Hypothalamic orexin neurons and tanycytes: Functional and molecular aspects in a mouse model of temporal

lobe epilepsy

By Harrison Morgen Walling Roundtree

iii

Table of contents

CHAPTER I. BACKGROUND AND OBJECTIVES1

Epilepsy and Sleep Disorder 1

Sleep Regulation 2

Lateral Hypothalamus and Orexin 4

Adenosine 5

Tanycytes of the third ventricle 10

Mouse Model 13

Hypothesis and Objectives 14

CHAPTER II. OREXIN NEURONS AND PATHOLOGY IN THE LATERAL HYPOTHALAMUS

Introduction 16

Materials and Methods 18

Results 25

Discussion 49

CHAPTER III. ADENOSINE SYSTEM IN THE LATERAL HYPOTHALAMUS

Introduction 57

Materials and Methods 59

Results 63

Discussion 674

CHAPTER IV. TANYCYTES OF THE THIRD VENTRICLE

Introduction 78

Materials and Methods 79

Results 81

Discussion 92

CHAPTER V. SUMMARY AND CONCLUSIONS 96

REFERENCES 99

1

Chapter 1: Introduction and Background

Epilepsy and Sleep Disorder

Epilepsy is a neurological disorder characterized by recurrent and

unprovoked seizures. Seizures and epilepsy are generally considered to be

caused by an increase in excitatory or decrease in inhibitory tone in the brain,

allowing neurons to become hyperactive and hypersynchronous. This imbalance

can have a variety of underlying causes, including traumatic brain injury or

genetic defects. Epilepsy has a lifetime incidence of 1:26 in the United States

(Hesdorffer et al. 2011). While severity of the disorder varies, epilepsy can have

a significant impact on a patient’s quality of life. Approximately a third of epileptic

patients are refractory to available epilepsy treatment.

Epilepsy is associated with increased risk for many comorbid conditions

(Manni et al. 2010; Gilliam et al. 2005). An estimated 30% or more epileptic

patients are thought to be afflicted with sleep disorders that cause insufficient

sleep. This includes symptoms such as increased latency to sleep onset,

reduced sleep duration, and increased fragmentation of sleep. Insufficient sleep

on its own can contribute to the development of cognitive impairments and

psychological disorders. It’s also capable of reducing seizure threshold,

worsening seizure frequency and/or severity, and exacerbates other co-

morbidities of epileptic patients (Kotagal et al. 2008; Grigg-Damberger et al.

2014; Malow 2005; Malow et al. 1997; Rocamora et al. 2008; de Weerd et al.

2

2004; Steinsbekk et al. 2013; Jacoby et al. 2015; Killgore et al. 2010; Pilhal et al.

1997).

Here, we have divided the research topics into three sections. Part I

discusses orexin neurons in the lateral hypothalamus role in sleep. Part II

discusses adenosine in the lateral hypothalamus, an important regulator of orexin

neuron activity. Part III discusses tanycytes of the third ventricle, a cell type

involved in regulation of the hypothalamus.

Sleep Regulation

Sleep states and wakefulness are characterized by unique brain rhythms

that can be identified with electroencephalography (EEG) recordings (reviewed in

Brown et al. 2012). Wake is primarily associated with faster oscillations in the

Beta rhythms (12-30 Hz) when alert and Alpha rhythms (8-12 Hz) when more

relaxed. Sleep is broken up into two major categories, non-rapid eye movement

(NREM) sleep and rapid eye movement (REM) sleep. NREM sleep can be

characterized by slower oscillations such as theta (4-8 Hz) and delta (less than 4

Hz) rhythms, as well as special oscillations known as sleep spindles. REM sleep,

however, is characterized by faster oscillations and appears similar to wake, but

is also associated with muscle tone that differentiates it from wakefulness.

3

Figure 1: Orexin neurons, located in the LH/P, are an integral step in sleep/wake

regulation. Orexin neurons have wake-promoting projections to Reticular Activating

System (RAS), including the Raphe Nucleus (RN), Locus Coeruleus (LC) and

Pedunculopontine Tegmental nucleus (PPT), and the Tuberomamillary Nucleus (TMN)

and cortex. Other regions include the Ventrolateral Preoptic nucleus (VLPO), which

sends GABAergic projections to many of the same regions to promote sleep; and the

Suprachiasmatic Nucleus (SCN), which maintains a circadian rhythm of activity to act as

a pacemaker (adapted from Saper et al. 2005).

4

Control of sleep is regulated by various brain nuclei (Fig. 1) working in

tandem to respond to environmental stimuli as well as work in accordance with

the body’s circadian rhythm (reviewed in Saper et al. 2005). These nuclei are

primarily located in the hypothalamus, thalamus, and brainstem with a

complicated network of connections between the nuclei. In the hypothalamus, the

suprachiasmatic nucleus (SCN), located dorsal of the optic chiasm, maintains

circadian rhythm and responds to stimuli from the retina. The ventrolateral

preoptic nucleus (VLPO) releases inhibitory neurotransmitters, such as GABA, to

promote sleep. It sends projections to orexin neurons (also known as hypocretin)

in the lateral hypothalamus and perifornical region (LH/P), the tuberomamillary

nucleus (TMN, histaminergic) in the posterior nucleus, and the reticular activating

system (RAS) of the brain stem. The RAS is made up of the pedunculopontine

nucleus (PPT, acetycholinergic), the locus coeruleus (LC, noradrenergic), and

raphe nucleus (RN, serotonergic).

-Lateral Hypothalamus and Orexin

The orexin neurons in the LH/P are spontaneously active neurons that

release orexin, a neuropeptide that acts on the orexin-1(OX-1) and orexin-2 (Ox-

2) G-protein couple receptors (Sakurai 2007). The LH/P is considered to be a

region that integrates various inputs to regulate energy balance. As part of this

function, orexin neurons promote feeding behavior in addition to promoting

wakefulness and arousal. It receives inhibitory input from the VLPO and

excitatory input from the SCN, and sends excitatory projections to the RAS,

which relays the signal to the cortex (Fig 1).

5

Adenosine

Adenosine, a purine nucleoside, and the purine family of molecules are

involved in a variety of functions (reviewed in Fredholm et al. 2001). Adenosine

triphosphate (ATP) is used in energy transfer in the cell, acting as an energy

currency. ATP and other adenosine nucleotides (adenosine diphosphate, ADP;

adenosine monophosphate, AMP) are phosphorylated and dephosphorylated in

a constant cycle (Fig 2), storing and providing energy to cellular machinery. ATP

is necessary for loading vesicles, a process required for synapses to transmit

signals and action potentials.

Adenosine and the purine family can also act as signaling molecules.

Cyclic AMP (cAMP) is a secondary messenger derived from ATP. It acts as an

intermediate step in the signaling cascade of G-protein coupled receptors

(GPCR) and activates protein kinase A (PKA). There are also purinergic

receptors. Purinergic receptors are grouped according to the activating molecule.

P2 receptors are activated by nucleotides. P2Y receptors are a group of 8

GPCRs activated by ATP and ADP as well as uridine based nucleotides. P2X

receptors are ligand-gated ion channels activated by ATP.

P1 receptors are also GPCRs, but are activated by adenosine rather than

adenine nucleotides, leading to them also be referred to as adenosine receptors.

There are four subtypes of adenosine receptors: A1 (Gi/o), A2A (Gs), A2B (Gs),

and A3 (Gi/o). All of the adenosine receptors are involved in regulation of the

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respiratory and circulatory systems in the periphery, but A1 and A2A are the

primary subtypes expressed in the CNS.

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Figure 2: Adenosine concentration is regulated by a futile enzymatic cycle. ADK is

primarily responsible for reducing the adenosine pool and is mainly expressed by

astrocytes in adult brain (Studer et al. 2006; figured based on Chikihasa and Séi 2011;

ADA: Adenosine Deaminase, NTD: Nucleotidase).

8

Adenosine concentration is tightly regulated in concert with the other

purine molecules by various enzymes (Fig 2). The two enzymes directly

responsible for reducing adenosine concentrations are adenosine deaminase

(ADA) and adenosine kinase (ADK). ADA converts adenosine to inosine by

irreversibly deaminating it. ADK phosphorylates adenosine to AMP. While both

are capable of reducing adenosine concentrations, ADK has a lower KM than

ADA and so is thought to be primarily responsible for controlling adenosine

concentrations. Studies of adenosine regulation in disease states further support

this as altering ADK activity or expression has a more pronounced effect than

similar changes to ADA. ADK is primarily expressed in astrocytes in the adult

brain.

A specific role of adenosine is the regulation of orexin neurons (Sakurai

2007). Adenosine acts on the A1 receptor pre- or post-synaptically to inhibit

spontaneously active orexin neurons, promoting sleep (Fig 3). Adenosine levels

increase during wakefulness or sleep deprivation, and it is considered to be a

way to promote sleep drive (Porkka-Heiskanen et al. 1997).

9

Figure 3: Adenosine inhibits spontaneously active orexin neurons.

Adenosine inhibits hypocretin neurons via the A1 receptor by both inhibiting presynaptic

glutamatergic neurons and postsynaptically on orexin neurons themselves.

10

Tanycytes of the third ventricle

Medially located in the hypothalamus is the third ventricle, one of the

cavities in the brain filled with cerebrospinal fluid (CSF). The walls of ventricles

are lined with ependymal cells, a type of neuroglia that makes an endothelial like

layer with a variety of functions. Specialized ependymal cells in the choroid

plexus produce CSF, while most ependymal cells have their apical surface

covered in cilia to encourage the circulation of CSF, and microvilli to absorb the

CSF. The ependymal layer forms a selective barrier between CSF and the brain

parenchyma due to modified tight junctions between the cells.

Another cell type located in the walls of the third ventricle and floor of the

fourth ventricle are tanycytes (review in Dale 2011). Tanycytes are located in the

ependymal layer, but also share similarities with other glial cell types, including

astrocytes and radial glia. In contrast with ependymal cells, tanycytes are

unciliated. However, the basal surfaces of tanycytes in the third ventricle have

long processes that extend into the parenchyma of the hypothalamus.

The functions of tanycytes aren’t yet fully understood. Similar to

astrocytes, processes from tanycytes contact blood vessels in the hypothalamus

and evidence suggests they help maintain the blood-brain barrier. They also help

maintain the barrier of the ventricle wall by forming tight junctions with

neighboring cells. However, tanycytes express transporters capable of absorbing

molecules from the CSF, including glucose transporters and aquaporin water

channels. The expression of glucose transporters in tanycytes led to the

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hypothesis that tanycytes are capable of acting as glucose sensing cells. In fact,

tanycytes have been shown to produce transient increases in calcium

preferentially in response to increased glucose in the third ventricle. Finally,

tanycytes are capable of neurogenesis in the adult brain, similar to radial glia,

with increased neurogenesis occurring in animals fed a high-fat diet. While the

functions of tanycytes in healthy brain are being investigated, little research has

looked at tanycytes in disease state.

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Figure 4: Tanycytes of the third ventricle are made up of anatomically

distinct subpopulations. Tanycyte subpopulations are outlined on a

representative image of skeletonized vimentin staining. α1 tancytes are the most

dorsal of the subpopulations, followed by α2 tanycytes. β tanycytes line the

ventral most wall of the third ventricle, near the median eminence. Here, α2

tanycytes are further separated as ventral and dorsal subgroups, similar to other

studies (Robins et al. 2012).

α1 tanycytes

Dorsal α2 tanycytes

Ventral α2 tanycytes

β tanycytes

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Subtypes of tanycytes have been characterized, predominantly based on

differential protein expression and anatomical location (Fig. 4). α tanycytes are

typically separated into α1 and α2 tanycytes, with α1 tanycytes approximately

level with the ventromedial nucleus of the hypothalamus and α2 tanycytes

located ventral to the α1 tanycytes, adjacent to the arcuate nucleus. β tanycytes

are located ventral of α2 tanycytes, lining the median eminence and the ventral

wall of the third ventricle. β tanycytes can also be further separated into β1 and

β2 tanycytes.

All subsets of tanycytes express the marker vimentin. GFAP, however, is

reported as primarily expressed in more dorsal tanycytes, including α1 and some

α2 tanycytes. β tanycytes (specifically β2) have been identified as acting as a

neurogenic niche in adult mice that responds to high fat diet, with progeny that

include orexin-positive neurons (Lee et al. 2012). A subpopulation of α2

tanycytes have also been identified as being capable of forming neurospheres in

adult mice, with increased proliferation in response to fibroblast growth factors

(Robins et al. 2013). While these differences seem to indicate that

subpopulations are capable of responding to different stimuli, functional

differences in tanycyte subpopulations is still being investigated.

Mouse model:

The experiments in the presented studies utilize Kcna1-null and wild-type

mice on a C3HeB/FeJ background. Kcna1 codes for the alpha-subunit of the

Kv1.1 voltage-gated delayed rectifier potassium channel. The strain was initially

14

generated by Bruce L. Tempel and colleagues at the University of Washington.

Kcna1-null mice are spontaneously epileptic, displaying spontaneous seizures

starting at approximately postnatal day 21 (Simeone et al. 2013). Severity of

disease gradually increases over the lifespan of Kcna1-null mice, eventually

resulting in mortality, generally before postnatal age of two months. Mice are

bred from heterozygous pairs, allowing use of wild-type littermates to serve as

controls, and all animals are genotyped at P12. Wild-type mice do not exhibit

spontaneous seizures or a high rate of mortality at an early age.

Hypothesis:

I hypothesize that Kcna1-null mice display pathological changes in the

hypothalamus, including increased astrogliosis, increased blood-brain barrier

permeability, decreased adenosinergic tone, and increased orexinergic tone,

resulting in a decreased sleep phenotype comorbid with epileptic phenotype. I

additionally hypothesize that tanycytes of the third ventricle, a cell type that has

not been previously investigated in epilepsy, have altered morphology, as well as

altered expression of GLUT1 and increased neurogenesis capability.

Objectives:

The overall goal of my study is to investigate changes in the physiology of

regions of the hypothalamus in Kcna1-null mice. Understanding changes

occurring in the orexin system originating in the lateral hypothalamus will further

understanding of comorbid sleep disorders in epilepsy. I also examined tanycytes

of the third ventricle, a cell class with a close association with function of the

15

hypothalamus that has not been previously implicated as contributing to disease

progression in epilepsy. Utilizing the Kcna1-null mouse model, the following

objectives were pursued:

1. Investigate signs of pathophysiology in the LH, including astrogliosis and

blood brain barrier permeability, and changes in wake-promoting orexin

neurons located in the LH.

2. Investigate changes in the adenosine regulation of orexin neurons in the

LH, including adenosine concentration and adenosine receptor

expression.

3. Investigate changes in tanycytes of the third ventricle, including

morphological alteration, protein expression and neurogenesis.

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Chapter 2: Orexin Neurons and Pathology in the Lateral Hypothalamus INTRODUCTION

Epilepsy is a serious neurological disorder characterized by recurrent,

unprovoked seizures that has a lifetime incidence of 1:26 people in the United

States (Hesdorffer et al. 2011). Epilepsy is associated with increased risk for

many comorbid conditions including sleep disorders (Manni et al. 2010; Gilliam et

al. 2005). The resulting insufficient sleep is associated with worsening seizure

frequency and/or severity, thus exacerbating the core syndrome (Kotagal et al.

2008; Grigg-Damberger et al. 2014). Insufficient sleep can also further reduce

the quality of life of people with epilepsy by promoting other comorbid conditions,

including cognitive impairments and/or psychological disorders, which are also

dependent on sleep efficiency (Kotagal et al. 2008; Malow 2005; Malow et al.

1997; Rocamora et al. 2008; de Weerd et al. 2004; Steinsbekk et al. 2013;

Jacoby et al. 2015; Killgore et al. 2010; Pilhal et al. 1997). Thus, there is a critical

need to better understand and ameliorate the sleep disorder symptoms

experienced by patients with epilepsy. Restoring sleep may improve the seizure

profile and/or reduce other comorbid conditions.

Symptoms of sleep disorders in epilepsy frequently include (i) increased

latency to sleep onset, (ii) reduced sleep duration and (iii) increased sleep

fragmentation (Kotagal et al. 2008; Malow 2005; Malow et al. 1997). Similar

symptoms in people without epilepsy and in animal models are associated with

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changes in orexin neurotransmission (Prober et al. 2006; Brisbare-Roch et al.

2007; Horvath et al. 2005; Vgontzas et al. 2002). Orexin neurons are located in

the lateral hypothalamus and perifornical region (LH/P) and when activated,

promote wakefulness, attention and arousal18. Orexin neurons funnel diverse

afferent signals and send projections to the ascending reticular activating system

to increase activation of wake-promoting norepinephrine, acetylcholine,

serotonin, histamine and dopamine neuronal populations (Sakurai 2007;

Szymusiak and McGinty 2008; Sakurai et al. 2010; Peyron et al. 1998).

Orexin neurons are also directly and indirectly connected to the seizure-

generating hippocampal network via mono- and poly-synaptic efferent and

afferent projections (Wang et al. 2008; Dube et al. 2001; Yoshida et al. 2006;

Kukkonen et al. 2002). Considering that (i) orexin activation promotes

wakefulness, (ii) wakefulness is increased in people with epilepsy and sleep

disorders and (iii) orexin neurons are connected to the hippocampus, we

hypothesized that dysregulated orexin neurotransmission plays a role in the

sleep disorders associated with epilepsy.

To test this hypothesis, we used Kcna1-null mice, a model of multiple

epilepsy syndromes (Simeone et al. 2013), that we have previously

demonstrated have highly variable diurnal rest activity patterns, specifically their

peak activity and diurnal period (Fenoglio-Simeone et al. 2009a; Fenoglio-

Simeone et al. 2009b). Here, we further determined that Kcna1-null mice express

additional sleep disorder symptoms including increased latency to rest onset and

rest fragmentation, and reduced non-rapid eye movement (NREM) and REM

18

sleep during periods of rest. Thus, Kcna1-null mice represent an appropriate

model of epilepsy with comorbid sleep disorder symptoms to test the hypothesis

that dysregulated orexin neurotransmission plays a role in the sleep disorders

associated with epilepsy.

SUBJECTS/MATERIALS AND METHODS

Animals: C3HeB/FeJ Kcna1-null and wild-type littermates were bred and

reared at Creighton University. Mice were entrained to a strict 12 h light/dark

cycle with access to food and water ad libitum. Lights on occurred at zeitgeber

time (ZT) 00:00 h. Tails clips were collected on postnatal day (P) 12-15 and

genotype was determined by Transnetyx, Inc (Cordova, TN, USA). On ~P21

mice were weaned onto standard diet. Adult mice (P35-P50) were used for these

experiments. All experiments conformed to NIH guidelines in accordance with the

United States Public Health Service’s Policy on Humane Care and Use of

Laboratory Animals and were approved by Creighton University’s Institutional

Animal Care and Use Committee.

Drugs and reagents: All reagents were purchased from Sigma unless

otherwise noted. Almorexant was provided by Acetlion Pharmaceuticals (San

Francisco, CA, USA).

Electroencephalography (EEG) and electromyography (EMG)

neurosurgeries: Neurosurgeries were conducted as we have previously

described (Simeone et al. 2014a). Adult Kcna1-null mice (~P35) were

anesthetized with isoflurane (5% initiation and 3% maintenance). EEG and EMG

19

electrodes were implanted in all mice. For cortical EEG surgeries, two subdural,

ipsilateral cortical electrodes were implanted at 1.2 mm anterior to Bregma and 1

mm lateral to midline, and at 1.5 mm posterior to bregma and 1 mm lateral to

midline. A reference electrode was implanted 1.5 mm posterior to bregma and 1

mm lateral to midline, contralateral from recording electrodes. For surgeries that

implanted both cortical and depth electrodes, the anterior cortical electrode was

implanted (as described above) and a depth electrode (PFA-coated tungsten

wire, A-M Systems) was placed 1 mm posterior to bregma and 1 mm lateral to

midline at a depth of 5 mm. EMG electrode wires were inserted into the nuchal

muscles in all mice. Electrodes were soldered to the headmount (Pinnacle

Technologies, Lawrence, KS, USA), and secured to the skull with dental cement.

Mice were allowed to recover for one week. During recovery and throughout the

experiment, mice were housed individually and were given access to food and

water ad libitum.

Dual orexin receptor antagonist (DORA) experimental design: The design

for the experiment using the dual orexin receptor drug almorexant is outlined in

Figure 5. Due to the shortened lifespan of these mice, we use a timeline similar

to those we have previously reported (Fenoglio-Simeone et al. 2009a; Fenoglio-

Simeone et al. 2009b; Simeone et al. 2014a; Simeone et al. 2014b; Kim et al.

2015). Adult mice (~P30) were placed in an 8” x 8” x 16” transparent plexiglass

cage and allowed to habituate for 5 d. On ~P35, mice were implanted with

EEG/EMG electrodes as described above. Following a 5-7 d recovery, mice were

injected with vehicle (i.p., 25% DMSO in sterile saline) once daily for three

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consecutive days, followed by three consecutive days of once daily 100 mg kg-1

almorexant (i.p., 25 mg/mL in vehicle). Injections began at ZT 00:00 h. For each

treatment, time-synchronized video-EEG-EMG recordings were started

immediately after the injection on the second day and continued for 48 h. To

allow the animals to habituate to and reduce the confounding effects of being

tethered, handling and injections, recordings from the third day of each treatment

were analyzed 30 min after the injection. The analyses were limited to the first

5.5 h (~ZT 00:30–06:00 h) due to the previously reported pharmacokinetics and

dynamics of almorexant (Morairty et al. 2012; Morairty et al. 2014; Black et al.

2013).

Sleep analyses: Using Sirenia Sleep Pro (v1.4.2, Pinnacle Technologies,

Lawrence, KS, USA), recordings were divided into 10s epochs for sleep state

scoring and analyzed using a semi-automated method. Recordings were initially

scored into different sleep states based on EEG power in the delta band of the

anterior cortical electrode, located over the motor cortices and the corpus

callosum, and EMG power (10-50 Hz). Sleep states were defined as follows:

Wake = low delta power, high EMG; NREM = high delta power, low to medium

EMG power; REM = low delta power, low EMG. Scoring based on EEG and EMG

was then manually verified with video recording. The epochs during which an

animal was seizing was excluded from the analyses. The percentage of epochs

Kcna1-null mice spent seizing was minimal when compared to the total number

of analyzed epochs (vehicle: 0.98% ± 0.44%; almorexant: 0.48% ± 0.08%).

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Figure 5: Schematic of the experimental design for the in vivo almorexant

study. The ages for each experimental procedure are listed along the bottom.

Animals were placed in recording cages on ~P30 and allowed to habituate for 5 d

before surgery. After 7 d of post-surgical recovery, mice were injected once daily

with vehicle (for 3 d) then almorexant (for 3 d). Continuous video-EEG-EMG

recordings were conducted for 48 hrs, starting on the second day of treatment.

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Several endpoints were analyzed for wake, NREM sleep and REM sleep:

(1) the number of epochs in each state of vigilance (NREM sleep, REM sleep or

wake), normalized to the total number of epochs analyzed within each animal; (2)

the number of bouts; and (3) the number of transitions. Bouts were defined as 3

or more consecutive epochs uninterrupted by a bout of a different state.

Transitions were defined as any change between states and categorized based

on initial and subsequent state. (4) The length of bouts for NREM sleep and (5)

the latency to REM sleep were also measured. The latency to REM sleep was

defined as the amount of transpired time until the first REM sleep epoch following

the start of the analysis.

Seizure analyses: Seizures were identified based on ictal cortical EEG

activity, high EMG activity and previously defined seizure behaviors for this

mouse strain (Fenoglio-Simeone et al. 2009a; Fenoglio-Simeone et al. 2009b;

Simeone et al. 2014a). Seizure behavior was scored using a modified Racine

scale, as we have previously described (Fenoglio-Simeone et al. 2009a;

Fenoglio-Simeone et al. 2009b; Simeone et al. 2014a): Stage 1-myoclonic jerk;

Stage 2-head stereotypy; Stage 3-forelimb/hindlimb clonus, tail extension, a

single rearing event; Stage 4-continuous rearing and falling; Stage 5-severe

tonic-clonic seizures. Stage 1 behavior is associated with an EEG spike wave

discharge. Stages 2-5 are associated with a concordant and sustained increase

in EMG activity and EEG amplitude and time-dependent frequencies. Seizure

burden scores (SBS) were generated using the following equation: SBS = Σ(σi

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θi), where σ indicates the seizure severity score using the modified Racine scale, θ

indicates the duration of each seizure, and i indicates each seizure.

Immunoglobulin G (IgG) immunohistochemistry: Mice were quickly

anesthetized with isoflurane, transcardially perfused with ice-cold 0.9% saline

followed by 4% paraformaldehyde in 0.1 M PB (pH 7.4), decapitated and brains

were post-fixed overnight. Brains were cryoprotected in 15% and 30%

sucrose/PB solutions before frozen in methyl-butane on dry ice. Sections

containing the lateral hypothalamus (-1.30 thru -2.06 mm Bregma) were

examined. Sections affixed to slides were fixed in 4% paraformaldehyde in 0.1 M

PB (pH 7.4), dehydrated and rehydrated in a series of ethanol baths, washed

with 0.01 M PBS/0.3% Triton X-100, pH 7.4 (PBS-T), blocked with 10% normal

goat serum/PBS-T, and incubated with AF-350 conjugated anti-mouse IgG

(1:2000, Invitrogen, Grand Island, NY, USA) overnight at room temperature.

Sections were scored on an intensity scale of punctate and diffuse IgG-positive

staining (1-5 with 1=low intensity, 5=high intensity) by five blinded investigators.

Sections with less punctate and more diffuse IgG are considered to have a leaky

or more permeable BBB as previously described33. Data were expressed as the

mean punctate intensity.

GFAP and orexin immunohistochemistry: Mice were quickly anesthetized

with isoflurane, decapitated, brains were removed and frozen in methyl-butane

on dry ice. Sections affixed to slides were fixed in 4% paraformaldehyde in 0.1 M

PB (pH 7.4), washed with 0.01 M PBS/0.3% Triton X-100, pH 7.4 (PBS-T) and

blocked with 10% normal goat serum/PBS-T. Sections incubated with rabbit anti-

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orexin (1:1000, Peninsula Laboratories, San Carlos, CA, USA) overnight at room

temperature followed by AF-594 conjugated goat anti-rabbit IgG (1:600,

Invitrogen, Grand Island, NY, USA) for 3 h at room temperature. In one cohort,

sections were immediately cover-slipped with mounting media containing DAPI

and orexin was quantified using stereology (described below). In the second

cohort, sections were incubated additionally in AF-488 conjugated mouse anti-

GFAP (1:500, Invitrogen, Grand Island, NY, USA) overnight at room temperature

and cover-slipped with DAPI mounting media. GFAP is used as a marker for

astrocytes. The number of reactive astrocytes in the LH/P was quantified.

Reactive astrocytes were operationally defined as astrocytes with morphological

hypertrophy and extended processes, as previously described (Sofroniew 2009;

Wenzel et al. 2007).

Stereology: LH/P sections with orexin positive labeling were selected

using unbiased systematic random sampling (West 2012). The total number of

immunopositive cells was estimated using the NV method: NV=(ΣQ-/#Q-)/DAt;

NV, numerical density; ΣQ-/#Q-, average number of cells per dissector; DAt,

dissector area over the specified thickness, t.

Statistics: Sleep data were analyzed using an ANOVA with Sidak’s post

hoc test. Actigraphic, immunohistochemical and mitochondrial data were

analyzed using an unpaired student’s t test. Statistical dependence was

determined using Spearman’s non-parametric rank correlation coefficient. A p-

value < 0.05 was considered statistically significant.

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RESULTS

Kcna1-null mice have reduced NREM sleep and REM sleep epochs during

periods of rest.

We have reported that Kcna1-null mice are more active during the rest

period based on measurements using infrared beam actigraphy (Roundtree et al.

2015). However, the increased time spent active during the rest period

suggested that Kcna1-null mice may either have longer durations of time spent

awake or may transition to wake more often than wild-type controls. To

distinguish between these possibilities, we examined the sleep architecture of

Kcna1-null mice, and analyzed NREM sleep, REM sleep and wakefulness using

continuous time-synchronized video-EEG-EMG recordings. In this experiment,

mice were treated with vehicle for 3 d followed by a treatment with an orexin

receptor antagonist for 3 d. Analyses were conducted using two-factor ANOVA.

The differences between genotypes (i.e. the vehicle data) will be discussed in

this section. The differences between treatments will be discussed in the next

section. To avoid plotting vehicle data twice, Figures 7-9 will be referenced in

both sections.

Raw EEG and EMG traces were divided into 10 s epochs. As depicted in

Figure 6, dominant frequencies and EMG activity were used to classify each

epoch as NREM sleep (high EEG delta power with low to medium EMG power),

REM sleep (low EEG delta power with low EMG activity) or awake (low EEG

delta power with high EMG power). Hypnograms of vehicle-treated wild-type and

Kcna1-null mice are depicted in Figure 7A. The amount of time mice spent in

26

each behavioral state was determined by totaling the number of epochs and

normalizing to the total number of epochs analyzed for that animal. During the

rest period, Kcna1-null mice spent more time awake (F(1,9) = 42.2, p<0.0005)

and less time in both NREM sleep (F(1,9) = 20.3, p<0.005) and REM sleep

(F(1,8) = 229.3, p<0.0001) when compared to wild-type controls (Figure 8A, left

two bars in each graph). These data indicate that Kcna1-null mice have a

deficiency in both NREM sleep and REM sleep.

27

Figure 6: Representative traces of behavioral states. Sleep architecture was

scored using EEG-EMG-video data in Sirenia Sleep Pro employing a semi-

automated method. Each automated EEG-EMG score was manually verified with

time-synchronized video data. Traces from time-matched subdural EEG

electrodes (blue and orange traces) and EMG electrode (green) show typical

characteristics of wake (low amplitude EEG, high amplitude EMG), NREM sleep

(high amplitude EEG, low-medium amplitude EMG), and REM sleep (low

amplitude EEG and EMG). EEG rhythms for each example epoch are shown in

corresponding spectrograms.

28

29

Figure 7: Representative hypnograms of Kcna1-null and wild-type mice.

Kcna1-null and wild-type mice were treated with vehicle for 3 d then almorexant

for 3 d (100mg/kg, i.p.). Video-EEG-EMG data were scored in 10s epochs as

wake, NREM sleep, or REM sleep for this first 5.5 h of the rest phase, 30 min

following injection (ZT 0:30-6:00 hr). (A) Sleep architecture is shown in

representative hypnograms of a wild-type mouse and Kcna1-null mouse on

vehicle, compared to (B) the same mice during almorexant treatment. Each

hypnogram data point identifies the behavioral state of the mouse at each epoch

over time (i.e. during each 10 s epoch from ZT 00:30-6:00 hr).

30

We began to assess how fragmented the rest period was by comparing

(1) the number of bouts of and (2) the number of transitions among the three

vigilant states. One bout was defined as three or more consecutive epochs of the

same state. Kcna1-null mice had significantly more wake bouts (F(1,9) = 10.64,

p<0.01) as well as a large reduction in number of REM sleep bouts (F(1,9) = 200,

p<0.0001) when compared to wild-type mice (Figure 8B, left two bars in each

graph).

The transitions between behavioral states (NREM sleep-wake, NREM

sleep-REM sleep, wake-REM sleep and vice versa) were sorted into six

categories based on the initial and subsequent behavioral states (Figure 9).

Kcna1-null mice had significantly more transitions from NREM sleep to wake

(F(1,9) = 61.3, p<0.0001) when compared to wild-type mice (Figure 9A). Kcna1-

null mice also had significantly fewer transitions between REM sleep and NREM

sleep (REM vs. NREM: F(1,9) = 260.5, p<0.0001 and NREM vs. REM: F(1,9) =

421.5, p<0.0001, Figure 9B), and between REM sleep and wake (F(1,9) = 24.0,

p<0.001, Figures 9C). This is likely reflecting the REM sleep deficiency apparent

during the period of analysis. These data suggest that Kcna1-null mice may have

more fragmented rest when compared to wild type mice.

31

Figure 8: Almorexant increases NREM sleep and reduces wake during the

rest phase of Kcna1-null mice. The total percentage of epochs spent in each

behavioral state was analyzed using two-way ANOVA with Sidak’s post hoc. (A)

Kcna1-null mice spend significantly less time in NREM sleep and REM sleep and

more time awake compared to wild-type. Almorexant treatment restores NREM

sleep of Kcna1-null mice to wild-type levels and significantly decreases time

awake. (B) Bar graphs depicting the total number of bouts for each genotype and

treatment. A bout is defined as three or more consecutive 10 s epochs of the

same behavioral state. Kcna1-null mice have a greater number of wake bouts

and fewer REM sleep bouts than wild-type mice. Data are mean ± SEM; n=5-6; *

p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

32

Blocking orexin receptors improves the NREM sleep in Kcna1-null mice

To determine whether orexin activation contributed to sleep disturbances,

following vehicle treatment, Kcna1-null mice were treated with the DORA

almorexant (100mg/kg, i.p.; Morairty et al. 2014; Black et al. 2013). Based on the

pharmacokinetics and pharmacodynamics of almorexant in rodent (Morairty et al.

2012), the analyses were limited to the first 5.5 h, 30 min following injection.

Representative EEG-EMG hypnograms of wild-type and Kcna1-null mice treated

with vehicle then almorexant are depicted in Figures 7A and 7B, respectively.

Blocking orexin receptors during periods of rest significantly reduced the

percentage wake epochs (F(1,9) = 15.2, p<0.01) and increased the percentage

of NREM sleep epochs in Kcna1-null mice (F(1,9) = 17.1, p<0.001) to levels that

were similar wild-type controls (Figure 8A). The deficit in REM sleep was not

rescued by three days of almorexant treatment in Kcna1-null mice. Almorexant

did not have a significant effect on the percentage of wake, NREM sleep, or REM

sleep epochs in wild-type mice during the rest phase. Unexpectedly, these data

suggest the dose of almorexant increased the amount of NREM sleep of Kcna1-

null mice, however did not have the same effect in wild-type controls.

Next, we determined whether almorexant influenced the number of bouts

of or transitions among states of vigilance. There was no change in the number

of NREM sleep or wake bouts of Kcna1-null or wild type mice (Figure 8B).

Further, almorexant treatment had no effect on the number of transitions among

vigilant states of Kcna1-null mice. In contrast, almorexant-treated wild type mice

had significantly more REM sleep bouts (F(1,9) = 12.9, p<0.01) and more

33

transitions between NREM and REM (NREM to REM: F(1,9) = 21.8, p<0.001;

REM to NREM: F(1,9) = 16.6, p<0.01) (Figure 9B).

The increased number of NREM sleep epochs and the lack of change in

NREM bout incidence suggest that the duration of each bout may be increased.

Figure 7A depicts the distribution of the NREM sleep bout length. Goodness-of-

fit-analysis indicates that treatment with almorexant increased the probability of

longer NREM sleep bout durations in Kcna1-null mice (p<0.0001). Almorexant

did not change NREM sleep duration in wild-type controls. These data indicate

that almorexant may improve the ability of Kcna1-null mice to remain in NREM

sleep.

In contrast to actigraphy data, there was no significant difference in

latency to sleep onset (Figure 10B), defined as the time that transpired between

the start of analysis and the first epoch of NREM sleep or REM sleep epoch (in

all but one case, NREM sleep occurred first). This is likely due to the

conservative definition of 30 min of continuous rest used in the actigraphy study.

However, Kcna1-null mice do have an increased latency to REM sleep (Figure

10C, p<0.0001 compared to wild-type on vehicle), defined as the time that

transpired between the start of analysis and the first epoch of REM sleep only.

(Note: as there was no statistical difference in the latency to sleep onset, the

latency to REM sleep onset was similar whether defined with respect to the start

of analysis or the start of sleep onset. Thus, we used the start of the analysis to

maintain focus on REM sleep alone). While it had no effect on latency to REM

sleep in wild-type mice, almorexant appeared to reduce the latency to REM sleep

34

onset when compared to vehicle treated Kcna1-null mice (Figure 10C, also

apparent in the hypnograms in Figure 7). Using the extreme studentized deviate

(ESD) method, analyses of the percent reduction in latency to REM sleep onset

following almorexant treatment identified one subject (which had an increase in

latency to REM onset during almorexant treatment) as a statistical outlier

(p<0.05, identified by a dashed line in Figure 7B). Quantification of the remaining

four Kcna1-null mice indicated that the latency to REM sleep onset was

significantly reduced during almorexant treatment (F(1,7) = 161.2, p<0.0001).

REM sleep rhythms were absent in one animal during vehicle treatment and then

became apparent during almorexant treatment (Figure 10B, the data point above

almorexant with no line). This interesting observation will be discussed more in

the ‘Correlations between seizure and sleep profiles’ section. These data suggest

that almorexant may reduce the latency to REM sleep onset, specifically in the

Kcna1-null mice with prolonged onset when compared to wild-type controls.

35

Figure 9: The transitions between behavioral states in Kcna1-null mice

differ from wild-type controls. Transitions between behavioral states were

grouped into six categories. Kcna1-null mice have significantly more transitions

between wake and NREM sleep, but have fewer transitions to or from REM

sleep. Almorexant treatment does not affect the number of transitions between

any behavioral states in Kcna1-null mice. Almorexant treatment does increase

the number of transitions between REM sleep and NREM sleep in wild-type

controls. Data are mean ± SEM; n=5-6; ** p<0.01, *** p<0.001, **** p<0.0001.

36

Figure 10: Almorexant increases the probability of longer NREM sleep bout

durations and reduces the latency to REM sleep onset in Kcna1-null mice.

(A) Probability curves of NREM sleep bout durations were plotted for each

genotype and treatment. Kcna1-null mice are less likely to have longer duration

of continuous NREM sleep. Almorexant increases the probability of longer NREM

sleep bout durations in Kcna1-null mice. (B) The latency to sleep onset was

determined as the time before the first occurrence of NREM sleep or REM sleep

in each animal. The within-subject comparisons are plotted. (C) The latency to

37

REM sleep was determined as the time before the first occurrence of REM sleep

in each animal and within-subject comparisons are plotted. The latency to REM

sleep in Kcna1-null mice was higher in all but one animal (statistical outlier;

dashed line) compared to wild-type controls. In all Kcna1-null mice with

increased latency to REM sleep, almorexant reduced the time to REM sleep

onset. REM sleep was not detected in one subject during vehicle, however was

apparent during almorexant treatment (data point with no associated line). Data

are mean ± SEM; n=5-6; *** p<0.001, **** p<0.0001.

38

Seizure-like activity and pathology is apparent in the wake-promoting LH/P

of Kcna1-null mice

The improvement in NREM sleep following almorexant suggested that

activation of wake-promoting orexin neurocircuitry may be increased in Kcna1-

null mice. Thus, we analyzed seizure propagation, markers of pathology and

orexin expression in the LH/P.

First, we determined whether seizures propagate to the orexin-rich LH/P.

Subdural cortical electrodes and an LH/P depth electrode were implanted in

Kcna1-null mice. Seizure activity, which originates from the hippocampus and

propagates to cortex of Kcna1-null mice35, was detected in the wake-promoting

LH/P. Figure 11 depicts representative cortical and depth electrode traces of a

seizure that electro-clinically manifested from a stage 1 to a stage 4 event. In all

seizures analyzed, epileptiform activity was apparent in both the cortical as well

as the depth electrode traces during severe seizures (stages 3-5), while the less

severe seizures (stages 1-2) did not consistently propagate to the LH/P.

39

40

Figure 11: Severe seizures in Kcna1-null mice propagate to the LH/P.

Representative LH/P and cortical EEG traces of one seizure that manifests from

a less severe stage 1 to a severe stage 5 seizure. Arrows indicate an EEG event

with a behavioral correlate. Seizure severity scores are in parentheses. A single

arrow indicates the event was detected in the subdural channel. Double arrows

indicate the event was detected in both subdural and LH/P electrodes. Stage 5

severe tonic clonic period detected in both electrodes is indicated with brackets.

Cort, subdural cortical electrode trace; LH, lateral hypothalamic and perifornical

region depth electrode trace.

41

The hyperexcitability and hypersynchrony associated with seizures can

induce pathology that significantly changes the neurobiological and metabolic

landscape, which are notably apparent in the hippocampus of Kcna1-null mice

(Simeone et al. 2013; Simeone et al. 2014a; Wenzel et al. 2007; Simeone et al.

2014b). Pathological markers were used to assess injury in the LH/P of Kcna1-

null mice. Blood-brain barrier (BBB) permeability and astrocyte morphology were

examined with immunofluorescent histochemistry, and mitochondrial function

was assessed with oxygen polarography (Wenzel et al. 2007). There was a

significant reduction of punctate immunostaining and a concordant increase in

IgG extravasation, indicative of an increase in BBB permeability in the LH/P of

Kcna1-null mice (p<0.05, Figure 12A). There was also an increase in the

incidence of reactive astrocytes in this region of Kcna1-null mice when compared

to wild-type controls (78.6±7.9% compared to 34.8±10.2%, p<0.01, Figure 12B).

Collectively, these data suggest that the more severe seizures propagate to the

LH/P and that injury is apparent in the hypothalamus of Kcna1-null mice.

42

Figure 12: Pathology is apparent in the LH/P of Kcna1-null mice. (A)

Photomicrographs depict IgG labeling (white/blue) in LH/P parenchyma. In wild-

type animals, punctate staining (arrows) is more pronounced, compared to

diffuse labeling in Kcna1-null mice. Bar graph of punctate mouse IgG staining as

scored on an arbitrary 1-5 scale by five blinded investigators (n=4 animals/group,

3-4 LH/P sections/animal). (B) Reactive astrocytes are GFAP-positive (green)

and exhibit extended processes and hypertrophy (see the Kcna1-null darkfield

photomicrograph) when compared to typical astrocytes as depicted in the wild-

type section. Coronal sections containing LH/P had a significantly higher

43

incidence of reactive astrocytes compared to wild-type LH/P (n=4 animals/group,

3-4 LH/P sections/animal). Sections are double-labeled for orexin (red).

44

Kcna1-null mice have significantly more orexin-positive neurons at P40,

but not at younger ages

Finally, we assessed orexin protein levels in the LH/P to support or refute

the hypothesis that orexin activity may be increased in Kcna1-null mice. The

number of neurons expressing orexin throughout the anterior-posterior axis of the

LH/P was significantly increased in Kcna1-null mice when compared to wild-type

controls (p<0.05, Figure 13). This increase existed in neurons both in the medial

(p<0.05) and lateral (p<0.05) aspects of the posterior LH/P between -1.0mm and

-1.2mm Bregma (right inset). Collectively, these data indicate long-term changes

in the neurobiological environment of the LH/P that supports the notion of

increased orexinergic tone of Kcna1-null mice. Orexin expression was also

assessed at other ages to further investigate differences in orexin ontogeny and

determine if changes in orexin-positive neurons are reflective of a developing

pathology in Kcna1-null mice (Figure 14). Animal at P12, before seizure onset;

P21, the approximate start of spontaneous seizures in this mouse model; and

P30 showed no significant differences in number of orexin neurons. This

indicates that the increase in orexin-positive neurons in Kcna1-null mice

compared to wild-types occurs over time and after seizure onset.

45

Figure 13: The number of orexin-positive neurons is increased in Kcna1-

null LH/P at P40. The distribution of orexin-positive neurons along the anterior-

posterior axis for coronal brain sections of wild-type and Kcna1-null mice is

shown. Kcna1-null mice show significantly more total orexin-positive cells. Left

inset: Darkfield photomicrograph depicting orexin-positive (red)

immunofluorescence in the LH/P. Right inset: The increase of orexin-positive

neurons occurred in both the medial and lateral aspects of the LH/P (n=4-5

animals, 3-4 sections/animal). Data are mean ± SEM, * p<0.05.

46

Figure 14: The number of orexin-positive neurons in Kcna1-null mice is not

significantly different at younger ages. While orexin neurons were significantly

increased in Kcna1-null mice at P40 (see Fig. 13), they were not increased at

P12, P21, or P30 when compared to age-matched wild-type controls. Data are

mean ± SEM, n=6 animals per group.

47

Dual orexin receptor antagonism decreases seizures in Kcna1-null mice

The association between sleep deprivation and the worsening of seizure

frequency and/or severity suggests that improving the sleep profile may be

associated with an improved seizure profile. We examined the same time period

used for the sleep analyses to analyze seizure frequency, duration and severity

using the modified Racine scale, as we have described (see methods) (Fenoglio-

Simeone et al. 2009a; Fenoglio-Simeone et al. 2009b; Simeone et al. 2014a).

Data were stratified into two groupings based on seizure severity (less

severe stage 1 seizures and moderate/severe stage 2-5 seizures). We observed

a 75 ± 6% percent reduction in the incidence of stage 2-5 seizures during

almorexant treatment in 4/5 mice (p<0.01; ESD analysis identified the mouse

with the lowest seizure profile during vehicle as an outlier, dashed line in Figure

15A). The incidence of stage 1 seizures did not change during almorexant

treatment (Figure 15B). Seizure durations range from less than 30s to several

minutes. To take into account the severity and duration of each seizure, a seizure

burden score was calculated for each animal (see methods). Seizure burden was

reduced by 75 ± 9% during almorexant treatment in the same cohort (4/5 mice,

p<0.01, Figure 15C). Plotting the initial seizure burden scores against the

percent reduction during almorexant treatment revealed a significant correlation

(Spearman’s r=1.00, p<0.05, Figure 15D, fit with a logarithmic growth curve).

These data indicate a potentially greater anti-seizure efficacy of almorexant with

more severe seizures.

48

Figure 15: Almorexant reduces severe seizures in Kcna1-null mice. Seizure

incidence and severity during vehicle and almorexant treatment were scored and

seizure burden was calculated. Within scatterplots depict (A) the incidence of

severe seizures (stages 2-5), (B) the incidence of the less severe stage 1

seizures and (C) seizure burden during vehicle and almorexant treatments. (D)

Percent reduction of seizure burden following treatment showed a positive

Spearman correlation with initial seizure burden and was fit using a logarithmic

growth curve. (E) Latency to REM sleep also showed a positive Spearman

correlation with a linear fit with seizure burden both before and after treatment

with almorexant. Dashed lines indicate an outlier, n=5, ** p<0.01.

49

Correlation between seizure and sleep profiles

A final interesting observation was in the extreme seizure and sleep

profiles: (1) The animal with the highest seizure burden lacked REM sleep during

vehicle treatment and (2) the mouse with the least severe seizure profile had a

latency to REM sleep onset that resembled wild-type latencies. Indeed, there

was a positive linear correlation between REM sleep onset and seizure severity

(r=0.82, p<0.05, Figure 15E).

DISCUSSION

A connection between epilepsy and sleep has long been recognized.

Seizures and sleep disorders have an interdependent relationship where the

occurrence of one can exacerbate the other. Research has primarily focused on

the relationship of brain EEG rhythms (e.g. slow wave sleep, sharp waves, sleep

spindles, ripples, etc.) and the development of seizures. Here, we used a novel

strategy and focused on the sleep disorder co-morbidity itself in an effort to

increase our understanding of the sleep and seizure dynamics and to identify a

novel anti-seizure and somnogenic treatment.

To our knowledge this study is the first to report a detailed characterization

of the sleep architecture deficiencies of Kcna1-null mice, further supporting the

use for this animal model to examine sleep disorder symptoms associated with

epilepsy. Treatment with a dual orexin receptor antagonist significantly increases

NREM sleep and reduces the latency to REM onset in Kcna1-null mice. The

more severe seizures propagate to the wake-promoting LH/P where astrogliosis

and blood-brain barrier permeability are apparent. The number of orexin-positive

50

neurons is increased in the LH/P. Blocking orexin receptors with almorexant

reduces the severity of seizures and importantly, there is a significant correlation

between latency to REM onset and seizure burden in Kcna1-null mice.

Seizure Propagation and neurobiological changes in the LH/P

Kcna1-null mice lack the potassium channel Kv1.1 alpha subunit and are

a genetic model of temporal lobe epilepsy. This model offers diverse and severe

seizure phenotypes involving multiple daily myoclonic jerks, head movement

stereotypies, clonus and tonic-clonic seizures (Simeone et al. 2013; Fenoglio-

Simeone et al. 2009a; Fenoglio-Simeone et al. 2009b; Simeone et al. 2014a;

Wenzel et al. 2007; Simeone et al. 2014b; Kim et al. 2015). Seizures arise in the

hippocampus and propagate to the cortex of Kcna1-null mice35. Seizure severity

and incidence increase with age and are associated with hippocampal sclerosis

involving cell death, astrogliosis, mossy fiber sprouting and mitochondrial

impairment (Simeone et al. 2014a; Wenzel et al. 2007; Kim et al. 2015).

To our knowledge, we are the first to provide evidence that severe

seizures propagate to the LH/P. Consistent with the damage reported in the

Kcna1-null hippocampus, we identified similar pathology including astrogliosis

and BBB permeability in the LH/P. Additionally, we have reported impairment of

hypothalamic mitochondrial function (Roundtree et al. 2015). Our findings

support a limited number of previous studies, which have noted seizure-related

injury in the hypothalamus and LH/P. Increased metabolic activity and injury have

been reported in the hypothalamus following induction of status epilepticus in the

pilocarpine epilepsy mode (Wang et al. 2008; Fernandes et al. 1999). Studies

51

using kainic acid and lithium-pilocarpine to induce severe seizures have

demonstrated neurodegeneration and cell death throughout the hypothalamus;

injury specifically in the LH/P was noted in mice subjected to kainic acid-induced

seizures (Covolan and Mello 2009). The limited available human data from MRI

studies of mesial temporal lobe epilepsy indicates a strong concordance between

changes in the hippocampus and degeneration of its major efferent tract, the

fornix, and subsequent targets in the hypothalamus (Ng et al. 1997; Karakas

2001; Ozturk et al. 2001).

In addition to the presence of pathology, we also found an increase in the

number of orexin-positive neurons in the LH/P. Previous studies have reported

mechanisms that promote orexin expression. Our data indicate one of two

possibilities. First, there may be an increase in the amount of orexin protein

expressed within neurons that previously expressed sub-detectable levels. Many

signals can influence the degree of orexin expression within a neuron. Prepro-

orexin mRNA and plasma orexin protein levels are significantly increased under

different metabolic conditions, such as 48 h of fasting or acute (6 hr)

hypoglycemia (Cai et al. 1999). Levels of orexin are also significantly higher in

younger rats compared to older rats (Desarnaud et al. 2004). A second possibility

is that the number of orexin expressing cells may be increased. Temporal lobe

seizures are associated with increased neurogenesis in the hippocampus (Parent

et al. 1997). The number of orexin neurons can be increased by the stress

hormone corticosterone (Jalewa et al. 2014). It is well documented that seizure

activity promotes an acute spatial-temporal change in the metabolic landscape in

52

the hippocampus and hypothalamus (Wang et al. 2008; Dube et al. 2001;

Covolan and Mello 2000; Ng et al. 1997; Ozturk et al. 2008), including enhancing

stress-regulating hormones and changing mitochondrial metabolism and function

(Simeone et al. 2014a; Sullivan et al. 2003; Patel et al. 2005). Whether seizures

increase expression in a low-expressing pool of neurons or increases the number

of neurons is currently under investigation.

The number of orexin-positive neurons was only observed to be

significantly higher in Kcna1-null mice at p40 and not at P12, before seizure

onset, or P21, the approximate age when seizure onset occurs. However, the

difference was still not apparent at P30, after seizure onset. Orexin-positive

neurons in rats, which show similar patterns as mice (Blumberg et al. 2007),

achieve adult size around approximately P5 and continue to increase in number

and axonal density until at least P21 (Steininger et al. 2004). Studies using

orexin-knockout mice suggest that orexin tone’s effect on circadian rhythm

doesn’t become significant until approximately P21 as well (Blumberg et al.

2007). While orexin-positive neurons were not increased at P30 in Kcna1-null

mice, seizure burden increases with age, indicating that the difference may be a

result of disease progression.

Regardless, the overall increase in orexin and other changes in the

neurobiological landscape of the LH/P in adult Kcna1-null mice will influence

neurotransmission. Similar changes within the Kcna1-null tri-synaptic

hippocampal network promote excitability (Simeone et al. 2013; Simeone et al.

2014a; Simeone et al. 2014b). Orexin neurons project to wake-promoting regions

53

containing norepinephrine, acetylcholine, serotonin, histamine and dopamine

neurons. Previous studies report that experimental increase in orexin receptor

activation induces wakefulness (Sakurai 2007). Increased drive of this circuitry

may participate in enhancing the propensity to wake in Kcna1-null mice during

periods of rest.

Sleep and seizures

Sleep disorder symptoms including increased latency to sleep onset,

increased awakenings and reduced sleep duration in humans and animal models

without epilepsy are associated with a dysregulated orexinergic system (Prober

et al. 2006; Brisbare-Roch et al. 2007; Horvath et al. 2005; Vgontzas et al. 2002),

a system responsible for promoting wakefulness (Sakurai 2007). If the Kcna1-null

orexinergic system were overactive during rest periods, we would predict that

sleep architecture changes may include increased time awake and decreased

NREM sleep and/or REM sleep. Further, we would predict that administration of

a DORA may restore some or all aspects of sleep architecture to resemble that

of wild-type mice.

Indeed, Kcna1-null mice have increased wake during periods of rest. We

previously reported that epileptic Kcna1-null mice exhibit highly variable diurnal

rest-activity patterns, specifically their peak activity and diurnal period (Fenoglio-

Simeone et al. 2009a; Fenoglio-Simeone et al. 2009b). Here, we further

determined that Kcna1-null mice express additional sleep disorder symptoms

during periods of rest including latency to REM sleep onset and reduced NREM

sleep and REM sleep (as determined using video/EEG/EMG recording). This

54

phenotype resembles sleep disorder symptoms described in people with

epilepsy, which include increased latency to sleep onset, increased entries into

wakefulness, and decreased sleep efficiency and NREM sleep (Kotagal and

Yardi 2008; Malow 2005; Malow et al. 1997). These sleep disorder symptoms

resemble those associated with a dysregulation of the orexinergic system

(Prober et al. 2006; Brisbare-Roch et al. 2007). Therefore, we further tested the

hypothesis that orexin plays a role in the sleep deficiency of Kcna1-null mice by

administering a dual orexin receptor antagonist, almorexant. We conducted

experiments during the rest phase when Kcna1-null mice are more active

compared to wild-type mice. Almorexant treatment reduced the time spent awake

and reversed the NREM sleep deficiency by improving the ability to maintain

NREM sleep (i.e. by increasing bout durations). While almorexant reduced the

latency to REM onset in Kcna1-null mice, the acute treatment was unable to

restore the number of REM sleep epochs.

Interestingly, these improvements to sleep following almorexant treatment

occurred in the Kcna1-null mice only. Wild-type mice did not experience a

change in the number of wake and NREM sleep epochs, or a change in latency

to REM sleep onset. This is not surprising considering DORAs are typically

tested in wild-type mice during the wake phase, a time when orexin neurons are

more active and an inhibitory effect would be maximal (Morairty et al. 2014; Black

et al. 2013). During normal sleep, orexinergic activity is suppressed, and hence

the inhibitory effect of almorexant would be minimal during the rest phase. This

55

may explain why acute almorexant treatment did not have a significant effect on

these endpoints in wild type mice.

In addition to improving sleep, almorexant treatment reduced the

incidence of the more severe seizures (stage 2-5) and seizure burden by 75%.

Whether almorexant exerted indirect or direct anti-seizure effects has yet to be

determined. Almorexant may have indirectly reduced seizures by improving

sleep. It is well known that inadequate sleep/sleep deprivation worsens seizures

in people with epilepsy (Kotagal and Yardi 2008; Grigg-Damberger and Ralls

2014; Ni et al. 2014). We specifically found that longer REM sleep latencies

positively correlated with greater seizure burden. In one study, sleep deprivation

associated with exacerbating seizures was also associated with an increase

orexin concentration in cerebral spinal fluid (Ni et al. 2014). Thus, almorexant

may have indirectly reduced seizures by improving sleep.

A second possibility is that almorexant directly reduced seizures. Orexin

neurons project to a multitude of neuronal populations including the hippocampus

(Peyron et al. 1998; Selbach et al. 2004) raising the possibility that almorexant

directly dampens seizure-generating mechanisms in the hippocampus. Excitatory

orexin receptors OX1R and OX2R are coupled to Gs and Gq/Gs proteins,

respectively, and are expressed in CA1-3 and the dentate gyrus (Sakurai 2007;

Selbach et al. 2004; Marcus et al. 2001). In vivo intracerebroventricular (i.c.v.)

injection of orexin excites CA1 pyramidal cells and in vitro application induces

LTP at the Schaffer collateral-CA1 synapse (Selback et al. 2004; Riahi et al.

2015). Furthermore, i.c.v injection of high dose of orexins causes behavioral

56

seizure activity in rats and bilateral hippocampal injection of selective OX1R or

OX2R antagonists reduces acute pentylenetetrazole-induced seizures (Ida et al.

1999; Goudarzi et al. 2015). Studies to distinguish the indirect and/or direct

mechanism of the seizure-dampening effects of almorexant are currently

underway.

Conclusion

Our findings suggest that pathology in the LH/P of Kcna1-null mice,

possibly caused by seizure propagation from the hippocampus, reflects long-term

changes in the neural environment that may contribute to comorbid sleep

disorders and the worsening of seizures in Kcna1-null mice. Inhibiting orexin

receptors circumvents the pathology and reverses the NREM deficit and reduces

seizure severity. DORAs are currently under evaluation for clinical use in sleep

disorders. One example is suvorexant, which is approved by the FDA for use in

treating insomnia. Here, we provide evidence that DORAs may be an effective

sleeping aid in epilepsy, and warrants further study on their effects in other

epilepsy models, both to improve co-morbid sleep disturbances as well as reduce

seizure severity.

57

Chapter 3: Adenosine System in the Lateral Hypothalamus INTRODUCTION

Epilepsy, a serious neurological disorder characterized by recurrent,

unprovoked seizures, afflicts 1 in 26 people in the US at some point in their lives

(Hesforffer et al. 2011). While epilepsy research has led to the identification of

many antiseizure treatments, an estimated 30% of all epilepsy patients are

considered refractory (NICE 2012). Further, approximately one third of all

epileptic patients experience sleep disruptions/disorders. This high rate of

comorbidity is especially important as insufficient sleep can exacerbate the core

syndrome (Kotagal et al. 2008; Grigg-Damberger et al. 2014). Current treatment

is largely symptomatic and unable to improve the comorbid sleep disorder

(Schachter 2002). Thus, the identification of novel antiseizure and somnogenic

targets is necessary.

The importance of adenosine in epilepsy research has been established

after adenosine was identified as an endogenous antiseizure molecule.

Adenosine acts on a family of G-protein coupled receptors, with the type-1 (A1)

and type-2A (A2A) being the most prevalent in the brain (Fredholm et al. 2001).

Evidence has shown that A1 agonists exert antiseizure effects which can be

blocked by A1 receptor antagonism, suggesting that adenosine’s anticonvulsant

action works through the A1 receptor (Gouder et al, Epilepsia 2003).

In the brain, adenosine and the purine family of molecules have their

concentration and action tightly regulated by several enzymes and receptors

58

(Boison, 2006; Chikahisa and Sei, 2011; see Chapter 1, Fig 2). Evidence

suggests that this regulation is altered in epilepsy. Adenosine kinase (ADK) is an

enzyme that phosphorylates adenosine, reducing the adenosine pool.

Upregulation of ADK has been reported in hippocampal lesions of kainate-

induced epileptic mouse models (Gouder et al 2004), and hippocampus and

cortex of rats of electrically induced status epilepticus that develop spontaneous

seizures (Aronica et al. 2011), as well as in astrocytic tumors (de Groot et al.

2012) and resected hippocampal and cortical human tissue (Aronica et al. 2012)

from epileptic patients. Additionally, experimental overexpression of ADK in an

animal model can lead to seizure development (Gouder et al. 2004).

Adenosine also exerts somnogenic actions in sleep promoting regions of

the brain (Bjorness and Greene 2009). Adenosine inhibits wake promoting orexin

neurons, also via acting on the A1 receptor on presynaptic glutamatergic neurons

and on orexin neurons themselves (see Chapter 1, Fig 3). Orexin is a peptide

produced in this population of spontaneously firing neurons found in the lateral

hypothalamus/perifornical region (LH/P). Orexin neurons target many of the brain

structures integral to arousal and attention, including those that comprise the

ascending reticular activating system (see Chapter 1, Fig 1).

We recently reported that orexin is upregulated in the LH/P of Kcna1-null

mice, a model of epilepsy with sleep disorders (Roundtree et al, Sleep 2015).

Kcna1-null mice lack the α subunit of the Kv1.1 voltage-gated delayed rectifier

potassium channel and have a severe epilepsy phenotype modeling several

epilepsy syndromes and presenting pathology similar to temporal lobe epilepsy.

59

Kcna1-null mice also have insufficient NREM and REM sleep. We recently

reported that the severe seizures propagate to the LH and there are signs of

chronic pathology in the LH/P, including astrogliosis, impaired mitochondrial

function, and increased blood-brain permeability. The number of orexin-positive

neurons was increased in the LH/P of Kcna1-null mice compared to wild-type

controls. Further, we reported that blocking orexin during periods of rest with a

dual orexin receptor antagonist increased NREM sleep and reduced seizure

severity.

Here, we report that adenosinergic inhibition in the LH is also altered in

Kcna1-null mice. Specifically, we report reduced pharmacological estimates of

adenosine in Kcna1-null LH/P compared to wild-type controls using in vitro brain.

Additionally, A1 receptor expression in LH/P tissue micro-punches is reduced in

Kcna1-null mouse tissue compared to wild-type controls. Spontaneous action

potentials in the LH/P of in vitro brain sections from Kcna1-null mice also show a

differential response to CPA compared to wild-type LH/P. Collectively, these data

suggest a reduction of adenosine tone in the LH/P in an animal model of

epilepsy.

METHODS

Animals: C3HeB/FeJ Kcna1-null and wild-type littermates were bred and

reared at Creighton University. Mice were entrained to a strict 12 h light/dark

cycle with access to food and water ad libitum. Lights on occurred at zeitgeber

time (ZT) 00:00 h. Tails clips were collected on postnatal day (P) 12-15 and

60

genotype was determined by Transnetyx, Inc (Cordova, TN, USA). On ~P21

mice were weaned onto standard diet. Adult mice (P35-P50) were used for these

experiments. All experiments conformed to NIH guidelines in accordance with the

United States Public Health Service’s Policy on Humane Care and Use of

Laboratory Animals and were approved by Creighton University’s Institutional

Animal Care and Use Committee.

Drugs and reagents: All reagents were purchased through Sigma unless

otherwise noted.

Acute slice preparation and multi-electrode array recordings: WT and

Kcna1-null mice (P34-P45) were anesthetized with isoflurane, decapitated, and

their brains removed and quickly placed into ice cold, oxygenated (95% O2/5%

CO2) sucrose-aCSF containing (in mM): 206 sucrose, 2.8 KCl, 1 CaCl2, 1 MgCl2,

2 MgSO4, 26 NaHCO3, 1.25 NaH2PO4, and 10 glucose, 0.4 ascorbic acid (pH

7.4). Coronal lateral hypothalamic slices (350 nm) were prepared on a Leica

VT1200 and transferred for 1 hr to a holding chamber with warm oxygenated

aCSF (34°C) containing (in mM): 125 NaCl, 3.0 KCl, 2.4 CaCl2, 2.5 MgSO4, 26

NaHCO3, 1.25 NaH2PO4, and 25 glucose (pH 7.4). Slices were placed over the

entire electrode grid of a MED64 probe (Alpha Med Systems, Osaka, Japan;

each electrode: 50x50 mm; interpolar distance: 150 mm; 8x8 electrode grid).

Slices were perfused (2 ml min-1) with in-line pre-warmed (32°C) oxygenated

aCSF. Slices were positioned with the 64 electrode array centered on the LH/P

and images were taken of electrode placement before and after experiment to

ensure appropriate electrodes were used. Spontaneous events from all 64

61

channels were recorded in continuous, gap-free modes with Mobius software

(AlphaMed Systems) and acquired at a 20 kHz sampling rate with a bandwidth of

10 Hz–10 kHz. Individual or multi- channel data was imported into Spike2 (v6)

software (Cambridge Electronic Design, Cambridge, England) or MATLAB7 (The

MathWorks, Natick, MA, USA) for analysis.

In separate sets of experiments, slices were perfused with 8-(p-

sulfophenyl)theophylline, dipropylcyclopentylxanthine, or N6-

cyclopentyladenosine. For all experiments involving drug application, slices were

allowed to equilibrate and adhere to the electrode dish for one hour followed by a

five minute recording of baseline spontaneous activity. Drugs were then

administered by bath application for one hour before a second five minute

recording was taken. A final recording was taken after one hour of drug washout

to ensure recorded neurons were still viable.

Western blotting and LH/P punch: Adult WT and Kcna1-null mice (n=6 per

group) were anesthetized with isoflurane, decapitated and coronal brain slices

(350 µm) containing the LH/P were prepared as above. Bilateral micro-punches

(approx. 1 mm diameter) of lateral hypothalamus tissue were taken from

consecutive sections immediately after sectioning (total thickness of 2.45 mm,

bilateral volume of 3.84 mm3), pooled for each animal and stored in -80°C until

lysed. Proteins were separated on a Mini-Protean 10% TGX gel (Bio-Rad,

California) and transferred to a polyvinyl difluoride membrane (Immobilon PVDF-

FL, 0.454 µm, Millipore, Massachusetts) with 68 mA/50cm2. Membranes were

blocked in Odyssey blocking buffer (LiCor, Lincoln, NE) and incubated overnight

62

at 4oC with the primary antibody (ADK: 1:8000 Rb anti-ADK, a generous gift from

Dr. Detlev Boison; or A1 receptor: 1:2000 Rb anti-A1 receptor, Millipore, Billerica,

MA; β-actin for housekeeping: 1:4000 Mouse anti- β-actin, Licor, Lincoln, NE) in

the blocking solution. Following washes, membranes were incubated in

secondary antibody (ADK: 1:8000 IRDye 680 donkey anti-rabbit; or A1 receptor:

1:7000 IRDye 680 donkey anti-rabbit; β-actin for housekeeping: IRDye 800

donkey anti-mouse; LiCor, Lincoln, NE). Membranes were imaged using the

Odyssey Imaging System (LiCor, Lincoln, NE).

ADK immunohistochemistry: Mice were quickly anesthetized with

isoflurane and decapitated, and brains were removed and frozen in methyl-

butane on dry ice. 30 µm thick sections affixed to slides were fixed in 4%

paraformaldehyde in 0.1 M PB (pH 7.4), washed with 0.01 M PBS/0.3% Triton X-

100, pH 7.4 (PBS-T) and blocked with 10% normal goat serum/PBS-T. Sections

incubated with rabbit anti-ADK (1:1000) overnight at room temperature followed

by AF-594 conjugated goat anti-rabbit IgG (1:600, Invitrogen, Grand Island, NY)

for 3 h at room temperature. Sections were immediately cover-slipped with

mounting media containing DAPI. ADK staining intensity was measured using

ImageJ software (NIH).

Statistics: The distributions of spontaneous single units were analyzed

using Fisher’s exact test. The distributions of single units frequency change in

response to CPA were analyzed using chi-square contingency test. HPLC,

adenosine estimation, and A1 receptor and ADK protein expression were

63

analyzed using t-test with Welch’s correction for unequal variance. A p-value <

0.05 was considered statistically significant.

RESULTS

Spontaneous neuronal activity in Kcna1-null LH/P

Previously, we reported an increase in the number of orexin-positive cells

in the LH/P of Kcna1-null mice compared to wild-type controls. Orexin neurons

are usually reported to spontaneously fire at low frequencies in vitro (≤ 10 Hz; Liu

and Gao, J Neurophysiology 2007; Tsunematsu et al, J Neurosci 2011).

Spontaneous action potentials (single units) of LH/P neurons were detected in in

vitro coronal slices with the LH/P placed atop a 64 extracellular electrode array.

Individual neurons were identified with principal components analysis shown in

Figure 16A, neurons were further classified as either interneurons or principal

cells using spike width and asymmetry, as we have described previously

(Simeone et al. 2014). Wild-type principal cells fired 3.74 ± 0.62 Hz (range 0.98

to 9.44 Hz, n=20 neurons from 3 slices from 2 mice, average of 6.7 neurons per

slice; Figure 16B). The firing frequency of Kcna1-null principal neurons of 2.33 ±

0.26 Hz (range 0.21 to 8.63 Hz, n=56 neurons from 5 slices, from 3 mice,

average of 11.2 neurons per slice; Figure 16B) did not differ from wild-type. The

distribution of LH/P neurons from Kcna1-null slices which fired faster than and

slower than 5 Hz was significantly different from wild-type firing distribution (see

dashed line in Figure 16B, p<0.05, Fisher’s exact test). Sixty-five percent of wild-

type neurons (13/20) had a frequency of less than 5 Hz (range of 0.98 to 3.412

Hz), with the remaining frequencies between 5 and 7 Hz (5/20 neurons), and 9

64

and 10 Hz (2/20 neurons). In contrast, more Kcna1-null principal neurons (87.5%

of neurons, 49/56) fired at a frequency slower than 5 Hz, with only 12.5% (7/56

neurons) firing faster than 5 Hz (compared to 35% of wild-type principal

neurons). There was no difference in the number of spontaneously firing neurons

per slice (Figure 16C).

Adenosine tone is reduced in the LH/P

Spontaneously firing orexin neurons in the LH/P are inhibited by local adenosine

acting on the A1 receptor. Adenosine is a purinergic nucleoside that is readily

converted into ATP via a futile enzymatic cycle, making local concentrations

difficult to quantify (see Chapter 1, Fig 3). Thus, we determined the adenosine

tone in Kcna1-null mice brain by calculating pharmacological estimates of

adenosine levels locally in the LH/P using A1 receptor antagonists and

extracellular multielectrode array recordings. Fractional increase of spontaneous

firing of principal cells following application of saturating concentrations of A1

receptor antagonist 8-(p-sulfophenyl)theophylline (8SPT; Ki= 1µM) or

dipropylcyclopentylxanthine (DPCPX; Ki= 1nM) were calculated. This fractional

increase was used to estimate endogenous adenosine concentrations using the

following equation from Dunwiddie and Diao (JPET 1994):

[𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴𝐴]𝑒𝑒𝑒 = (𝐹𝐹)1 𝐻⁄ × 𝐸𝐸50

where FI is the fractional increase in response to application of an antagonist, H

is the Hill slope (1.52, estimated in rat by Dunwiddie and Diao, JPET 1994), and

EC50 is the adenosine concentration that produces a half-maximal response (610

nM, estimated in rat by Dunwiddie and Diao, JPET 1994). 8-SPT and DPCPX

65

were used in separate cohorts to independently estimate endogenous adenosine

concentrations.

66

Figure 16: Spontaneously firing principal cells in the LH/P have a similar

firing frequency in Kcna1-null mice and wild-type controls, but a differential

distribution of individual neuron frequencies. (A) Both principal cells and

interneurons were recorded and identified in in vitro slices. Individual neurons

were identified using principal components analysis and then identified as

principal cells or interneurons based on spike width and symmetry. (B) Principal

cells in wild-type and Kcna1-null mice have similar mean population firing

frequencies (3.74 ± 0.62 Hz, range of 0.98 to 9.44 Hz, and 2.33 ± 0.26 Hz, range

of 0.21 to 8.63 Hz, respectively; p>0.05). However, the distribution of the

frequency of individual principal cells is significantly different with more frequency

principal neurons firing below 5 Hz in Kcna1-null slices (p<0.05, Fisher’s exact

test). (C) While more neurons were recorded in Kcna1-null slices, there was no

significant difference in the number of neurons recorded per slice to account for

the difference in frequency distribution (wild-type: n=20 neurons from 3 slices,

67

average of 6.7 neurons per slice; Kcna1-null: n=56 neurons from 5 slices,

average of 11.2 neurons per slice).

68

Using 10 µM 8-SPT, adenosine was estimated to be 302.4±79.82 nM in

wild-type LH/P neurons and 106.7±24.63 nM in Kcna1-null LH/P neurons; the

estimated adenosine concentration is significantly lower in the Kcna1-null LH/P

compared to the estimation in wild-type LH/P (p<0.05, t-test with Welch’s

correction; Figure 17A). In a separate cohort using 1 µM DPCPX, adenosine

was estimated to be 461.1±76.05 nM in wild-type LH/P principal neurons and

229.0±51.59 nM in Kcna1-null LH/P principal neurons; again, the estimated

adenosine concentration is significantly lower in the Kcna1-null LH/P compared

to the estimation in wild-type LH/P (p<0.05, t-test with Welch’s correction; Figure

17B). These data suggest that adenosine levels are significantly reduced in

Kcna1-null LH/P when compared to wild-type.

69

Figure 17: Kcna1-null mice have lower estimated adenosine in the LH/P.

Extracellular adenosine was estimated in the LH/P using a method based on

Dunwiddie et al. using (A) 8-SPT and (B) DPCPX. Data are mean ± SEM; n= 6

neurons from 3 slices and 12 neurons from 5 slices for wild-type and Kcna1-null,

respectively; * p<0.05.

70

Adenosine kinase levels are similar in Kcna1-null and wild type mice.

Levels of adenosine are primarily determined by ADK (Pak et al,

Neuropharmacology 1994; Huber et al, PNAS 2001). ADK is expressed in

astrocytes and its expression has been shown to increase during astrogliosis

associated with epileptic pathology in the hippocampus (Gouder et al. 2004;

Aronica et al. 2011). Previously, we have reported an increased incidence of

reactive astrocytes in the LH/P of Kcna1-null mice. Despite the increased

astrogliosis (Roundtree et al, Sleep 2015) and reduced estimated adenosine

concentration we found in the LH/P, we did not detect a significant difference in

ADK expression in IHC of coronal sections (n=6 animals per group; Figure 18A).

We further found no difference in ADK protein levels of bilateral micro-dissected

LH/P punches (3.84 mm3 volume) from coronal sections using western blot (n=6

animals per group, normalized to β-actin; Figure 18B). These data suggest that

ADK is not changed in the LH/P of Kcna1-null mice and may not play a critical

role in reducing adenosine concentrations in LH/P.

Adenosine type-1 receptor expression and function is decreased in LH/P of

Kcna1-null mice

Adenosine inhibits orexin neurons via acting through the A1 receptor, a

Gi/Go-protein coupled receptor. We examined total A1 receptor levels in bilateral

micro-dissected LH/P punches from Kcna1-null and wild-type coronal sections

(3.84 mm3 tissue volume). A1 receptor protein levels were significantly reduced

when compared to wild-type (p<0.005, n= 6 animals per group, normalized to β-

actin; Figure 18C).

71

A1 receptor function was determined in the LH/P of in vitro coronal slices

following application of A1 receptor agonists and assessing the degree of single

unit inhibition using multielectrode array recordings. Single units of principal

neurons were analyzed during baseline and following application of saturating

concentrations of A1 receptor-specific agonist N6-cyclopentyladenosine (CPA,

10uM; Yang et al. 2014). Mean firing frequency and change in firing frequency for

each neuron are both depicted in Figure 19. A majority (10/11 or 91%) of wild-

type principal neurons showed a greater than 20% reduction in firing frequency.

Similarly, a majority of Kcna1-null principal cells also showed a greater than 20%

reduction in frequency (19/27 or 70%). Interestingly, all of the wild-type neurons

that slowed in response to CPA had a significant reduction in firing frequency (i.e.

more than a 20% reduction, (n= 11 wild-type neurons, from 3 slices), whereas

22% Kcna1-null principal cells (6/27) showed a less than 20% reduction in

frequency (27 Kcna1-null neurons, from 5 slices). This data may suggest that

there is a population of spontaneously firing Kcna1-null LH/P neurons that are

not as sensitive to A1 receptor agonists when compared to wild type neurons.

72

Figure 18: Adenosine kinase levels are similar, but A1 receptor expression

is decreased in Kcna1-null mice compared to wild-type controls. ADK

expression was measured in Kcna1-null and wild-type mice using (A)

immunohistochemistry and (B) western blot. (C) A1 receptor expression was

significantly lower in Kcna1-null mice compared to wild-type when measured by

western blot. Data are mean ± SEM; n for immunohistochemistry = 6 animals per

group, 4 slides per animal; n for western blots = 6 animals per group. * p<0.05

73

Figure 19: LH/P principal cells respond differentially to CPA. Individual

neurons were identified from extracellular electrodes placed in the LH/P based

on PCA analysis and sorted as principal cells or interneurons (see Fig 16). The

frequency of spontaneous action potentials from principal cells were recorded at

baseline and following treatment with 10 µM CPA. Principal cells were further

sorted into 3 groups based on the degree of response to CPA. Data for individual

neurons is superimposed on group means ± SEM.

74

Discussion

Adenosine’s role as a regulator of neurotransmission is well-known and

has been extensively studied in relation to both epilepsy and sleep disorder.

Adenosine regulation of sleep shares common mechanisms as the proposed

anticonvulsive mechanism, acting via the A1 receptor. However, little research

has attempted to look at changes of adenosinergic regulation of the LH/P in

epileptic models and the effects on sleep. Here, we report that the estimated

concentration of adenosine in the Kcna1-null mouse LH/P is reduced compared

to wild-type. We also report that A1 receptor expression in LH/P is reduced in

Kcna1-null mice compared to wild-type LH/P. Finally, we report that principal

cells in the LH/P may respond differentially to an A1 receptor agonist. This data

supports the notion that adenosine tone may be reduced in Kcna1-null mice.

Adenosine’s role in epilepsy and sleep disorder

Adenosine’s inhibitory action via the A1 receptor has been well-

documented and is an important area of research in epilepsy models. Adenosine

has been proposed as a possible endogenous anticonvulsant and mediator of

seizure arrest. Identification of adenosine’s role in epileptic models is confounded

by the difficulty in reliably measuring in vivo brain adenosine concentration and

instead often relies on indirect measures of adenosine concentration and tone.

For example, ADK, the primary regulator of adenosine concentration, has been

shown to be increased in epileptic animal models and patients (Gouder et al.

2004; de Groot et al. 2012; Aronica et al. 2012), leading to the hypothesis that

adenosine would also be reduced.

75

Here, we estimated the local concentration of adenosine in the LH/P using

a pharmacologic method (as outlined in Dunwiddie and Diao, 1994). The

estimation of adenosine in the LH/P was found to be significantly lower in Kcna1-

null mice compared to wild-type controls in two separate experiments. Lower

adenosine concentration in the LH/P may contribute to increased orexin tone and

sleep disorder seen in Kcna1-null mice. Unfortunately, a difference in expression

of A1 receptor between genotypes could be a confounding factor in estimating

adenosine this way. Further studies will need to either measure adenosine

directly by methods like HPLC, or attempt to control for difference in receptor

expression.

Adenosine and regulation of LH/P

Orexin-producing neurons located in the LH/P are spontaneously active

neurons that promote wakefulness and attention, as well as being involved in

regulation of mood and appetite. This group of neurons is tightly regulated by

various signals of body state. For example, increased glucose (Yamanaka et al.

2003) can inhibit orexin neurons, as can decreases in CO2 or increases in pH, a

possible consequence of hypoxic conditions following seizures (Williams et al.

2007), providing a mechanism for the hypothalamus to respond to and integrate

changes in energy balance and respiration. Similarly, adenosine has been

proposed as a molecular modulator of sleep demand: adenosine levels are

higher during wakefulness and lower during sleep, and increase following

prolonged wakefulness (Porkka-Heiskanen et al. 1997). Interestingly, we found

reduced estimations of adenosine in the LH/P in Kcna1-null mice despite these

76

mice also having reduced amount of sleep, further suggesting a pathological

change in the regulation of adenosine.

Adenosine inhibits orexin neurons, mediated through A1 receptors (Li and

Gao, 2007; Thakkar et al 2008). Here, we report Kcna1-null mice have

significantly lower A1 receptor expression in tissue punches taken of LH/P. A

reduction in the expression of A1 receptor could be responsible for decreased

regulation of orexin neurons and an increase in orexin tone, contributing to

insufficient sleep in Kcna1-null mice. However, this study looked at total protein

expression. Future studies could look at surface expression of A1 to determine

any change in functional receptor.

Function of the A1 receptor was investigated using CPA, a selective A1

receptor agonist. Application of CPA would be expected to decrease

spontaneous activity in neurons expressing A1 receptor, including orexin

neurons. Principal cells in the LH/P predominantly showed decrease firing

frequency in response to application of CPA. Interestingly, while all wild-type

principal cells recorded from showed a greater than 20% decrease in firing

frequency following CPA application, some principal cells from Kcna1-null mice

showed a less than 20% decrease. While this may represent a decrease in

response to an A1 receptor agonist in Kcna1-null LH/P, further studies will need

to look at orexin neurons specifically and further characterize the response.

Here, we have reported evidence of a reduction in the adenosine tone in

the LH/P of epileptic Kcna1-null mice. Previous data has demonstrated

insufficient sleep, pathology and an increase in orexin-positive neurons in the

77

LH/P, and improvement in sleep and seizures following treatment with an orexin

receptor antagonist in Kcna1-null mice (see Chapter 2; Roundtree et al. 2015).

Together, these studies demonstrate alterations in orexin neuron regulation in

the LH/P of an epileptic model that may lead to comorbid sleep disorder. Future

work can further investigate changes in the adenosine system, including

differences in other purinergic receptors and enzymes.

78

Chapter 4: Tanycytes of the Third Ventricle Introduction

Tanycytes are bipolar glial cells located in the floor of the fourth ventricle and

surrounding the third ventricle. Alternatively classified either as a specialized

ependymal cell, the cell type that lines ventricles and is responsible for producing

and circulating cerebrospinal fluid (CSF), or as a subtype of astrocyte, the

function of these cells is still under investigation. Like astrocytes and some other

glial cells, tanycytes express both glial fibrillary acidic protein (GFAP) and

vimentin (Dale 2011). The basal surface of these cells form part of the surface of

ventricles and come into contact with the CSF while processes extend into the

brain parenchyma.

There are several current hypotheses regarding the function of tanycytes.

Tanycytes of the third ventricle have been proposed to act as glucosensors,

responding to the presence of glucose within the CSF and passing the signal

through their processes into the hypothalamus (Frayling et al. 2011). This has

been supported by the presence of glucose transporters in the basal surface of

tanycytes and increased intracellular calcium concentrations in response to an

experimental increase of glucose in the 3rd ventricle. In addition, tanycytes have

also been proposed as adult stem cells capable of increasing neurogenesis in

response to high fat diet and filling a neurogenic niche in the hypothalamus (Lee

et al. 2011). Finally, tanycytes express proteins involved in creating and

79

maintaining barrier properties along the third ventricle as well as along blood

vessels with which their apical extensions come into contact (Rodriguez et al.

2005). Together, these functions would implicate tanycytes as a cell type integral

to regulating functions of the hypothalamus, a brain structure which integrates

diverse signals in the body. Most research to date has focused on tanycytes in

the healthy CNS. Here, we report alterations in tanycyte morphology in the

Kcna1-null mouse model of spontaneous epilepsy.

Material and Methods

Animals: C3HeB/FeJ Kcna1-null and wild-type littermates were bred and

reared at Creighton University. Mice were entrained to a strict 12 h light/dark

cycle with access to food and water ad libitum. Lights on occurred at zeitgeber

time (ZT) 00:00 h. Tails clips were collected on postnatal day (P) 12-15 and

genotype was determined by Transnetyx, Inc (Cordova, TN, USA). On ~P21

mice were weaned onto standard diet. Adult mice (P35-P50) were used for these

experiments. All experiments conformed to NIH guidelines in accordance with the

United States Public Health Service’s Policy on Humane Care and Use of

Laboratory Animals and were approved by Creighton University’s Institutional

Animal Care and Use Committee.

Drugs and reagents: All reagents were purchased through Sigma unless

otherwise noted.

GFAP and vimentin Immunohistochemistry: Mice were quickly

anesthetized with isoflurane, decapitated, brains were removed and frozen in

methyl-butane on dry ice. Sections affixed to slides were fixed in 4%

80

paraformaldehyde in 0.1 M PB (pH 7.4), washed with 0.01 M PBS/0.3% Triton X-

100, pH 7.4 (PBS-T). For vimentin staining, sections were blocked with 5%

normal donkey serum/PBS-T. Sections were incubated with chicken anti-vimentin

(1:500, Millipore) overnight at room temperature followed by AF-488 conjugated

donkey anti-chicken IgG (1:500, Invitrogen, Grand Island, NY, USA) for 3 h at

room temperature and cover-slipped with DAPI mounting media. For GFAP

staining, sections were blocked in 10% normal goat serum/PBS-T, then

incubated in AF-488 conjugated mouse anti-GFAP (1:500, Invitrogen, Grand

Island, NY, USA) overnight at room temperature and cover-slipped with DAPI

mounting media. Black and white images were taken with an Evos microscope

(AMG) and converted to binary images then skeletonized using ImageJ software

(NIH). Mean intensity was either measured in ROIs of equal area for whole third

ventricle, or measured in ROIs for tanycyte subpopulations as determined by

anatomy and normalized to ROI area.

BrdU labeling: Mice were injected twice daily with sterile BrdU (50mg/kg,

BD Biosciences) at least six hours apart on either P28 or P38. On P30 or P40,

respectively, mice were quickly anesthetized with isoflurane and decapitated, and

brains were removed and frozen in methyl-butane on dry ice. Sections affixed to

slides were fixed in 4% paraformaldehyde in 0.1 M PB (pH 7.4), washed with

0.01 M PBS/0.3% Triton X-100, pH 7.4 (PBS-T) and blocked with 10% normal

goat serum/PBS-T. Sections were incubated with AF-488 conjugated mouse anti-

BrdU (1:50, Invitrogen, Grand Island, NY, USA) for 36 hours at room temperature

then cover-slipped with DAPI mounting media.

81

Results

Periventricular GFAP and vimentin are reduced in Kcna1-null mice

First, we identified changes in tanycyte morphology using the most

common markers for tanycytes, GFAP and vimentin. GFAP and vimentin were

expressed around the wall of the third ventricle as well as in processes extending

into the parenchyma of the hypothalamus (Figure 20B and 21B). GFAP and

vimentin stained cells surrounding the third ventricle in Kcna1-null mice display

altered morphology compared to wild-type, including shorter processes and

decreased staining of the third ventricle wall. To quantify the change in

morphology, images were skeletonized, creating pixel-wide lines with high fidelity

to the stained processes and eliminating confounding effects of staining intensity.

GFAP staining surrounding the third ventricle is reduced in Kcna1-null mice

compared to wild-type (n=6 animals per group, *p<0.05; Figure 20A). Vimentin

staining shows a similar reduction in staining in Kcna1-null mice (n=6 animals per

group, *p<0.05, Figure 21A).

Tanycyte subpopulations

Different subtypes of tanycytes surround the third ventricle and have been

reported to have differential protein expression, such as GFAP expression being

reduced in beta tanycytes located ventrally along the third ventricle. Additionally,

GFAP and vimentin are both expressed in other cell types around the third

ventricle, including other ependymal cells, despite being the most common

markers for tanycytes. To exclude other cell types and examine differences in

82

tanycyte subtypes, ROIs for α1, dorsal α2, ventral α2, and β tanycytes were

placed around the respective subtype populations as identified based on

previously reported anatomical location (Robins et al. 2013).

83

Figure 20: GFAP staining around the third ventricle is reduced in Kcna1-

null mice. A. Mean intensity of skeletonized GFAP images is reduced in Kcna1-

null mice compared to wild-type (n=6 animals per group). B. Representative

84

image of skeletonized GFAP around the third ventricle. C. Subpopulations of

tanycytes were selected with individual ROIs (see Chapter 1, Figure 4). Mean

intensity of GFAP was not significantly different in α1, dorsal α2 or β tanycytes,

but is significantly reduced in ventral α2 tanycytes in Kcna1-null mice. Data are

mean ± SEM, * P<0.05.

85

86

Figure 21: Vimentin staining around the third ventricle is reduced in Kcna1-

null mice. A. Mean intensity of skeletonized vimentin images is reduced in

Kcna1-null mice compared to wild-type (n=6 animals per group). B.

Representative image of skeletonized vimentin around the third ventricle. C.

Subpopulations of tanycytes were selected with individual ROIs (see Chapter 1,

Figure 4). Mean intensity of vimentin was not significantly different in α1, dorsal

α2 or β tanycytes. Vimentin staining was not significantly different for all ventral

α2 tanycytes, but when tanycytes were separated into population anterior and

posterior of the median eminence, vimentin staining was significantly reduced in

the anterior population. Data are mean ± SEM, * P<0.05.

87

When tanycyte subpopulations were analyzed individually, GFAP labeling

was not significantly different in α1, dorsal α2 or β tanycytes. However, GFAP

labeling was reduced in ventral α2 tanycytes in Kcna1-null mice (*p<0.05, Figure

20C). Similarly, there was no significant change in vimentin labeling in α1, dorsal

α2 or β tanycytes, though there was a trend to reduced labeling in the dorsal α2

and β tanycytes. When looking at all coordinates, there wasn’t a significant

difference in vimentin labeling in ventral α2 tanycytes, but when only looking at

ventral α2 tanycytes anterior of the median eminence, vimentin labeling was

significantly reduced in Kcna1-null mice (*p<0.05, Figure 21C). Together, this

suggests that the ventral α2 subpopulation of tanycytes may be primarily altered

in this model of epilepsy.

GLUT1 expression is not significantly different in third ventricle of Kcna1-null

mice

One of the reported functions of tanycytes is to act as a class of

glucosensing cells capable of responding to increased concentrations of glucose

in the CSF of the third ventricle (Frayling et al. 2011). In support of this, tanycytes

have been shown to express the glucose transporter GLUT1. We assessed

GLUT1 expression in the third ventricle. GLUT1 expression was primarily found

in the cells lining the wall of the third ventricle, but not in tanycyte processes.

There was no significant change in GLUT1 expression in Kcna1-null mice in the

wall of the third ventricle (Figure 22A).

88

In addition to the expression in the ependymal layer, GLUT1 was

expressed in the parenchyma of the hypothalamus with a distinctive pattern of

thin lines that don’t appear to originate from the third ventricle. While no co-

labeling was performed, based on morphology these GLUT1 positive structures

are likely blood vessels. While GLUT1 is expressed in the endothelium of blood

vessels in the brain (Lee and Klip 2012), tanycytic processes have been shown

to come in contact with blood vessels in the parenchyma and possibly act to

maintain normal function. However, there was no significant difference in the

number of periventricular GLUT1 blood vessels in Kcna1-null mice (Figure 22B).

Periventricular BrdU positive cells are significantly reduced at P40, but not P30,

in Kcna1-null mice

Several recent studies have reported the ability for tanycytes to act as

neural progenitors in the adult brain (Lee et al 2012; Robins et al. 2012; Haan et

al. 2013). One method for monitoring new cell division is administration of BrdU,

which will substitute for thymidine and be incorporated into DNA of dividing cells.

Kcna1-null mice and wild-type littermates were injected with sterile BrdU

(50mg/kg) twice on one day at least six hours apart and euthanized one the

second day following injections. Two age groups were used, with mice either

sacrificed on postnatal day 30 or 40. These ages were chosen as they occur

after the development of seizures. Additionally, tanycyte progeny have been

shown to co-label with orexin, and these ages were investigated to coincide with

the observed orexin expression as described in Chapter 2.

89

At P30, there was no significant difference in the number of BrdU-positive

cells in Kcna1-null mice compared to wild-type (n=5 animals per group, Figure

23A). However, at P40, Kcna1-null mice had fewer BrdU positive cells than wild-

type animals (p<0.05, n=5 animals per group, Figure 23B).

90

Figure 22: Periventricular GLUT1 is not significantly different in Kcna1-null

mice. A. The mean intensity of skeletonized GLUT1 surrounding the third

ventricle is not significantly different in Kcna1-null mice. B. GLUT1 is also

expressed in a distinct pattern in the parenchyma, likely labeling blood vessel

endothelium. The number of labeled vessels was not significantly different in

Kcna1-null mice. Data are mean ±SEM.

91

Figure 23: Periventricular BrdU-positive cells are reduced at P40, but not

P30, in Kcna1-null mice. BrdU (50 mg/kg) was administered twice daily to

Kcna1-null and wild-type animals at P28 or P38. Animals were then sacrificed on

the second day following BrdU administration, at P30 or P40 respectively. A.

There was no significant difference in the number of BrdU positive cells at P30.

B. Kcna1-null mice have significantly reduced periventricular BrdU-positive cells

compared to wild-type animals. Data are mean ± SEM, *p<0.05.

92

Discussion

The functions of tanycytes of the third ventricle are still poorly understood.

They have been hypothesized to act as a neurogenic niche in the hypothalamus

and to transport signals between the CSF and local structures, assisting the

hypothalamus in signal integration (reviewed in Dale 2011). Research regarding

tanycytes’ role in disease states is scarce. Their involvement in epilepsy has so

far been limited to being part of ependymoma (Kambe et al. 2014; Richards et al.

2004; del Mar Ortiza et al. 2014). Additionally, little research has looked at

involvement of tanycytes in sleep or sleep regulation, though the third ventricle is

centrally located to several important sleep nuclei. Here, we identify changes in

tanycyte structure and attempt to identify possible functional alterations in the

epileptic Kcna1-null mouse.

GFAP and vimentin staining surrounding the third ventricle is reduced in

Kcna1-null mice. This may be a result of either a reduction in the expression of

these proteins, or an alteration in the morphology or number of tanycytes. As

tanycytes have not been extensively researched in disease models, there is little

available data on how morphology can change in disease states. Aged rats show

a decrease in the number of GFAP positive processes extending into the arcuate

nucleus with a simultaneous increase in GFAP immunoreactivity interpreted as

hypertrophy (Zoli et al. 1994).

Several subpopulations of tanycytes have been identified. While

differences in function between subtypes are still being investigated, individual

93

populations have been observed to act as unique neurogenic niches (Lee et al.

2012; Robins et al. 2012) that may respond to different stimuli. Only the α2

tanycytes showed a significant decrease in GFAP and vimentin staining when

subpopulations were analyzed, with a decrease in dorsal α2 and the anterior

portion of ventral α2 for each marker, respectively. α2 tanycytes are capable of

acting as neural progenitors in adult rodents in response to FGF (Robins et al.

2012), but further insight into unique functions of this subpopulation is still

needed. Additionally, the cause of the change in morphology is not identified with

current studies, as it may either be a direct consequence of the potassium

channel knockout or related to the epileptic pathology. Similar studies performed

in other models of epilepsy could determine if altered tanycytes are ubiquitous or

limited to this model alone.

Glucose transporters, including GLUT1, have been identified to play a role

in certain epilepsy diseases. GLUT1 deficiency syndrome is a rare neurological

disorder that usually has seizures starting at a young age (Klepper and

Leiendecker 2007). GLUT1 expression in the wall of the third ventricle and the

periventricular parenchyma did not show a significant difference in Kcna1-null

mice. However, this alone does not discount the possibility that tanycytes may

have a reduced ability to act as glucosensing cells. We showed Kcna1-null mice

have impaired mitochondrial function in the hypothalamus (Roundtree et al.

2015); mitochondria are intermediate to the glucosensing paradigm seen in

pancreatic cells. Additionally, tanycytes may have an alternative mechanism of

94

glucosensing (Frayling et al 2011). Future studies will need to directly determine

the ability of tanycytes to respond to glucose in Kcna1-null mice.

Kcna1-null mice have increased orexin-positive neurons at P40 (see

Chapter 2 Fig. 13), but not at younger ages even after seizures have initiated

(see Chapter 2 Fig. 14). The mechanism for this increase is still under

investigation. However, tanycytes have been shown to be capable of acting as

orexin neuron progenitors (Xu et al 2005). Neurogenesis around the third

ventricle at two time points was investigated using BrdU administration: P40,

after the observed increase in orexin-positive neurons in Kcna1-null mice; and

P30, where no difference in orexin-positive neurons is observed, but seizures

have started. While BrdU positive cells were not significantly different at P30,

Kcna1-null mice had a reduced number of BrdU positive cells at P40. However,

due to the short incubation time following BrdU administration, these labeled cells

are still located close to the third ventricle and have not migrated. Further studies

with longer incubation following BrdU administration will be necessary to observe

migration of new cells into the hypothalamus and how tanycyte neurogenesis

may relate to orexin ontogeny in Kcna1-null mice.

While we observed that tanycyte morphology in Kcna1-null mice is altered,

the functional consequences of changes in tanycytes still need to be more

rigorously investigated. As tanycytes are still being described in detail, the effects

of potassium channel knockout are unknown. Future studies will need to

determine if similar changes in tanycytes are observable in other epileptic

models, including other genetic models or induced epileptic models, such as

95

chemoconvulsant or viral induced epilepsies. Several of the potential functions of

tanycytes are promising avenues for involvement in epilepsy or sleep disorder in

Kcna1-null mice. The alteration in tanycyte morphology also further supports

previous evidence of pathological changes in the hypothalamus of Kcna1-null

mice.

96

Chapter 5: Summary and conclusions

Epilepsy has an estimated lifetime incidence of 1:26 in the United States.

While many anticonvulsants are available, approximately a third of patients are

refractory to available treatments. Additionally, an estimated 30% of epileptic

patients are afflicted with sleep disorder resulting in insufficient sleep. The

mechanism of comorbid sleep disorders in epilepsy is poorly understood. Here,

we report on evidence of pathology and an altered environment in the Kcna1-null

mouse model of epilepsy in a region of the hypothalamus involved in maintaining

sleep.

Epileptic Kcna1-null mice display an insufficient sleep phenotype

characterized by decreased NREM and REM sleep and increased wake as

monitored with EEG/EMG recording. Treatment with a dual orexin receptor

antagonist, almorexant, was capable of increasing the amount of NREM sleep

and decreasing the amount of wake, as well as reducing the latency to REM

sleep. Almorexant treatment also decreased seizure burden. Kcna1-null mice

showed a positive correlation between the latency to REM and seizure severity,

suggesting a possible mechanistic link.

We report that seizures are capable of propagating to the LH, and

pathology was evidenced in the LH where orexin neurons are located. Blood-

brain barrier permeability was increased as measured by decrease in punctuate

IgG staining, and the incidence of reactive astrocytes was increased. Other

97

studies have also shown that mitochondrial function is compromised in the LH

(Roundtree et al 2015). Orexin-positive neurons are increased in animals at P40,

but not P12, P21, or P30.

The spontaneous activity of neurons in the LH was recorded using an

extracellular multielectrode array. The number of spontaneously active neurons

and the firing frequency of the neurons did not significantly differ between Kcna1-

null and wild-type in vitro slices, however Kcna1-null principal neurons were

significantly more likely to fire at a frequency less than 5 Hz.

Adenosine concentration in the LH was pharmacologically estimated in in

vitro slices using 8SPT and DPCPX. Kcna1-null slices were estimated to have

significantly less adenosine than wild-type controls. A1 receptor expression was

also decreased in the LH. Together, this suggests reduced adenosine tone which

could contribute to increased orexin activity.

Tanycytes of the third ventricle have not been previously examined in

epileptic models. However, tanycytes are implicated in regulation and

maintenance of the hypothalamic environment. Tanycytes of the third ventricle

showed an altered morphology as evidenced by reduced GFAP and vimentin

staining in Kcna1-null mice. This reduction in staining primarily affects the ventral

α2 subpopulation of tanycytes. GLUT1 did not show a significant difference in

Kcna1-null mice. BrdU-positive cells near the third ventricle showed no significant

change in P30 Kcna1-null mice, but were reduced at P40 compared to wild-type

controls.

98

Taken together, these data show an altered environment in the

hypothalamus and especially in the LH where orexin neurons reside. The

compromised environment of the LH in Kcna1-null mice may contribute to their

comorbid sleep disorder, suggesting a novel mechanism for comorbid sleep

disorder in epilepsy. This is further supported by the ability of treatment with an

orexin receptor antagonist to improve sleep and decrease seizure burden.

99

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