Reagan Stress BLA

Activation of Phenotypically DistinctNeuronal Subpopulations in the Anterior

Subdivision of the Rat BasolateralAmygdala Following Acute and Repeated

Stress

LEAH R. REZNIKOV, LAWRENCE P. REAGAN,* AND JIM R. FADEL

Department of Pharmacology, Physiology and Neuroscience, School of Medicine,University of South Carolina, Columbia, South Carolina 29208

ABSTRACTThe effects of acute and repeated stress on expression of the early immediate gene c-fos

in the basolateral amygdala have previously been reported; however, characterization ofwhich neuronal subpopulations are activated by these stimuli has not been investigated. Thisquestion is of considerable relevance, insofar as the basolateral amygdala houses a hetero-geneous population of neurons, including those of �-aminobutyric acid (GABA)-ergic andglutamatergic phenotypes that may be subcategorized based on their expression of variouscalcium-binding proteins, including parvalbumin, calbindin, calretinin, and the calcium-sensitive enzyme calcium/calmodulin-dependent kinase II. Characterization of these sub-populations has revealed unique differences in their physiology, synaptology, and morphol-ogy, suggesting that each distinct phenotype may have profound effects on the local circuitryof the amygdala. Therefore, we examined the effects of acute and repeated restraint stress onexpression of the immediate early gene c-fos in neurons containing parvalbumin, calbindin,calretinin, or calcium/calmodulin-dependent kinase II in the basolateral amygdala. Double-label immunohistochemistry revealed that acute restraint stress activated a proportion ofparvalbumin-, calbindin-, or calcium/calmodulin-dependent kinase II-positive neurons. Priorexposure to repeated restraint stress markedly attenuated acute-stress mediated activationof these neuronal populations, although not equally. Expression of c-Fos protein was notdetected in calretinin-positive neurons in any experimental group. These results demonstratethat distinct neuronal phenotypes in the basolateral amygdala are activated by acute re-straint stress and that prior repeated restraint stress differentially affects this response.J. Comp. Neurol. 508:458 – 472, 2008. © 2008 Wiley-Liss, Inc.

Indexing terms: mood disorders; c-Fos; parvalbumin; calbindin; calretinin; calcium/calmodulin-

dependent kinase II

Alterations in amygdalar activity (Drevets, 1999;Thomas et al., 2001; Hull, 2002) and structure (Frodl etal., 2002; Brambilla et al., 2003) accompany various mood-related disorders, and stress can precipitate or provokeepisodes of such illnesses in certain patient populations(Williamson et al., 2005; Phillips et al., 2006). Under-standing how these phenomena relate to one another hashighlighted the basolateral amygdalar complex (BLC) as aregion of interest (Braga et al., 2002). The BLC serves asa critical site for memory-modulating effects of stress hor-mones (Donley et al., 2005) as well as the consolidation offear-related memories (Pare, 2003). The BLC also housestwo morphologically and functionally distinct classes of

neurons, namely, pyramidal and nonpyramidal neurons(McDonald, 1982, 1992a,b; Carlsen, 1988). Pyramidal

Grant sponsor: University of South Carolina Research Foundation (toL.P.R., J.R.F.).

*Correspondence to: Lawrence P. Reagan, PhD, Department of Pharma-cology, Physiology and Neuroscience, University of South Carolina Schoolof Medicine, 6439 Garner’s Ferry Road, D40, Columbia, SC 29208.E-mail: [email protected]

Received 19 April 2007; Revised 25 July 2007; Accepted 29 January 2008DOI 10.1002/cne.21687Published online in Wiley InterScience (www.interscience.wiley.com).

THE JOURNAL OF COMPARATIVE NEUROLOGY 508:458–472 (2008)

© 2008 WILEY-LISS, INC.

neurons are predominantly spine-dense projection neu-rons that utilize glutamate for neurotransmission and areidentifiable via the presence of the calcium-sensitive en-zyme calcium/calmodulin-dependent kinase II (CaMKII;McDonald et al., 2002). Conversely, nonpyramidal neu-rons are spine-sparse, utilize �-aminobutyric acid (GABA),and function primarily as interneurons. The expression ofvarious calcium-binding proteins, including parvalbumin(PV; Celio, 1990; Smith et al., 2000), calbindin (CB; Celio,1990; McDonald and Betette, 2001), and calretinin (CR;McDonald and Mascagni, 2001), has allowed for furtherclassification of BLC inhibitory neurons in which distinctphysiological properties (Rainnie et al., 2006) and/or syn-aptology (Muller et al., 2003, 2005, 2006, 2007) have beennoted. These observations have led to the suggestion thatphenotypic differences represent functional differencesthat may be critical for the regulation of local amygdalarcircuitry and subsequent amygdalar function (Rainnie etal., 2006).

Taking these observations into account, we hypothe-sized that stress would activate specific neuronal popula-tions in the BLC and that prior repeated stress wouldalter this response. Induction of the immediate early genec-fos has been used extensively to assess neuronal activa-tion following stressful stimuli (Cullinan et al., 1995;Chowdhury et al., 2000; Day et al., 2005). Accordingly, theaim of our current study was to determine the effects ofacute restraint stress (ARS) and repeated restraint stress(RRS) on induction of the immediate early gene proteinproduct c-Fos in PV-positive (PV�), CB-positive (CB�),CR-positive (CR�), and CaMKII-positive (CaMKII�) sub-populations of neurons in the BLC.

Materials and Methods

Animals

Eight week-old adult male Sprague Dawley rats (CDstrain; Charles River) weighing approximately 225–250 gwere provided ad libitum access to food and water, inaccordance with all guidelines and regulations of The Uni-versity of South Carolina Animal Care and Use Commit-tee. Animals were maintained in a temperature-controlledroom, with a light/dark cycle of 12/12 hours (lights on at0700 hours). Rats were thoroughly handled daily for 1week prior to onset of experimentation.

Stress procedures

Animals were divided into four groups: nonstress con-trols (NSC; n � 6), nonstress controls subjected to a 2-houracute restraint stress challenge (NSC � ARS; n � 4), ratssubjected to repeated restraint stress (RRS; n � 5), andanimals subjected to repeated restraint stress and chal-lenged with an acute 2-hour restraint stress session(RRS � ARS; n � 5). RRS was performed as described

previously (Reagan et al., 2004). Briefly, animals wereplaced in flexible wire mesh restrainers with protectiverubberized edges and were restrained for 6 hours/day for10 consecutive days in their home cages. Restrainers werefastened using clips that allowed enough space to compen-sate for slight movements of the animal within the re-straining apparatus. Both the NSC and the NSC � ARSgroups were handled daily during this duration. Followingday 10 of stress or day 10 of handling (day 11), NSC � ARSand RRS � ARS groups were subjected to an acute 2-hourrestraint stress challenge followed by immediate death byisoflurane inhalation and transcardial perfusion withphosphate-buffered saline and 4% paraformaldehyde.Control animals for both groups were also killed on thesame day utilizing the same procedure. All brains wereremoved, cryoprotected, and coronally sectioned (45 �m)on a cryostat.

Rational for selection of stress duration andparadigm

Peak expression of stimulus-induced c-Fos expressionhas been reported to occur approximately 1–3 hours fol-lowing onset of acute stress (Kovacs, 1998). Our initialpilot studies were supportive of these findings, such thatexamination of peak c-Fos expression following variousdurations of ARS challenge was observed at the 2-hourtime point. Based on these findings, as well as other stressstudies using a 2-hour time point (Ryabinin et al., 1995;Chowdhury et al., 2000; Lino-de-Oliveira et al., 2001), a2-hour ARS challenge was chosen to examine the effects ofstress on c-Fos protein expression.

Stress consisting of 6 hours of daily restraint for 10 dayshas been shown to induce a strong trend for enhancedamygdala-dependent behavior in mice (Kim and Han,2006), suggesting that 6 hours of daily restraint stress for10 days represents a critical transitory period duringwhich stress-associated pathological processes are just be-ing initiated. In support of this, a recent study examiningthe effects of various restraint stress durations and timecourses on hippocampal structure and function also con-cluded that 6-hour restraint stress for 10 days representeda potential transitory period during which stress-mediated effects on hippocampal function were just man-ifesting, an observation not noted with 2 hours of dailyrestraint for 10 days and in the absence of hippocampalmorphological changes (McLaughlin et al., 2007). Al-though this study did not examine amygdalar parameters,it is possible that alterations in amygdalar function par-tially contributed to this event, in that evidence suggeststhat the amygdala is capable of modulating hippocampalfunction during stress (Kim et al., 2001, 2005). In light ofthese findings, we postulated that a 6-hour restraintstress for 10 days paradigm may provide critical knowl-edge regarding pathological processes occurring in theamygdala that contribute to the stress-mediated alter-ations amygdalar function and subsequent regulation ofmood.

Immunohistochemistry

Immunohistochemical procedures followed were similarto those previously described (Fadel et al., 2005). Briefly,free-floating sections from a 1:6 series (i.e., every serialsection was 270 �m apart) through the rostral-caudalextent of the BLC were incubated with a rabbit anti-c-Fosantibody [1:15,000; 24 hours at room temperature (RT);

Abbreviations

BLa basolateral amygdaloid nucleus anteriorBLC basolateral amygdalar complexBLP basolateral amygdaloid nucleus posteriorLaVL lateral amygdaloid nucleus ventrolateralLaVM lateral amygdaloid nucleus ventromedialopt optic tractPir piriform cortex

The Journal of Comparative Neurology

459EFFECTS OF STRESS ON BASOLATERAL AMYGDALAR ACTIVATION

Calbiochem, La Jolla, CA; product No. PC38, lot No.D31912]. This incubation was followed by a biotinylateddonkey anti-rabbit secondary (1:1,000; 1.5 hours at RT;Jackson Immunoresearch, West Grove, PA; product No.711-065-152, lot No. 70285) and horseradish peroxidase-conjugated streptavidin (1:1,600; 1 hour at RT; JacksonImmunoresearch; product No. 016-030-084, lot No.70753). Fos immunoreactivity was visualized by develop-ing sections in a nickel sulfate-cobalt chloride-intensifieddiaminobenzidine solution with hydrogen peroxide, yield-ing a blue-black reaction product confined to the nucleusof c-Fos-positive cells. Sections were next incubated at 4°Cfor 48 hours in mouse primary antisera directed againstPV (1:8,000; Sigma, St. Louis, MO; product No. P3088, lotNo. 075K4794), CB-D-28K (1:8,000; Sigma; product No.C9848, lot No. 015K4826), CaMKII � subunit (1:500; Up-state, Lake Placid, NY; product No. 05-532, lot No. 26534),or goat anti-CR (1:8,000; Chemicon, Temecula, CA; prod-uct No. AB1550, lot No. 0603024584), followed by an un-labeled donkey anti-mouse (1:100; Jackson Immunore-search; product No. 715-005-151, lot No. 71333) or donkeyanti-goat secondary antibody (1:100; Jackson Immunore-search; product No. 705-005-003, lot No. 70713) andmouse (1:250; Sternberger Monoclonals, Lutherville, MD;product No. SMI-4050L, lot No. 14833501) or goat perox-idase antiperoxidase (1:250; Sternberger Monoclonals;product No. SMI-4020L, lot No. 53).

All four markers were visualized by developing the sec-tions in a plain diaminobenzidine solution with hydrogenperoxide, resulting in light brown cytoplasmic antigen-specific labeling. After mounting and coverslipping, slideswere viewed with a Nikon E600 microscope equipped witha Photometrics color digital camera (Roper Scientific,Trenton, NJ). Photomicrographic images were capturedwith IP Lab Software. Minor adjustments to color, con-trast, and brightness were made in Adobe Photoshop CS2.

Antisera

The antiserum for c-Fos was raised against a syntheticpeptide corresponding to amino acids 4–17 of humanc-Fos. Western blot analysis from previous characteriza-tion indicated that the antibody recognizes 55-kDa c-Fosand 62-kDa v-Fos proteins but does not cross-react withthe 39-kDa Jun proteins (manufacturer’s technical infor-mation). Additionally, a commercially available goat anti-c-Fos antibody raised against a peptide mapping to resi-dues 3–16 of the amino terminus of human c-Fos (SantaCruz Biotechnology, Santa Cruz, CA; product No. sc-52-G,lot No. 02506) in stress tissues resulted in a stainingpattern identical to that produced by the rabbit anti-c-Fosantibody used in this study (data not shown).

The antiserum for PV was derived from a PARV-19hybridoma produced by fusion of mouse myeloma cells andsplenocytes from an immunized mouse (manufacturer’sinformation). Purified frog muscle parvalbumin was usedas the immunogen. Previous characterization of this anti-body indicated that it reacts with a 12-kDa PV protein, butnot with other members of the calcium-binding proteinfamily such as calmodulin, intestinal calcium-binding pro-tein, S100A2, and calcyclin (manufacturer’s information).The labeling of neuronal bodies produced by this antibodywas consistent with previous studies (Petrie et al., 2005;Woodruff and Sah, 2007) as well as with studies utilizingother PV antisera (McDonald and Betette, 2001).

Similarly, antiserum for CB-D-28K was derived fromthe CB-955 hybridoma produced by the fusion of mousemyeloma cells and splenocytes from the BALB/c immu-nized with a purified bovine kidney CB-D-28K (manufac-turer’s information). Western blot analysis from previouscharacterization indicated that CB-D-28K does not cross-react with other members of the calcium-binding proteinfamily, including CB-D-9K, CR, PV, myosin light chain,S-100A, and calcyclin (manufacturer’s information). Thelabeling of neuronal bodies produced by this antiserumwas consistent with previous studies (Berdel and Morys,2000) as well as with studies utilizing other CB antisera(McDonald, 1997).

The antiserum for CR was raised against recombinantrat full-length protein (manufacturer’s information) andreacts specifically with CR from human, rat, mouse, andguinea pig. Specificity was established by previous West-ern blots and positive controls (information provided byChemicon) and immunohistochemically by adsorptioncontrols in fixed tissue (Winsky and Kuznicki, 1996). Thelabeling of neuronal bodies by this antibody was consis-tent with previous studies (Perrotti et al., 2004; Pinard etal., 2005) as well as studies utilizing other CR antisera(Mascagni and McDonald, 2007).

A partially purified rat CaMKII � subunit clone 6G9served as the immunogen for the CaMKII � subunit anti-serum (manufacturer’s information). Prior characteriza-tion via Western blot analysis demonstrated a high spec-ificity and affinity for the 50-kDa � subunit of CaMKII(manufacturer’s information). The labeling pattern of neu-ronal bodies produced by this antibody was consistentwith previous reports (Erondu and Kennedy, 1985; Mulleret al., 2007) as well as studies utilizing other CaMKIIantisera (McDonald et al., 2002).

Fluoro-Jade (FJ) histochemistry

Fluoro-Jade B (Histo-Chem Inc., Jefferson, AR; productNo. 1FJB, lot No. 53) is an anionic tribasic fluoresceinderivative that specifically identifies neurons actively un-dergoing degeneration (Schmued et al., 1997). Studies ex-amining the specificity and selectivity of FJ in variousneurotoxic insult models indicate that FJ results in astaining pattern corresponding to that observed with sup-pressed silver techniques. These studies also indicatedthat FJ did not stain healthy neurons, myelin, vascularelements, or neuropil (Schmued et al., 1997). We elected touse this marker as a means of assessing potential activeneuronal death following stress conditions. FJ histochem-istry was performed utilizing methods as previously de-scribed (Reagan, 2002; Grillo et al., 2005). Briefly, sectionsencompassing the rostral-caudal extent of the BLC weremounted on gelatin-coated slides in 10 mM PB and al-lowed to dry briefly. Slides were then washed in 100%ethanol for 3 minutes, 95% ethanol for 3 minutes and thenwashed in HPLC-grade H2O for 1 minute. Slides wereincubated in 0.05% KMnO4 for 15 minutes at RT withgentle shaking and then washed in HPLC-grade H2O.Slides were incubated in 0.001% FJ B for 30 minutes atRT with gentle shaking. Slides were washed in HPLC-grade H2O, dried on a slide warmer, treated with xylenes,and coverslipped. Sections encompassing the hippocam-pus of animals subjected to adrenalectomy served as pos-itive controls (Sloviter et al., 1993).


460 L.R. REZNIKOV ET AL.

Cell counts

Double-labeled (PV�c-Fos, CB�c-Fos, CR�c-Fos,CaMKII�c-Fos) and single-labeled (PV, CB, CR, CaMKII,or c-Fos) cell profiles were manually counted by an ob-server blind to treatment groups using a square reticleencompassing an area of 0.1225 mm2 at �20 magnifica-tion. Cell profile counts were performed bilaterally andtargeted toward the medial-caudal extent of the anteriorsubdivision of the basolateral amygdalar nucleus (BLa)corresponding to approximately 2.3–3.30 mm caudal toBregma (Paxinos and Watson, 1998). Systematic randomsampling was used to guide reticle placement within theBLa in which the third section from the most rostral onewhere the BLa first appeared was designated as the initialsection on which to perform counts. Reticle placement wasalso aligned with the ventrolateral edge of the BLa in anonstereotyped manner to ensure random and represen-tative sampling of the total population of BLa neurons.Because immunohistochemistry was performed on a 1:6series (i.e., every serial section was 270 �m apart), thesampling of five sections was possible within the definedlimits of our analysis (Fig. 1). Sampling occurred once,within each hemisphere, at each anatomical level. Initialexamination of tissues revealed no hemispheric differ-ences in stress-mediated c-Fos induction. Therefore, datafrom each hemisphere were averaged to provide a repre-sentative mean per each section examined. All cell countswere corrected using the Ambercrombie correction for-mula (Abercrombie, 1946).

Rationale for restricting cell counts to themedial-caudal BLa

The rat BLC consists of three main nuclei: 1) the lateralnucleus, 2) the basolateral nucleus, and 3) the basomedialnucleus (McDonald and Mascagni, 2001). Each of thesenuclei may be further subdivided into two or more regions.One subdivision that forms a core component of the BLC isthe anterior subdivision of the basolateral amygdalar nu-cleus (BLa). In this study, the BLa was chosen as a regionof interest because of its connections with the striatumand prefrontal cortex (McDonald, 1991), two stress-responsive regions implicated in the etiology of variousmood-related disorders (Kennedy et al., 1997; Soares andMann, 1997; Drevets, 2000; Kristiansen and Meador-Woodruff, 2005; Epstein et al., 2006). Moreover, extensiveliterature regarding the phenotypic characteristics andsynaptic connectivity of BLa neurons exists (McDonald,1982, 1994, 1996, 1997; McDonald and Betette, 2001),thereby providing a solid foundation for interpretation ofsubsequent results.

It has also been suggested that a functional heteroge-neity exists within the amygdala throughout the rostral-caudal extent (Kantak et al., 2002), such that projectionsfrom more rostral amygdalar sites terminate preferen-tially on the rhinal fissure and in the lateral and themedial agranular cortex, whereas those originating frommore caudal sites terminate preferentially in the medialsurface of the frontal cortex (Kita and Kitai, 1990). Con-sidering that the frontal cortex is important for integrat-ing cognitive-affective information and displays alteredactivity and structure in a variety of mood-related disor-ders (Marchand et al., 2005), examination of more caudalamygdalar sites seemed most appropriate for the scope ofthis study.

Data analysis

Double-label data were expressed as percentage of PV�,CB�, CR�, or CaMKII� neurons expressing c-Fos immu-noreactivity and analyzed by using a one-way ANOVA totest for significant main effects as defined by P � 0.05.Single-label counts were also analyzed by one-wayANOVA. Planned comparisons with a Bonferroni post hoctest were then performed on the means contributing to thesignificant one-way ANOVAs. All statistical analyses wereperformed in Statview for Windows (V 5.0.1; Statview,Cary, NC).

Results

Effects of stress on single-labeled c-Fosnuclei in the basolateral amygdala

Fos immunoreactivity was consistent with previousstudies from our laboratory (Pasumarthi et al., 2006), inwhich distinct staining confined to the nucleus was ob-served (Fig. 2A,B). Minimal expression of single-labeledc-Fos was detected under basal conditions (Fig. 2A,C).Subsequent cell count analysis revealed a significant maineffect of treatment condition (F3,16 � 33.530, P � 0.0001).Planned comparisons revealed a significant increase inthe number of single-labeled c-Fos-positive nuclei inNSC � ARS animals compared with NSC animals (Fig.2C), a finding consistent with previous studies(Chowdhury et al., 2000). RRS slightly elevated basalc-Fos expression, although this effect was not statisticallysignificant. Further comparisons between NSC � ARSand RRS � ARS groups revealed that prior RRS signifi-cantly decreased ARS-mediated c-Fos expression, a phe-nomenon also previously reported for the amygdala (Chenand Herbert, 1995; Stamp and Herbert, 2001). Finally, nodifferences were detected among NSC, RRS, and RRS �ARS groups, although a trend for significance was notedbetween NSC and RRS � ARS groups (P � 0.0377) as wellas between RRS and RRS � ARS groups (P � 0.0677).

Phenotypic characterization of BLa neuronsexpressing c-Fos

Parvalbumin. The staining pattern observed in PV�

neurons was consistent with earlier reports (McDonaldand Betette, 2001), such that size and shape fell withinthe previously described ranges (Fig. 3B–D). Tissues fromNSC animals revealed minimal basal expression of c-Fosin PV� neurons (Fig. 3B). Examination of tissues fromanimals subjected to ARS demonstrated that c-Fos expres-sion was increased in PV� neurons (Fig. 3C,D). Subse-quent cell count analysis revealed significant main effectsamong experimental groups (F3,16 � 38.117, P � 0.0001).Further analysis indicated that the expression of c-Fosprotein in PV� neurons increased from roughly 2.5% un-der basal conditions to approximately 20% following anARS challenge, resulting in a significant difference be-tween NSC and NSC � ARS groups (Fig. 4A).

Animals subjected to RRS displayed slightly lower basallevels of c-Fos in comparison with NSC animals (Fig. 4A),although this effect was not significant. However, inRRS � ARS animals, approximately 6% of PV� neuronsexpressed c-Fos, resulting in a significant decrease in com-parison with NSC � ARS groups. Moreover, the degree ofattenuation was so dramatic that no differences were ob-



served among the numbers of double-labeled neuronspresent in NSC, RRS, and RRS � ARS groups, althoughstrong trends for significance were noted between bothNSC and RRS � ARS (P � 0.0696) and RRS and RRS �ARS (P � 0.0131).

Calbindin. Immunoreactivity for CB� neurons re-sulted in a staining pattern that labeled oval to spheroidneurons ranging in size from 10 to 20 �m (Fig. 5B–D), anobservation consistent with previous reports (McDonald,1997). Examination of NSC animals revealed minimalexpression of c-Fos protein in CB� neurons under basalconditions (Fig. 5B). However, in NSC � ARS tissues,c-Fos expression was increased in CB� neurons (Fig.5C,D). Cell count analysis revealed significant main ef-fects among experimental groups (F3,16 � 57.874, P �0.0001). Post hoc comparisons indicated that ARS in-creased c-Fos expression in CB� neurons to approximately24%, resulting in a significant increase in comparison withNSC animals (Fig. 4B).

Further analysis of CB� neurons indicated that basalexpression of c-Fos protein was slightly elevated in RRSanimals in comparison with NSC, although this effect wasnot significant (Fig. 4B). An attenuation of c-Fos expres-sion was also observed in RRS � ARS animals in compar-ison with NSC � ARS, such that only approximately 5% ofCB� neurons expressed c-Fos protein in the RRS � ARSgroup. This was in stark contrast to the amount of double-labeling observed in NSC � ARS; analysis revealed asignificant decrease in the amount double-labeled neuronsbetween NSC � ARS and RRS � ARS groups. Similar toobservations described in PV� neurons, the impact of thisattenuation was great enough that no differences in thenumber of double-labeled neurons were observed amongNSC, RSS, and RRS � ARS groups, although a strongtrend for significance was noted between NSC and RRS �ARS groups (P � 0.0324).

Calretinin. The immunoreactivity for CR� neuronswas consistent with previous reports (McDonald, 1994),such that small nonpyramidal neurons were labeled (Fig.6B–D). No basal expression of c-Fos protein was detectedin CR� neurons (Fig. 6B). Examination of tissues fromNSC � ARS animals also revealed no expression of c-Fosin CR� neurons (Fig. 6C,D). In fact, no expression of c-Fosprotein was detected in CR� neurons following any stresscondition examined in this study.

Calcium/calmodulin-dependent kinase II. The stain-ing pattern observed in CaMKII� neurons was consistentwith that previously described (McDonald et al., 2002).Multipolar neurons of pyramidal or piriform shape weredetected (Fig. 7B–D). The size and density of this popula-tion were also representative of those previously reported.

Examination of NSC tissues revealed minimal c-Fosexpression in CaMKII� neurons (Fig. 7B). Similar to ob-servations in neurons expressing calcium-binding pro-teins, animals subjected to an ARS challenge displayed anincrease in the percentage of CaMKII� neurons express-

Fig. 1. Cell profile count analysis schematic. Depiction of the an-atomical levels of the basolateral amygdalar complex analyzed in thisstudy. All analysis occurred within the anterior subdivision of thebasolateral nucleus (BLa). The shaded box represents the approxi-mate size and location of the sampling area. For abbreviations see list.Reproduced from Paxinos and Watson (1998).



ing c-Fos (Fig. 7C,D). Cell count analysis revealed signif-icant main effects among experimental groups (F3,16 �21.108, P � 0.0001). Subsequent post hoc comparisonsindicated that c-Fos expression increased from 2.4% underbasal conditions to 14.5% following ARS, resulting in asignificant increase in the percent of double-labeled neu-rons between NSC and NSC � ARS groups (Fig. 4C).

Examination of tissues from animals subjected to RRSrevealed no significant differences in the percentage ofCaMKII� neurons expressing c-Fos in comparison withNSC animals (Fig. 4C), indicative that RRS did not resultin increased basal activation of CaMKII� neurons. How-ever, in RRS � ARS animals, a significant decrease inc-Fos expression was observed in CaMKII� neurons com-pared with NSC � ARS animals, such that only 8.7% ofCaMKII� neurons expressed c-Fos protein following thistreatment (Fig. 4C). This habituation effect was not asdramatic as that observed in neurons expressing eitherPV or CB, insofar as significant differences were detectedbetween NSC and RRS � ARS animals as well as betweenRRS and RRS � ARS animals, findings that were notnoted in either the PV� or the CB� populations.

Examination of active neuronal deathfollowing stress

Cell count analysis revealed that changes in ARS-induced c-Fos expression did not result from a decrease inthe number of PV�, CB�, CR�, or CaMKII� neurons inthe BLa following stress conditions (Table 1). Consistentwith this observation, no FJ-positive (FJ�) neurons wereobserved in any of the stress conditions, indicating that noactive neuronal death occurred in the BLa at any of thetime points examined under our experimental conditions(Fig. 8B).

Discussion

The present study indicates that ARS elicits c-Fos ex-pression in PV�, CB�, and CaMKII� neuronal subpopu-lations in the BLa but is without effect on CR� neurons.Prior RRS decreased ARS-mediated activation of thesepopulations, with the greatest effect observed in PV� andCB� populations (Fig. 4D). Collectively, these findingssuggest that ARS and RRS elicit activation of phenotypi-cally distinct BLa populations, a phenomenon that mayhave profound effects on local amygdalar circuitry andsubsequent amygdalar function.

Although numerous studies have utilized induction ofthe immediate early gene c-fos to assess neuronal activa-tion following stressful stimuli (Cullinan et al., 1995;Chowdhury et al., 2000; Day et al., 2005), a few limitationsto this approach exist. The absence of c-Fos protein cannot

Fig. 2. Effects of stress on c-Fos expression. A: Representativephotomicrograph illustrating single-labeled c-Fos expression in thebasolateral amygdala of NSC animals. B: Demonstration of acuterestraint stress-mediated induction of c-Fos in the basolateral amyg-dala of NSC � ARS animals. C: Acute restraint stress resulted in asignificant increase in single-labeled c-Fos expression. This effect wasmarkedly reduced in RRS animals whether or not they were subjectedto an ARS challenge. For A and B, arrowheads represent single-labeled c-Fos nuclei. *P � 0.0083 compared with NSC; #P � 0.0083compared with NSC � ARS. Scale bar � 30 �m.



be explicitly interpreted to indicate lack of activation(Figueiredo et al., 2003). Expression of immediate earlygenes relies on the temporal properties of action poten-tial patterns, so it is possible that longer intervals be-tween bursts may result in minimal expression of suchgenes (Fields et al., 1997). However, despite these lim-itations, c-Fos expression remains a valuable tool withwhich to gain insight into stimulus-induced neural ac-tivation (Figueiredo et al., 2003), especially when con-sidering that not all stressful stimuli result in the samedegree and pattern of c-Fos expression (Herman andCullinan, 1997; Bratincsak and Palkovits, 2004; Day etal., 2005).

ARS-mediated expression of c-Fos:implications for activation of local

amygdalar circuits

Fos expression can be mediated through a number offactors, including increases in intracellular Ca2� (Tsuda,1996), phosphorylation of the cAMP response element-binding protein (CREB; Sassone-Corsi et al., 1988), andstimulation of Ras and mitogen-activated pathways(Herdegen and Leah, 1998). Although the exact mecha-nisms underlying stress-mediated c-Fos expression arenot fully understood, previous work suggests that circa-dian rhythms (Girotti et al., 2007), increases in mitogen-

Fig. 3. Stress-mediated c-Fos expression in parvalbumin neurons.A: Representative photomicrograph illustrating the basolateral amyg-dala. B: Representative labeling of PV� neurons in NSC animals.C: Representative labeling for c-Fos expression in PV� neurons inNSC � ARS animals. D: Higher power magnification of C demonstrat-

ing c-Fos expression in PV� neurons. For B–D, the star representsexamples of single-labeled PV� neurons. Arrows indicate examples ofPV� neurons expressing c-Fos. Arrowheads represent single-labeledc-Fos nuclei. For abbreviations see list. Scale bars � 1.2 mm in A; 30�m in B,C; 15 �m in D.



activated protein kinase (MAPK) activity via N-methyl-D-aspartate (NMDA) glutamate receptor-stimulated Ca2�

influx (Xia et al., 1996), and various actions of stress-related hormones (Funk et al., 2003; Fevurly and Spencer,2004) may in part underlie this event. Therefore, anycombination of these factors may contribute to stress-elicited activation of amygdalar neurons.

In the present study, examination of c-Fos indicatedthat some PV� neurons in the BLa were activated inresponse to ARS. Studies indicate that PV� neurons in theBLa preferentially terminate on neighboring pyramidalneurons (McDonald and Betette, 2001; Muller et al., 2006)and other PV� cells (Muller et al., 2005), suggesting thatactivation of PV� neurons affects the activity of local

amygdalar circuits via modulation of both excitatory andinhibitory transmission. Anatomical and electrophysiolog-ical evidence suggests that PV� neurons of the BLa areassociated with synchronization of the firing activity oflocal pyramidal neurons (Muller et al., 2005, 2006; Rain-nie et al., 2006), an occurrence thought to be essential forconsolidation processes (Pelletier and Pare, 2004) as wellas recall of fear-related memory (Seidenbecher et al.,2003). More recent evidence from Woodruff and Sah(2007) illustrates that electrical coupling occurs preferen-tially between distinct subclasses of PV� interneurons inthe BLC and that action potentials evoked in PV� inter-neurons synapsing onto pyramidal cells result in a time-locked hyperpolarizing response in the postsynaptic prin-

Fig. 4. Quantification of stress-mediated c-Fos expression in baso-lateral amygdalar neurons. A: Acute restraint stress significantlyincreased the percentage PV� neurons expressing c-Fos. Prior RRSlead to a significant decrease in ARS-mediated activation of thisneuronal population. B: A significant increase in c-Fos expression inCB� neurons was observed in response to ARS. However, the percent-age of CB� neurons expressing c-Fos was decreased in animals sub-jected to RRS � ARS. C: Acute restraint stress elicited a significant

increase in c-Fos expression in CaMKII� neurons. This effect wassignificantly decreased in animals subjected to RRS � ARS. D: Bargraph presents c-Fos expression in RRS � ARS groups as a percent-age of initial stress response observed in NSC � ARS animals. Dataindicate that both PV� and CB� populations are more greatly affectedby RRS than are CaMKII� neurons. *P � 0.0083 compared with NSC;#P � 0.0083 compared with NSC � ARS; @P � 0.0083 compared withRRS.



cipal neuron. Collectively, these studies suggest that PV�

cells of the BLC are well-suited to modulate local amyg-dalar activity, including synchronization of principal neu-rons, and, because of this, ARS-mediated activation ofPV� neurons may be important for both consolidation andrecall processes.

The neurons expressing CB represent approximately60% of the total GABAergic interneurons in the BLa (Mc-Donald and Mascagni, 2001; Muller et al., 2003). Studiesindicate that CB� terminals innervate other populationsof BLa interneurons at a frequency of 15%, suggestingthat the primary target of CB� terminals is pyramidalneurons. These data are congruent with the finding that

66% of the CB� neurons in the BLC colocalize with PV(McDonald and Mascagni, 2001). Therefore, consideringthat these two populations greatly overlap, it is likely thatactivation of CB� neurons results in modulation of amyg-dalar circuitry and information processing similar to thatdescribed for PV� neurons. However, roughly 34% of CB�

neurons do not colocalize with PV, and a small percentageinnervates other interneurons. Furthermore, extensive co-localization of the neuropeptide somatostatin (SOM) withCB, but not PV, has been reported in the BLa (McDonaldand Mascagni, 2002). Anatomical evidence indicates thatBLa SOM� neurons project to and interact with excitatoryinputs converging on pyramidal cell distal dendrites (Mul-

Fig. 5. Effects of stress on c-Fos expression in calbindin neurons.A: Representative photomicrograph illustrating the basolateral amyg-dala. B: Representative labeling of CB� neurons in NSC animals.C: Representative labeling for c-Fos expression in CB� neurons inNSC � ARS animals. D: Higher power magnification of C illustrating

c-Fos expression in CB� neurons. For B–D, the star represents exam-ples of single-labeled CB� neurons. Arrows indicate examples of CB�

neurons expressing c-Fos, whereas arrowheads represent examples ofsingle-labeled c-Fos nuclei. For abbreviations see list. Scale bars � 1.2mm in A; 30 �m in B,C; 15 �m in D.



ler et al., 2007), suggesting that this population may playan important role in the regulation of synaptic plasticity.Therefore, further differentiation of which CB� neuronsare activated by ARS is necessary to assess fully thefunction that this population assumes in the stress re-sponse.

The lack of c-Fos expression in CR� neurons could alsoaffect local amygdalar circuitry. Human studies suggestthat CR� neurons of the BLC preferentially synapse ontoCB� neurons (Sorvari et al., 1998), with similar observa-tions noted in the rat hippocampus (Gulyas et al., 1996).Muller and colleagues (2003) have also reported thatmany of the neurons containing vasoactive intestinal pep-

tide (VIP) in the BLC, which display approximately 75%colocalization with CR, innervate CB� neurons. Accord-ingly, a lack of ARS-mediated c-Fos expression in CR�

neurons could indicate an absence of inhibition on CB�

neurons. This assumes that the absence of c-Fos proteinequates to lack of neuronal activation (Figueiredo et al.,2003), which, as discussed previously, is not necessarilytrue. It is possible that the frequency at which CR� neu-rons are stimulated by ARS does not exceed the thresholdof stimulation required for expression of c-Fos protein.

It is also important to note that a lack of c-Fos expres-sion in CR� neurons does not indicate that this populationis incapable of expressing c-Fos. Previous studies demon-

Fig. 6. Lack of c-Fos expression in calretinin neurons. A: Repre-sentative photomicrograph illustrating the basolateral amygdala.B: Representative labeling of CR� neurons in NSC animals. C: Rep-resentative labeling of c-Fos expression in NSC � ARS animals.D: Higher power magnification of C illustrating the lack of c-Fos

expression in CR� cells. For B–D, the star represents examples ofsingle-labeled CR� neurons. Arrowheads represent examples ofsingle-labeled c-Fos nuclei. No arrows are present, because no double-labeling was observed in the BLa. For abbreviations see list. Scalebars � 1.2 mm in A; 30 �m in B,C; 15 �m in D.



strate that blockade of GABAergic transmission in themedial prefrontal cortex elicits c-Fos expression in CR�

neurons of the BLC (Berretta, 2003). Additionally, al-

though it is not known whether CR� neurons in the BLCexpress other immediate early genes in response to RRS,evidence suggests that Fos-B and �FosB are elevated in

TABLE 1. Number of Parvalbumin�, Calbindin�, Calretinin�, and Calcium/Calmodulin-Dependent Kinase II� Neurons per Square Millimeter1

NSC NSC � ARS RRS RRS � ARS ANOVA results

PV 79.3 5.8 84.6 4.9 73.6 6.1 80.6 8.5 F3,16�0.43,P�0.7331CB 90.7 5.6 93.7 3.9 82.7 7.2 96.0 2.7 F3,16�0.66,P�0.5850CR 46.7 1.9 47.7 4.6 46.6 1.8 49.1 2.6 F3,16�0.21,P�0.8904CaMKII 299.8 8.2 301.8 10.9 291.8 10.7 285.2 7.4 F3,16�0.66,P�0.5867

1No differences across experimental stress conditions were observed among the number of neurons for any phenotypic marker. These results indicate that the reduction in thepercentage of neurons expressing c-Fos did not result from a decrease in number of individual neurons (data expressed as number of neurons/mm2 SEM).

Fig. 7. Stress-mediated expression of c-Fos in calcium/calmodulin-dependent kinase neurons. A: Representative photomicrograph illus-trating the basolateral amygdala. B: Representative labeling ofCaMKII� neurons in NSC animals. C: Representative labeling forc-Fos expression in CaMKII� neurons in NSC � ARS animals.

D: Higher power magnification of C demonstrating c-Fos expressionin CaMKII� neurons. For B–D, the star represents examples of single-labeled CaMKII� neurons. Arrows indicate examples of CaMKII�

neurons expressing c-Fos, whereas arrowheads represent examples ofsingle-labeled c-Fos nuclei. For abbreviations see list. Scale bars � 1.2mm in A; 30 �m in B,C; 15 �m in D.



the BLC following similar RRS protocols (Stamp and Her-bert, 2001; Perrotti et al., 2004). Fear stimuli and ARShave also been shown to elevate early-growth response 1gene (egr-1; also called zif268, ngfi-a, krox 24, and tis-8)expression in the rat (Rosen et al., 2005) and mouse (Rya-binin et al., 1999) BLC, respectively, whereas ARS in-creases expression of activity-regulated cytoskeletal-associated protein (Arc) in the rat medial prefrontal cortex(Mikkelsen and Larsen, 2006). Finally, it is possible thatdifferent subdivisions within the BLC other than the BLamay show stress-mediated induction of c-Fos in CR� neu-rons. Thus, although the present data indicate that ARSdoes not elicit c-Fos expression in CR� neurons in theBLa, this cannot be explicitly interpreted to indicate thatCR� neurons are not responsive to stress.

The presumed pyramidal cell population labeled withCaMKII represents approximately 85% of the total neu-rons in the BLa (McDonald, 1992b), most of which projectto the prefrontal cortex and ventral striatum (McDonald,1991, 1996). These connections make the CaMKII� neu-rons appealing to study, in that recent evidence suggeststhat individuals with mood-related disorders display al-tered activity and function of striatal (Drevets, 2000; Kris-tiansen and Meador-Woodruff, 2005; Epstein et al., 2006)and prefrontal cortical areas (Kennedy et al., 1997; Soaresand Mann, 1997). Additionally, the prefrontal cortex playsan important role in the regulation of stress responses(Diorio et al., 1993) and also serves as a target of its effects(Radley et al., 2006). Bearing in mind the proposed asso-ciation of stress and onset or precipitation of mood-relateddisorders (Williamson et al., 2005; Phillips et al., 2006),understanding how stress affects CaMKII� neurons of theBLa is of considerable interest.

Conditioned freezing elicits c-Fos expression inCaMKII� cells in the BLC (Burghardt et al., 2006), afinding that may be interpreted to indicate that activationof CaMKII� neurons is important for fear-related behav-

ior and memory. However, studies suggest that the BLCserves more closely as a critical interface linking emotion-ally relevant stimuli with intentional action vs. mediatingreflexive conditioned responses (Killcross et al., 1997).Therefore, one interpretation of the current study may bethat activation of CaMKII� neurons of the BLa in re-sponse to ARS may be important for fear-related memoryand behavior by mediating the appropriate intentionalaction or behavioral response following a stressful stimu-lus.

Consequences of RRS-mediated decreases inc-Fos expression

Repeated exposure to glucocorticoids (GCs) has beenassociated with attenuated c-Fos expression in a variety ofregions, including the BLC (Stamp and Herbert, 2001).This effect may be explained by the observation that GCreceptors can directly suppress the c-fos promoter (Herd-egen and Leah, 1998). Although it is not known whetherrepeated stress affects GC receptor expression and/orbinding in the BLC, investigation of this relationship mayprovide a potential mechanism for the results that wedescribe below.

In the present study, c-Fos induction in response to anARS challenge was significantly reduced in animals sub-jected to prior RRS, a finding that did not result from aloss of cell bodies as indicated by cell profile count analysis(Table 1) and FJ staining (Fig. 8B). We did not detect anyactive neuronal death at the chosen time points, but it ispossible that FJ� staining could be present at other timepoints not examined. However, were this the case, cellprofile count analysis should reflect a change in the esti-mated number of cell bodies, a phenomenon that we didnot observe.

Previous work suggests that reduced inhibitory neuro-transmission in the BLC is involved in the enhancementof conditioned fear behavior in animals subjected to pre-

Fig. 8. Lack of Fluoro-Jade labeling in the basolateral amygdala following repeated stress. A: FJ�

labeling identifying degenerating dentate gyrus granule neurons in the rat hippocampus 7 days followingadrenalectomy. B: Image of basolateral amygdala illustrating no detection of FJ labeling in RRS animals.Scale bars � 30 �m.



vious stress (Rodriguez Manzanares et al., 2005), whereaselectrophysiological data indicate that repeated adminis-tration of the stress-related hormone corticotropin-releasing factor agonist urocortin leads to hyperexitabilityof the BLC network, a phenomenon attributed to activa-tion of CaMKII� neurons (Rainnie et al., 2004). Our datatend to support these observations, insofar as both PV�

and CB� neurons were more affected in comparison withCaMKII� neurons (see Fig. 4D). Thus, a resulting effectmay include decreased inhibitory tone coupled to in-creased excitatory transmission and, perhaps, chronic ex-citation of CaMKII� neurons. Therefore, these findingsmay provide a mechanism through which repeated stresscan lead to exaggerated amygdala-dependent behaviors(Vyas et al., 2002).

Alternatively, it is interesting to speculate that de-creased expression of c-Fos in BLa interneuronal popula-tions, and specifically in the PV� population, may modu-late synchronization of the firing activity of localpyramidal neurons. Although there are no studies directlyexamining the effects of stress on synchronization of BLCpyramidal firing, it is known that altered synchronizationof ongoing electroencephalograms are observed in patientswith schizophrenia (Winterer et al., 2000) and anxiety(Knyazev et al., 2005). Coincidentally, both schizophreniaand anxiety can be precipitated or provoked by stressfullife events (Williamson et al., 2005; Phillips et al., 2006)and are associated with enhanced amygdalar activity (Et-kin et al., 2004; Taylor et al., 2005).

Prior experiments in cerebellar Purkinje cells indicatedthat deficits in CB and PV resulted in increased neuronalspine length and volume in these cells (Vecellio et al.,2000; Chen et al., 2006). Although we did not find anychanges in the actual number of PV� or CB� neurons inresponse to RRS, it interesting to note that these twopopulations displayed the most profound deficits in c-Fosexpression. Because of this, it is possible that the pre-sumed diminution of inhibitory tone elicited by RRS re-sults in or enables chronic excitation of CaMKII� neurons.We highlight this because previous morphological studiesindicate that type I and type II pyramidal neurons of theBLC display increased spine density and dendritic lengthin response to repeated stress (Vyas et al., 2002). Giventhat many PV�, SOM�/CB�, and VIP�/CR� cells makesynaptic contacts at distal dendrites and spines of pyra-midal neurons (Muller et al., 2003, 2006, 2007), it is in-teresting to speculate that our current findings suggest asynaptic neuronal mechanism that may help to explainrepeated stress-elicited hypertrophy of pyramidal neu-rons.

It has recently been shown that exposure to a singleacute 2-hour immobilization stress (in which the animal iscompletely immobilized) elicits changes in spine density ofBLa neurons 10 days later, a phenomenon that occurs inthe absence of increased dendritic length observed in an-imals subjected to 2 hours of daily immobilization for 10days (Mitra et al., 2005). Significantly, this finding wasassociated with increases in anxiety-like behavior, sug-gesting that a single exposure to acute immobilizationstress results in functional changes that are manifested atlater times. Thus, it is possible that a single 6-hour re-straint stress session could have long-lasting effects onARS-mediated c-Fos expression. However, this assump-tion warrants caution, especially when considering thatthe RRS paradigm used in this study utilizes a different

stress technique, duration, and intensity as well as differ-ent rat strain and age from that described by Mitra andcolleagues. Still, we cannot be certain that 10 days of6-hour daily restraint stress is necessary to attenuateARS-mediated c-Fos expression. We conceive that it ispossible that the initial 6-hour restraint period on day 1 issufficient to trigger a cascade of events that may ulti-mately attenuate ARS-mediated c-Fos expression ob-served 10 days later. Therefore, more extensive studiesexamining the time course necessary for restraint stress-induced cellular changes to occur is an important futuredirection.

Conclusions

The results of the current study support the hypothesisthat subpopulations of BLC neurons differentially re-spond to acute and repeated stress. The pattern of c-Fosexpression elicited by RRS is intriguing, in that it mayidentify potential cellular targets involved in stress-mediated alterations of amygdalar behavior and morphol-ogy. Although the specific roles that PV�, CB�, CR�, orCaMKII� neurons of the BLC play in mediating stressresponses remain to be fully elucidated, a simple explana-tion is that activation (or lack there of) is important forcontrolling the flow on information into and out of theamygdala (Davis et al., 1994), which in turn influences thegeneration of an emotional response and fear-related be-havior and memory. Therefore, further studies are neces-sary to explore more thoroughly the potential roles thatthese subpopulations might have in modulating amyg-dalar function, especially as it relates to stress.

ACKNOWLEDGMENTS

The authors thank Dr. Alex McDonald for his helpfuladvice and suggestions regarding the final preparation ofthe manuscript. The authors also thank Dr. Gerardo Piroliand Dr. Claudia Grillo for helpful suggestions and com-ments and Dani Frederick-Duus for excellent technicalassistance.

LITERATURE CITED

Abercrombie M. 1946. Estimation of nuclear population from microtomesections. Anat Rec 239–247.

Berdel B, Morys J. 2000. Expression of calbindin-D28k and parvalbuminduring development of rat’s basolateral amygdaloid complex. Int J DevNeurosci 18:501–513.

Berretta S. 2003. Local release of GABAergic inhibition in the medialprefrontal cortex induces immediate-early genes in selective neuronalsubpopulations in the amygdala. Ann N Y Acad Sci 985:505–507.

Braga MF, Aroniadou-Anderjaska V, Post RM, Li H. 2002. Lamotriginereduces spontaneous and evoked GABAA receptor-mediated synaptictransmission in the basolateral amygdala: implications for its effects inseizure and affective disorders. Neuropharmacology 42:522–529.

Brambilla P, Harenski K, Nicoletti M, Sassi RB, Mallinger AG, Frank E,Kupfer DJ, Keshavan MS, Soares JC. 2003. MRI investigation of tem-poral lobe structures in bipolar patients. J Psychiatr Res 37:287–295.

Bratincsak A, Palkovits M. 2004. Activation of brain areas in rat followingwarm and cold ambient exposure. Neuroscience 127:385–397.

Burghardt PR, Pasumarthi RK, Wilson MA, Fadel J. 2006. Alterations infear conditioning and amygdalar activation following chronic wheelrunning in rats. Pharmacol Biochem Behav 84:306–312.

Carlsen J. 1988. Immunocytochemical localization of glutamate decarbox-ylase in the rat basolateral amygdaloid nucleus, with special referenceto GABAergic innervation of amygdalostriatal projection neurons.J Comp Neurol 273:513–526.



Celio MR. 1990. Calbindin D-28k and parvalbumin in the rat nervoussystem. Neuroscience 35:375–475.

Chen G, Racay P, Bichet S, Celio MR, Eggli P, Schwaller B. 2006. Defi-ciency in parvalbumin, but not in calbindin D-28k upregulates mito-chondrial volume and decreases smooth endoplasmic reticulum surfaceselectively in a peripheral, subplasmalemmal region in the soma ofPurkinje cells. Neuroscience 142:97–105.

Chen X, Herbert J. 1995. Regional changes in c-fos expression in the basalforebrain and brainstem during adaptation to repeated stress: correla-tions with cardiovascular, hypothermic and endocrine responses. Neu-roscience 64:675–685.

Chowdhury GM, Fujioka T, Nakamura S. 2000. Induction and adaptationof Fos expression in the rat brain by two types of acute restraint stress.Brain Res Bull 52:171–182.

Cullinan WE, Herman JP, Battaglia DF, Akil H, Watson SJ. 1995. Patternand time course of immediate early gene expression in rat brain fol-lowing acute stress. Neuroscience 64:477–505.

Davis M, Rainnie D, Cassell M. 1994. Neurotransmission in the rat amyg-dala related to fear and anxiety. Trends Neurosci 17:208–214.

Day HE, Nebel S, Sasse S, Campeau S. 2005. Inhibition of the centralextended amygdala by loud noise and restraint stress. Eur J Neurosci21:441–454.

Diorio D, Viau V, Meaney MJ. 1993. The role of the medial prefrontalcortex (cingulate gyrus) in the regulation of hypothalamic-pituitary-adrenal responses to stress. J Neurosci 13:3839–3847.

Donley MP, Schulkin J, Rosen JB. 2005. Glucocorticoid receptor antago-nism in the basolateral amygdala and ventral hippocampus interfereswith long-term memory of contextual fear. Behav Brain Res 164:197–205.

Drevets WC. 1999. Prefrontal cortical-amygdalar metabolism in majordepression. Ann N Y Acad Sci 877:614–637.

Drevets WC. 2000. Neuroimaging studies of mood disorders. Biol Psychi-atry 48:813–829.

Epstein J, Pan H, Kocsis JH, Yang Y, Butler T, Chusid J, Hochberg H,Murrough J, Strohmayer E, Stern E, Silbersweig DA. 2006. Lack ofventral striatal response to positive stimuli in depressed vs. normalsubjects. Am J Psychiatry 163:1784–1790.

Erondu NE, Kennedy MB. 1985. Regional distribution of type II Ca2�/calmodulin-dependent protein kinase in rat brain. J Neurosci 5:3270–3277.

Etkin A, Klemenhagen KC, Dudman JT, Rogan MT, Hen R, Kandel ER,Hirsch J. 2004. Individual differences in trait anxiety predict the re-sponse of the basolateral amygdala to unconsciously processed fearfulfaces. Neuron 44:1043–1055.

Fadel J, Pasumarthi R, Reznikov L. 2005. Stimulation of cortical acetyl-choline release by orexin A. Neuroscience 130:541–547.

Fevurly RD, Spencer RL. 2004. Fos expression is selectively and differen-tially regulated by endogenous glucocorticoids in the paraventricularnucleus of the hypothalamus and the dentate gyrus. J Neuroendocrinol16:970–979.

Fields RD, Eshete F, Stevens B, Itoh K. 1997. Action potential-dependentregulation of gene expression: temporal specificity in ca2�, cAMP-responsive element binding proteins, and mitogen-activated proteinkinase signaling. J Neurosci 17:7252–7266.

Figueiredo HF, Bruestle A, Bodie B, Dolgas CM, Herman JP. 2003. Themedial prefrontal cortex differentially regulates stress-induced c-fosexpression in the forebrain depending on type of stressor. Eur J Neu-rosci 18:2357–2364.

Frodl T, Meisenzahl E, Zetzsche T, Bottlender R, Born C, Groll C, Jager M,Leinsinger G, Hahn K, Moller HJ. 2002. Enlargement of the amygdalain patients with a first episode of major depression. Biol Psychiatry51:708–714.

Funk D, Li Z, Shaham Y, Le AD. 2003. Effect of blockade of corticotropin-releasing factor receptors in the median raphe nucleus on stress-induced c-fos mRNA in the rat brain. Neuroscience 122:1–4.

Girotti M, Weinberg MS, Spencer RL. 2007. Differential responses of HPAaxis immediate early genes to corticosterone and circadian drive. En-docrinology (in press).

Grillo CA, Piroli GG, Wood GE, Reznikov LR, McEwen BS, Reagan LP.2005. Immunocytochemical analysis of synaptic proteins provides newinsights into diabetes-mediated plasticity in the rat hippocampus. Neu-roscience 136:477–486.

Gulyas AI, Hajos N, Freund TF. 1996. Interneurons containing calretininare specialized to control other interneurons in the rat hippocampus.J Neurosci 16:3397–3411.

Herdegen T, Leah JD. 1998. Inducible and constitutive transcription fac-tors in the mammalian nervous system: control of gene expression byJun, Fos and Krox, and CREB/ATF proteins. Brain Res Brain Res Rev28:370–490.

Herman JP, Cullinan WE. 1997. Neurocircuitry of stress: central control ofthe hypothalamo-pituitary-adrenocortical axis. Trends Neurosci 20:78–84.

Hull AM. 2002. Neuroimaging findings in post-traumatic stress disorder.Systematic review. Br J Psychiatry 181:102–110.

Kantak KM, Black Y, Valencia E, Green-Jordan K, Eichenbaum HB. 2002.Dissociable effects of lidocaine inactivation of the rostral and caudalbasolateral amygdala on the maintenance and reinstatement ofcocaine-seeking behavior in rats. J Neurosci 22:1126–1136.

Kennedy SH, Javanmard M, Vaccarino FJ. 1997. A review of functionalneuroimaging in mood disorders: positron emission tomography anddepression. Can J Psychiatry 42:467–475.

Killcross S, Robbins TW, Everitt BJ. 1997. Different types of fear-conditioned behaviour mediated by separate nuclei within amygdala.Nature 388:377–380.

Kim JJ, Lee HJ, Han JS, Packard MG. 2001. Amygdala is critical forstress-induced modulation of hippocampal long-term potentiation andlearning. J Neurosci 21:5222–5228.

Kim JJ, Koo JW, Lee HJ, Han JS. 2005. Amygdalar inactivation blocksstress-induced impairments in hippocampal long-term potentiationand spatial memory. J Neurosci 25:1532–1539.

Kim KS, Han PL. 2006. Optimization of chronic stress paradigms usinganxiety- and depression-like behavioral parameters. J Neurosci Res83:497–507.

Kita H, Kitai ST. 1990. Amygdaloid projections to the frontal cortex andthe striatum in the rat. J Comp Neurol 298:40–49.

Knyazev GG, Savostyanov AN, Levin EA. 2005. Anxiety and synchrony ofalpha oscillations. Int J Psychophysiol 57:175–180.

Kovacs KJ. 1998. c-Fos as a transcription factor: a stressful (re)view froma functional map. Neurochem Int 33:287–297.

Kristiansen LV, Meador-Woodruff JH. 2005. Abnormal striatal expressionof transcripts encoding NMDA interacting PSD proteins in schizophre-nia, bipolar disorder and major depression. Schizophr Res 78:87–93.

Lino-de-Oliveira C, Sales AJ, Del Bel EA, Silveira MC, Guimaraes FS.2001. Effects of acute and chronic fluoxetine treatments on restraintstress-induced Fos expression. Brain Res Bull 55:747–754.

Marchand W, Dilda V, Jensen CR. 2005. Neurobiology of mood disorders.Hosp Physician 43:17–26.

Mascagni F, McDonald AJ. 2007. A novel subpopulation of 5-HT type 3Areceptor subunit immunoreactive interneurons in the rat basolateralamygdala. Neuroscience 144:1015–1024.

McDonald AJ. 1982. Neurons of the lateral and basolateral amygdaloidnuclei: a Golgi study in the rat. J Comp Neurol 212:293–312.

McDonald AJ. 1991. Organization of amygdaloid projections to the prefron-tal cortex and associated striatum in the rat. Neuroscience 44:1–14.

McDonald AJ. 1992a. Cell types and intrinsic connections of the amygdala.In Aggleton JP, editor. The amygdala. New York: Wiley-Liss. p 67–96.

McDonald AJ. 1992b. Projection neurons of the basolateral amygdala: acorrelative Golgi and retrograde tract tracing study. Brain Res Bull28:179–185.

McDonald AJ. 1994. Calretinin immunoreactive neurons in the basolateralamygdala of the rat and monkey. Brain Res 667:238–242.

McDonald AJ. 1996. Glutamate and aspartate immunoreactive neurons ofthe rat basolateral amygdala: colocalization of excitatory amino acidsand projections to the limbic circuit. J Comp Neurol 365:367–379.

McDonald AJ. 1997. Calbindin-D28k immunoreactivity in the rat amyg-dala. J Comp Neurol 383:231–244.

McDonald AJ, Betette RL. 2001. Parvalbumin-containing neurons in therat basolateral amygdala: morphology and co-localization of calbindin-D(28k). Neuroscience 102:413–425.

McDonald AJ, Mascagni F. 2001. Colocalization of calcium-binding pro-teins and GABA in neurons of the rat basolateral amygdala. Neuro-science 105:681–693.

McDonald AJ, Mascagni F. 2002. Immunohistochemical characterizationof somatostatin containing interneurons in the rat basolateral amyg-dala. Brain Res 943:237–244.

McDonald AJ, Muller JF, Mascagni F. 2002. GABAergic innervation ofalpha type II calcium/calmodulin-dependent protein kinase immunore-active pyramidal neurons in the rat basolateral amygdala. J CompNeurol 446:199–218.



McLaughlin KJ, Gomez JL, Baran SE, Conrad CD. 2007. The effects ofchronic stress on hippocampal morphology and function: an evaluationof chronic restraint paradigms. Brain Res (in press).

Mikkelsen JD, Larsen MH. 2006. Effects of stress and adrenalectomy onactivity-regulated cytoskeleton protein (Arc) gene expression. NeurosciLett 403:239–243.

Mitra R, Jadhav S, McEwen BS, Vyas A, Chattarji S. 2005. Stress durationmodulates the spatiotemporal patterns of spine formation in the baso-lateral amygdala. Proc Natl Acad Sci U S A 102:9371–9376.

Muller JF, Mascagni F, McDonald AJ. 2003. Synaptic connections of dis-tinct interneuronal subpopulations in the rat basolateral amygdalarnucleus. J Comp Neurol 456:217–236.

Muller JF, Mascagni F, McDonald AJ. 2005. Coupled networks ofparvalbumin-immunoreactive interneurons in the rat basolateralamygdala. J Neurosci 25:7366–7376.

Muller JF, Mascagni F, McDonald AJ. 2006. Pyramidal cells of the ratbasolateral amygdala: synaptology and innervation by parvalbumin-immunoreactive interneurons. J Comp Neurol 494:635–650.

Muller JF, Mascagni F, McDonald AJ. 2007. Postsynaptic targets ofsomatostatin-containing interneurons in the rat basolateral amygdala.J Comp Neurol 500:513–529.

Pare D. 2003. Role of the basolateral amygdala in memory consolidation.Prog Neurobiol 70:409–420.

Pasumarthi RK, Reznikov LR, Fadel J. 2006. Activation of orexin neuronsby acute nicotine. Eur J Pharmacol 535:172–176.

Paxinos G, Watson C. 1998. The rat brain in sterotaxic coordinates. SanDiego: Academic Press.

Pelletier JG, Pare D. 2004. Role of amygdala oscillations in the consolida-tion of emotional memories. Biol Psychiatry 55:559–562.

Perrotti LI, Hadeishi Y, Ulery PG, Barrot M, Monteggia L, Duman RS,Nestler EJ. 2004. Induction of deltaFosB in reward-related brain struc-tures after chronic stress. J Neurosci 24:10594–10602.

Petrie KA, Schmidt D, Bubser M, Fadel J, Carraway RE, Deutch AY. 2005.Neurotensin activates GABAergic interneurons in the prefrontal cor-tex. J Neurosci 25:1629–1636.

Phillips LJ, Francey SM, Edwards J, McMurray N. 2006. Stress andpsychosis: Toward the development of new models of investigation.Clin Psychol Rev (in press).

Pinard CR, Mascagni F, McDonald AJ. 2005. Neuronal localization ofCav1.2 L-type calcium channels in the rat basolateral amygdala. BrainRes 1064:52–55.

Radley JJ, Rocher AB, Miller M, Janssen WG, Liston C, Hof PR, McEwenBS, Morrison JH. 2006. Repeated stress induces dendritic spine loss inthe rat medial prefrontal cortex. Cereb Cortex 16:313–320.

Rainnie DG, Bergeron R, Sajdyk TJ, Patil M, Gehlert DR, Shekhar A. 2004.Corticotrophin releasing factor-induced synaptic plasticity in theamygdala translates stress into emotional disorders. J Neurosci 24:3471–3479.

Rainnie DG, Mania I, Mascagni F, McDonald AJ. 2006. Physiological andmorphological characterization of parvalbumin-containing interneu-rons of the rat basolateral amygdala. J Comp Neurol 498:142–161.

Reagan LP. 2002. Glucose, stress and hippocampal neuronal vulnerability.Int Rev Neurobiol 51:289–324.

Reagan LP, Rosell DR, Wood GE, Spedding M, Munoz C, Rothstein J,McEwen BS. 2004. Chronic restraint stress up-regulates GLT-1 mRNAand protein expression in the rat hippocampus: reversal by tianeptine.Proc Natl Acad Sci U S A 101:2179–2184.

Rodriguez Manzanares PA, Isoardi NA, Carrer HF, Molina VA. 2005.Previous stress facilitates fear memory, attenuates GABAergic inhibi-tion, and increases synaptic plasticity in the rat basolateral amygdala.J Neurosci 25:8725–8734.

Rosen JB, Adamec RE, Thompson BL. 2005. Expression of egr-1 (zif268)

mRNA in select fear-related brain regions following exposure to apredator. Behav Brain Res 162:279–288.

Ryabinin AE, Melia KR, Cole M, Bloom FE, Wilson MC. 1995. Alcoholselectively attenuates stress-induced c-fos expression in rat hippocam-pus. J Neurosci 15:721–730.

Ryabinin AE, Wang YM, Freeman P, Risinger FO. 1999. Selective effects ofalcohol drinking on restraint-induced expression of immediate earlygenes in mouse brain. Alcohol Clin Exp Res 23:1272–1280.

Sassone-Corsi P, Visvader J, Ferland L, Mellon PL, Verma IM. 1988.Induction of proto-oncogene fos transcription through the adenylatecyclase pathway: characterization of a cAMP-responsive element.Genes Dev 2:1529–1538.

Schmued LC, Albertson C, Slikker W Jr. 1997. Fluoro-Jade: a novel fluo-rochrome for the sensitive and reliable histochemical localization ofneuronal degeneration. Brain Res 751:37–46.

Seidenbecher T, Laxmi TR, Stork O, Pape HC. 2003. Amygdalar andhippocampal theta rhythm synchronization during fear memory re-trieval. Science 301:846–850.

Sloviter RS, Sollas AL, Dean E, Neurbort S. 1993. Adrenalectomy-inducedgranule cell degeneration in the rat hippocampal dentate gyrus: char-acterization of an in vivo model of controlled neuronal death. J CompNeurol 330:324–336.

Smith Y, Pare JF, Pare D. 2000. Differential innervation of parvalbumin-immunoreactive interneurons of the basolateral amygdaloid complexby cortical and intrinsic inputs. J Comp Neurol 416:496–508.

Soares JC, Mann JJ. 1997. The functional neuroanatomy of mood disor-ders. J Psychiatr Res 31:393–432.

Sorvari H, Miettinen R, Soininen H, Paljarvi L, Karkola K, Pitkanen A.1998. Calretinin-immunoreactive terminals make synapses on cal-bindin D28k-immunoreactive neurons in the lateral nucleus of thehuman amygdala. Brain Res 783:355–358.

Stamp J, Herbert J. 2001. Corticosterone modulates autonomic responsesand adaptation of central immediate-early gene expression to repeatedrestraint stress. Neuroscience 107:465–479.

Taylor SF, Phan KL, Britton JC, Liberzon I. 2005. Neural response toemotional salience in schizophrenia. Neuropsychopharmacology 30:984–995.

Thomas KM, Drevets WC, Dahl RE, Ryan ND, Birmaher B, Eccard CH,Axelson D, Whalen PJ, Casey BJ. 2001. Amygdala response to fearfulfaces in anxious and depressed children. Arch Gen Psychiatry 58:1057–1063.

Tsuda M. 1996. Cascade of gene expression induced by Ca2� signals inneurons. Neurochem Int 29:443–451.

Vecellio M, Schwaller B, Meyer M, Hunziker W, Celio MR. 2000. Alter-ations in Purkinje cell spines of calbindin D-28 k and parvalbuminknock-out mice. Eur J Neurosci 12:945–954.

Vyas A, Mitra R, Shankaranarayana Rao BS, Chattarji S. 2002. Chronicstress induces contrasting patterns of dendritic remodeling in hip-pocampal and amygdaloid neurons. J Neurosci 22:6810–6818.

Williamson DE, Birmaher B, Dahl RE, Ryan ND. 2005. Stressful lifeevents in anxious and depressed children. J Child Adolesc Psychophar-macol 15:571–580.

Winsky L, Kuznicki J. 1996. Antibody recognition of calcium-binding pro-teins depends on their calcium-binding status. J Neurochem 66:764–771.

Winterer G, Ziller M, Dorn H, Frick K, Mulert C, Wuebben Y, HerrmannWM, Coppola R. 2000. Schizophrenia: reduced signal-to-noise ratio andimpaired phase-locking during information processing. Clin Neuro-physiol 111:837–849.

Woodruff AR, Sah P. 2007. Networks of parvalbumin-positive interneuronsin the basolateral amygdala. J Neurosci 27:553–563.

Xia Z, Dudek H, Miranti CK, Greenberg ME. 1996. Calcium influx via theNMDA receptor induces immediate early gene transcription by a MAPkinase/ERK-dependent mechanism. J Neurosci 16:5425–5436.



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