolite however is only
part of the mechanism regulating myelination since other metabolites of progesterone
also regulate myelin related proteins. Baulieu's group simultaneously confimed
Melcangi's findings (and came to the same conclusions) by showing that in addition to its
effects on Schwann cells, progesterone also increases the expression of myelin-specific
proteins in oiigodendrocytes(0nowo et al., 1980).
Evidence from ontological expression and activity of 5-alpha reducatase also
supports its role myelin formation and maintenance. In an early experiment, Melcangi's
group f o n d that 5-alpha reductase activity in pwified myelin from rats was highest in the
third week of life(Me1cangi et al., 1988). Lauber and Lichtensteiger preformed more
detailed studies on the ontogeny of 5-alpha reductase type 1 and they found three distinct
patterns of its mRNA expression(Lauber and Lichtensteiger, 1996). 1) In the embryonic
stage, on GD 12- 1 8, 5-alpha reductase is expressed in the germinal and ventncular zones.
2) During the late fetal and early postnatal development 5-alpha reductase levels
gradually decrease in the ventricular zone. but are expressed in differentiating regions of
the brain including the cortical plate, the thalamus, the cerebellum, and the pyramidal ce11
layer of the hippocarnpus with the subiculurn. 3) From PND 15 and into adulthood lower
levels of the enzyme are detected primarily in white matter structure. This last finding is
in agnement with Melcangi's findings and hypothesis. The distribution of 5-alpha
reductase in pre and early postnatal rat however, suggests a role in proliferation and
differentiation(Lauber and Lichtensteiger, 1996).
A second isoform 5-alpha reductase has been found(Russell and Wilson, 1994). It
is called type 2. 5-alpha reductase type 2 has a much higher affinity for various
substrates than the type 1 isoform, however it is active only in a very narrow PH range
around 5. The type 2 isoform has a different ontological profile from the type 1 enzyme.
It is absent at GD14. It is detected at GD18. It reaches its peak levels at PNDZ, and then
decreases to extremely low levels or becomes absent altogether(Po1etti et al., 1998). In
adutt rats the type 2 enzyme is detected primarily in the hypothalarnas(Po1etti and
Martini, 1999). Unlike 5-alpha reductase type 1, the type 2 isoform gene is highly
inducible by testosterone, but only in cells derived from the hypothalamus. This is
intriguing because both isofoms have a glucocorticoid response element in their
promoter regions. Differences in presence or absence of cofactors might explain
differrntial responses to testosterone. The correlation between the levels of 5-alpha
reductase type 2 during the 'critical period' of sexual dittèrentiation and it's pattern of
inducability by testosterone has led Poletti to hypothesizes that the type 2 isofomi may be
involved in sexual differentiation. Their studies to date however, have been in male rats
only(Po1etti et al., 1998). None-the-less there is strong support for this idea fkom the
observation that a genetically defective type 2 enzymes in the male human results in
pseudohemaphroditism(Imperato-McGinley et al., 1 974;Katz et al.. 1 995).
Recently Kellogg and Frye showed that the activity of 5-alpha reductase is
different in infants and adults(Kellogg and Frye, 1999). In the fetal rat brain (fiom the
last five days of gestation) the levels of progesterone metabolites are twenty fold higher
than the levels of progesterone, whereas the levels of testosterone metabolites are ten fold
lower than the levels of testosterone. In adults however, the main substrates for 5-alpha
reductase seem to change. The conversion of progesterone to its metabolites becomes
more inefficient, but the levels of testosterone 5-alpha reduced metabolites are three to
ten folds higher than the levels of the parent hormone.
A2.3 3-alpha hydroxysteroid dehydrogenase (3-alpha HSD)
3-alpha HSD metabolizes a number of hormones, including 5-alpha reduced
progesterone. 3-alpha HSD is not found in neurons(Me1cangi et al., 1993). 3-alpha HSD
is found primarily in type 1 astrocytes and to a lesser degree in
oligodendrocytes(Melcangi et al., 1994). Al1 regions of the rat brain express 3-alpha
HSD. The highest levels of 3-alpha HSD however, are found in the olfactory bulb(Cheng
et al., 1994a). It is curious that the olfactory bulb also contains the highest concentration
of intemewons in the brain. The distribution of 3-alpha HSD is consistent with the role
of 3-alpha modified hormones in modulating the activity and genomic expression of
GABA-a receptors(Bele1li et al., 1990;Srnit.h et al., 1998a).
The promoter region of 3-alpha HSD contains response elements for estrogen,
glucocorticoids and progesterone(Penning et al., 1997).
3-alpha HSD has a substrate affinity in the low micro molar range for 5-alpha
reduced homiones. It has specific activity that is a 100 fold higher than 5-alpha
reductase(Pe~ing et al., 1985). This indicates that in vivo, 5-alpha reductase is the rate
limiting step in producing 5-alpha,3-alpha modified steroids.
3-alpha HSD is responsible for giving testosterone and progesterone metabolites
GABA-modulating activity. There are however, important interspecies differences in the
activity of this and other enzymes in the brain. Costa's group established the levels of
various metabolites of progesterone in the braîns of rats, mice and monkeys(Korneyev et
al., 1993). In the rat the major metabolites are 5-alpha dihydroprogesterone, and 3-
alpha,S-alpha tetrahydroprogesterone. In the mouse the major metabolites are 5-alpha
dihydroprogesterone, and 20-alpha dihyrdroprogesterone. In the monkey the main
metabolite is 20-alpha di hydroprogesterone.
Appendix 3:
The Role of Sex Hormones in Some Important
Aspects of Neuronal Plasticity
Appendix 3: The Role of Sex Hormones in Some Important Aspects of Neuronai
Plasticity
A3.1 Some Behavionl Changea Dunng the Fertillty Cycle
A number of behavioral changes that occur during the estrous cycle, in pregnancy,
at puberty or at menopause have provided insights into the role of sex hormones on brain
plasticity. Figure 2 dernonstrates the variation in levels of progesterone and estrogen in
the estrous cycles of hurnan and rat respectively.
Premenstnial syndrome is correlated temporally to progesterone withdrawal.
Smith's group suggests that premenstrual imtability is due to the effect of progesterone
withdrawal on the genomic expression of the alpha4 subunit of the GABA-a
receptor(Smith et al., 1998a). The differential expression of various GABA-a subunits
altea the electrophysiological properties of the GABA-a cornplex. GABA-a complexes
containing alpha-4 subunits have decreased chloride currents. A brain rich in alpha-4
subunits is more susceptible to seimres(Smith et al., 1998a) and is insensitive to the
potentiating effect of benzodiazepines(Reddy and Rogawski, 2000; Smith et al., 1 998~).
Smith suggests that postpartum depression and postmenopausal dysphoria also share the
same mechanism as premenstmai syndrome.
Tiredness is linked to elevated levels of progesterone. This phenornenon rnay be
apparent in the late luteal phase; however, it is much mon pronounced in pregnancy
where progesterone levels increase to more than ten times luteal levels(Behrenz and
Monga, 1999). There is a close relationship between plasma levels of progesterone and
allopregnanolone(Concas et al., 1998). Tiredness during pregnancy is due to the
agonistic affects of allopregnanolone on the GABA-a receptor(Rupprecht and Holsboer,
1999). The generai sedative effects of progesterone can manifest itself in depression as
experienced by some women during pregnancy or during hormonal therapy(Herzog,
1 986a).
The behavioural change that is of particular interest to this thesis is the catarnenial
pattern of seizures. If pathological changes in the brin are an extention of neuronal
placicity (McEachem and Shaw, 1999), then catamenial exacerbation too is a fonn of
neuronal plasticity in response to hormones. In Catamenial epilepsy seizure fiequency is
increased during the estrous cycle at times of high estrogen levels or with progesterone
withdrawal, and it seems to decrease at times of high progesterone levels(Herzog et al.,
1 997b).
Rat behavior in response to hormones is often similar to human behavior. There
is a notewonhy exception. In the human female the height of sexual receptivity generally
occurs at ovulation(Singer and Singer, 1972;Stanislaw and Rice, 1988). This corresponds
to a peak in estrogen levels. In rats, sexual receptivity also occurs at ovulation; however,
it is not pnmarily mediated by estrogen. This event occurs on estrous at the height of the
progesterone peak(1chikawa et al., 1972). - . Estrogen priming in proestous however, is a
necessary prelude for the lordosis behavior(Freeman et ai., 1976).
A3.2 Neuronal Plasticity and the Fertility Cycle: Cellular Changes
It is possible to think of epilepsy as a pathologicai form of neuronal plasticity. In
this paradigm plasticity is a continuum encompassing development of neuronal circuitry,
learning, memory, and pathological disorders of the CNS(McEachem and Shaw, 1999).
Hence a number of cellular and molecular markers of neuronal plasticity can be used to
assess the extent of changes in the brain in response to various stimuli. Sex hormones
induce a number of cellular and molecular changes that are associated with neuronal
plasticity .
LTP and LDP are standard measurements of synaptic plasticity. LTP is very
simply a semi-permanent enhancement in synaptic strength while LDP describes the
inverse process. Estrogen enhances long-terni potentiation (LTP) and attenuates long-
term depression (LTD)(Good et al., 1999;Warren et al., 1995). This effect is mediated by
NMDA receptors(Foy et al., 1999). The effect of progesterone on these parameters is not
clear. Allopregnanolone however, decreases LTP(Dubrovsky et al., 1993). LTP and LDP
are also correiated with seinue activity. Electrical and chernical kindling enhance
LTP(Ben Ari and Gho, 1988;Suhila and Steward, 1986). Likewise, LTP speeds the
kindling process(Sutu1a and Steward, 1987).
In rats increased neurogenesis is obsemed during proestrous (Taubal1 Md
Lindstrom, 1993). Neurogenesis is a naturai process that occurs at a basal rate in the
dentate gyrus and around the third ventricle(Kuhn and Svendsen, 1999). Many of the
new neurons that are produced die soon after through apoptosis. Estrogen is an anti-
apoptotic agent(Garcia-Segura et al., 2001), and it increases the number of new neurons
by increasing cell s d v a l and also by increasing the rate of production(Tanapat et al.,
1 999). Proges terone' s e ffect on neurogenesis is unclear.
New dentate granule neurons have a greater capacity for long-terni
potentiation(Wang et al., 2000). They also have less GABA-ergic inhibition than older
dentate granule neurons(Wang et al., 2000). Therefore, enhanced neurogenesis
potentially enhances hippocampal excitability. Gould has proposed that neurogenesis is a
potential mechanism through which hormones affect spatial learning and memory(Gou1d
et al., 1999). Recently, abnormal neurogenesis has been linked to ôoth the onset and
recovery fiom depression(Jacobs et al., 2000). Also, various methods of inducing
seizures have been shown to enhance neurogensis(Parent et al., 1998;Scott et al., 2000).
Sex hormones affect spine density of the pyramidal neurons of the
hippocampus(Woo1ley and McEwen, 1993). Spines are outgrowths of dendrites ont0
which afferent axons synapse. Woolley's group found that estrogen increases spine
density by activating NMDA receptors(Wool1ey and McEwen, 1994). Hence increased
spine density is associated with increased neuronal excitability. In the rat estrous cycle,
spine density is greatest in proestrous. corresponding to an estrogen peak, and drop
sharply in estrous corresponding to a progesterone peak(Wooi1ey and McEwen, 1994).
Hormonal regulation of mossy fiber sprouting is similar to the regulation of
pyramidal ce11 spine densities. Mossy tibers are axons of dentate granule cells that
synapse onto the dendrites of pyramidal cells in CA3 region of the hippocampus(Sutu1a et
al., 1992). Estrogen enhances sprouting while the effects of progesterone have not been
examined so far(Teter et al., 1999). Also, various foms of inducing seinires have been
show to enhance mossy fiber sprouting(Sutu1a et al., 1992).
Recently Garcia-Segura proposed that estrogen increases synaptic excitability by
altering synaptic architecture(Garcia-Seguni et al., 1 999). This process involves the
interaction between glia and neurons(M0r et al., 1999). On the afkemoon of proestrous
estrogen activates astroglia by increasing the synthesis of GFAP. Activated astroglia
ensheathe the soma of glutametergic neurons in their vicinity. In doing so they
tem poraril y force the disinhi bit ion of axonosomat ic synapses b y intemeurons.
A3.3 Neuronal Plasticity and the Fertility cycle: Some Molecular Changes
A3.3.1 GABA
GABA is the primary inhibitory amino acidin the &tain. GABA plays an
important role in neuronal plasticity by regulating the expression its receptors or receptor
subunits. Two alpha, two beta, and one gamma subunits make up the GABA receptor
complex and each subunit is present in a multitude of isoforms(Mohler et al., 1996).
Abnonnalities in GABA-ergic response underlie the GABA hypothesis of epilepsy.
Loscher's group did not see any long-tenn changes in the number of GABA-ergic
neurons in the hippocampus after kindling(Lehmann et al., 1996). Reductions however,
were seen in the ipsilateral pirifom cortex(Lehrnann et al., 1998). A~so, regardless of the
number of GABA-ergic neurons, kindling causes a long-lasting decrease in inhibitory
effect of GABA in the hippocampus(Kamphuis et al., 1991). GABA receptor subunits
are altered in various seizure models including kindling(Fol1esa et al., 1999;Kokaia et al.,
1 994;Poulter et al., 1 999).
Estrogen's effects on the GABA complex are still clouded in the literature.
Canonco has argued that estrogen decreases GABA receptor levels in the brain(Canonaco
et al., 1993). Herbison on the other hand has argued that in areas of the bmin containing
estrogen receptors, estrogen significantly increases the levels of some GABA receptor
subunits(Herbison and Fenelon, 1995). Herbison's work is in agreement with Perez's
findings that estmgen changes the function of GABA-a receptors by increasing the
binding of GABA agonists in specific regions of the brain(Perez et al., 1988). This
enhancement of GABA-ergic activity however, does not increase protection against
convulsants(Perez et al., 1 988).
Progesterone's effects on G AB A receptors are ptunanly indirect and involve
progesterone's tetra-hydroxy metabolite, also called allopregnanolone(Concas et al.,
1999). Allopreganaolone binds io the alpha subunit of the GABA-a receptor with a
hundred fold greater afEnity than barbiturates(Gee et al., 1988). Allopregiuurolone
enhances the effects of GABA and can alter subunit expression as well. Progesterone
withdrawal increases the formation of the alpha-4 subunit of the GABA-a receptor(8mith
et al., 1998b). This makes the complex less responsive to agonists of the alpha subunit.
A3.3.2 Glutamate
Glutamate is the primary excitatory amino acid in the brain. It plays an important
role in neuronal plasticity via the AMPA, kainite, and NMDA receptors. Abnonnalities
in glutametergic response underlie the glutamate hypothesis of epilepsy.
Wooley and McEwen have s h o w that estrogen enhances certain excitatory
glutamergic pathways. Estrogen increases NMDA agonist binding directly by regulating
the agoinst binding siteWeiland et ai., 1997). Estrogen also increases the nurnber of
synapses containing glutamatergic receptors(Wool1ey and McEwen, 1994). These
axonodendritic synapses are made on denciritic spines. The estrogen-induced spine
formation in the hippocampus during proestrous contain elevated levels of NMDA-1
receptors, but they lack AMPA receptors(Woolley, 1 998).
Progesterone does not alter the expression or bind directly to glutametergic
receptors. It may however, inhibit NMDA receptors indirectly through it's action on the
sigma receptor(M0nnet et al., 1995). Sigma receptor upon agonist binding potentiates
neuronal response to NMDA(Bergeron et al., 1999). Progesterone is a Sigma receptor
antagonist. Progesterone's effect on NMDA receptors is probably more apparent through
cross-talk with estmgen receptors. Woolley showed that initially, progesterone acts
synergistically with estrogen to enhance CA 1 dendritic spines containing NMDA
receptors(Wool1ey and McEwen, 1993). Eighteen hours after progesterone treatment
however, spine density is significantly decreased.
A3.3.3 Connexin
Gap junctions mediate a non-synaptic mechanism of cell-to-ce11
communication by pemitting direct electrical coupling between cells(Rozenta1 et al.,
2000b). Gap junctions are comprised of a large family of connexins. Six connexins form
a hemichannel complex called a connexon at the cell surface(Dermietzel, 1998).
Co~exons aggregate together into plaques. DisuIfide bonds adjoin connexons fiom
plaques on adjacent cells(Foote et al., 1998;Manjunath et al., 1987). Gap junctions are
involved in neuronal placticity and they are regulated by sex hormones(Perez et al.,
1990). Estrogen, alone or in combination with progesterone, increases connexin
expression(Lye et al., 1993;Shinohara et al., 2000). Progesterone withdrawal also
increases c o ~ e x i n expression(Micevych et al.. 1996). The combination of mechanical or
electrical stimulation and estrogen synergistiacally increase gap junction mRNA
expression. While electrical stimulation alone, or pmgesterone without estrogen
however, do not increase connexin expression (Edwards and Maclusky, unpublished
data).
C o ~ e x i n 43 is the first isoform to be expressed in the brain(Chang and Balice-
Gordon, 2000). In the rat it is expressed at GD 12, peaks at PND2 1 and levels remain
high into adulthood. It is primarily a glial gap junction although it is also found in some
neurons such as the CA 1 pyramidal neurons(Rozenta1 et al., 2000a). Neuronal comexins
such as connexin 32 and 36 are expressed most prominently in late gestation and drops to
adult levets in the first few weeks of life(Bel1iveau and Naus, 1995;Rozental et al.,
2000~). An exception is the neuronal connexin 26, which is primarily expressed in the
developing brain.
Outside the brain, gap junctions have a wide range of physiological functions.
C o ~ e x i n 43 is responsible for the synchronous contractions of the heart. Gap junctions
also synchronize labor contractions. In the brain, they mediate high frequency
oscillations (100-200 Hz) in the hippcarnpus@raguhn et al., 1998;Draguhn et al., 2000).
These high fiequency oscillations seem to involve pyramidal neurons interconnected by
axoaxonal gap junctions(Traub et al., 1999). Gap junctions also mediate slow calcium
waves arnong astrocytes(Giaurne and Venance, 1998;Rottingen and Iversen, 2000).
Temporal expression of gap junctions suggests that they rnay also play a role in neuronal
migration(Naus and Bani-Yaghoub, 1998). Hence, gap junctions may provide a general
rnechanism through which excitatory or inhibitory pathways operate in a synchmnued
fashion.
There is some evidence that gap junctions may be responsible for synchrony of
epileptic seizures. Carlen's group showed that in absence of synaptic transmission, gap
junctions mediate epileptiform activity in hippocampal slices(Carlen et al., 2000;Perez
Velazquez and Carlen, 2000). Ironically, in animal seinire models, connexin expression
is decreased or remains unchanged(E1isevich et al., 1997a;Elisevich et al., 1998;Khurgel
and Ivy, 1996). Likewise, hippocampal tissue From human epileptic patients does not
show an increase in c o ~ e x i n expression(Elisevich et al., 1997b). Chang however,
showed that following axonal injury there is increased gap junction coupling without an
increase in expression(Chang et al ., 2000).
A3.3.4 c-fos
Sex hormones alter the expression of number of early response genes, such as c-
fos. Both estrogen and progesterone enhance c-fos expression(Auger and Blaustein,
1997). c-fos expression is one of the hallmarks of neuronal plasticity and it is commonly
used as a molecular market of seizure activity(Andre et al., 1998;Applegate et al.,
199S;Dragunow et al., 1988;Samonski et al., 1998;Simler et al., 1999). There is some
evidence that c-fos is a convulsant agent and is involved in the spread of seizures in the
kainic acid seizure model(Panegyres and Hughes, 1997). On the other hand, c-fos seems
to be anticonvulsive in the kindling model(Rocha and Kaufman, 1998).
A3.3.5 BDNF
Another family of early response genes is the neurotrophic factors. Neurotrophic
factors are an important part of the maintenance and repair mechanism of newons and
gl ia(Mackay -Sima and C huahb, 2000). Estrogen alters the expression of nurnber of
neurotrophic factors, such as brain-derived neurotrophic factor (BDNF). The response
appears as an increase or decrease in expression depending on brain location or time
interval afier application of estrogen(Gibbs, 1999;Murphy and Segal, 2000).
Progesterone can act in synergy with estrogen to enhance BDNF expression(Gibbs,
1999). Murphy's group has shown that BDNF prevents estrogen-induced dendritic spine
formation in hippocampal neurons(Murphy et al., 1 998). Furthemore, progesterone
prevents spine formation through a non-BDNF mechanism(Murphy and Segal, 2000).
Consistent with Murphy's findings, BDNF decreases seinire susceptibility in the kindling
model(0sehobo et al., 1999;Reibel et al., 2000). Ironically, BDNF enhances LTP(Lu and
Chow, 1999).
Reference List
Aiken,S.P., Brown, W.M., 2000. Treatment of epilepsy: existing therapies and future developments. Front Biosci 5, E124-E152.
Albright,P.S., Bumharn,W.M., 1980. Development of a new phamiacological seizure model: effects of anticonvulsants on cortical- and arnygdala-kindled seinires in the rat. Epilepsia 2 1, 68 1-689.
Albright,P.S., Bumham,W.M., 1983. Effects of phenytoin, carbarnazepine, and clonazepam on coriex- and arnygdala-evoked potentials. Exp Neurol 8 1,308-3 19.
Andre,V., Pineau,N ., Motte, J.E., Marescaux,C., Nehlig, A., 1998. Mapping of neuronal nctworks underlying generalized seinires induced by increasing doses of pentylenetetrazol in the immature and adult rat: a c-Fos immunohistochemical study. Eur J Neurosci 10,2094-2 106.
Apgar,B.S., Greenbrg,G., 2000. Using progestins in clinical practice. Am Fam Physician 62, 1839-1850.
Applegate,C.D., Pretel,S., Piekut,D.T., 1995. The substantia nigra pars reticulata, seinires and Fos expression. Epilcpsy Res 20, 3 1-39.
Auger,A.P., Blaustein,J.D., 1997. Progesterone treatment incrcascs Fos-imrnunoreactivity within some progestin receptor-containing neurons in localized regions of female rat forebrain. Brain Res 746, 164- 170.
Avanzini,G., 1995. Anima1 models relevant to human epilepsies. Ital J Neurol Sci 16, 5-8.
Backstrom,T., 1976. Epileptic seinires in wornen reiated to plasma estrogen and progesterone during the menstrual cycle. Acta Neurol Scand 54,32 1-347.
Backstrom,T., Zetterlund,B., Blom,S., Rornano,M ., 1 984. E ffects of intravenous progesterone in fiisions on the epileptic discharge fkquency in women with partial epilepsy. Acta Neurol Scand 69,240-248.
Baird,D.T., McNeilly,A.S., 198 1. Gonadotrophic control of follicular development and hinction during the oestrous cycle of the ewe. J Reprod Fertil Suppl30, 1 19-133.
Baldi,E., Luconi,M., Muratori,M., Forti,G., 2000. A novel functional estrogen receptor on human spenn membrane interferes with progesterone effects. Mol Cell Endocrinol 16 1,3 1-35,
Behrenz,K.M., Monga,M., 1999. Fatigue in pregnancy: a comparative study. Am J Perinatol 16, 185-188.
Belelli,D., Lan,N.C., Gee,K.W., 1990. Anticonvulsant steroids and the GABA/benzodiazepine receptor- chloride ionophore cornplex. Neurosci Biobehav Rev 14,3 15-322.
Belliveau,D.J., Naus,C.C., 1995. Cellular localization of gap junction mRNAs in devetoping rat brain. Dev Neurosci 17,8 1-96.
Ben AR,Y ., Gho,M., 1988. Long-lasting modification of the synaptic properties of rat CA3 hippocampal neurones induced by kainic acid. i Physiol (Lond) 404,365-384.
Berg,A.T., Testa,F.M., Levy,S.R., Shinnar,S., 1996. The epidemiology of epilepsy. Past, present, and future. Neurol Clin 14, 383-398.
Bergeron,R., de Montigny,C., DebonneI,G., 1999. Pregnancy reduces brain sigma receptor function. Br J E - Phannacoi 127,1769-1776.
Berruti,G., 1998. Signaling events during male germ cell diflerentiation: bases and perspectives. Front Biosci 3, D1097-D1108.
Blankenstein,M.A., Koehorsf S.G., van der Kallen,C.J., Jacobs,H.M., van Spriel,A.B., Donker,G.H., van? Verlaat, J. W., Biaauw,G., Thijssen, J.H., 1 995. Oestrogen receptor independent expression of progestin receptors in hurnan rneningioma--a review. J Steroid Biochern Mol Biol53,361365.
Bonuccclli,U., Melis,G.B., Paoletti,A.M., Fioretti,P., Murri,L., Muratori0.A.. 1989. Unbalanced progesterone and estradiol secretion in catarnenial epilepsy. Epilepsy Res 3, 100- 106.
Bourgeois,B.F., 1998. New antiepileptic dmgs [see comments]. Arch Ncurol55, 1 181- 1 183.
Brann,D.W., Rao,I.M., Mahesh,V.B., 1988. Antagonism of estrogen-induced prolactin release by progesterone. Biol Reprod 39, 1067- 1073.
Brenner,P.F., Guerrero,R., Cekan,Z., Diczfalusy,E., A radioimrnunoassay method for six steroids in plasma. pp. 25 1-64. In: Crosignani PG , James VH , ed Recent progress in reproductive endocrinology London, Academic Press, 1974 W3 SE4779W no 4 1973.
Britton,K.T., Koob,G.F., 1998. Neurophannacology. Prernenstrual steroids? [news; comment]. Nature 392, 869-870.
Brown,T.J., Yu,J., Gagnon,M., Sharma,M., MacLusky,N.J., 1996. Sex differences in estrogen receptor and progestin receptor induction in the guinea pig hypothalamus and preoptic area. Brain Res 725,374.
Buterbaugh,G.G., 1987. Acquisition of amygdala-kindled seizures in female rats: relationship behveen the effect of estradiol and intra-amygdaloid electrode location. Pharmacol Biochem Behav 28,291-297.
Calogero,A.E., Bumllo,N., Barone,N., Palenno,l., Grasso,U., DIAgata,R., 2000. Effects of progesterone on spenn function: mechanisms of action. Hum Reprod 15 Suppl 1,2845.
Carnacho-ArroyoJ., Guerra-Araiza,C., Cerbon,M.A., 1998b. Progesterone receptor isofonns are differentially regulated by sex steroids in the rat forebrain. Neworepon 9,3993-3996.
Camacho-Arroyo,l., Guerra-Araiza,C., Cerbon,M.A., 1998a. Progesterone receptor isofonns are differcntiaiiy regulated by s a steroids in the rat forebtain. Neuroreport 9,39933996,
Canonaco,M., Tavolaro,R., Maggi,A., 1993. Steroid hormones and receptors of the GABAA supramolecular complex. II. Progesterone and estrogen inhibitory effects on the chloride ion channel receptor in different forebrain areas of the female rat. Neuroendocrinology 57,974-984.
Carlen,P.L., Skinner,F., Zhang,L., Naus,C., Kushnir,M., Perez Velazquez,J.L., 2000. The role of gap Junctions in seizures. Brain Res Brain Res Rev 32,235-24 1.
Carter,R.B., Wood,P.L., Wieland,S., Hawkinson,J.E., Belelli,D., Larnbert,J.J., White,H.S., Wulf,H.H., Mirsadeghi,S., Tahir,S.H., Bolger,M.B., Lan,N.C., Gee,K.W., 1997. Characterization of the anticonvulsant properties of ganaxolone (CCD 1042; 3alpha-hydroxy-3beta-methyl-Salpha-pregnan-2O-one), a selective, high-affinity, steroid rnodulator of the gamma-aminobutyric acid( A) receptor. J Phmacol Exp Ther 2 80, 1284- 1295.
Chang,Q., Balice-Gordon,R.J., 2000. Gap junctional communication among developing and injured motor neurons. m i n Res Brain Res Rev 32,242-249.
Chang,Q., Pereda,A., Pinter,M.J., Balice-Gordon,R.J., 2000. Ncrve injury induces gap junctional coupling among axotomized adult motor neurons. J Neurosci 20.674484.
Cheng,K.C., Lee,J., KhannqM., Qin,K.N., 1994b. Distribution and ontogeny of 3 alpha-hydroxysteroid dehydrogenase in the rat brain. J Steroid Biochem Mol Biol50,85-89.
Cheng,K.C., Lee,J., Khanna,M., Qin,K.N., 1994a. Distribution and ontogeny of 3 alpha-hydroxysteroid dehydrogenase in the rat brain. J Steroid Biochem Mol Biol 50,85089.
Compagnone,N.A., Mellon,S.H., 2000. Neurosteroids: biosynthesis and fiinction of these novel neuromodulators. Front Neuroendocrinol2 1, t -56.
Concas,A., Follesa,P., Barbaccia,M.L., Purdy,R.H., Biggio,G., 1999. Physiological modulation of GABA(A) receptor plasticity by progesterone metabolites. Eur J Phannacol 375,225-235.
Concas,A., Mostallino,M.C., Porcu,P., Follesa,P., Barbaccia,M.L., Trabucchi,M., Purdy,R.H., Grisenti,P., Biggio,G., 1998. Role of brain alloprcgnanolone in the plasticity of gamma-arninobutyric acid type A receptor in rat brain during pregnancy and aftcr delivery. Proc Natl Acad Sci U S A 95, 13284-1 3289.
Cooke,l.D., 1988. The corpus luteum. Hum Reprod 3, 153- 1 56.
Coulter,D.A., 1997. Antiepileptic drug cellular mechanisms of action: where does lamotrigine fit in? J Child Neurol 12 Suppl 1, S2-S9.
Coulter,D.A., Huguenard,J.R., Prince,D.A., 1989. Characterization of ethosuximide reduction of low- threshold calcium current in thalarnic neurons. Ann Neurol23,582-593.
Cummings,L.N., Giudicc,L., Morrell,M.J., 1995. Ovulatory function in epilepsy. Epilepsia 36, 355-359.
Darne,F.J., McGarrigle,H.H., Lachelin,G,C., 1989. Diumal variation of plasma and saliva oestrogen, progesterone, cortisol and plasma dehydroepiandrosterone sulphate in late pregnancy. Eur J Obstet Gynecol Reprod Biol32,57-66.
Davies,J.A., 1995. Mechanisms of action of antiepileptic drugs. Seizure 4,267-27 1.
Delaunay,F., Pettersson,K., Tujague,M., GustafssonJ.A., 2000- Functional Werences betwecn the amino- terminal domains of estrogen receptors alpha and beta. Mol Pharmacol 58,584-590.
Dermietzel,R., 1998. Gap junction wiring: a 'new' principle in cell-to-cell communication in the nervous system? Brain Res Brain Res Rev 26, 176- 183.
Desmedt,L.K., Niemegeers,C.J., Lewi,P.J., Janssen,P.A., 1976. Antagonism of maximal metrazol seinires in rats and its relevance to an experimental classification of antiepileptic drugs. Ameimittelforschung 26, 1 592- 1603.
Devinsky,O., Kelley,K., Porter,R.J., Theodore,W.H., 1988. Clinical and electroencephalographic features of simple partial seizures. Neurology 38, i 347- 1352.
Doble,A., 1999, New insights into the mechanism of action of hypnotics. J Psychopharmacol 13, S 1 1 -S20.
Draguhn,A., Traub,R.D., BibbigA., Schmitz,D., 2000. Ripple (approximately 200-Hz) oscillations in tmiporat stntctures f hr Process Citation]. I Cfin Neuroptiysïot 17,36ï-376.
Draguhn,A., Traub,R.D., Schmitz,D., Jefferys,J.G., 1 998. Electrical coupling underlies high-frequency oscillations in the hippocampus in vitro [see comments]. Nature 394, 189-192.
Dragun0w.M.. Robertson,H.A., Robertson,G.S., 1988. Arnygdala kindling and c-fos protein(s). Exp Neurol 1 02.26 1 -263.
Dubrovsky,B., Gijsbers,K., Filipini,D., Binningharn,M.K., 1993. Effects of adrenocortical steroids on long-term potentiation in the limbic system: basic mechanisms and behavioral consequences. Cell Mol Neurobiol 13, 399-4 14.
Edwar&,H.E., Burnham,W.M., MacLusky,N.J., 1999a. Testosterone and its metabolites affect aficrdischarge thresholds and the development of amygdala kindled seizures. Brain Res 83 8, 1 5 1 - 1 57.
Edwards,H.E., Bumharn, W.M., Mendonca,A., Bowlby,D.A., MacLusky,N.J., 1999b. Steroid hormones affect limbic allerdischarge thresholds and kindling rates in adult femalc rats. Brain Res 838, 136-1 50.
Edwards,H.E., Bumham, W.M., Mendonca,A., Bowlby,D.A., MacLusky,N.J., 1999c. Steroid hormones affect limbic afterdischarge thresholds and kindling rates in adult female rats. Btain Res 838, 136-1 50.
Elisevich,K., Rempel,S.A., Smith,B., Allar,N., 1997a. Connexin 43 mRNA expression in two experimental models of epilepsy. Mol Chem Neuropathol32,75-88.
Eliscvich,K., Rempel,S.A., Smith,B., Hirst,K., 1998. Temporal profile of connexin 43 mRNA expression in a tetanus toxin- induced seizure disorder. Mol Chem Neuropathol35.23-37.
Eliscvich,K., Rernpel,S.A., Smith,B.J., Edvardsen,K., 1997b. Hippocampal connexin 43 expression in human complex partial seizure disorder. Exp Neurol 145, 154-164.
Engel,J., Jr., 1996. Excitation and inhibition in epilepsy. Can J Neurol Sci 23, 167- 174.
Engel,J., Jr., Starkman,S., 1994. Overview of seizures. Emerg Med Clin North Am 12,895-923.
Ferrendelli,J.A., Holland,K.D., McKeon,A.C., Covey,D.F., 1989. Comparison of the anticonvulsant activitics of ethosuximide, valproate, and a new anticonvulsant, thiobutyrolactone. Epilepsia 30,6 17-622.
Feuer,G., 1983. Drug control of steroid metabolism by the hepatic endoplasmic reticuluni. Dnig Metab &v 14, 11 19-1 144.
Finn,D.A., Gee,K. W., 1994. The estrus cycle, sensitivity to convulsants and the anticonvulsant effect of a neuroactive steroid. J Pharmacol Exp Ther 27 1, 164- 170.
Fisher,R.S., 1989. Animal models of the epilepsies. Brain Res Brain Res Rev 14,245-278.
Follesa,P., Tarantino,A., Floris,S., Mallei,A., Porta& Tuligi,G., Cagetti,E., Caddeo,M., Mura,A., Sem,M., Biggio,G., 1999. Changes in the gene expression of GABAA mceptor subunit mRNAs in the septum of rats subjected to pentylenetetrazol-induced kindling. Brain Res Mol Brain Res 70, 1-8.
Foote,C.I., Zhou,L., Zhu,X., Nicholson,B.J., 1998. The pattern of disuifide linkages in the extracellular loop regions of connexin 32 suggests a mode1 for the docking interface of gap junctions. J Cell Biol 140, 1 187-1 197.
Foresta$., Rossato,M., Di Virgilio,F., 1993. Ion fluxes through the progesterone-activated channel of the spenir plasma membrane. Biochem J 294 CR Ij, 279-283.
Foy,M.R., Xu,J., Xie,X., Brinton,R.D., Thompson,R.F., Berger,T. W., 1999. t 7beta-estradiol enhances NMDA receptor-mediated EPSPs and long-term potentiation. J Neurophysiol8 1,925-929.
Frang W.B., 111, 1988. Basic review: endocrinology of the normal menstrual cycle. Prim Care 15,607-6 16.
Freeman,M.C., Dupke,K.C., Croteau,C.M., 1976. Extinction of the estrogen-induced daily signal for LH release in the rat: a role for the proestrous surge of progesterone. Endocrinology 99,2230229.
Friedlander,W.J., 1986a. Puhum, Merritt, and ihe discovery of Dilantin. Epilepsia 27 Suppl 3, S 1-20.
Friedlander,W.J., 1986b. Who was 'the father of bromide treatmcnt of epilepsy'? Arch Neurol43,SOS-507.
Frucht,M.M., Quigg'M., Schwaner,C., Fountain,N.B., 2000. Distribution of seizure precipitants among epilepsy syndromes [In Process Citation]. Epilepsia 4 1, 1334-1 539.
Frye,C.A., Bayon,L.E., 1999. Cyclic withdrawal from cndogcnous and exogenous progesterone increases kainic acid and petforant pathway induced seizures. Phannacol Biochcm Behav 62,3 15-32 1.
Garcia-Segura,L.M., AzcoitiaJ., DonCarlos,L.L., 2001. Neuroprotection by estradiol [In Process Citation]. Prog Neurobiol63,29-60.
Garcia-Segura,L.M., Naf?olin,F., Hutchison,J.B., Azcoitia,l., Chowen,J.A., 1999. Role of astroglia in estrogen regulation of synaptic plasticity and brain repair. J Neurobiol40,574-584.
Gee,K. W., Bolger,M.B., Brinton,R.E., Coirini,H., McEwcn,B.S., 1988. Steroid modulation of the chloride ionophore in rat brain: structure- activity requircments, regional dependencc and mechanism of action. J Pharmacol Exp Ther 246,803-8 12.
George,F. W ., OjedqS.R., 1982. Changes in aromatase activity in the rat brain during embryonic, neonatal, and infantile development. Endocrinology 1 I 1, 522-529.
Giannakopoulou,M., Bozas,E., Philippidis,H., Stylianopoulou,F., 2001. Pmtooncogene c-fos Involvement in the Molecular Mechanism of Rat Brain Sexual Differentiation. Neuroendocrinology 73,387-396.
Giaume,C., Venance,L., 1998. Intercellular calcium signaling and gap junctional communication in astrocytes. Glia 24, 50-64.
Gibbs, R. B., 1 999. Treatment w ith estrogen and progesterone affects relative levek of brain-derived neurotrophic factor mRNA and protein in different regions of the adult rat brain. Brain Res 844,20-27.
Gilbert,M.E., Cain,D.P., 198 1. A developmental study of kindling in the rat. Brain Res 254,321-328.
Glazko,A.J., 1986. Discovery of phenytoin. Ther Dmg Monit 8,490-497.
Goddard,G.V., McIntyre,D.C., Leech,C.K., 1969. A permanent change in brain function resulting from daily electrical stimulation. Exp Neurol25,295-330.
Good,M., Day,M., Muir,J.L., 1999. Cyclical changes in endogenous levels of oestrogen modu lace the induction of LTD and LTP in the hippocampal CA1 region. Eur J Neurosci I 1,447614480.
Gould,E., Bey1in.A.. TanapaSP., Reeves,A., Shors,T.J., 1999. Learningenhances adult neurogenesis in the hbpocampal formation [see comments]. Nat Neurosci 2,260-265.
Graves,N.M., Leppik,l.E., 1993. Advances in pharmacotherapy: recent developments in the treatment of epilepsy. J Clin Pham Ther 18,227-242.
Gu,Q., Korach,K.S., Moss,R.L., 1999. Rapid action of 17beta-estradiol on kainate-induced currents in hippocampal neurons lacking intracellular estrogen receptors. EndocrinoIogy 140,660-666.
Guerra-Amiza,C., Cerbon,M.A., Morimoto,S., Camacho-Arroyo,l., 2000. Progesterone receptor isofoms expression pattern in the rat brain during the estrous cycle. Life Sci 66, 1 743- 1752.
Hagihara,K., Hirata,S., Osada,T., Hirai,M., Kato,J., 1992. Distribution of cells containing progesterone receptor mRNA in the female rat di- and telencephalon: an in situ hybridization study. Brain Res Mol Brain Res 14,239-249.
Hall,S.M., 1977. Treatment of mensmal epilepsy with a progesterone-only oral contraceptive. Epilepsia 18,235-236.
Harden,C.L., Pulver,M.C., Ravdin,L., Jacobs,A.R., 1999. The effcct of menopause and perimenopause on the course of epilepsy. Epilepsia 40, 1402- 1407.
Hauser, W.A., Annegers,J.F., Kurland,L.T., 199 1. Prevalence of epilepsy in Rochester, Minnesota: 1940- 1980. Epilepsia 32,429-445.
Herbison,A.E., Fenelon,V.S., 1995. Estrogen regulation of GABAA receptor subunit mRNA expression in preoptic area and bed nucleus of the stria terminalis of female rat brain. J Neurosci 15,2328-2337.
Herzog,A.G., 1986b. Intermittent progesterone therapy and fiequency of complex partial seizures in women with menstruat disorders. Neurology 36, 1607-1 6 10.
Herzog,A.G., 1986a. Intermittent progesterone therapy and frequcncy of complex partial seinires in women with mensnual disorders. Neurology 36, 1607- 16 10.
Herzog,A.G., 199 1. Reproductive endocrine considerations and hormonal therapy for women with epilepsy. Epilepsia 32 Suppl6, S27433.
Herzog,A.G., 1995a. Progesterone therapy in women with complex partial and secondary generalized seiwres. Neurology 45, 1660-1662.
Henog,A.G., 1995b. Progesterone therapy in women with complex partial and secondary generalized seizures. Neurology 45, 1660- 1662.
Herzog,A.G., 1999a. Progesterone therapy in women with epilepsy: a 3-year follow-up. Neurology 52, 1917-1918.
Herzog,A.G., 1999b. Psychoneuroendocrine aspects of temporolimbic epilepsy. Part Il: Epilepsy and reproductive steroids. Psychosomatics 40, 102- 108.
Herzog,A.G., Klein,P., Ransil,B.J., 1997a. Three patterns of catarnenial epilepsy. Epilepsia 38, 1082- 1088.
Herzog,A.G., K1ein.P.. Ransi1,B.J.. 1997b. Three patterns of catarnenial epilepsy. Epilepsia 38, 1082- 1088.
Holmes,G.L., Weber,D.A., 1984. The effect of progesterone on kindling: a developmental study. Brain Res ?tg* 45-53.
Howard,G., Peng,L., Hyde,J.F., 1997. An estrogen receptor binding site within the human galanin gene. Endocrinology 138,46494656.
Ichikawa.S., Morioka,H., Sawada,T., 1972. Acute effect of gonadotrophins on the secretion of progestins by the rat ovary. Endocrinology 90, 1356- 1362.
Imperato-McGinley,J., Guerrero,L., Gautier,T., Peterson,R.E., 1974. Steroid Salpha-reductase deficiency in man: an inherited form of male pseudohermaphroditism. Science 1 86, 12 13- 1 2 15.
Jacobs,B.L., Praag,H., Gage,F.H., 2000. Adult brain neurogcnesis and psychiatry: a novel theory of depression. Mol Psychiatry S. 262-269.
Joels,M., 1997. Steroid hormones and excitability in the mammalian brain. Front Neuroendocrinol 18-2- 48.
Kamphuis,W., Gorter,.l.A., da Silva,F.L., 199 1. A long-lasting decrease in the inhibitory effect of GABA on glutamate responses of hippocampal pyramidal neurons induced by kindling epileptogenesis. Neuroscience 4 1,42543 1.
Katz,M.D., Cai,L.Q., Zhu,Y.S., Herrera,C., DefilIo-Ricart,M., Shackleton,C.H., Imperato-McGinley,J., 1995. The biochernical and phenotypic characterization of females homozygous for 5 alpha-teductase-2 deficiency . J Clin Endocrinol Metab 80,3 160-3 167.
Kellogg,C.K., Frye,C.A., 1999. Endogenous levels of 5 alpha-reduced progestins and androgens in fetal vs. adult rat brains. Brain Res Dev Brain Res 1 15, 17-24.
KepaJK., Jacobsen,B.M., Boen,E.A., Prendergast,P., Edwards,D.P., Takimoto,G., Wieman,M.E., 1996. Direct binding of progesterone receptor to nonconsensus DNA sequences represses rat GnRH. Mol Cell Endocrinol 1 17,27-39.
Kerrigan,J,F., Shields, W.D., Nelson,T.Y., Bluestone,D.L., Dodson, W.E., Bourgeois,B.F., Pellock,J.M., Morton,L.D., Monaghan,E.P., 2000. Ganaxotone for treating intractable infantile spasms: a rnulticenter, open-label, add-on trial [In Process Citation]. Epilepsy Res 42, 133- 139.
Khisti,RT., Mandhane,S.N., Chopde,C.T., 1998. The neurosteroid 3 alpha-hydroxy-5 alpha-pregnan-20- one induces catalepsy in mice- Neurosci Leû 25L,85-88.
Khurgel,M., Ivy,G.O., 1996. Astrocytes in kindling: relevance to epileptogenesis. Epilepsy Res 26, 163- 175.
Kokaia,M., Pratt,G.D., Elrner,E., Bengzon,J., Fritschy,J.M., Kokaia,Z., tindvall,O., Mohler,H., 1994. Biphasic differential changes of GABAA receptor subunit mRNA ievels in dentate gyrus granule cells following recurrent kindling-induced seizures. Brain Res Mol Brain Res 23,323-332.
Kokate,T.G., Cohen,A.L., Karp,E., Rogawski,M.A., 1996. Neuroactive steroids protect against pilocarpine- and kainic acid- induced limbic seizures and status epilepticus in mice. Neuropharmacology 35, 1049- 1056.
Kokate,T.G., Juhng,K.N., Kirkby,R.D., Llamas,J., Yamaguchi,S., Rogawski,M.A., 1999. Convulsant actions of the neurosteroid pregnenolone sulfate in mice. ûrain Res 83 1, 1 19- 124.
Kokate,T.G., Svensson,B.E., Rogawski,M.A., 1994. Anticonvulsant activity of neurosteroids: correlation with gamma- aminobutyric acid-evoked chioride curent potenfration. 1 PFiarmacoI Exp nier 270, 1223- 1229.
Kokka,N., Sapp,D.W., Witte,U., Olsen,R.W., 1992. Sex differences in sensitivity to pentylcnetetrazol but not in GABAA receptor binding. Pharmacol Biochem Behav 43,441-447.
Komeyev,A., Costa,E., 1996. Allopregnanolone (THP) mediates anesthetic cffécts of progesterone in rat brain. Hom Behav 30,37-43.
Korneyev,A., Guidotti,A., Costa,E., 1993. Regional and interspecies difletences in brain progesterone metabolism. J Neurochem 6 1,204 1-2047.
Krall,R.L., Penry,J.K., Kupferberg,H.J., Swinyard,E.A., 1978. Antiepileptic drug development: 1. History and a program for progress. Epilepsia 19,393408.
Kuhn,H.G., Svendsen,C.N., 1999. Origins, functions, and potential of adult neural stem cells. Bioessays 2 1, 625-630.
Landgren,S., AasiyJ., Backstrom,T., Dubrovsky,B., Danielsson,E., 1987. The eflect of progesterone and its metabolites on the interictal epileptiform discharge in the cat's cerebral cortex. Acta Physiol Scand 13 1, 33-42.
Lauber,A.H., Romano,G.J., Pfaff,D.W., 1991. Sex difference in estradiol regulation of progestin receptor mRNA in rat mediobasal hypothalamus as demonstrated by in situ hybridization. Neuroendocrinology 53, 608-6 1 3.
Lauber,M.E., Lichtensteiger, W ., 1994. Pre- and postnatal ontogeny of aromatase cytochrome P450 messenger ribonucleic acid expression in the mate rat brain studied by in situ hybridization. Endocrinology 135, 1661-1668.
Lauber,M.E., LichtensteigeqW., 1996. Ontogeny of 5 alpha-reductase (type 1) messenger ribonucleic acid expression in rat brain: early presence in germinal zones. Endocrinolagy 137,27 18-2730.
Lauber,M.E., Sarasin,A., Lichtensteiger, W., 1997. Sex di fferences and androgen-dependent regulation of aromatase (CYP19) mRNA expression in the developing and adult rat brain. J Steroid Biochem Mol Bi01 61,359-364.
Laxer,K., Blum,D., Abou-Khalil,B. W., Morretl,M.J., Lee,D.A., Data.J.L., MonaghaaEP, 2000. Assessment of ganaxolone's anticonvulsant activity using a randomized, double-blind, presurgical trial design. Ganaxolone Presurgical Study Group. Epilepsia 4 1, 1 187- 1 194.
Lehmann,H., Ebert,U., Loscher, W., 1996. Immunocytochemical localization of GABA immunoreactivity in dentate granule cells of normal and kindled rats. Neurosci Lett 212,4144,
Lehmann,H., Ebert,U., Loschet, W., 1998. Amygdala-kindling induces a lasting reduction of GABA- immunoreactive neurons in a discrete area of the ipsilatetal pirifonn cortex. Synapse 29,299-309.
Lephart,E.D., Sirnpson,E.R., McPhaul,M.J., Kilgore,M. W., Wilson,J.D., OjedqS .R., 1992. Brain aromatase cytochrorne P-450 messenger RNA levels and enzyme activity during prenatal and perinatal development in the rat. Brain Res Mol Brain Res 16, 187-192.
Leman-Sagie,T., Lerman,P., 1999. Phenobarbital still has a role in epilepsy treatrnent. J Child Neurol 14, 820-82 1 .
Leyendecker,G., Geppert,G., Nocke, W., Ufer,J., 1975. [Estradial- l7beta, estrone, LH and FSH in serum after administratron ofestradior-17beta, estradiolbenzoate, estradiol-valeriate and estradiol- undecylate in the female (author's transi)]. Geburtshilfe Frauenheilkd 35,370-374.
London,E.D., Buterbaugh,G.G., 1978. Modification of electroshock convulsive responses and thresholds in neonatal rais afier brain monoamine reduction. J Phannacol Exp Ther 206,8 1-90.
Loscher-W., 1993. Effects of the antiepileptic dnig valproate on metabolism and function of inhibitory and excitatory amino acids in the brain. Neurochem Res 18.485-502.
Loscher,W., 1998. New visions in the pharmacology of anticonvulsion. Eur J Pharmacol 342, 1-1 3.
Lu,B., Chow,A., 1999. Neurotrophins and hippocampal synaptic transmission and plasticity. J Neurosci Res 58, 76-87.
Lye,S.J., Nichotson,B.J., Mascarenhas,M., MacKenzie,L., Petrocelli,T., 1993. lncreased expression of conncxin-43 in the rat myometrium during labor is associated with an increase in the plasma estr0gen:progesterone ratio. Endocrinology 132,2380-2386.
Macdonald,R.L., Kelly,K.M., 1995. Antiepileptic drug mechanisms of action. Epilepsia 36 Suppl2, S2-12.
Macdonald,R.L., McLean,M.J., 1986. Anticonvulsant drugs: mechanisms of action. Adv Neurol44,7 13- 736.
Mackay-Sima,A., Chuahb,M.I., 2000. Neurotrophic factors in the primary olfactory pathway [In Process Citation]. Prog Neurobiol62,527-559.
MacLusky,N.J., Philip,A., Hurlburt,C., Naftolin,F., 1985a. Estrogen formation in the developing rat brain: sex differences in aromatase activity during eariy pst-natal lifc. Psychoneuroendoctinology 10,355-361.
MacLusky,N.J., Philip,A., Hurlburt,C., Naftolin,F., 1985b. Estrogen formation in the developing rat brain: sex differences in aromatase activity during early post-natal life. Psychoneurocndocrinology 10,355-36 1.
MacLusky,N.J., Walters,M.J., Clark,A.S., Toran-Allerand,C.D., 1994. Aromatase in the cerebral cortex, hippocampus, and mid-brain: ontogeny and devclopmental implications. Mol Cell Neurosci 5.69 1-698.
Maione,S., Berrino,L., Vitagliano,S., Leyva,J., Rossi,F., 1992. Pregncnolone sulfate increases the convulsant potency of N-methyl-D- aspartate in mice. Eur J Pharmacol 2 19,477-479.
Maitra,R., Reynolds,J.N., 1998. Modulation of GABA(A) receptor function by neuroactive steroids: cvidence for heterogeneity of steroid sensitivity of recombinant GABA(A) receptor isoforms. Can J Physiol Pharmacol 76,909-920.
Majewska,M.D., 1992. Neurosteroids: endogenous bimodal modulators of the GABAA receptor. Mechanism of action and physiological significance. Prog Neurobiol38, 379-395.
Majewska,M.D., Mienville,J.M., Vicini,S., 1988. Neurosteroid pregnenolone sulfate antagonizes electrophysiotogical responses to GABA in neurons. Neurosci Lett 90,279-284.
Majewska,M.D., Schwartz,R.D., 1987. Pregncnolone-sulfate: an endogenous antagonist of the gamma- aminobutyric acid receptor complex in brain? Brain Res 404,355-360.
Manjunath,C.K., Goings,G.E., Page,E., 1987. Human cardiac gap junctions: isolation, ultrastructure, and protein composition. J Mol Cell Cardiul 19, 13 1 - 134.
Marson,A.G.. Kadir,Z.A.. Hutton,J.L., Chadwick,D.W.. 2000. Gabapentin addsn for dmg-resistant partial epilepsy (Cochrane review) pn Process Citation]. Cochrane Database Syst Rev CD00 14 15.
Mattson,R.H., Cramer,J.A., Caldwell,B.V., Siconolfi,B.C., 1984b. Treatment of seizures with medroxyprogesterone acetate: preliminary report. Neurology 34, 1255- 1258.
Mattson,R.H., Cramer,l.A., Caldwell,B.V., Siconolfi,B.C., l984a. Treatment of seizures with medroxyprogesterone acetate: pwliminary report. Neurology 34, 1255- 1258.
McEachern,J.C., Shaw,C.A., 1999. The plasticity-pathology continuum: defining a role for the LTP phenornenon. J Neurosci Res 58.42-6 1.
McEwen,B.S., 1992. What makes a steroid a neurosteroid? Neurosteroids and Brain Function, sponsored by the Fidia Research Foundation, New Orleans, LA, USA, November 8-9, 199 1. New Biol4.2 12-2 16.
McEwen,B.S., 1999. Stress and hippocampal plasticity. Annu Rev Neurosci 22. 105- 1 22.
McEwen,B.S., Alves,S.E., Bulloch,K., Weiland,N.G., 1997. Ovarian steroids and the brain: implications For cognition and aging. Neurology 48, S8-15.
McLcan,M.J., 1999. Gabapentin in the management of convulsive disorders. Epilepsia 40 Supp16, S39- S50.
McNatty,K.P., 198 1. Hormonal correlates of follicular development in the human ovary. Aust J Biol Sci 34,249-268.
McNeilly,A.S., 1988. The control of FSH secretion. Acta Endocrinol Suppl (Copenh) 288,3 1-40,
McNicol,D., Jr., Crews,D., 1979. Estrogedprogesterone synergy in the control of female sexual receptivity in the lizard, Anolis carolinensis. Gen Comp Endocrinol38,68-74.
Medlock,K,L., Branharn, W.S., Sheehan,D.M., 1992. Long-term effects of postnatal exposure to diethylstilbestrol on uterine estrogen receptor and growth. J Steroid Siochem Mol Biol42,23-28.
Medlock,K.L., Forrester,T.M., Sheehan,D.M., 199 1. Short-tenn effects of physiological and pharrnacological doses of estradiol on estrogen reccptor and uterine growth. J Recept Res I 1,743-756.
Melcangi,R.C., Celotti,F., Ballabio,M., Castanosp., Po1etti.A.. Milan& Martini,L., 1988. Ontogenetic development of the 5 alpha-reductasc in the rat brain: cerebal cortex, hypothalamus, purifid myelin and isolated oligodendrocytes. Brain Res Dev Brain Res 44, 18 1 - 1 88.
Melcangi,R.C., Celotti,F., Castano,P., Martini,L., 1993. Differcntial localization of the 5 alpha-reductase and the 3 alpha- hydroxysteroid dehydrogenase in neuronal and glial cultures. Endocrinology 132, 1252- 1259.
Melcangi,R.C., Celotti,F., Martini,L., 1994. Ptogesterone 5-alpha-reduction in neuronal and in different types of glial ce11 cultures: type 1 and 2 astrocytes and oligodendrocytes. Brain Res 639,202-206.
Mermelstein,P.G., Becker,J.B., Surmeier,D.J., 1996. Estradiol reduces calcium currents in rat neostriatal neurons via a membrane receptor. J Neurosci 16,595-604.
Micevych,P.E., Popper,P., Hanon,G.l., 1996. Connexin 32 mRNA levels in the tat supraoptic nucleus: up- regulation prior to parturition and during lactation. Neuroendocrinology 63,3945.
Mienville,J.M., Vicini,S., 1989. Pregnenolone sulfate antagonizes GABAA receptor-mediated currents via a reduction of chan& opening frquency . &sin Res 489, t WB&.
Mirsky,A.F., Duncan,C.C., Mystobodsky,M.S., 1986. Petit mal epilepsy: a review and iniegration of recent information. J Clin Neurophysiol3, 179-208.
Mohammad,S., Abolhassan,A., Pourgholami,M.H., 1998a. Evaluation of the anticonvulsant profile of progesterone in male amygdala-kindled rats. Epilepsy Res 30, 195-202.
Mohammad,S., Abolhassan,A., Pourgholami,M.H., 1998b. Evaluation of the anticonvulsant profile of progesterone in male amygdala-kindled rats. Epilepsy Res 30, 195-202.
Mohler,H., Fritschy,J.M., Luscher,B., Rudolph,U., Benson,J., Benke,D., 1996. The GABAA receptors. From subunits to diverse functions. Ion Channels 4,8901 13.
Mohier,H., Okada,T., 1977. Benzodiazepine receptor: demonstration in the central nervous systcm. Science 198, 849-85 1.
Monnet,F.P., Mahe,V., Robel,P., Saulieu,E.E., 1995. Neurosteroids, via sigma receptors, modulate the [3H]norepinephrine release evoked by N-methyl-D-aspartate in the rat hippocampus. Proc Natl Acsd Sci U S A 92,3774-3778.
Mor,G., Ni lsen,J., H0rvath.T.. Bechmann,l., Brown,S., Garcia-Segura,L.M., Naftolin,F., 1999. Estrogen and microglia: A regulatoiy system that affects the brain. J Neurobiol40,484-496.
Moran,M.H., Smith,S.S., 1998. Progesterone withdrawal 1: pro-convulsant effects. Brain Res 807,84-90.
Morozova,T.M . , Levashova,Z.B ., NagibnevaJ .N ., Rau,V.A ., S idorkina,O.M., 1 990. [The participation of transrnembrane messenger systems in the action of steroid hormones on target cells]. Fiziol Zh SSSR Im 1 M Sechenova 76, 1 1 79- 1 186.
Morrison,T., Waggoner,L., Whitworth-Langley,L., Stith,B.J., 2000. Nongenomic action of progesterone: activation of Xenopus oocyte phospholipase C through a plasma membrane-associatcd tyrosine kinase. Endocrinology 14 1.2 145-2 1 52.
Murphy,D.D., Cole,N.B., Sega1.M.. 1998. Brain-derived neurotrophic factor mediates estradiol-induced dendritic spine formation in hippocampal neurons. Proc Natl Acad Sci U S A 95, 1 14 12- 1 14 17.
Murphy,D.D., Segal,M., 2000. Progesterone prevents estradiol-induced dendritic spine formation in cultured hippocampaI neurons [In Process Citation], Neuroendocrinology 72, 13 3- 143.
Murphy,K., Delanty,N., 2000. Primary Generalized Epilepsies. Curr Treat Options Neurol2, 527-542.
Murri,L., Galli,R,, 1997. Catamenial epilepsy, progesterone and its metabolites. Cephalalgia 17 Suppl20, 46-47.
Narbone,M.C., Ruello,C., Oliva,A., Baviera,G., D8Amico,D., Bramanti,P., Di Perri,R., 1990. Hormonal disregulation and catamenial epilepsy. Funct Neurol5,49-53.
Naus,C.C., Bani-Yagh0ub.M.. 1998. GAP JUNCTIONAL COMMUNICATION IN THE DEVELOPING CENTRAL NERVOUS SYSTEM. Cell Biol tnt 22,75 1-763.
Nicoletti,F., Speciale,C., Sortino,M.A., Summa,G., Caruso& Patti,F., Canonico,P.L., 1985. Comparative effects of muadiot b e n e , the mthmgm ctamipttme citrate, aircttk pragcstin medroxyprogesterone acetate on kainic acid- induced seizures in male and female rats. Epilepsia 26,252-257.
NissinenJ., Halonen,T.. Koivisto,E., Pitkanen,A., 2000. A new model of chronic temporal lobe epilepsy induced by electrical stimulation of the amygdala in rat. Epilepsy Rcs 38, 177-205.
O'Conner,J.L., Wade,M.F., PrendergashP., Edwards,D.P., Boonyaratanakomkit,V., Mahesh,V.B., 1 997. A 36 1 base pair region of the rat FSH-beta promoter contains multiple ptogesterone receptor-binding sequences and confers progesterone responsivencss. Mol Cell Endocrinol 136.67-78.
Orikasa,C., McEwen,B.S., Hayashi,H., Sakuma,Y., Hayashi,S., 2000. Estrogcn receptor alpha, but not bcta, is expressed in the intemeurons of the hippocampus in prepubertal rats: an in situ hybridization study. Brain Res Dev Brain Res 120,245-254.
0riow0,M.A.~ Landgren,B.M., Stenstrom,B., Diczfalusy,E., 1980. A cornparison of the pharmacokinetic properties of three estradiol esters. Contraception 2 1,4 15-424.
Osehobo, P., Adams, B., Sazgar,M., Xu,Y ., Racine,R.J., Fahnestock,M., 1999. Brain-derived neurotrophic factor infusion delays amygdala and perforant path kindling without affecting paired-pulse measures of neuronal inhibition in adult rats. Neuroscience 92, 1367- 1375.
Osterlund,M.K., KellefiE., Hurd,Y.L., 2000. The human forebrain has discrete estrogen receptor alpha messcnger RNA expression: high levels in the arnygdaloid cornplex. Neuroscience 95,333-342.
Panegyres,P.K., Hughes,J., 1997. The anticonvulsant properties of antisense c-fos oligodeoxynucleotides in kainic acid-induced seizures. J Neurol Sci 153, 12-19.
Parent,J.M., Janumpalli,S., McNamara,J.O., Lowenstein,D.H., 1998. lncreased dentate granule cell neurogenesis following amygdala kindling in the adult rat. Neurosci Lett 247,9-12.
Pelletier,G., Luu-The,V., Labrie,F., 1994. lmmunocytochemical localization of 5 alpha-reductase in rat brain. Mol Cell Neurosci 5, 394-399.
Pellmar,T.C., Wilson,W.A., 1977. Synaptic mechanism of pentylenetetrazole: selectivity for chloride conductance. Science 197,9 12-9 14.
Penning,T.M., Pawlowski,J.E., Schlegel,B.P., Jez,J.M., Lin,H.K., Hoog,S.S., Bennett,M.J., Lewis,M., 1997. "Mammalian 3 alpha-hydroxysteroid dehydrogenases". Steroids 62,455-456.
Penning,T.M., Sharp,R.B., Krieger,N.R., 1985. Purification and properties of 3 alpha-hydroxysteroid dehydrùgenase from rat brain cytosol. Inhibition by nonsteroidal anti-inflamrnatory drugs and progestins. J Biol Chem 260, 15266- 15272.
Perez Ve1azquezJ.L.. Carlen,P.L., 2000. Gap junctions, synchrony and seizures. Trends Neurosci 23,68- 74.
PerezJ., Tranque,P.A., Naftolin,F., Garcia-Segura,L.M., 1990. Gap junctions in the hypothalarnic arcuate neurons ~Fovariectomized and estradiol-treated rats. Neurosci Lett 1 08, 1 7-2 1,
Perez,J., Zucchi,l., Maggi,A., 1988. Estrogen modulation of the gamma-arninobutyric acid receptor com plex in the central nervous systern of rat. J Phamiacol Exp Ther 244, 1005- 10 10.
Perucca,E., 1996. Established antiepileptic drugs. Baillieres Clin Neurol 5,693-722.
Perucca,E., 1999. The spectrum of the new antiepileptic drugs. Acta Neurol Belg 99, 23 1-238.
Petraglia,F., de Micheroux,A.A., Florio,P., Salvatori,M., Gallinelli,A., Cela,V., Palumbo,M.A., Genazzani,A.R., 1995. Steroid-protein interaction in hurnan placenta. J Steroid Biochem Mol Biol53,227- 23 1.
Pine1,J.P.. Skelton,R., Mucha,R.F., 1976. Kindling-related changes in afterdischarge "thresholds". Epilepsia 17, 197-206.
Poletti,A., Martini,L., 1999. Androgen-activating enzymes in the central nervous system. J Steroid Biochem Mol Bi01 69, 1 17- 122.
Poletti,A., Negri-Cesi,P., Rabuffetti,M.. Colciago,A., Celotti,F., Martini,L., 1998. Transient expression of the Salpha-reductase type 2 isozyme in the rat brain in late fetal and early postnatal life. Endocrinology 139,2171-2178.
Poulter,M.O., Brown,L.A., Tynan,S., Willick,G., Williarn,R., Mclntyre,D.C., 1999. Differential expression of alphal, alpha2, alpha3, and alpha5 GABAA receptor subunits in seinire-prone and seinire-resistant rat models of temporal lobe epilepsy. J Neurosci 19,4654-466 1.
Racine,R.J., 1972. Modification of seimre activity by electrical stimulation. 1. After- discharge threshold. Electroencephalogr Clin Neurophysiol32,269-279.
Ramirez,V.D., Zheng,J., Siddique,K.M., 1996. Membrane teceptors for estrogen, progestcrone, and testosterone in the rat brain: fantasy or reality. Cell Mol Neurobiol 16, 175-198.
Reddy,D.S., Kulkarni,S.K., 1998. Proconvulsant effects of neurosteroids pregnenolone sulfate and dehydroepiandrosterone sulfate in mice. Eur J Pharmacol 345,5559.
Reddy,D.S., Rogawski,M.A., 2000. Enhanced anticonvulsant activity of ganaxolone afier neurosteroid withdrawal in a rat model of catamenial epilepsy. J Pharmacol Exp Ther 294,909-91 5.
Reibel,S., LarmeGy., Le,B., Carnahan,J., Marescaux,C., Depaulis,A., 2000. Brain-derived neurotrophic factor delays hippocarnpal kindling in the rat [In Process Citation]. Neuroscience 100, 777-788.
Reindollar,R.H., Moretuzzo,R. W., Ellegood,J.O., Mahesh,V.B., McDonough,P.G., 1985. The effects of single-dose luteinizing hormone-releasing hormone on ovulatory menstrual function: support for a single luteinizing hormone- and follicle-stimulating hormone-releasing factor. Fertil Steril43, 185- 195.
Richard,S., Zingg,H.H., 1990. The human oxytocin gene promoter is regulated by estrogens. J Bi01 Chem 265,6098-6 1 03.
Rocha,L., Kaufinan,D.L., 1998. In vivo administration of c-Fos antisense oligonucleotides accelerates amygdala kindling. Neurosci Lett 24 1, 1 1 1-1 14.
Rogvi-Hansen,B., Gram,L., 1995. Adverse effects of established and new antiepileptic drugs: an atternpted cornparison. Pharmacol Ther 68,425434.
Rosse,R.B., Mastropaolo,J., Novitzki,M.R., Deutsch,S.I., 1990. Depot testosterone attenuates the anticonvulsant effect of flurazepam in mice. Psychoneuroendocrinology 15,83-85.
Rottingen,J., Iversen,J.G., 2000. Ruled by waves? lntracellular and intercellular calcium signalling. Acta Physiol Scand 169,203-2 19.
Rozental,R., Giaume,C., Spray,D.C., 2000a. Gap junctions in the nervous system. Brain Res Brain Res Rev 32, 1145.
Rozental,R., Giaume,C., Spray,D.C., 2000b. Gap junctions in the nervous system. Brain Res Brain Res Rev 32, 11-15.
Rozental,R., Srinivas,M., Gokhan,S., Urban,M., Dennietzel,R., Kessler,J.A., Spray,D.C., Mehler,M.F., 2000c. Temporal expression of neuronal connexins during hippocampal ontogeny. Brain Res Brain Res Rev 32, 57-7 1.
Rupprecht,R., Holsbocr,F., 1999. Neuropsychopharmacological propeflies of neuroactive steroids. Steroids 64,83-9 1.
Russell,D. W., WiIson,J.D., 1994. Steroid 5 alpha-reductase: hvo genesbo enzymes. Annu Rev Biochem 63,25-61.
Sackeim,H., Decinqp., Prohovnik,I., Malitz,S., 1987. Seizure threshold in electroconvulsive therapy. Effects of sex, age, electrode placement, and number of treatments. Arch Gen Psychiatry 44,355-360.
Samoriski,G.M., Piekut,D.T., Applegate,C.D., 1998. Regional analysis of the spatial patterns of Fos induction in brain following flurothyl kindting. Neutoscience 84, 1209-1222.
Sanghera,M.K., Simpson,E.R., McPhaul,M.J., Kozlowski,G., Conley,A.J., Lephm,E.D., 199 1 a. lmmunocytochemical distribution of aromatase cytochrome P450 in the rat train using peptide-generated polyclonal antibodies. Endocrinology 129,2834-2844.
Sanghera,M.K., Simpson,E.R., McPhaul,M.J., Kozlowski,G., Con!ey,A.J., Lephart,E.D., 199 1 b. lmmunocytochemical distribution of aromatase cytochrome P450 in the rat brain using peptide-generated polyclonal antibodies. Endocrinology 129,2834-2844.
Scher,M.S., 1997. Seinires in the newbom infant. Diagnosis, treatment, and outcorne. Clin Perinatol24, 735-772.
Scon,B.W., Wojtowicz,J.M., Burnham,W.M., 2000. Neurogenesis in the dentate gyrus of the rat following electroconvuisive shock seizures [In Process Citation]. Exp Neurol l65,23 1-236.
Selye,H., 1970. Resistance to picrotoxin poisoning induced by catatoxic steroids. Agents Actions 1, 133- 135.
Shank,R.P., Gardocki,J.F., Streeter,A.J., Maryanoff,B.E., 2000. An overview of the preclinical aspects of topiramate: pharmacology, pharrnacokinetics, and mechanism of action. Epilepsia 4 1 Suppl 1, S3-S9.
Shinoda,K., 1 994. Brain aromatization and its associated structures. Endocr J 4 1, 1 1 5- 1 3 8.
Shinohara,K., Funabashi,T., Mitushirna,D., Kimura,F., 2000. Effects of estrogen on the expression of connexin32 and connexin43 mRNAs in the suprachiasrnatic nucleus of female rats. Neurosci Lett 286, 107-1 IO.
Shorvon,S.D., 1996. The epidem iology and treatment of chronic and refractory epilepsy . €pi lepsia 37 SUPPI 2, S 1 -S3.
Shughrue,P.J., Lane,M.V., Merchenthaler.1.. 1997. Comparative distribution of estrogen receptor-alpha and -beta mRNA in the rat central nervous system. J Comp Neurol388,507-525.
Shughrue,P.J., Lane,M.V., Scrimo,P.J., Merchenthaler,l., 1998a. Comparative distribution of estrogen recepbr-alpha (ER-alpha) mdbettr f ER-beta)mRNEt in- the rat ph-, ganad, and nptoda&e tract. Steroids 63,498-504.
Shughrue,P.J., Scrimo,P.J., Merchenthaler.1.. 1998b. Evidence for the colocaiization of estrogen receptor- beta mRNA and estrogen receptor-alpha immunoreactivity in neurons of the rat forebrain. Endocrinology 139,5267-5270.
Simler,S., Vergnes,M., Marescaux,C., 1999. Spatial and temporal relationships between C-Fos expression and kindling of audiogenic seimres in Wistat rats. Exp Neurol 1 57, 106- 1 19.
Singer,[., SingerJ., 1972. Periodicity of sexual desire in relation to time of ovulation in women. J Biosoc Sci 4,471-48 1.
Sm ith,S.S., 199 1. Progesterone administration attenuates excitatory amino acid responses of cerebellar Purkinje cells. Neuroscience 42,309-320.
Smith,S.S., Gong,Q.H., Hsu,F.C., Markowitz,R.S., f'fiench-Mullen,J.M., Li,X., 1998b. GABA(A) receptor alpha4 subunit suppression prevents withdrawal properties of an endogenous steroid [see comments). Nature 392,926-930.
Smith,S.S., Gong,Q.H., Hsu,F.C., Markowitz,R.S., ffrench-Mullen,J.M., Li,X., 1998a. GABA(A) receptor alpha4 subunit suppression prevents withdrawal properties of an endogenous steroid [see comments]. Nature 392,926-930.
Smith,S.S., Gong,Q.H., Li,X., Moran,M.H., Bitran,D., Frye,C.A., Hsu,F.C., 1998c. Withhwal fiom 3alpha-OH-Salpha-pregnan-20-One using a pseudopregnancy mode1 alters the kinetics of hippocampal GABAA-gated current and increases the GABAA receptor alpha4 subunit in association with increased anxiety. J Neurosci 18, 5275-5284.
Snead,O.C., 111, 1998. Ganaxolone, a selective, high-affinity steroid modulator of the gamma- aminobutyric acid-A receptor, exacerbates seimres in animal models of absence. Ann Neurol44,688-69 1.
So,E.L., 1995. Classifications and epidemiologic considerations of epileptic seizures and epilepsy. Neuroimaging Clin N Am 5, 5 13-526.
Spector,S., Cull,C., Goldstein,L.H., 2000. Seizure precipitants and perceived self-control of seizures in adults with poorly-controlled epilepsy. Epilepsy Res 38,207-216,
Stanislaw,H., Rice,F.J., 1988. Correlation between sexual desire and menstrual cycle characteristics. Arch Sex Behav 17,499-508.
Stefan0,G.B.. Cadet,P., Breton,C., Goumon,Y., Prevot,V., Dessaint,J.P., Beauvillain,J.C., Roumier,A.S., Welters,l., Salzet,M., 2000. Estradiol-stimulated nitnc oxide rclease in human granuiocytes is dependent on intracellular calcium transients: evidence of a cell surtace estrogen receptor. Blood 95,395 1-3958.
Steiner,M., 1992. Female-specific mood disorders. Clin Obstet Gynecol35,599-6 1 1.
Stitt,S.L., Kinnard,W.J., 1968a. The effect of certain progestins and estrogens on the threshold of electrically induced seizure patterns. Neurology 18,2 13-2 16.
Stitt,S.L., Kinnard,W.J., 1968b. The effect of certain progestins and estrogens on the threshold of electrically induced seizure patterns. Neurology 18,2 13-2 16.
Stunn,G., Haberlein,H., BauerJ., Plaum,T., Stalker,D.J., 199 1. Mass spectrometric and high-performance liquid chromatogragbic sndies of medcoxyprogesterone m a t e metabotites in hmart plasma. i Chromatogr 562,35 1-362.
Sutula,T., Steward,O., 1986. Quantitative analysis of synaptic potentiation during kindling of the perforant path. J Neurophysiol56.732-746.
Sutula,T., StewardBo., 1987. Facilitation of kindling by prior induction of long-term potentiation in the perforant path. Brain Res 420, 109- 1 17.
Sutula,T.P., Golarai,G., Cavazos,J., 1992. Assessing the tirnctional significance of mossy fiber sprouting. Epilepsy Res Suppl7,251-259.
Suzuki,S., Rogawski,M.A., 1989. T-type calcium channels mediate the transition between tonic and phasic firing in thalarnic neurons. Proc Natl Acad Sci U S A 86,722807232.
Swoboda,K.J., Drislane,F. W.. 1994. Seizure disorders: syndromes, diagnosis, and management. Compr Ther 20.67-73.
Szabo,M., Kilen,S.M., Nho,S.J,, Schwartz,N.B., 2000. Progesterone receptor A and B messenger ribonucleic acid levels in the anterior pituitary of rats are regulated by estrogen. Biol Reprod 62,9S- 102.
Tanapat,P., Hastings,N.B., Reeves,A.J., Gould,E., 1999. Estrogen stimulates a transient increase in the number of new neurons in the dentate gynis of the itdult female rat. J Neurosci 19,5792-5801.
Tauboll,E., Lindstrom,S., 1993. The effect of progesterone and its metabolite 5 alpha-pregnan-3 alpha- ol- 20-one on focal epileptic seizures in the cat's visual cortex in vivo. Epilepsy Res 14, 17-30.
Teter,B., Harris-White,M.E., Frautschy,S.A., Cole,G.M., 1999. Role of apolipoprotein E and estrogen in mossy fiber sprouting in hippocampal slice cultures. Neuroscience 9 1, 1009- IO 16.
Thavendiranathan,P., Mendonca,A., Dell,C., Likhodii,S.S., Musa,K., Iracleous,C., Cunnane,S.C., Bumham,W.M., 2000. The MCT ketogenic diet: effects on animal seizure models. Exp Neurol 161,696- 703.
Ticku,M.K., Olsen,R.W., 1978. Interaction of barbiturates with dihydropicrotoxinin binding sites related to the GABA receptor-ionophore system. Life Sci 22, 1643- 165 1.
Traub,R.D., SchmitqD., Jefferys,J.G., Draguhn,A., 1999. High-frequency population oscillations are predicted to occur in hippocampat pyramidal neuronal networks interconnected by axoaxonal gap junctions. Neuroscience 92,407-426.
Velisek,L., Kubova,H., Pohl,M., Stankova,L., Mares,P., Schickerova,R., 1992. Penty lenetetrazol-induced seizures in rats: an ontogenetic study. Naunyn Schmiedebergs Arch Pharmacol 346,588-591.
Wada,J.A., The clinical relevance of kindling: species, brain sites and seizure susceptibility. pp. 369-88. In: Livingston KE , Hornykiewicz O , ed Limbic mechanisms New York , Plenum Press, 1978 WL 3 14 L733L 1976.
Wada,J.A., Mizoguchi,T., Osawa,T., 1978. Secondarily generalized convulsive seizures induced by daily amygdaloid stimulation in rhesus monkeys. Neurology 28, 1026- 1036.
Wahnschaffe,U., Loscher, W., 1992. Lack of changes in seizure susceptibility during the estrous cycle in kindled rats. Epilepsy Res 13, 199-204.
Wang& Scott,B.W., Wojtowicz,J.M., 2000. Heterogenous properties of dentate granule neurons in the rtduk rat. k Ncurobiot 42,248-257.
Warren.S.G., Humphreys,A.G., Juraska,J.M., Greenough,W.T., 1995. LTP varies across the estrous cycle: enhanced synaptic plasticity in proestrus rats. Brain Res 703.26-30.
Weiland,N.G., Orikasa,C., Hayashi,S., McEwen,B.S., 1997. Distribution and hormone regulation of estrogen receptor immunoreactive cefls in the hippocampus of male and female rats. J Comp Neurol388, 603-6 12.
Williams,R.F., Barber,D.L., Cowan,B.D., Lynch,A., Manit, EL., Hodgen,G.D., 198 1. Hyperprolactinemia in monkeys: induction by an estrogen-progesterone synergy. Stetoids 38,32 1-33 1.
Willow,M., Canerall,W.A., 1982. lnhibition of binding of [3H]batrachotoxinin A 20-alpha-benzoate to sodium channels by the anticonvulsant drugs diphenylhydantoin and carbarnazepine. Mol Pharmacol 22, 627-635.
Wong,M,, Thompson,T.L., Moss,R.L., 1996. Nongenomic actions of estrogen in the brain: physiological significance and cellular mechanisms. Crit Rev Neurobiol 10, 189-203.
Woolley,C.S., 1998. Estrogen-mediated structural and hnctional synaptic plasticity in the female rat hippocampus. Hom Behav 34, 140- 148.
Woolley,C.S., McEwen,B.S., 1993. Roles of estradiol and progesterone in regulation of hippocampal dendritic spine density during the estrous cycle in the rat. J Comp Neurol336,293-306.
Woolley,C.S., McEwen,B.S., 1994. Estradiol regulates hippocampal dendritic spine density via an N- methyl- D-aspartate receptor-dependent mechanism. 1 Neurosci 14,7680-7687.
Woolley,C.S., Schwartzkroin,P.A., 1998. Hormonal effects on the brain. Epilepsia 39 Suppl 8, 52-58.
Wu,F.S., Gibbs,T.T., Farb,D.H., 1991. Pregnenolone sulfate: a positive allosteric modulator at the N- methyl-D- aspartate receptor. Mol Pharmacol 40,333-336.
Xiong,F., Liu,D., Le Drean,Y., Elsholtz,H.P., Hew,C.L., 1994. Differential recruitment of steroid hormone response elements may dictate the expression of the pituitary gonadotropin II beta subunit gene during salmon maturation. Mol Endocrinol8,782-793.
Y ukawa,E., 2000. lnvestigation of phenobarbital-carbarnazepine-valproic acid interactions using population pharrnacokinetic analysis for optimisation of antiepi leptic drug therapy : an overview. Drug Metabol Drug lnteract 16, 86-98.
Zhou,Y., Watters,J.J., Dorsa,D.M., 1996. Estrogen rapidly induces the phosphorylation of the CAMP response element binding protein in rat brain. Endocrinoiogy 137.2 163-2 166.
Zimmerrnan,A. W., Holden,K.R., Reiter,E.O., Dekaban,A.S., 1973. Medroxyprogesterone acetate in the treatment of seizures associated with menstruation. The Journal of Pediatrics 83,959-963.
Zwain,l.H., Yen,S.S., 1999. Neurosteroidogenesis in astrocytes, oligodendrocytes, and neurons of cerebral cortex of rat brain. Endocrinology 140, 3843-3552.