www.elsevier.com/locate/brainresBrain Research 1005 (2004) 154–163

Tonic and phasic alteration in amygdala 5-HT, glutamate and GABA

transmission after prefrontal cortex damage in rats

Luis E. Gonzalez*, Belkis Quinonez, Alejandra Rangel, Silvano Pino, Luis Hernandez

Laboratory of Behavioral Physiology, Department of Physiology, School of Medicine, Los Andes University, Av. Don Tulio. Nivel calle 33,

Merida 5101A, Apartado 109, Merida, Venezuela

Accepted 28 January 2004

Abstract

The relationship between the ventromedial prefrontal cortex and the amygdala during the presentation of an unconditioned fear stimulus

was assessed. Rats underwent bilateral ibotenic acid or vehicle administration into the ventromedial prefrontal cortex. Five weeks later, the

behavior as well as the neurochemical changes in the amygdala was evaluated before and after a brief cat presentation. Lesioned animal

freezing behavior increased 10 times when compared to controls. In the right basolateral amygdala, basal concentrations of 5-HT, 5-HIAA,

glutamate and serine were elevated but basal level of GABAwas diminished in lesioned animals relative to controls. Sham but not lesioned

animals increased 5-HT and decreased GABA and serine levels after cat presentation. Phasic changes in glutamate were not detected either in

lesioned or shams but the difference in amygdala glutamate between lesioned and shams persisted after cat presentation. These data show that

increased serotonin and glutamate tone and decreased gabaergic tone in the amygdala correlate to elevated fear and anxiety after prefrontal

cortex ibotenic acid lesion. The lesion also seems to produce a failure of adaptive changes in neurotransmitter systems revealing lost of

control of the ventromedial prefrontal cortex over the amygdala in frightening situations.

D 2004 Elsevier B.V. All rights reserved.

Theme: Neural basis of behavior

Topic: Motivation and emotion

Keywords: Anxiety; Prefrontal cortex; Amygdala; 5-HT; Glutamate; GABA; Serine

1. Introduction

Several experimental and clinical observations have

revealed that medial prefrontal cortex (MPFC)–amygdala

interactions are involved in fear, anxiety and depression

[9,10,30]. Anatomically, corticofugal and corticopetal con-

nections between the MPFC and the amygdala have been

described [5,6,27,40,49,56,67]. Functionally, positron-emis-

sion tomography measurements of glucose metabolism re-

veal that individual differences in metabolic activity in the

amygdala are associated with levels of distress or dysphoria,

i.e. the more activity, the greater the negative affect. In

contrast, metabolic activity in the medial prefrontal cortex

is inversely related to levels of activity in the amygdala, i.e.

the greater the activity level in the medial prefrontal cortex

(predominantly in the left hemisphere) the more positive the

0006-8993/$ - see front matter D 2004 Elsevier B.V. All rights reserved.

doi:10.1016/j.brainres.2004.01.048

* Corresponding author. Tel.: +58-74-403110; fax: +58-74-638304.

E-mail address: gorey@ing.ula.ve (L.E. Gonzalez).

person’s emotional state. Thus, a major locus of the ability

to regulate negative affect appears to be the circuit between

the prefrontal cortex and the amygdala [9]. Neuroimaging

studies have also shown abnormalities of resting blood flow

and glucose metabolism in the amygdala and MPFC in

major depressive disorder patients. These abnormalities

reverse after antidepressant drug treatment [10,11]. There

are scarce reports on neurochemical alterations in the

amygdala following MPFC experimental manipulations

[30,49]. Nonetheless, substantial evidence supports that

emotional responses are mediated for several types of

neurotransmitters converging in the amygdala [2,8,12,16,

19,20,24,25,32,34,39,51,58,61]. Therefore, a concurrent

multiple neurotransmitter analysis in the amygdala in com-

bination with behavioral assessment during fear situations in

rats with MPFC deficit may cast light on MPFC–amygdala

neurochemistry correlates of emotional behavior.

Abundant indirect evidences strongly suggest that

GABA, glutamate, serine, serotonin and dopamine sys-

tems play a role in MPFC–amygdala control of emotional

L.E. Gonzalez et al. / Brain Research 1005 (2004) 154–163 155

behavior. Electrophysiological tests have shown that elec-

trical stimulation of the MPFC activates MPFC–amygdala

glutamate pathways, which in turn excite GABAergic

interneurons in basolateral amygdala [18,49]. Changes in

amygdala extracellular levels of amino acids have been

implicated in emotional control. Rats with greater cardio-

vascular and behavioral response to stress displayed

exaggerated amygdaloid glutamate release in response to

acute stress [58]. In contrast, there was a reduction of

extracellular GABA in mouse amygdala during and fol-

lowing presentation of conditioned fear stimulus [61] and

reduced GABA-stimulated chloride influx in rat amygdala

during and after restraint experience [39]. Consistent with

these data, administration of benzodiazepines and GABAA

agonists to the amygdala induced anxiolytic effects in

several animal models [16,19,25].

It is believed that D-serine, an endogenous agonist at the

glycine site of N-methyl-D-aspartate (NMDA) receptors,

modulates excitatory neurotransmission [55]. D-Serine is

concentrated in astrocytes and released by a glutamate-

dependent mechanism [48,55]; the pattern of its brain

distribution closely correlates with that of the NMDA

receptor [21,22]. Little is known on the functional correlates

of amygdala D-serine in emotional alterations. Yet, it was

found that 10-min handling, a mild stressful stimulus,

increased serine levels in three limbic-related areas, i.e.

the ventral tegmental area, prefrontal cortex and locus

coeruleus mimicking glutamate release changes [64]. Con-

current exposition to noise, intense light and inescapable

electric shock, strong stressful stimuli, increased release of

serine and several other amino acids including glutamate

and GABA in the locus coeruleus [31]. However, studies

assessing amygdala D-serine release in emotional responses

have not been reported so far.

MPFC neurons innervate monoamine cell bodies within

the midbrain that, in turn, project to the basolateral amyg-

dala and, reciprocally, to the MPFC. Anatomical and

functional studies show direct MPFC pathways to the

ventral tegmental area [3,29]. The mesoamygdaloid dopa-

mine system comprises dopamine cells from ventral teg-

mental area projecting to amygdala nuclei including

basolateral amygdala [33]. The increase in amygdala

homovanillic acid (HVA) concentrations, an index of

dopamine neuron activity, induced by conditioned stress

was antagonized by low anxiolytic doses of diazepam [8].

This suggested that dopaminergic neurotransmission in the

amygdala is associated to fear and anxiety. Anatomical

evidence also indicates that MPFC neurons project to the

dorsal raphe nucleus [66] and electrical stimulation of the

MPFC modified the activity of dorsal raphe 5-HT neurons

[7]. Serotonin pathway ascending from dorsal raphe nucle-

us to the amygdala and frontal cortex are likely to be

involved in the mediation of anxiogenic responses (for

review, see Refs. [19,54]). In particular, anxiogenic states

have been associated with elevated serotonin release in the

amygdala [12,20,32,35], whereas 5-HT depletion in the

amygdala resulted in a specific anxiolytic effect as mea-

sured by a punished drinking paradigm [60]. Electrical

stimulation of the MPFC enhances extracellular serotonin

in the amygdala. This effect was highly region specific,

because stimulation of the lateral part of the prefrontal

cortex, the medial precentral area, the primary motor cortex

or the parietal cortex had not effect whatsoever on extra-

cellular 5-HT in the amygdala [30]. Although acute sero-

tonin release seems to activate GABAergic neurons, high

chronic levels of serotonin have opposite effects [46].

Anxiolytic [17,38,57,63], anxiogenic [26,28], no effects

[37,63] or anxiolytic followed by anxiogenic effects [47]

have been reported following lesions in the MPFC. These

discrepancies have usually been attributed to the nature of

lesion inductor, lesion size, sub-region affected within the

MPFC and animal model category. However, we advanced

the idea that time length after lesion, a factor overlooked in

most behavioral studies, is critical in determining the

direction of behavioral and physiological changes [47].

We have previously reported a sequence of anxiolytic

and anxiogenic effects in the rat social interaction test of

anxiety following bilateral damage in the ventromedial

prefrontal cortex [47]. Because the anxiogenic stage was a

consistent finding after 30 days of lesioning, we suggested

disinhibition of amygdala responses on the fifth week after

surgery as a late result of denervation from MPFC. Simi-

larly, spinal transection induces motor activity suppression

followed by disinhibition and lack of motor control reveal-

ing inhibitory influences from higher motor centers. MPFC

inhibitory influence over the amygdala is revealed by

increased neural activity and lack of control for adaptive

responses in the amygdala during the delayed phase of

MPFC lesion.

The present investigation was aimed to characterize

behavioral and amygdaloid neurochemical parameters dur-

ing the anxiogenic state on the fifth week after ibotenic acid

administration to the ventral region of the MPFC. Because

the anxiogenic effects of ibotenic acid microinjections in

ventromedial prefrontal cortex were detected only in ani-

mals with bilateral lesions [47], we study the effects of

bilateral lesions. Animals received MPFC bilateral lesions

and a microdialysis probe was placed in the right basolateral

amygdala. The right amygdala was probed because greater

activation of right amygdala circuits has been associated

with fear and anxiety. Thus, increased defensive response in

cats after administration of the anxiogenic agent FG 7142

predominantly increased neural transmission in right amyg-

dala pathways as measured by evoked potential techniques

[1]. In rats, an increased right/left 5-HT release ratio was

related to the anxiogenic response in the plus-maze [2].

Dialysates were analyzed for serotonin (5-HT), 5-hydrox-

yindoleacetic acid (5HIAA) and homovanillic acid (HVA)

by HPLC coupled to electrochemical detection and for

glutamate, serine and GABA by capillary electrophoresis

coupled to laser induced fluorescence detection (CE-LIFD).

The neurotransmitter basal measures were obtained from

L.E. Gonzalez et al. / Brain Research 1005 (2004) 154–163156

freely moving rats during the 25 min that preceded a brief

cat encounter (pre-stress sample) and a second sample was

collected during the 25 min after cat presentation (post-

stress sample). The rat freezing response following cat

encounter was evaluated.

2. Materials and methods

2.1. Animals and surgery

Male Wistar rats weighing 250 to 300 g were individ-

ually housed with food and water ad libitum, and the room

temperature was kept at 22 jC. Lights went on at 07:00

and off at 19:00 h. Animals were anesthetized by co-

administration of ketamine and pentothal (110 and 10 mg/

kg i.p., respectively) and positioned on a stereotaxic frame

(David-Kopf Instruments). The skull was exposed and

leveled by adjusting the incisor bar. To produce the

bilateral lesion of the ventromedial prefrontal cortex, a

device comprising two injector needles was positioned at

2.7 mm anterior to bregma, F0.7 mm lateral to the midline

and 5.4 mm ventral to skull surface. Ibotenic acid dis-

solved in 0.1 M phosphate-buffered saline (10 mg/ml) was

microinjected (0.5 ml) at the flow rate of 0.25 ml/min.

Phosphate-buffered saline was given to sham-operated

animals using the same microinjection procedure and

coordinates mentioned above.

A guide shaft made of 10-mm-long pieces of 21-gauge

stainless steel tubing was stereotaxically implanted aiming

to the right basolateral amygdala. The tip of the guide shaft

was positioned at 3.0 mm posterior to bregma, 5.0 mm

lateral to the midline and 4.2 mm ventral to the skull.

Microdialysis procedure started on the fifth week after

surgery.

The Ethical Commission from Los Andes University

Scientific and Humanistic Development Counsel approved

the experimental procedures of this report.

2.2. Drugs

Ibotenic acid (Sigma, St. Louis, MO, USA) was dissolve

in 0.1 M phosphate-buffered saline at pH 7.4.

2.3. Microdialysis procedure and behavioral score

Laboratory-made microdialysis probes [23] protruded 5

mm off the tip of the guide shaft. The effective length of

the cellulose fiber was 2 mm. Artificial cerebral spinal

fluid (135 mM NaCl, 3.7 mM KCl, 1.2 mM CaCl2, 1.0

mM MgCl2 and 10 mM NaHCO3, at pH 7.4) was

injected into the probe by a syringe pump at a flow rate

of 1 ml/min.

The microdialysis probe was gently inserted into the

guide tubing at 7:00 PM. Perfusion was conducted over

night at the flow rate of 0.2 ml/min. On the next day, the

flow rate was elevated to 1 ml/min at 7:00 AM. After 3 h, a

basal sample (25 ml) was obtained between 10:00 AM and

11:00 AM. When the collection of the second sample began,

the rat was subjected to a brief cat encounter. A researcher

was inside the room during the experiment and indicated

when the cat owner should introduce the cat into the

experimental room. The cat owner stayed in the room just

for cat presentation. The cat was held in the arms of its

owner in front of the microdialysis cage preventing any of

its movement that could alter rat typical approaching be-

havior. In general, the rat slowly moved toward the cat as

soon as it was close to the grid wall. Time spent in

spontaneous rat approaching did not differ (T-test, p=0.2)

between shams (7.5F3.2 s) and lesioned (8.1F3.4 s)

animals. The rat smell the cat through a metal grid wall

and time spent smelling did not differ (T-test, p=0.2)

between shams (4.0F0.5 s) and lesioned (3.7F0.7 s) rats.

Immediately after that, rats fast withdrew and most of them

adopted a frozen posture. At this point, the cat was retired

from the room and rat freezing behavior time was scored for

5 min by an observer unaware of the rat treatment. The cat

never vocalized during the experiment. Dialysis carried on

for 20 further minutes without interruption so that the

second dialysate sample achieved a 25 Al volume. From

each 25 Al sample (the pre-stress or post-stress sample), 20

Al were used for 5-HT, 5HIAA and HVA determination by

HPLC coupled to electrochemical detection and 5 Al wereused for amino acid (GABA, serine and glutamate) analysis

by CE-LIFD.

The HPLC system was a double piston Water model 510

HPLC pump (Millipore/Waters CA) with a standard head

and a model 7125 valve (Rheodyne, Cotati, CA) equipped

with a 20-ml loop. Separation was made in a 10-cm-long,

3.2 mm bore, 3 Am particles, ODS Brownlee column

(Perkin Elmer, Applied Biosystems, Woburn, MA). The

mobile phase was a 0.116 M acetate buffer with 100 mM

EDTA, 1 mM octanesulfonic acid and 3% v/v acetonitrile at

pH 2.9.

5-HT, 5-HIAA and HVA were detected in a Water 464

electrochemical detector (Millipore/Water CA) equipped

with a glassy carbon working electrode, a stainless steel

auxiliary electrode and a Ag–AgCl reference electrode. The

chemicals were oxidized at 600 mV applied between the

working and the reference electrode. The sample com-

pounds of interest were measured by comparing their peak

heights with standard solutions.

For glutamate determination, each sample was mixed

with 20 mM carbonate buffer at pH 9.4 and 2.57 mM

fluorescein isothiocianate isomer I (FITCI) in acetone in a

proportion 5:1:1 (v/v/v). The procedure was the same for

GABA and serine determination except that the samples

were mixed with borate buffer for micellar electrokinetic

chromatography as detailed elsewhere [65]. The mixture

was placed in a water-saturated chamber for 24 h in the

dark. Then the mixture was diluted 5-fold with water and

injected into a model R2D2-1 CZE-LIFD instrument (Mer-

Fig. 1. (A) Photomicrograph of a typical coronal section (2.5 mm anterior to

bregma) stained for thionin showing detailed excitotoxic damage and

gliosis in the right ventromedial prefrontal area. (B) Diagram of lesioned

area in the ventromedial prefrontal cortex in all the animals included in this

study. (C) Diagram of a coronal section with the target probes position in

basolateral amygdala at approximately 3.0 mm posterior to bregma.

L.E. Gonzalez et al. / Brain Research 1005 (2004) 154–163 157

idialysis C.A Merida, Venezuela). Separation of analytes

was carried out in a 27-Am ID and 360-Am OD fused silica

capillary column filled with 20 mM carbonate buffer. The

two ends of the column were immersed in buffer reservoirs

with Pt-Ir electrodes. A high voltage (20 kv) was applied for

10 min. Fluorescence was excited by the 488-nm line of an

argon ion laser, collected through an objective and focused

on a photomultiplier tube (PMT). The output current of the

PMT was acquired and processed by means of the ONICERsoftware (Dialdemo C.A, Merida, Venezuela) in a PC.

Glutamate, GABA and serine were identified by migration

time and spiking.

2.4. Histology

At the end of the microdialysis procedure, all animals

were overdosed with chloroform and the brains fixed

through perfusion with 0.9% saline followed by 4% form-

aldehyde solution. Brains were removed from the skull,

included in paraffin and coronal sections of 25 Am were

stained for thionin to evaluate the position and extensions of

the lesion [44].

2.5. Statistics

Behavioral scores and basal neurotransmitter levels were

compared by unpaired t-test. The response to cat presenta-

tion for each neurotransmitter in sham or lesioned animals

was analyzed by repeated one-way ANOVA test. The

interaction between the lesion and the response to cat

presentation was analyzed by two-way ANOVA with treat-

ment (sham vs. bilateral lesion) treated as the between-

subjects variable and sample time (before vs. after cat

encounter) treated as the within-subject variable.

Fig. 3. Mean (FS.E.M.) of 5-HT, 5-HIAA, HVA concentrations in

dialysates from right amygdala before and after cat presentation (on the fifth

week after surgery) by MPFC lesioned or sham-operated rats. Unpaired

Student’s t-test for basal levels (n/group=12): *P<0.05. For further details

L.E. Gonzalez et al. / Brain Research 1005 (2004) 154–163158

3. Results

3.1. Excluded animals from data analysis

There were eight animals lost during the experiment.

Two animals microinjected with ibotenic acid died within 2

h after surgery. Probes flow was blocked in two animals.

There were two animals with wrong amygdala probes

placement. Animals with incorrect lesion area were only

two. In one of them, the double needle for bilateral injection

fell down in the right side and in the other the damage was

found at the level of the dorsal MPFC spearing part of the

ventral MPFC.

3.2. Histology (Fig. 1)

A typical damage of the ventral area of the MPFC

produced by bilateral ibotenic acid microinjection is shown

in Fig. 1A. The extension of the damage was evaluated in all

the animals (Fig. 1B). A diagram describing probes location

in the amygdala is shown in Fig. 1C.

3.3. Freezing behavior (Fig. 2)

Lesioned animals adopted the freezing posture for longer

period of time compared with shams in response to cat

presentation (unpaired t-Test, p<0.001). Freezing behavior

was displayed immediately after cat presentation and fol-

lowed by exploratory walking and risk assessment behav-

iors (stretching, sniffing and rearing) (Fig. 2).

3.4. Serotonin, 5-HIAA and HVA in the amygdala (Fig. 3)

Basal 5-HT concentration was elevated in lesioned ani-

mals relative to shams (unpaired t-test, p<0.01). Increased 5-

HT level was observed in shams [F(1,11)=5.8, p<0.03] but

not in lesioned animals [F(1,11)=0.3, p=0.7] after cat

presentation. Repeated two-way ANOVA showed that the

Fig. 2. Mean (FS.E.M.) time (seconds) spent in freezingafter cat presentation

byMPFC lesioned or sham-operated rats. A domestic cat was held in front of

the microdialysis cage for few seconds. In general, the rat approached to

investigate the cat’s body and smell it through a metal grid; afterward, the rat

withdrew to a cage corner and adopted a frozen posture. This behavioral

observation took place during microdialysis on the fifth week after surgery.

Unpaired t-test (n/group=12): *P<0.001.

on data analysis, see Results.

rat condition affected 5-HT changes after cat presentation

[Treatment factor, F(1,22)=7.3, p<0.01, Treatment�time,

F(1,22)=3.1, p=0.05]. Similarly, basal concentration of 5-

HIAA was elevated in lesioned animals relative to shams

(unpaired t-test, p<0.01). Cat presentation increased 5-

HIAA in both sham [F(1,11)=9.1, p<0.01] and lesioned

animals [ F(1,11)=12.8, p<0.004]. Repeated two-way

ANOVA showed that differences in 5-HIAA levels associ-

ated with the rat condition persisted after cat presentation

[Treatment factor, F(1,22)=5.2, p<0.03; Treatment�time,

F(1,22)=0.2, p=0.7]. HVA basal concentration was signifi-

cantly elevated in lesioned animals as compared with shams

(unpaired t-test, p<0.005). Cat presentation induced signif-

icant HVA increases in both lesioned animals [F(1,11)=

48.9, p<0.0001] and shams [F(1,11)=55.6, p<0.0001]. The

HVA increase differed between sham and lesioned animals

L.E. Gonzalez et al. / Brain Research 1005 (2004) 154–163 159

[treatment factor, F(1,22)=10.7, p<0.003; treatment�time,

F(1,22)=8.2, p<0.01] (Fig. 3).

3.5. Glutamate, GABA and serine in the amygdala before

and after cat presentation (Fig. 4)

Glutamate basal level was greater in lesioned animals as

compared with shams [unpaired t-test, p<0.02]. Cat presen-

tation did not increase glutamate level either in sham [s]

[F(1,11)=0.9, p=0.8] or lesioned animals [F(1,11)=0.6,

p=0.8]. Two-way ANOVA showed that the difference in

glutamate levels between groups remained after cat presenta-

tion [treatment factor, F(1,22)=4.8, p<0.03; treatment�time,

F(1,22)=0.007, p=0.9]. GABA basal level was higher in

shams as compared with lesioned rats (unpaired t-test,

p<0.02). GABA levels diminished after cat presentation in

shams [F(1,11)=6.7, p<0.02] but not in lesioned animals

[F(1,11)=0.15, p=0.7]. Two-way ANOVA confirmed that

Fig. 4. Mean (FS.E.M.) of glutamate, GABA and serine concentrations in

dialysates from right amygdala before and after cat presentation (on the fifth

week after surgery) by lesioned or sham-operated rats. Unpaired Student’s

t-test (n/group=12): *P<0.05. For further details on data analysis, see

Results.

GABA changes after cat presentation depended on whether

the animal was sham or lesioned [treatment�time,

F(1,22)=5.4, p<0.03; Treatment factor, F(1,22)=3, p<0.05].

Serine basal level was diminished in lesioned rats (unpaired t-

test, p<0.01). Serine significantly decreased in shams

[ F(1,11)=12.1, p<0.005] but not in lesioned animals

[F(1,11)=0.001, p=1.0] after cat presentation. Two-way

ANOVA showed that serine changes after cat presentation

depended on whether the animal was sham or lesioned

[treatment�time, F(1,22)=7.9, p<0.01; treatment factor,

F(1,22)=3.0, P<0.05] (Fig. 4).

4. Discussion

Freezing scores show that animals with ventromedial

prefrontal damage were more reactive to an unconditioned

fear stimulus. This finding is in accordance with the

augmented anxiety inferred from the social interaction test

on the fifth post-lesion week [47]. A possible explanation

for this result is that sub-cortical structures such as the

amygdala, normally subjected to inhibitory tone by the

MPFC become disinhibited following MPFC lesions. Pre-

vious reports have indicated that MPFC might act to inhibit

amygdaloid circuits. Thus, decreased conditioned fear re-

sponse was found following electrical stimulation of MPFC

[41] and ventromedial prefrontal damage slow down extinc-

tion of learned aversive response in rats [42,45]. This effect,

however, seems to have a high degree of anatomical

specificity because Gewirtz et al. [14] failed to detect any

effect of ventromedial prefrontal lesions that missed the

infralimbic nucleus. Posterior experiments by Quirk et al.

[45] confirmed delayed fear conditioned extinction when the

lesions included the rostral part of ventromedial prefrontal

cortex.

Lesioned animals were frozen during almost the whole 5-

min session indicating their inability to cope with predator

risk. By contrast, sham-operated animals were able to

engage explorative behaviors. This suggests that the integ-

rity of the MPFC is required for assessing predator risk.

Presence of the cat induced a sequence of flight, freezing

and risk assessment behaviors. Transition from freezing to

risk assessment behaviors might be associated with fear

attenuation through MPFC–amygdala inhibitory pathways.

Blanchard et al. [4] have also described the sequence of

flight, freezing and risk assessment behaviors in wild rats

after cat presentation. It is worth noting, however, that

laboratory-bred rats used in the present experiments are

much less prone to recognize predator risk compared with

wild rats. This explains the low fear reactivity in shams.

Increased amygdaloid 5-HT levels have been associated

with increased anxiety [12,20,32,35,68] whereas amygdaloid

5-HT depletion by local 5,7-dihydroxytryptamine lesions had

anxiolytic effects [60]. Administration of 5HT or 5-HT1A

receptor agonists to the amygdala increased anxiety as

measured by conflict models and the social interaction test

L.E. Gonzalez et al. / Brain Research 1005 (2004) 154–163160

[16,25,43]. Furthermore, humans expressing the short allele

of the 5-HT transporter (5-HTT) that causes increased syn-

aptic 5-HT had increased fMRI signal in the amygdala during

perception of fearful stimuli [20]. This collection of data fit

well with the anxiolytic effects induced by inhibition of the

dorsal raphe nucleus, the 5-HT cell cluster that project to the

amygdala [13,15]. Therefore, several lines of evidence sup-

port the association found in the present report of an increased

amygdaloid 5-HT basal concentration in lesioned animals

with exaggerated fear reactivity.

Similarly, lesioned animals had increased basal glutamate

levels in the amygdala and increased glutamate transmission

or administration of glutamate receptors agonists in the

amygdala has been associated with anxiogenesis, whereas

blockade of glutamate transmission leads to anxiolysis

[34,51,52,69].

Prefrontal lesioned animals show diminished amygdala

basal GABA level, which might be associated with anxio-

genesis. Decreased extracellular GABA in mouse amygdala

was detected during and following presentation of a condi-

tioned fear stimulus [61] and reduced GABA-stimulated

chloride influx in rat amygdala was found during and after a

restraint experience [39]. Further, local administrations of

GABA or benzodiazepine receptor agonists to the amygdala

decrease anxiety [16,25,70], while intra-amygdala adminis-

tration of a GABAA receptor blocker led to anxiogenesis

[52,53]. Interestingly, mice with high trait anxiety exhibited

low expression of benzodiazepine receptors exclusively in

the amygdala [24].

A second relevant finding here is the lack of the phasic

response in amygdaloid GABA and 5-HT dialysate concen-

trations after cat presentation in lesioned animals as com-

pared with shams. Basal concentrations of GABA and 5-HT

in lesioned animals were comparable to post-stress concen-

trations in shams. Further, the pattern of the basal change

induced by the lesion predicts the direction of the phasic

change in shams induced by the fear stimulus. Thus,

amygdala 5-HT and GABA overflows increased and de-

creased, respectively, after cat presentation resembling the

direction of the basal changes induced by the MPFC-lesion,

whereas the increased 5-HT and decreased GABA basal

levels in the amygdala of lesioned animals remained un-

modified after cat presentation. This suggests a ceiling effect

in lesioned animals because their neurotransmitter release

was at a maximum. Similarly, it was reported [47] that basal

plasma corticosterone concentration was elevated in le-

sioned animals while the stress response (plasma cortico-

sterone increase) was blunted. Therefore, it is possible that

persistent basal changes induced by the lesion entail dimin-

ished capacity of response for neurotransmitter downstream

mechanisms in the amygdala and hypothalamic–pituitary–

adrenal axis.

Data from electrophysiological studies in micro-dissected

amygdala indicates the existence of pre synaptic serotoner-

gic inhibition on GABAergic synaptic transmission [36],

which may in part explain our findings. This serotonergic

inhibition might be both tonic and phasic. Indeed, basal 5-

HT was significantly higher and basal GABA was signifi-

cantly lower in amygdala of MPFC-lesioned animals. Fur-

thermore, after cat presentation amygdala extracellular 5-HT

increased and GABA decreased in sham animals.

GABA receptors blockade increases glutamate neuro-

transmission in the basolateral amygdala as inferred from

pharmacological studies [52,59]. This is in agreement with

the pattern of diminished basal GABA and elevated basal

glutamate in lesioned rats. The lack of glutamate phasic

response might due to inhibitory effects of increased 5-HT.

It was found in the lateral amygdala that 5-HT exerts

inhibitory effects on glutamate release [62]. Nonetheless,

caution should be exerted because of the 25-min intervals

for sample collection in the present study. Extracellular

changes in glutamate levels occur as fast as in 1 s [50]

and rapid variations might be missed in a cumulative

dialysate that corresponds to a 25-min time scale.

Two distinctive glutamate inputs from MPFC and sen-

sory association cortex to the amygdala have been charac-

terized. Amygdala-mediated affective behaviors are driven

by sensory stimuli transmitted from sensory association

cortical regions, whereas prefrontal cortical pathway impo-

ses inhibitory actions on amygdala-mediated behaviors [49].

For that reason, the pattern of high glutamate and low

GABA amygdaloid basal levels in lesioned rats does not

necessarily exclude the existence of MPFC glutamate input

on the GABA interneurons inhibiting amygdala output

[18,49]. New sprouting of glutamate axons from sensory

association regions innervating amygdala neurons other

than GABA accounts for the high glutamate levels. Where-

as, the selective damage of glutamate MPFC–pathway may

contribute to lower GABA levels and enhanced fear reac-

tivity because the animal lost MPFC-depended inhibitory

mechanisms.

Basal level of a dopamine metabolite, homovanillic

acid (HVA) was higher in lesioned animals suggesting

tonic elevation of dopamine turnover. Cat presentation

further increases HVA levels in lesioned animals suggest-

ing that dopamine turnover increase after MPFC damage.

Conditioned stress increased amygdaloid homovanillic

acid (HVA) level, which was blocked by low anxiolytic

doses of diazepam because increased dopamine turnover

might be associated with augmented fear and anxiety [8].

Electrophysiological data also suggest that dopamine

facilitates amygdala function [18]. This indicates that a

larger amygdala dopamine turnover in MPFC-lesioned

animals might contribute to the freezing behavior en-

hancement found in the present experiment.

Decreased basal concentration of amygdala serine in

lesioned animals could result of increased NMDA receptor

stimulation. Perfusion of NMDA or kainate in rat striatum

caused a significant decrease in D-serine suggesting that D-

serine could be taken up by the astrocytes following

synaptic activation [22]. Amygdala serine decreased after

cat presentation in shams. This phasic change in shams was

L.E. Gonzalez et al. / Brain Research 1005 (2004) 154–163 161

in the same direction as the basal change in lesioned rats.

Thereby, our observations indicate that diminished serine

levels in the amygdala are related to increased fear reactiv-

ity. Yet again, MPFC damage induced a lack of adaptive

responses in serine release.

Although the present data are compatible with amygdala

disinhibition from MPFC, this finding does not rule out that

direct or indirect MPFC–amygdala pathways activate

amygdala neurons. The anxiolysis detected on the second

post-lesion week indicated the lost of an excitatory influence

on the amygdala [47].

In conclusion, we observed increased fear reactivity and

altered amygdala transmission in ventromedial prefrontal

damaged animals on the fifth week after lesion. This

indicated amygdala disinhibition because (1) amygdala

basal alterations in 5-HT, GABA and serine in lesioned

animals were in the same direction as changes elicited by a

fear stimulus in shams and (2) the lack of adaptive responses

in the amygdala neurotransmitter systems in lesioned ani-

mals. The association of altered amygdala neurotransmis-

sion with augmented fear reactivity fits well with a bulk of

previous observations. From an ethological view, this study

showed that the integrity of the MPFC is required for

displaying adaptive strategies against predator threats.

Acknowledgements

Grants G-97000820 from FONACIT and M653-9903A

from CDCHT-ULA supported this work.

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