Physiology & Behavior 104 (2011) 653–658

Contents lists available at ScienceDirect

Physiology & Behavior

j ourna l homepage: www.e lsev ie r.com/ locate /phb

Kindling of the lateral septum and the amygdala: Effects on anxiety in rats

Earl Thomas ⁎, Deborah J. GuntonDepartment of Psychology, Bryn Mawr College, 101 N. Merion Avenue, Bryn Mawr, PA 19010, United States

⁎ Corresponding author. Tel.: +1 610 526 5013; fax:E-mail address: ethomas@brynmawr.edu (E. Thoma

0031-9384/$ – see front matter © 2011 Elsevier Inc. Aldoi:10.1016/j.physbeh.2011.07.005

a b s t r a c t

a r t i c l e i n f o

Article history:Received 9 March 2011Received in revised form 5 July 2011Accepted 6 July 2011

Keywords:Long term kindlingAmygdalaLateral septumEmotionalityConflict

Long-term kindling of limbic system structures may produce substantial changes in emotional behavior inrats. This study examined long-term changes in two kindled structures that have opposite effects on anxiety,the lateral septum and the central nucleus of the amygdala. The purpose of the experiment was to examinethe specificity of the emotional effects of kindling by employing a double dissociation design. Animals weretested in two common animal models of anxiety, the water-lick conflict test and the elevated plus-maze. Inthe conflict test amygdala-kindled animals demonstrated a significant anxiolytic effect when compared withsham-kindled animals. This effect was potentiated by chlordiazepoxide. Septally-kindled animals exhibited asignificant anticonflict effect when compared to sham-kindled animals in the first session. Septally-kindledanimals spent significantly more time on the open arms of the elevated plus-maze than did sham-kindledanimals. Observed changes persisted 6 weeks after the termination of 150 kindling sessions. The effects oflong-term kindling were highly consistent with those of disruption rather than facilitation.

+1 610 526 7476.s).

l rights reserved.

© 2011 Elsevier Inc. All rights reserved.

1. Introduction

Kindling is a phenomenon in which repeated exposure to initiallysubconvulsive brain stimulation results in the development andprogressive intensification of elicited motor seizures [1]. Prolongedexperimental kindling has been reported to induce permanent neuro-logical changes both on a structural and a molecular level [2–4].Kindling of limbic system structures has been reported to producelong lasting changes in emotional behavior in animals and as such hasbecome a widely utilized animal model for the study of seizure-associated changes in fear and anxiety-like behavior underlying thebehavioral and emotional problems experienced by temporal lobeepileptics. Long-term kindling, typically defined as that which yieldsmajor seizure activity on 60 or more trials, has an especially profoundand long lasting effect on behavior. The capacity for kindling tomediate enduring neurological changes also has a number of potentialimplications for the manipulation of emotionality. In particular,kindling offers the opportunity to examine more thoroughly therole and function of specific brain structures relevant to anxietybehavior.

One rather puzzling aspect of the effect of long-term kindling iswhether it engenders a facilitation of the kindled structure or adisruption of the functioning of the structure, akin to a lesion. Botheffects of kindling have been reported in the literature. So, forinstance, kindling of the amygdala has been reported to be bothanxiogenic, indicating facilitation of function [5,6] and anxiolytic,

indicating disruption of function [7,8]. Hippocampal kindling has alsobeen reported as producing a disruption of functioning [9].

Our understanding of anxiety mechanisms in the brain hasbenefited greatly from a number of paradigms that have been usedto assess the behavioral effects of anxiolytic drugs, especially thebenzodiazepine anxiolytics. Two highly validated models are theconflict paradigm [10] and the elevated plus-maze [11]. In combina-tion with benzodiazepine drugs, these models implicated twoputative neuronal structures involved in the expression of anxiety,namely the amygdala and lateral septum.

Widespread evidence supports an anxiety-generating or anxio-genic function of the amygdala. Thus, electrical stimulation of theamygdala induces anxiety-associated behavior in rats [12]. Lesions ofthe amygdala suppress the typical affective reactions associated withfear- or anxiety-provoking stimuli [13–20]. Injection of benzodiaze-pines directly into the amygdala has a profound anxiolytic effect [21–25]. Direct projections of the central nucleus of the amygdala into thebrainstem areas possibly playing a role in fear or anxiety responseshave been described [26–30].

There is less agreement about the role of the lateral septum in themediation of anxiety. However, a variety of reports suggest an anxiety-relieving or anxiolytic functionof the lateral septum, including electricalstimulation studies [31–41], lesioning studies [42,43], and single-unitrecording studies [42–47].

Given the contradictory reports of kindling-related changes,describing either excitation [5,6] or disruption of function [7,8],stimulating two distinct structures with different anxiety-relatedfunctions may permit the clarification of the impact of long-termkindling in these models. It would also provide for a double dis-sociation control for general effects of kindled seizures. For example, if

654 E. Thomas, D.J. Gunton / Physiology & Behavior 104 (2011) 653–658

kindling has an excitatory effect, then stimulation of the amygdalawould result in intensified anxiogenic function. Similarly, theactivating impact of kindling on the lateral septum would also leadto heightened anxiolytic function. However, in the event that long-term kindling has a disruptive effect, then normal function in bothstructures would be disrupted with the amygdala-kindled animalsbehaving in a fashion consistent with decreased anxiety and septally-kindled animals exhibiting a reduced capacity to obtain relief fromfear. The purpose of the current study was to address the long-termeffects on emotionality induced by the kindling procedure in twostructures involved in the expression of anxiety, the lateral septumand the central nucleus of the amygdala. We examined the effects ofkindling in both the conflict paradigm and the elevated plus-maze. Inaddition, we examined the effect of a typical benzodiazepineanxiolytic, chlordiazepoxide, upon the kindled behavior.

2. Materials and methods

2.1. Subjects

The subjects were male Sprague–Dawley rats (Harlan, Frederick,MD)weighing 250–300 g. Animals were individually housed in a lightand temperature controlled colony. A 12-hour light/dark cycle wasmaintained along with ad lib food availability. Water availability wasrestricted to 30 min daily several days prior to training for the conflictparadigm. Care and use of animals were approved by the InstitutionalAnimal Care and Use Committee and experiments were carried out inaccordance with the National Institutes of Health Guide for the Care andUse of Laboratory Animals.

2.2. Apparatus

The kindling chamber was a 34 cm×26 cm×22 cm clear Plexiglaschamber with a stainless steel grid floor. The conflict chamber wasidentical to the kindling chamber except that a water bottle wasmounted on the outside of the front panel with a metal tubeprotruding into the chamber in the center of the panel, 5 cm above thefloor. The apparatus was housed in a sound-attenuating cubiclecontaining a house light and speaker. White noise (70 dB SPL) was fedinto the chamber throughout the experiment except during pre-sentations of the tone. A solid state contact relay was utilized to detectlicking. Shocks were delivered through the grid floor using a constantcurrent shock generator. Activation of the experimental sequence andrecording of the results were controlled by a computer and solid stateequipment.

The elevated plus-maze (EPM)wasmade of black opaque Plexiglasand raised approximately 70 cm above the ground. Two opposing“open arm” sections, approximately 50 cm×10 cm in surface area,were open on the sides. Two opposing “closed arm” sections wereequal in surface area to the open arms; however, these sections wereadditionally enclosed by walls (42 cm in height). The center was10 cm2.

2.3. Surgery

Animals were randomly assigned to one of three groups: (1) lateralseptum-kindled (n=14); (2) amygdala-kindled (n=16); and (3) sham-kindled (lateral septum (n=6) and amygdala (n=6)). A single bipolarelectrode (Plastics One) was implanted in the left lateral septum orthe central nucleus of the left amygdala of each rat under sodiumpentobarbital anesthesia (40 mg/kg, i.p.). The coordinates for lateralseptal implants were 0.3 mm anterior to bregma, 0.7 mm lateral tomidline, and 6.0 mm from the surface of the skull. The coordinates for theamygdala implants were 2.2 mm posterior to bregma, 4.0 mm lateral tomidline, and 8.0 mm from the surface of the skull [48]. Electrodes weresecured to the skull with four stainless steel screws and dental acrylic.

2.4. Stimulation procedures

Following a post surgical recovery period of at least 12 days, ratsdesignated for kindling received electrical stimulation. Stimulationconsisted of a one-second train of biphasic square wave pulses with apulse duration of 0.1 ms. Stimulation was presented at 100 Hz with aconstant current of 0.3 mA. The interval between the two pulses ofbiphasic stimulation was 0.1 ms. Stimulation was generated by aGrass Stimulator (Model S88) in combination with a stimulus isolatorand constant current generator (Model A365, World PrecisionInstruments). Kindling occurred 5 days per week, 3 times daily, foran experimental total of 150 stimulations. There was an interval of atleast 2 h between consecutive stimulations. Sham animals weresimilarly connected to the stimulation cable; however, the animalswere not stimulated.

Behavioral responses to stimulations of both areas were initiallylimited to momentary behavioral arrest; however, after 10 daysalmost every stimulation elicited a clonic convulsion characterizedby facial and forelimb clonus, rearing and loss of equilibrium. Themeasure of convulsion severity was expressed as the convulsion classelicited by each stimulation. The convulsion classwas scored accordingto Pinel and Rovner's [49] extension of Racine's [4] widely used scale.The classes we used were designated as follows: (1) Class 1, headnodding only; (2) Class 2, head nodding and jaw clonus; (3) Class 3,head nodding, jaw clonus and forelimb clonus; (4) Class 4, headnodding, jaw clonus, forelimb clonus, and rearing; (5) Class 5, a Class 4response plus falling once; and (6) Class 6, a Class 5 response withmultiple falling episodes. The criterion for inclusion in the study wasa full seizure corresponding to at least a Class 5 response.

2.5. Behavioral testing

In the conflict paradigm, water availability was restricted to 0.5 hdaily beginning on thefifth day of kindling. Four days later, the animalsbegan training in the conflict test. The conflict procedure was carriedout between 0800 and 1100 h, prior to the kindling procedure eachday. This was in order to preclude any direct effect of kindling onperformance on the conflict test. The conflict paradigm employed amodified water-lick suppression procedure in which animals wereexposed to four 2-minute time periods, the second and fourth ofwhichwere signaled by an 80 dB/1000 Hz tone. The total number of lickswithin each 2-minute component was counted. Habituation sessionsin which animals were exposed to the experimental apparatus andthe sequence of the stimuli without their behavior being punishedcontinued until a stable baseline was achieved. Subsequently trainingbeganwith the introduction of a 60 Hz AC shock delivered to the gridsevery 20th lick during the second and fourth signaled time periods.Current levels were initially 0.3 mA. The current was increased to0.4 mA in the 43rd session. Kindling was terminated after the 46thsession. For adequate training and to ensure that the behavior wasunder stimulus control it was advantageous to have the four-periodsequence. However, the relevant data are found in the first two timeperiods because the effects of satiation may possibly contaminate theresults particularly in the last punished time period.

Conflict behavior was further evaluated in the presence of thebenzodiazepine chlordiazepoxide (CDP). Drug tests were all con-ducted after the termination of kindling. Each animal was tested withthree doses of CDP, 5, 10, and 15 mg/kg administered intraperitone-ally. Animals had a “washout” period of at least 1 week following eachdrug session. The drug was administered 15 min prior to each conflictsession. The measure of anti-conflict or anxiolytic action of the drugwas a change in licking rate during the punished period. In addition,animals were given saline injections on the day preceding each of thethree drug sessions to control for drug-administration effects.

The elevated plus-maze (EPM) was employed 1 day after the finalkindling session following the conflict session on that day. Rats were

Fig. 1. Typical location of electrode from representative animals. Fixed sections taken at40 μmwere transilluminated and photographedwith a digital camera. The arrows pointto electrode tips implanted in the dorsolateral septum (A) or the central nucleus of theamygdala (B).

Sessions

16-18

Mea

n N

umbe

r of

Lic

ks

100

200

300

400

500

600sham

lateral septum

amygdala

38-4035-3732-3429-3126-2823-25222119-21

Fig. 2. Effects of footshock (0.3 mA) on punished responding in a water lick conflict test.Sessions are blocked in groups of three except for sessions 21 and 22, which representthe transition point between no shock and shock (marked by a vertical line). Error barsrepresent SEM. Kindled animals in both groups were less affected than controls bypunishment, with septum-kindled animals showing significantly elevated lickingduring the initial shock session (22) only, and amygdala-kindled animals showingsignificantly elevated licking when assessed over time.

655E. Thomas, D.J. Gunton / Physiology & Behavior 104 (2011) 653–658

placed in the center of the EPM facing one of the four arms with thedirection being rotated with each successive rat. The room wasdarkened to encourage exploratory behavior. The number of entriesinto each arm and total time spent on each arm were recorded. Inorder for a rat to qualify as having entered an arm, all four paws wererequired to have crossed over the entry line.

2.6. Histology

At the conclusion of behavioral testing animals were sacrificedwith an overdose of sodium pentobarbital. Brains were subsequentlyremoved and preserved in a formalin solution for at least 24 h. Thebrains were then frozen and sectioned through the structure ofinterest; 40 μm sections were wet-mounted on slides and photo-graphed using a digital camera. The position of each electrode tip wasestimated from the digital photograph.

2.7. Statistical analyses

Effects of kindling on behavior in the conflict paradigm wereassessed using independent samples t-tests for individual sessioncomparisons or repeated measures analysis of variance (ANOVA) foranalysis of group differences over time. The elevated plus-maze datawere analyzed using independent samples t-tests. The effects of CDPon behavior in the conflict paradigm were assessed using repeatedmeasures ANOVA for analysis of group differences at different drugconcentrations. The significance level for all comparisons was pb0.05.

3. Results

3.1. Histology

Examples of electrode tip locations are depicted in Fig. 1A (lateralseptum) and B (amygdala). Upon histological examination, mostelectrodes aimed at the septum were found to terminate in either thedorsolateral or intermediate lateral septum, while the majority ofelectrodes directed at the amygdala were found to terminate in orproximal to the central nucleus. Final behavioral analyseswere based on10 amygdala-kindled, 8 lateral septum-kindled, and 11 control animals.

3.2. Kindling effects on conflict behavior

All analyses of conflict behavior were performed on the firstunpunished and punished time periods. Fig. 2 depicts performanceduring the signaled punished period for 7 sessions prior to and 19sessions post shock. The sessions are blocked in groups of three, withthe exception of sessions 21 and 22, which were of particular interestas they represent the sessions immediately before and after theintroduction of footshock. As may be seen in Fig. 2 all animalsdemonstrated a diminished licking response with the introduction offootshock. Kindled animals in both groups appeared less impactedby punishment. During the initial session with footshock, punishedlicking rates of lateral septum-kindled animals were significantlyless suppressed than those of control animals (t (17)=2.56, p=.02).Amygdala-kindled animals revealed a similar behavioral profile,which failed to reach significance during the first session. How-ever, over the next 6 session blocks, the licking rate of amygdala-kindled animals was significantly higher than sham-kindled animals(F (1, 19)=4.91, p=.04), whereas the licking rate of LS kindledanimals was not significantly different from sham kindled animals.

As shown in Fig. 3, increasing the shock level in session 43 (0.4 mA)resulted in increased suppression in all groups. As may be seenamygdala-kindled animals continued to lick at a significantly higherrate than sham-kindled animals when compared over the same8 session blocks (F (1, 19)=5.75, p=.03). This punishment-releaseeffect persisted 6 weeks after kindling termination, strongly suggesting

that long-lasting neural changes had occurred within these kindledgroups. Taken together, these data suggest an initial anti-conflict effectof kindling on both experimental groups. Over time, however, this effectpersisted in amygdala- but not septal-kindled animals.

Session

42

Mea

n N

umbe

r of

Lic

ks

0

100

200

300

400

500sham

lateral septum

amygdala

74-7671-7366-6860-6257-5951-5348-5044-4643

Fig. 3. Effects of increased footshock (0.4 mA) and termination of kindling on punishedresponding in a water-lick conflict test. Sessions are blocked in groups of three exceptfor sessions 42 and 43, which represent the transition point between the 0.3 mA shockand the 0.4 mA shock (marked by a solid vertical line). Termination of kindling ismarked by a dashed vertical line. Error bars represent the SEM. The figure depicts theadditional proconflict effect of increased footshock (0.4 mA) on septal-kindled animals.Amygdala-kindled animals continued to show significantly elevated licking over time,which persisted 6 weeks after kindling. These results suggest that kindling exertedlong-lasting neural change.

Sham

Num

ber

of O

pen

Arm

Ent

ries

0.0

0.5

1.0

1.5

2.0

2.5

3.0

B

Tim

e S

pent

in O

pen

Arm

s (s

ec.)

0

20

40

60

80

100

A*

CeALS

Sham CeALS

656 E. Thomas, D.J. Gunton / Physiology & Behavior 104 (2011) 653–658

3.3. Drug effects

Fig. 4 presents a dose–response curve for the effects of CDP onpunished responding in amygdala- and septal-kindled animals as wellas in sham-kindled controls. The zero point is a mean of three sessionsin which vehicle alone (saline) was administered. Each of these salinecontrol sessions preceded the drug sessions, which were performedon the following day. A significant group effect was observed withamygdala-kindled animals licking at a significantly higher rate thansham-kindled animals (F (1, 19)=8.58, p=.01). A significant groupby dose interaction effect was also observed, suggesting a moremarked effect of the drug on amygdala-kindled animals (F (3, 57)=3.23, p=.03). Upon comparison of the rates of punished respondingin septal- and sham-kindled animals, a significant session effect wasobserved, suggesting that the drug treatment, although small, waseffective (F (3, 51)=3.13, p=.03). These data suggest that, while theamygdala-kindled animals demonstrated a heightened sensitivity tothe releasing effects of CDP, no significant alterations in sensitivitywere exhibited by septal-kindled animals.

[CDP] (mg/kg)

0

Mea

n N

umbe

r of

Lic

ks

0

100

200

300

400

500 shamlateral septum amygdala

5 10 14

Fig. 4. Dose–response curves for the effects of CDP on punished responding in kindledanimals in thewater-lick conflict test. The zero point is amean of three sessions inwhichvehicle alone (saline) was administered the day prior to each drug session. Error barsrepresent the SEM. Amygdala-kindled animals showed a substantially greater effect ofCDP than either the sham or septum-kindled animals.

3.4. Elevated plus-maze

The performance of the three groups on the EPM is summarized inFig. 5. The time spent in open arms is depicted in panel A and thenumbers of open arm entries are illustrated in panel B. Upon com-parison of the performances of sham- and septal-kindled animalsusing independent samples t-test analyses, a significant group dif-ference was evident with septal-kindled animals spending signifi-cantly more time on the open arms than the sham-kindled controls(t (17)=2.24, p=.04). Septal-kindled animals also demonstrated atrend toward more entries onto the open arms than control animals;however, this trend failed to reach significance in a t-test analysis(t (17)=1.63, p=.12). Amygdala-kindled animals also appeared tomake more entries onto the open arms than the control group;however, this trend failed to reach significance.

Tot

al N

umbe

r of

Arm

Ent

ries

0

2

4

6

8

10

12

C

Sham CeALS

Fig. 5. Effects of kindling on behavior on the elevated plus maze. Panel A depicts relativetime spent in open arms. The asterisk indicates a statistically significant difference.Panel B represents numbers of open arm entries. Panel C depicts the total number ofentries into all arms and serves as an index of changes in locomotor activity. Theseptum-kindled rats spent significantly more time on the open arms than did the sham-kindled rats. Both septum- and amygdala-kindled animals showed a trend towardmore entries onto the open arms, which did not reach significance when compared tosham-kindled controls. Kindling had a small and statistically insignificant effect ongeneral locomotor activity.

657E. Thomas, D.J. Gunton / Physiology & Behavior 104 (2011) 653–658

The question often arises as to whether increases in the amount oftime on the open arms or number of entries onto the open arms reflectchanges in anxiety or simply general changes in locomotor activity.This is typically assessed by looking at changes in the total number ofentries onto all of the arms which would reflect changes in generallocomotor activity. Fig. 5C depicts the number of entries onto all of thearms for the three groups. As may be seen general locomotor activityis not increased by kindling of either structure. If anything there is asmall and insignificant decrease in overall activity.

4. Discussion

The current findings are consistent with previous reports in theliterature in which long-term kindling resulted in a disruption offunction rather than facilitation. Amygdala-kindled animals demon-strated significant anti-conflict effects in the release of punishedlicking behavior when compared with sham-kindled animals in theconflict paradigm. This effect was significantly potentiated by CDP.Such anti-conflict effects and increases in CDP efficacy are in strikingagreement with previous findings in amygdala-lesioned animals [41].Similarly, septally-kindled animals also demonstrated behavioraleffects almost identical to those observed in septally-lesioned animals.Thus, in the conflict paradigm, septally-kindled animals exhibitedsignificantly greater levels of punished responding when compared tosham-kindled animals in the first session which disappeared on sub-sequent sessions. This very closely mirrors the behavior observed inseptally-lesioned animals [50]. Furthermore, septally-kindled animalsspent significantlymore time on the open arms of the EPM than sham-kindled animals, once again replicating the findings in septally-lesioned animals [51,52]. The apparent disparity between ourobservations in the conflict situation and the elevated plus-maze inthe septally kindled animals may seem puzzling. We have noted thesame disparity in our lesioning experiments [53]. We have suggestedthat the elevated plus-maze data are, in fact, consistent with a fear-inhibitory function of the lateral septum.While septal-kindled animalstend to enter the open arms of the elevated-plus maze more than thesham-kindled animals they nevertheless appear to be more anxious,hyperactive, and show increased defecation on the open arms. Theseptally-kindled animals with a deficit in the ability to inhibit fear nolonger benefit from the safety provided by the enclosed arms [53].Ultimately, the similarities of our observations on lesioned and kindledanimals support the idea that long-term kindling has a lesion-likedisruptive effect.

The mechanism of the lesion-like disruption is not currentlyknown. The appearance of such a disruption of function has beenreported in several models in addition to the current study, includingthe hippocampus [9], the amygdala [8] and the globus pallidus [54],suggesting a common pathway of action. It is of interest that theeffects of long-term kindling are remarkably similar to the effects ofdeep-brain stimulation in humans and may involve similar mecha-nisms such as depolarization block or mobilization of inhibitorytransmitters such as GABA [55].

A large body of evidence has suggested that the amygdala is criticalfor the learning of associations between neutral and aversive stimuliand for the expression of fear-related behaviors [56–59]. Thus, in theconflict paradigm, disruption of amygdala functionmay interfere withthe capacity to recognize the conflict tone as a discriminatory signal,thereby blocking the usual acquisition of the conditioned suppressionof licking.

Increased CDP efficacy was also observed in amygdala-kindledanimals, both in the additional release of punished responding andin the facilitation of sedating effects typically observed in unpun-ished responding. Evidence of increased benzodiazepine efficacy inamygdala-kindled animals further supports the reciprocity modelin which amygdala disruption leads to the release of a reciprocally-regulated structure such as the septum, resulting in increased

anxiolytic action and increased efficacy of benzodiazepines [41].Amygdala-kindled animals also showed a tendency toward increasedentries onto the open arms, a trend that did not reach significance butis consistent with other signs of increased anxiolytic action.

The effects of septal kindling are consistent with a hypothesis putforward by Yadin et al. that disruption of septal function leads to adeficit in fear relief which is necessary for the acquisition of passiveavoidance behavior as seen in the conflict paradigm. However, oncethe avoidance has been learned then increased fear resulting fromseptal disruption would result in a proconflict effect [50]. This wouldaccount for the initial continuation of punished licking seen on thefirst day of shock followed by a suppression of punished water lickingon subsequent sessions. By the same reasoning the observation thatseptally-kindled animals spent significantly more time on EPM openarms than control animals may be attributed to an inability to obtainrelief (and thus reinforcement) from remaining in the closed arms.

In conclusion, the behavioral effects of long-term kindling of boththe amygdala and the lateral septum provide a striking example of adouble dissociation, demonstrating that kindling can be highly sitespecific and not simply a general effect of epileptogenesis. The resultswere consistent with disruptive effect of kindling as assessed in theconflict paradigm, with the benzodiazepine CDP, and on the elevatedplus maze. Consistent with a number of lesioning studies, findingsfrom the current study uphold the notion that the amygdala andlateral septum are both involved in the control of anxiety; and thatthere is a reciprocal relationship, between the structures. Furtherwork is needed to clarify specific parameters.

References

[1] Goddard GV, McIntyre DC, Leech CK. A permanent change in brain functionresulting from daily electrical stimulation. Exp Neurol 1969;25:295–330.

[2] Adamec RE. Amygdala kindling and anxiety in the rat. Neuroreport 1990;1:255–8.[3] Goddard GV. Development of epileptic seizures through brain stimulation at low

intensity. Nature 1967;214:1020–1.[4] Racine RJ. Modification of seizure activity by electrical stimulation. II. Motor

seizure. Electroencephalogr Clin Neurophysiol 1972;32:281–94.[5] Kalynchuk LE, Pinel JP, Treit D, Kippin TE. Changes in emotional behavior produced

by long-term amygdala kindling in rats. Biol Psychiatry 1997;41:438–51.[6] Pinel JP, Treit D, Rovner LI. Temporal lobe aggression in rats. Science 1977;197:

1088–9.[7] Adamec RE, Morgan HD. The effect of kindling of different nuclei in the left and

right amygdala on anxiety in the rat. Physiol Behav 1994;55:1–12.[8] Witkin JM, Lee MA, Walczak DD. Anxiolytic properties of amygdaloid kindling

unrelated to benzodiazepine receptors. Psychopharmacology(Berl) 1988;96:296–301.[9] Hannesson DK, Howland J, Pollock M, Mohapel P, Wallace AE, Corcoran ME. Dorsal

hippocampal kindling produces a selective and enduring disruption of hippo-campally mediated behavior. J Neurosci 2001;21:4443–50.

[10] Vogel JR, Beer B, Clody DE. A simple and reliable conflict procedure for testing anti-anxiety agents. Psychopharmacologia 1971;21:1–7.

[11] Pellow S, Chopin P, File SE, Briley M. Validation of open:closed arm entries in anelevated plus-maze as a measure of anxiety in the rat. J Neurosci Methods1985;14:149–67.

[12] Davis M. The role of the amygdala in fear and anxiety. Annu Rev Neurosci 1992;15:353–75.

[13] Coover GD, Murison R, Jellestad FK. Subtotal lesions of the amygdala: the rostralcentral nucleus in passive avoidance and ulceration. Physiol Behav 1992;51:795–803.

[14] Grijalva CV, Levin ED, Morgan M, Roland B, Martin FC. Contrasting effects ofcentromedial and basolateral amygdaloid lesions on stress-related responses inthe rat. Physiol Behav 1990;48:495–500.

[15] Grossman SP, Grossman L, Walsh L. Functional organization of the rat amygdalawith respect to avoidance behavior. J Comp Physiol Psychol 1975;88:829–50.

[16] Jellestad FK, Bakke HK. Passive avoidance after ibotenic acid and radio frequencylesions in the rat amygdala. Physiol Behav 1985;34:299–305.

[17] Jellestad FK, Markowska A, Bakke HK, Walther B. Behavioral effects after ibotenicacid, 6-OHDA and electrolytic lesions in the central amygdala nucleus of the rat.Physiol Behav 1986;37:855–62.

[18] Kopchia KL, Altman HJ, Commissaris RL. Effects of lesions of the central nucleus ofthe amygdala on anxiety-like behaviors in the rat. Pharmacol Biochem Behav1992;43:453–61.

[19] Riolobos AS, Martin Garcia AI. Open field activity and passive avoidance responsesin rats after lesion of the central amygdaloid nucleus by electrocoagulation andibotenic acid. Physiol Behav 1987;39:715–20.

[20] Shibata K, Kataoka Y, Yamashita K, Ueki S. An important role of the centralamygdaloid nucleus andmammillary body in themediation of conflict behavior inrats. Brain Res 1986;372:159–62.

658 E. Thomas, D.J. Gunton / Physiology & Behavior 104 (2011) 653–658

[21] Hodges H, Green S, Glenn B. Evidence that the amygdala is involved inbenzodiazepine and serotonergic effects on punished responding but not ondiscrimination. Psychopharmacology (Berl) 1987;92:491–504.

[22] Nagy J, Zambo K, Decsi L. Anti-anxiety action of diazepam after intra-amygdaloidapplication in the rat. Neuropharmacology 1979;18:573–6.

[23] Scheel-Kruger J, Petersen EN. Anticonflict effect of the benzodiazepines mediatedby a GABAergic mechanism in the amygdala. Eur J Pharmacol 1982;82:115–6.

[24] Shibata K, Kataoka Y, Gomita Y, Ueki S. Localization of the site of the anticonflictaction of benzodiazepines in the amygdaloid nucleus of rats. Brain Res 1982;234:442–6.

[25] Thomas SR, Lewis ME, Iversen SD. Correlation of [3H]diazepam binding densitywith anxiolytic locus in the amygdaloid complex of the rat. Brain Res 1985;342:85–90.

[26] Abraham WC, Tate WP. Metaplasticity: a new vista across the field of synapticplasticity. Prog Neurobiol 1997;52:303–23.

[27] Gloor P. Amygdala. In: Field J, editor. Handbook of physiology. Washington, DC:American Physiological Society; 1960. p. 1395–420.

[28] Kapp BS, Gallagher M, Underwood MD, McNall CL, Whitehorn D. Cardiovascularresponses elicited by electrical stimulation of the amygdala central nucleus in therabbit. Brain Res 1982;234:251–62.

[29] Pascoe JP, Kapp BS. Electrophysiological characteristics of amygdaloid centralnucleus neurons during Pavlovian fear conditioning in the rabbit. Behav Brain Res1985;16:117–33.

[30] Sarter M, Markowitsch HJ. Involvement of the amygdala in learning and memory:a critical review, with emphasis on anatomical relations. Behav Neurosci 1985;99:342–80.

[31] Brayley KN, Albert DJ. Suppression of VMH-lesion-induced reactivity andaggressiveness in the rat by stimulation of lateral septum, but not medial septumor cingulate cortex. J Comp Physiol Psychol 1977;91:290–9.

[32] Covian MR, Antunes-Rodrigues J, O'Flaherty JJ. Effects of stimulation of the septalarea upon blood pressure and respiration in the cat. J Neurophysiol 1964;27:394–407.

[33] Grauer E, Thomas E. Conditioned suppression of medial forebrain bundle and septalintracranial self-stimulation in the rat: evidence for a fear-relief mechanism of theseptum. J Comp Physiol Psychol 1982;96:61–70.

[34] Holdstock TL. Effects of septal stimulation in rats on heart rate and galvanic skinresponse. Psychon Sci 1967;9:491–504.

[35] Malmo RB. Slowing of heart rate after septal self-stimulation in rats. Science1961;133:1128–30.

[36] Malmo RB. Classical and instrumental conditioning with septal stimulation asreinforcement. J Comp Physiol Psychol 1965;60:1–8.

[37] Rolls ET. The brain and reward. Oxford: Pergamon Press; 1975.[38] Routtenberg A, Olds J. Stimulation of dorsal midbrain during septal and

hypothalamic self-stimulation. J Comp Physiol Psychol 1966;62:250–5.[39] Siegel A, Skog D. Effects of electrical stimulation of the septum upon attack

behavior elicited from the hypothalamus in the cat. Brain Res 1970;23:371–80.

[40] Thomas E, Evans GJ. Septal inhibition of aversive emotional states. Physiol Behav1983;31:673–8.

[41] Yadin E, Thomas E, Strickland CE, Grishkat HL. Anxiolytic effects of benzodiaz-epines in amygdala-lesioned rats. Psychopharmacology (Berl) 1991;103:473–9.

[42] Lee EH, Lin YP, Yin TH. Effects of lateral and medial septal lesions on variousactivity and reactivity measures in rats. Physiol Behav 1988;42:97–102.

[43] Melia KR, Davis M. Effects of septal lesions on fear-potentiated startle, and on theanxiolytic effects of buspirone and diazepam. Physiol Behav 1991;49:603–11.

[44] Thomas E, Yadin E. Neural correlates of conditioning assessed by extracellular unitrecording: implications for neuroplasticity. In: Milgram NW,McCleod CM, Petit TL,editors. Neuroplasticity, learning and memory. New York: Allan R. Liss; 1987.p. 199–229.

[45] Thomas E, Yadin E, Strickland CE. Septal unit activity during classical conditioning:a regional comparison. Brain Res 1991;547:303–8.

[46] Yadin E, Thomas E. Septal correlates of conditioned inhibition and excitation inrats. J Comp Physiol Psychol 1981;95:331–40.

[47] Yadin E, Thomas E, VaughnMP. Effects of anxiolytic agents onfiring of lateral septalunits in acute and chronic preparations. Soc Neurosci Abstr 1986;12:1077–83.

[48] Paxinos G, Watson C. The rat brain in stereotaxic coordinates. London: AcademicPress; 1986.

[49] Pinel JP, Rovner LI. Experimental epileptogenesis: kindling-induced epilepsy inrats. Exp Neurol 1978;58:190–202.

[50] Yadin E, Thomas E, Grishkat HL, Strickland CE. The role of the lateral septum inanxiolysis. Physiol Behav 1993;53:1077–83.

[51] Treit D, Pesold C, Rotzinger S. Dissociating the anti-fear effects of septal andamygdaloid lesions using two pharmacologically validated models of rat anxiety.Behav Neurosci 1993;107:770–85.

[52] Treit D, Menard J. Dissociations among the anxiolytic effects of septal,hippocampal, and amygdaloid lesions. Behav Neurosci 1997;111:653–8.

[53] Yadin E, Thomas E. Limbic mechanisms of anxiety and stress. In: Levy A, Grauer E,Ben-Nathan D, de Kloet RE, editors. New frontiers in stress research: modulationof brain function. Amsterdam: Harwood Academic Publishers; 1998. p. 73–81.

[54] Dostrovsky JO, Hutchison WD, Lozano AM. The globus pallidus, deep brainstimulation, and Parkinson's disease. Neuroscientist 2002;8:284–90.

[55] Benazzouz A, Hallett M. Mechanism of action of deep brain stimulation. Neurology2000;55:S13–6.

[56] Davis M. Neurobiology of fear responses: the role of the amygdala. J NeuropsychiatryClin Neurosci 1997;9:382–402.

[57] Davis M. Functional neuroanatomy of anxiety and fear. In: Charney DS, Nestler EJ,editors. Neurobiology of mental illness. Second Edition. New York: OxfordUniversity Press; 2004. p. 584–604.

[58] LeDoux JE. Emotion circuits in the brain. Annu Rev Neurosci 2000;23:155–84.[59] Pare D, Quirk GJ, Ledoux JE. New vistas on amygdala networks in conditioned fear.

J Neurophysiol 2004;92:1–9.