Role of the amygdalo-hippocampal transition area in the fear expression: evaluation by behavior and immediate early gene expression

Neuroscience 124 (2004) 247–260

ROLE OF THE AMYGDALO-HIPPOCAMPAL TRANSITION AREA IN THE FEAR EXPRESSION: EVALUATION BY BEHAVIOR AND IMMEDIATE EARLY GENE EXPRESSION M. FUJISAKI,a,b* T. CHIBAa

K.

HASHIMOTO,b

M.

IYOb

AND

of the BM, the number of Egr-1 immunoreactive-positive cells was increased in both experiments, and it was possible that the activation of neurons with high basal levels of expression might be associated with memory retrieval or expression as a freezing behavior observed in the test session. © 2004 IBRO. Published by Elsevier Ltd. All rights reserved.

a Department of Anatomy and Neurobiology, Graduate School of Medicine, Chiba University, Chiba, Japan b Department of Psychiatry (K2), Graduate School of Medicine, Chiba University 1-8-1 Inohana, Chiba 260-8670, Japan

Key words: fear conditioning, freezing behavior, c-Fos, Egr-1, learning, memory.

Abstract—Using pre- and post-training lesions of the amygdalo-hippocampal transition area (AHi), the role of the AHi in the fear conditioning of rats was examined. Pretraining lesions by N-methyl-D-aspartate led to the enhancement of freezing behavior in auditory fear conditioning and contextual conditioning. However, the freezing of post-training-lesioned rats did not differ from that of the sham-lesioned rats. There were several regions of the brain observed in this study in which c-Fos and/or Egr-1 immunoreactive-positive cell expression changed in diverse manners after the test session. In the pretraining lesioned rats that were trained for auditory conditioning, the number of c-Fos and Egr-1 decreased in the infralimbic cortex (IL) and the number of Egr-1 increased in the basomedial amygdaloid nucleus (BM). In the pretraining AHi-lesioned rats that were trained for contextual conditioning, the number of c-Fos increased in the lateral periaqueductal gray (LPAG) and the number of Egr-1 increased in the BM. These results suggest that the AHi plays an important role in the acquisition of memory during conditioning alone, whereas it is improbable that the AHi had an effect on consolidation, retrieval, and expression in the case of either auditory or contextual fear conditioning. The findings also suggest that the freezing behavior was related to the changes in c-Fos and/or Egr-1 in the IL, BM, and LPAG. As in the case

Fear conditioning has been used as an animal model of fear and anxiety in numerous experiments (LeDoux, 1998, 2000; Zinebi et al., 2002). The amygdala is known to play a central role in fear and anxiety (Nishijo et al., 1988a,b; Muramoto et al., 1993; Uwano et al., 1995; Oakes and Coover, 1997). Conditioned fear depends strongly on the basolateral nucleus group of the amygdala (the lateral [La), basolateral [BL), and central [Ce) amygdaloid nucleus) is thought to be the principal output structures, mediating fear-related behavioral response (LeDoux, 1998, 2000; Davis and Whalen, 2001; Maren, 2001). The hippocampus also plays a crucial role in contextual conditioning (LeDoux, 1996). The hippocampal lesion selectively eliminated fear responses elicited by contextual stimuli without affecting fear responses elicited by a tone. One explanation for this would be that the tone stimulus reached the amygdala directly. The animals with hippocampal lesions were unable to form the contextual representation to be sent to the amygdala. In further support of this hypothesis, amygdala damage was shown to similarly interfere with both contextual conditioning and tone conditioning. These findings suggest that the hippocampus might be not required for auditory fear conditioning but for contextual conditioning and the amygdala appears to be necessary for both auditory fear conditioning and contextual conditioning. Crosby and Humphrey (1944) initially called particular attention to a relatively small band of neurons just deep to the angular bundle in the furthest caudal part of the amygdala of the shrew; they referred to this bundle as the corticoamygdaloid transition area. This structure is currently referred to as the amygdalo-hippocampal transition area (AHi) or as the posterior nucleus of the amygdala (Canteras et al., 1992). Canteras et al. (1992) reported the afferent connections with the cortical amygdaloid nucleus (Co), medial amygdaloid nucleus (Me), BL, La, Ce, and the ventral parts of hippocampal field CA1, etc., and the efferent connections with the Co, Me, basomedial amygdaloid nucleus (BM), BL, Ce, the ventral part of field CA1, the

*Correspondence to: M. Fujisaki, Department of Psychiatry (K2), Graduate School of Medicine, Chiba University 1-8-1 Inohana, Chiba 260-8670, Japan. Tel: ⫹81-43-226-2149; fax: ⫹81-43-226-2150. E-mail address: [email protected] (M. Fujisaki). Abbreviations: AcbC, accumbens nucleus core; AcbSh, accumbens nucleus shell; AHi, amygdalo-hippocampal transition area; ANOVA, analysis of variance; AP-1, activator protein-1; BL, basolateral amygdaloid nucleus; BM, basomedial amygdaloid nucleus; Ce, central amygdaloid nucleus; Co, cortical amygdaloid nucleus; CTX, contextual; DAB, 3,3⬘-diaminobenzidine; DLPAG, dorsolateral periaqueductal gray; DMPAG, dorsomedial periaqueductal gray; IEGs, immediate early genes; IL, infralimbic cortex; ITI, intertrial interval; La, lateral amygdaloid nucleus; LPAG, lateral periaqueductal gray; Me, medial amygdaloid nucleus; mPFC, medial prefrontal cortex; NMDA, N-methyl-D-aspartate; PAG, periaqueductal gray; PBS, phosphatebuffered saline; post-AHi/CTX, post-training AHi lesion/contextual conditioning; post-AHi/Tone, post-training AHi lesion/auditory fear conditioning; post-Sh/CTX, post-training sham lesion/contextual conditioning; post-Sh/Tone, post-training sham lesion/auditory fear conditioning; pre-AHi/CTX, pretraining AHi lesion/contextual conditioning; pre-AHi/Tone, pretraining AHi lesion/auditory fear conditioning; pre-Sh/CTX, pretraining sham lesion/contextual conditioning; pre-Sh/Tone, pretraining sham lesion/auditory fear conditioning; PrL, prelimbic cortex; TB, Tris buffer; VLPAG, ventrolateral periaqueductal gray.

0306-4522/04$30.00⫹0.00 © 2004 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2003.11.022

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nucleus accumbens, and the infralimbic cortex (IL), etc. We previously reported that the AHi sends axon collateral projections to both the hypothalamic nuclei and the prefrontal cortex, although little is known as regards the function of the AHi (Chiba, 2000). At present, it remains unknown how the AHi influences fear, anxiety, and the activity of the amygdala. Based on the role of the amygdala in the fear and anxiety, it is, therefore, of great interest to study the role of the AHi in fear conditioning experiments. The immediate early genes (IEGs), including c-Fos and Egr-1, have been used as markers of neural activity associated with conditioned fear (Pezzone et al., 1992; Beck and Fibiger, 1995a,b). IEG-encoded transcription factors are activator protein-1 (AP-1), composed of members of the fos/jun families and Egr-1 (early growth response-1; also referred to as Zif268, NGFI-A [nerve growth factor-induced clone A], TIS8, and Krox24). The members of the AP-1 and Egr transcription factor families possess different structural properties and may be subjected to different regulatory mechanisms. Whereas Fos protein has low basal levels of expression, Egr-1 protein is constitutively expressed at high basal levels in several brain regions. In fear conditioning, Fos and Egr-1 may reflect different representations. Therefore, the present study was undertaken in order to examine the role of the AHi in auditory and contextual fear conditioning models using behavioral evaluation and immunohistochemical analysis of IEGs, c-Fos and Egr-1. At pretraining or post-training, rats were lesioned on both sides of the AHi, and changes in freezing behavior and IEGs were investigated.

EXPERIMENTAL PROCEDURES Subjects The protocols for animal procedures in these experiments received approval from the ethics committees of our institutions following National Institute of Health guidelines for the Care and Use of Laboratory Animals 1996 revision and all efforts were made to minimize animal suffering and limit the number of animals used. Seventy-two male Sprague–Dawley rats (200 –260 g; Saitama Exp. Animal Supply Co., Ltd., Saitama, Japan) were used. Each group had six rats. All animals were individually caged in a temperature-controlled room (21 °C) with a 12-h light/dark cycle (lights on between 09:00 and 21:00 h). Food and water were available ad libitum. All experiments were conducted during the light phase of the cycle. After being housed, the rats were handled daily for 7 days to acclimate them to the researchers. All experimental procedures with animals were approved by the Animal Care Committee of the Chiba University Graduate School of Medicine.

Surgery procedures To study the function of the AHi, we lesioned the AHi itself. As the bilateral amygdala connects and interacts, we lesioned both sides. As even passage fiber is impaired by electrical lesion, N-methylD-aspartate (NMDA) neurotoxin was selected. NMDA neurotoxin produces a potent depolarization of the neuron through the glutamic acid receptor and, as a result, the neuron degenerates. Rats were anesthetized with an i.p. injection of Nembutal (sodium pentobarbital; 30 mg/kg body weight), and mounted in a stereotaxic apparatus (Narishige Co., Tokyo, Japan). The scalp was incised and retracted, and the head position was adjusted to place

the bregma and lambda in the same horizontal plane. Small burr holes (2 mm diameter) were drilled bilaterally with a dental drill in the skull for the placement of a 30-gauge cannula in the AHi. A 10 ␮l Hamilton syringe (Hamilton Company, Reno, NV, USA) was mounted on a microinjection pump (KDS 101, Linton Instrumentation, Norfolk, UK) and connected to the injection cannula with polyethylene tubing. NMDA (Sigma, St. Louis, MO, USA) was dissolved in 0.01 M phosphate-buffered saline (PBS; pH 7.4), to a final pH of 7.4, at a concentration of 20 ␮g/␮l, and was delivered at an infusion rate of 0.05 ␮l/min via an injection cannula. An infusion volume of 0.10 ␮l NMDA was delivered at depths of 8.0 mm below the skull surface at 4.3 mm posterior to the bregma and 4.0 mm lateral to the midline (Pezzone et al., 1992). After each infusion, 5 min were allowed for diffusion of the drug. Sham lesioning was performed using PBS infusions at the same coordinates and delivery volumes.

Fear conditioning apparatus Training and test sessions took place in two modular operant test chambers, each equipped with speaker modules (ENV-225S; MED Associates, Inc., GA, VT, USA), located in a controlled acoustic room. The two chambers differed in several regards: chamber A was rectangular (24 cm width⫻30.5 cm length⫻29 cm height; ENV-007CT; MED Associates, Inc.), whereas chamber B was octagonal (26.5 cm diameter⫻25 cm height). Chamber A had front and back walls made of clear Plexiglas and two side walls made of stainless steel plates, whereas in chamber B, all eight walls were constructed of clear Plexiglas. Finally, chamber A was placed in a wooden isolation box (55.9 cm width⫻35.6 cm length⫻55.9 cm height; ENV-018; MED Associates, Inc.) that was painted white, whereas chamber B was placed in a similar box that was painted black. The grid floor of chamber A was composed of 19 stainless steel bars (4.8 mm in diameter) spaced 16.0 mm center-to-center and wired to an electric shock generator (ENV411; MED Associates, Inc.). The floor of chamber B was composed of newspapers. The floor grid and base pan of each chamber were washed thoroughly with tap water and dried completely before the training and test sessions.

Conditioning and test session procedures This study relied on previously reported methods of conditioning (Lee et al., 2001). The experimental design used in this study is described below and presented schematically in Fig. 1. The behavior of the rats was recorded by videotape recorder with an infrared camera.

Experiment 1: pretraining AHi lesions and auditory fear conditioning To test the effects of pretraining AHi lesions on stress responses to auditory fear conditioning, animals were prepared with sham or excitotoxic AHi lesions. These groups were subdivided further into pretraining sham lesion/auditory fear conditioning (pre-Sh/Tone) and pretraining AHi lesion/auditory fear conditioning (pre-AHi/ Tone) groups. After a 2-week recovery from surgery, the animals were conditioned. The experiments took place over the course of 2 consecutive days. On day 1, rats were placed in chamber A. The chamber A cages were wiped with 5% ammonium hydroxide solution, and the overhead room light was on. After 1 min of being placed in chamber A, animals were presented with 10 coterminating tone–footshock pairings (tone, 2.8 kHz, 82 dB, 10 s; footshock, 1 mA, 1 s) with 1 min intertrial intervals (ITIs). Animals were removed 1 min after the last shock and returned to their home cages. After 24 h, rats were placed in chamber B. In chamber B, the overhead lights were turned off, and each internal chamber was wiped with a 1% acetic acid solution. These changes produced a reliable context shift. The tone test consisted of 1 min of

M. Fujisaki et al. / Neuroscience 124 (2004) 247–260

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Fig. 1. Experimental design for testing the effects of AHi lesions on the behavioral components of stress responses. A shows design of experiments 1 and 2 for testing the effects of pretraining AHi lesions on stress responses. B shows design of experiments 3 and 4 for testing the effects of post-training AHi lesions on stress responses. Note that in the post-training design, all animals were surgically naive at the time of conditioning.

baseline followed by 8 min of continuous tone. Animals were removed after the test and returned to their home cages.

Experiment 4: post-training AHi lesions and contextual conditioning

Experiment 2: pretraining AHi lesions and contextual conditioning

To test the effects of post-training AHi lesions on stress responses to contextual conditioning, animals were prepared with contextual conditioning. These groups were subdivided further into posttraining sham lesion/contextual conditioning (post-Sh/CTX) and post-training AHi lesion/contextual conditioning (post-AHi/CTX) groups. On day 1, rats were placed in chamber A and conditioned with a tone as described in experiment 2. Animals then were subjected to either sham or excitotoxic lesions of the AHi 24 h after conditioning. After lesioning, the animals were allowed to recover for 2 weeks before the contextual test session was conducted as described in experiment 2. Animals were removed after the test and returned to their home cages. Note that these post-training lesioned animals were surgically naive at the time of conditioning.

To test the effects of pretraining AHi lesions on stress responses to contextual conditioning, animals were prepared with sham or excitotoxic AHi lesions. These groups were subdivided further into pretraining sham lesion/contextual conditioning (pre-Sh/CTX) and pretraining AHi lesion/contextual conditioning (pre-AHi/CTX) groups. After a 2-week recovery period from surgery, the animals were conditioned. The experiments took place over the course of 2 consecutive days. On day 1, rats were placed in chamber A. The Chamber A cages were wiped with 5% ammonium hydroxide solution, and the overhead room light was on. After 1 min of being placed in chamber A, animals were presented with 10 footshocks (1 mA, 1 s) with 1 min ITIs. Animals were removed 1 min after the last shock and returned to their home cages. On the second day, rats were placed in chamber A again. The contextual test session consisted of 10 min. Animals were removed after the test and returned to their home cages.

Experiment 3: post-training AHi lesions and auditory fear conditioning To test the effects of post-training AHi lesions on stress responses to auditory fear conditioning, animals were prepared with auditory fear conditioning. These groups were subdivided further into posttraining sham lesion/auditory fear conditioning (post-Sh/Tone) and post-training AHi lesion/auditory fear conditioning (post-AHi/Tone) groups. On day 1, rats were placed in chamber A and conditioned with a tone as described in experiment 1. Animals then were subjected to either sham or excitotoxic lesions of the AHi 24 h after conditioning. After lesioning, the animals were allowed to recover for 2 weeks before the test session was conducted as described in experiment 1. Animals were removed after the test and returned to their home cages. Note that these post-training lesioned animals were surgically naive at the time of conditioning.

No-conditioning (experiment 0) For determination of the effects of AHi lesions on basal freezing behavior and IEG expression, we used animals without lesions (control) and animals that received excitotoxic AHi lesions as described above. The control animals did not undergo an operation, but were exposed to the tone (Control/Tone) or contextual (Control/CTX) training and a test session, as explained in the section describing the conditioning procedures of experiment 1 and 2; these animals did not undergo footshock and were returned to their home cages after the test session. Lesioned animals were prepared with excitotoxic AHi lesions and after a 2-week recovery period, they were exposed to the tone (AHi/Tone) or contextual (AHi/CTX) training and test session, as explained in the conditioning procedures used for experiments 1 and 2, but without the footshocks; the animals were then returned to their home cages after the test session.

Behavioral measurements A video recording of each test session was observed and the duration of freezing behavior was measured, whereby freezing was defined strictly as the absence of visible movement of the animal, including vibrissae, except that related to respiration. An observer who was blind to group assignment measured the freezing time

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Fig. 2. Sham and excitotoxic lesions of the AHi. A shows sham lesions of the AHi. B shows excitotoxic lesions of the AHi. Photomicrographs after formalin fix and Cresyl Violet stain. Scale bar⫽500 ␮m.

during ITIs in the training session and 8 min of the sounding of a continuous tone in the test session for auditory fear conditioning or 8 min from the beginning of the contextual conditioning test session, and calculated the percent freezing time as duration of 8 min.

Histological procedures Animals were deeply anesthetized 1 h after the test session and perfused transcardially with 50 ml of 0.9% saline, followed by 500 ml of cold 4% paraformaldehyde in 0.1 M phosphate buffer. The brains were removed and soaked in 20% sucrose overnight for cryoprotection at 4 °C. Coronal sections were cut at a thickness of 40 ␮m on a freezing microtome and were collected in 0.01 M PBS buffer. To assist in the identification of neural structures, some of these sections were mounted and stained with Cresyl Violet (Fig. 2). The extent of neuronal cell loss was assessed microscopically and transcribed onto atlas sections from Paxinos and Watson (1998). Histological criteria for inclusion in the lesions group required evidence of bilateral damage.

Immunohistochemistry Sections were placed in 0.01 M PBS buffer containing 10% normal goat serum and 0.3% Triton X-100 for 90 min at room temperature. They were then incubated for 24 h at 4 °C in the first antibody, anti-c-Fos (1:2000; Ab-5; Oncogene Research Products, Boston, MA, USA) or anti-Egr-1 (1:2000; C-19; Santa Cruz Biotechnology, Santa Cruz, CA, USA) in PBS with 0.1% Triton X-100 and 0.1% bovine serum albumin. Sections were then washed three times in PBS, incubated for 90 min in the second antibody to biotinylated goat anti-rabbit IgG, adsorbed against rat serum in PBS with 0.1% Triton X-100 and 0.1% bovine serum albumin. Then, sections were again washed three times in PBS and incubated with avidin– biotin complex reagent (Elite Vectastain Kit; Vector Laboratories, Burlingame, CA, USA) in PBS at room temperature for 90 min. Sections were rinsed once with PBS and twice with 0.05 M Tris buffer (TB; pH 7.6), and the reaction was made visible with 0.02% 3,3⬘-diaminobenzidine (DAB; Wako, Japan) and 0.25% nickel ammonium sulfate (DAB-nickel) in TB with 0.0015% hydrogen peroxide. The sections were then rinsed in PBS, mounted onto gelatin-coated glass slides and dried overnight. Finally, they were coverslipped with Eukitt (Kindler, Freiburg, Germany). The number of c-Fos and Egr-1 immunoreactive-positive cells was counted in each structure within a 0.5⫻0.5-mm area (0.25 mm2) by an observer who was blind to group assignment. The AP coordinates of sections included for detailed analysis (Paxinos and Watson, 1998) and associated

structures were as follows: AP⫹3.2, prelimbic cortex (PrL), IL; AP⫹1.2, accumbens nucleus core (AcbC), accumbens nucleus shell (AcbSh); AP ⫺1.4, paraventricular hypothalamic nucleus; AP ⫺2.8, dorsal CA1 field of hippocampus, dentate gyrus, La, BL, BM, Ce, Me, Co; AP ⫺6.0, dorsomedial periaqueductal gray (DMPAG), dorsolateral periaqueductal gray (DLPAG); AP ⫺8.7, lateral periaqueductal gray (LPAG), ventrolateral periaqueductal gray (VLPAG); AP ⫺10.0, locus coeruleus.

Statistical analysis Data are reported as the mean⫾S.E.M. To test for significant effects of the lesion, treatment, and lesion-by-treatment interaction, a twoway analysis of variance (ANOVA) statistical analysis was performed using data, on the training sessions percent freezing from the sham and the AHi lesion group of the conditioned group in experiments 1 and 2 and on the test sessions percent freezing and the number of IEGs positive cells from the control and the AHi lesion group of the no-conditioning group in experiment 0 and the sham and the AHi lesion group of the conditioned group in experiments 1 and 2. When significant main effects were found, additional analysis was performed using one-way ANOVA with post hoc comparisons conducted by Fisher’s protected least significant difference tests. In experiments 3 and 4, statistical analysis of the sham and the AHi lesion groups as regards percent freezing and the number of IEG positive cells was carried out using Student’s t-test. Differences were considered to be statistically significant at P⬍0.05.

RESULTS Histology Fig. 2 displays a photomicrograph of a representative lesion, and Fig. 3 illustrates the maximum and minimum extent of the lesions. The lesioned area included the AHi. Some animals also sustained damage unilaterally in a small part of the ventral hippocampus or the Co. Nuclei of the injection sites disappeared in the Nissl-stained photomicrograph. These findings suggest that neuronal degeneration or dropout occurred in the excitotoxic lesions (Goldstein et al., 1996). The PBS-injected sham-operated animals were not injured at any location in the AHi. Figs. 4 and 5 show representative photomicrographs in the IL or Me that reveal the expression of the c-Fos or Egr-1 protein, respectively. Immunoreactivepositive cells are seen as dark dots.

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Bregma -3.80mm

Bregma -4.80mm

Bregma -4.30mm

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Fig. 3. Representations of largest and smallest AHi lesions. In each case, the smallest lesion (stippled area) and the largest lesion (horizontal line pattern in addition to the enclosed stippled area) are drawn onto plates.

Two weeks after sham or excitotoxic lesioning of the AHi, the animals were subjected to auditory conditioning with a test session on the following day. The data presented in this section are derived from responses observed during the test session on day 2, when the animals were presented only with the tone (i.e. without footshock). These results were compared with those of the Control/Tone and AHi/Tone groups of experiment 0. The behavioral results are presented in Fig. 7A. Significant effects on freezing behaviors were observed for lesion, F(3, 20)⫽24.5, P⬍0.0001; treatment, F(3, 20)⫽282.1, P⬍0.0001; and le-

Experiment 1: effects of pretraining AHi and sham lesions on auditory conditioned stress-induced behavioral responses Fig. 6A shows the effects of pretraining AHi lesions during the 1 min baseline and during the intervening 10 toneshock parings of the 1 min ITIs on training of auditory fear conditioning. There was no significant difference between the mean percentage of the duration of freezing in the pre-AHi/Tone group and that in the pre-Sh/Tone group. The effects of pretraining NMDA lesions of the AHi on behavioral indices of auditory conditioning were assessed.

A

B

Fig. 4. Representative photomicrographs of coronal section showing the expression of the c-Fos protein in the IL. A, the pre-Sh/Tone group; B, pre-AHi/Tone group of the conditioning group. Immunoreactive-positive cells are seen as dark dots. Scale bar⫽200 ␮m.

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Fig. 5. Representative photomicrographs of coronal section showing the expression of the Egr-1 protein in the Me. A, the pre-Sh/CTX group; B, pre-AHi/CTX group of the conditioning group. Immunoreactive-positive cells are seen as dark dots. Scale bar⫽200 ␮m.

of treatment (F(3, 20)⫽0.53; P⫽0.48), and a significant interaction between lesion and treatment (F(3, 20)⫽7.93; P⫽0.01). In the Co, the pre-Sh/Tone group was statistically distinguishable from the other three groups. A twoway ANOVA comparing lesion and treatment found a significant main effect of lesion (F(3, 20)⫽7.04; P⫽0.02), a significant main effect of treatment (F(3, 20)⫽18.33; P⬍0.001), and a significant interaction between lesion and treatment (F(3, 20)⫽9.92; P⫽0.005). Fig. 9A shows the number of Egr-1 positive cells in all groups. A two-way ANOVA was used to assess the significance of the results of the pre-AHi/Tone group obtained from each anatomical structure. In the IL and BM, the pre-AHi/Tone group was statistically distinguishable from the other three groups. A two-way ANOVA comparing lesion and treatment in the IL found a significant main effect of lesion (F(3, 20)⫽3.27; P⫽0.09), a significant main effect of treatment (F(3, 20)⫽2.14; P⫽0.16), and a significant interaction between lesion and treatment (F(3, 20)⫽5.93; P⬍0.05); in the BM, there was a significant main effect of lesion (F(3, 20)⫽6.32; P⬍0.05), a significant main effect of treatment

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sion by treatment interaction, F(3, 20)⫽22.3, P⫽0.0001. Both the Control/Tone and AHi/Tone groups demonstrated low levels of freezing, with the following mean percentages of the duration of freezing: 5.9⫾3.7% and 6.8⫾4.2%, respectively. These two groups were statistically indistinguishable from each other. In contrast, the pre-Sh/Tone and pre-AHi/Tone groups exhibited marked freezing behavior (56.6⫾6.2% and 97.2⫾0.6%, respectively), and the results in both groups were statistically different from the results obtained with the Control/Tone and AHi/Tone groups (P⬍0.0001). Furthermore, the pre-AHi/Tone group showed an enhancement of the stress-induced freezing response, whereby P⬍0.0001 was obtained, in contrast to the results obtained with the pre-Sh/Tone group. Fig. 8A shows the number of c-Fos immunoreactive-positive cells per 0.25 mm2 in all groups. A two-way ANOVA was used to assess the significance of the results of the pre-AHi/ Tone group obtained from each anatomical structure. In the IL, the pre-AHi/Tone group was statistically distinguishable from the other three groups. A two-way ANOVA comparing lesion and treatment found a significant main effect of lesion (F(3, 20)⫽5.66; P⬍0.05), a significant main effect

intertrial intervals

Fig. 6. Mean⫾S.E.M. percentage of freezing on training sessions. A shows the effects of pretraining AHi lesions during the 1 min baseline and during the intervening 10 tone-shock pairings of the 1 min ITIs on training of auditory fear conditioning. B shows the effects of pretraining AHi lesions during the 69 s baseline and during the intervening 10 shocks of the 69 s ITIs on training of contextual conditioning.

M. Fujisaki et al. / Neuroscience 124 (2004) 247–260 **

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Fig. 7. Mean⫾S.E.M. percentage of freezing during an 8 min test session. A shows the effects of pretraining AHi lesions on stress-induced freezing behavior of auditory fear conditioning. B shows the effects of pretraining AHi lesions on stress-induced freezing behavior of contextual conditioning. C shows the effects of post-training AHi lesions on stress-induced freezing behavior of auditory fear conditioning. D shows the effects of post-training AHi lesions on stress-induced freezing behavior of contextual conditioning. Animals were subjected to bilateral sham (Sh) or excitotoxic lesions of the AHi (AHi) or were not subjected (Control). Tone and CTX indicate auditory fear conditioning or contextual conditioning, respectively. Conditioning and No-conditioning indicate experimental conditions in which animals were or were not exposed to shock during conditioning, respectively. ND, No statistically significant difference; * statistically significant difference, P⬍0.01; ** statistically significant difference, P⬍0.0001.

(F(3, 20)⫽19.76; P⬍0.001), and a significant interaction between lesion and treatment (F(3, 20)⫽7.51; P⬍0.05). Experiment 2: effects of pretraining AHi and sham lesions on contextual conditioned stress-induced behavioral response. Fig. 6B shows the effects of pretraining AHi lesions during the 69 s baseline and during the intervening 10 shock pairings of the 69 s ITIs on training of contextual conditioning. There was no significant difference between the mean percentage of the duration of freezing in the pre-AHi/CTX group and that in the pre-Sh/CTX group. The effects of pretraining NMDA lesions of the AHi on behavioral indices of contextual conditioning were assessed. Two weeks after sham or excitotoxic lesioning of the AHi, the animals were subjected to contextual conditioning with a test session on the following day. The data presented in this section are derived from responses obtained during the test session on day 2, when the animals were not presented with any stimulus (i.e. without tone and footshock). These results were compared with those of the Control/CTX and AHi/CTX groups of experiment 0. The behavioral results are presented in Fig. 7B. Significant effects on freezing behaviors were observed for lesion, F(3, 20)⫽4.7, P⫽0.04; treatment, F(3, 20)⫽714.7, P⬍0.0001; and lesion by treatment interaction, F(3, 20)⫽7.9, P⫽0.01. Both the Control/CTX and AHi/CTX groups demonstrated low levels of freezing, with the following mean percentages of the duration of freezing: 5.3⫾1.7% and 3.6⫾1.4%, respectively. These two groups were statistically indistinguishable from each other. In contrast, the pre-Sh/CTX and pre-AHi/

CTX groups exhibited marked freezing behavior (69.3⫾3.8% and 82.6⫾3.0%, respectively), and the results in both groups were statistically different from the results obtained with the Control/CTX and AHi/CTX groups (P⬍0.0001). Furthermore, the pre-AHi/CTX group showed an enhancement of the stress-induced freezing response, whereby P⬍0.01 was obtained, in contrast to the results obtained with the pre-Sh/CTX group. Fig. 8B shows the number of c-Fos immunoreactivepositive cells per 0.25 mm2 in all groups. A two-way ANOVA was used to assess the significance of the results of the pre-AHi/CTX group obtained from each anatomical structure. In the BL, Co, and LPAG, the pre-AHi/CTX group was statistically distinguishable from the other three groups. As regards the BL a two-way ANOVA comparing lesion and treatment found a significant main effect of lesion (F(3, 20)⫽18.00; P⬍0.001), a significant main effect of treatment (F(3, 20)⫽8.65; P⬍0.01), and a significant interaction between lesion and treatment (F(3, 20)⫽6.52; P⬍0.05); in the Co, there was a significant main effect of lesion (F(3, 20)⫽8.70; P⬍0.01), a significant main effect of treatment (F(3, 20)⫽5.21; P⬍0.05), and a significant interaction between lesion and treatment (F(3, 20)⫽5.60; P⬍0.05); in the LPAG, there was a significant main effect of lesion (F(3, 20)⫽3.10; P⫽0.09), a significant main effect of treatment (F(3, 20)⫽32.22; P⬍0.0001), and a significant interaction between lesion and treatment (F(3, 20)⫽9.06; P⬍0.01). Fig. 9B shows the number of Egr-1 positive cells in all groups. A two-way ANOVA was used to assess the significance of the results of the pre-AHi/CTX group obtained from each anatomical structure. In the

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Fig. 8. Number of c-Fos immunoreactive-positive cells (mean⫾S.E.M.). A shows the effects of pretraining AHi lesions on the expression of c-Fos positive cells in auditory fear conditioning. B shows the effects of pretraining AHi lesions on the expression of c-Fos positive cells in contextual conditioning. C shows the effects of post-training AHi lesions on the expression of c-Fos positive cells in auditory fear conditioning. D shows the effects of post-training AHi lesions on the expression of c-Fos positive cells in contextual conditioning. Animals were subjected to bilateral sham (Sh) or excitotoxic lesions of the AHi (AHi) or were not subjected (Control). Tone and CTX indicate auditory fear conditioning or contextual conditioning, respectively. Conditioning and No-conditioning indicate experimental conditions, i.e. animals were or were not exposed to shock during conditioning, respectively. * Statistically significant difference, P⬍0.05; ** statistically significant difference, P⬍0.01, by two-way ANOVA followed by one-way ANOVA, with Fisher’s protected least significant difference tests at A and B, and by Student’s t-test at C and D.

BM, Me, and Co, the pre-AHi/CTX group was statistically distinguishable from the other three groups. A two-way ANOVA comparing lesion and treatment in the BM found a significant main effect of lesion (F(3, 20)⫽6.49;

P⬍0.05), a significant main effect of treatment (F(3, 20)⫽163.44; P⬍0.0001), and a significant interaction between lesion and treatment (F(3, 20)⫽5.87; P⬍0.05); in the Me, there was a significant main effect of lesion

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Fig. 8. (C–D).

(F(3, 20)⫽5.28; P⬍0.05), a significant main effect of treatment (F(3, 20)⫽73.00; P⬍0.0001), and a significant interaction between lesion and treatment (F(3, 20)⫽4.36; P⬍0.05); in the Co, there was a significant main effect of lesion (F(3, 20)⫽7.06; P⬍0.05), a significant main effect of treatment (F(3, 20)⫽65.89; P⬍0.0001), and a significant interaction between lesion and treatment (F(3, 20)⫽4.38; P⬍0.05).

Experiment 3: effects of post-training AHi and sham lesions on auditory conditioned stress-induced behavioral responses The effects of post-training NMDA lesions of the AHi on behavioral indices of auditory conditioning were assessed. Two weeks after sham or excitotoxic lesioning of the AHi, the animals were subjected to a test session.

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Number of Egr-1 immunoreactive-positive cells

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Fig. 9. Number of Egr-1 immunoreactive-positive cells (mean⫾S.E.M.). A shows the effects of pretraining AHi lesions on the expression of Egr-1 positive cells in auditory fear conditioning. B shows the effects of pretraining AHi lesions on the expression of Egr-1 positive cells in contextual conditioning. C shows the effects of post-training AHi lesions on the expression of Egr-1 positive cells in auditory fear conditioning. D shows the effects of post-training AHi lesions on the expression of Egr-1 positive cells in contextual conditioning. Animals were subjected to bilateral sham (Sh) or excitotoxic lesions of the AHi (AHi) or were not subjected (Control). Tone and CTX indicate auditory fear conditioning or contextual conditioning, respectively. Conditioning and No-conditioning indicate experimental conditions, i.e. animals were or were not exposed to shock during conditioning, respectively. * Statistically significant difference, P⬍0.05; ** statistically significant difference, P⬍0.01, by two-way ANOVA followed by one-way ANOVA with Fisher’s protected least significant difference tests at A and B, and by Student’s t-test at C and D.

The data presented in this section are derived from responses observed during the test session when the animals were presented with only the tone (i.e. without footshock). Behavioral results are presented in Fig. 7C. There was no significant difference between the mean

percentage of the duration of freezing in the post-AHi/ Tone group (55.0⫾9.3%) and that in the post-Sh/Tone group (51.3⫾8.0%; Z⫽⫺0.320, P⬎0.05). Fig. 8C shows the number of c-Fos immunoreactive-positive cells. Student’s t-test was used to assess the significance of the

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Number of Egr-1 immunoreactive-positive cells

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Fig. 9. (C–D).

results in the post-AHi/Tone group obtained from each anatomical structure. A significant increase in the number of c-Fos positive cells was seen in the DMPAG and DLPAG of the post-AHi/Tone group, versus the post-Sh/ Tone group. A significant decrease in the number of c-Fos positive cells was seen in the Me of the post-AHi/ Tone group, versus the post-Sh/Tone group. Fig. 9C shows the number of Egr-1 positive cells. Student’s

t-test was used to assess the significance of the results of the post-AHi/Tone group obtained from each anatomical structure. A significant increase in the number of Egr-1 positive cells was seen in the AcbC, AcbSh, and Me of the post-AHi/Tone group, versus the post-Sh/ Tone group. There was no region in which the number of Egr-1 positive cells decreased in the post-AHi/Tone group, versus the post-Sh/Tone group.

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Experiment 4: effects of post-training AHi and sham lesions on contextual conditioned stress-induced behavioral responses The effects of post-training NMDA lesions of the AHi on behavioral indices of contextual conditioning were assessed. Two weeks after sham or excitotoxic lesioning of the AHi, the animals were subjected to a test session. The data presented in this section are derived from responses observed during the test session when the animals were not presented with any stimulus (i.e. without tone and footshock). Behavioral results are presented in Fig. 7D. There was no significant difference between the mean percentage of the duration of freezing in the post-AHi/CTX group (52.3⫾7.7%) and that in the post-Sh/CTX group (49.3⫾9.6%; Z⫽⫺0.320, P⬎0.05). Fig. 8D shows the number of c-Fos immunoreactive-positive cells. Student’s t-test was used to assess the significance of the results in the post-AHi/CTX group obtained from each anatomical structure. A significant increase in the number of c-Fos positive cells was seen in the Co of the pre-AHi/CTX group, versus the post-Sh/CTX group. There was no region in which the number of c-Fos positive cells decreased in the pre-AHi/CTX group, versus the post-Sh/CTX group. Fig. 9D shows the number of Egr-1 positive cells. Student’s t-test was used to assess the significance of the results of the post-AHi/CTX group obtained from each anatomical structure. A significant increase in the number of Egr-1 positive cells was seen in the BL, DMPAG, and DLPAG of the pre-AHi/CTX group, versus the post-Sh/CTX group. There was no region in which the number of Egr-1 positive cells increased in the pre-AHi/CTX group, versus the postSh/CTX group.

DISCUSSION The principal finding of this study was that bilateral excitotoxic AHi lesions before a training session enhanced freezing behaviors associated with stress, which were induced by re-exposure to stimuli paired previously with an unconditioned stressor. However, the freezing behavior of rats lesioned after training did not differ from that of the sham treatment rats. This finding suggests that the AHi is important in linking negative stimuli to the normally contingent behavioral responses to psychological stress. There were several regions observed in this study wherein c-Fos and/or Egr-1 expression changed in diverse manners after the test session. In this study, we applied auditory and contextual fear conditioning. There is a general consensus that neuronal processing in the amygdala is important for classical fear conditioning to explicit conditioned stimulus, (auditory fear conditioning was used for this purpose in the present study), although it has been suggested that the amygdala is partly involved in contextual conditioning. Some researchers have held the view that the amygdala modulates the formation and storage of the memory in other brain regions (Cahill et al., 1999), whereas others have considered the amygdala to be the site where the fear memories are formed and stored (Fanselow and LeDoux, 1999;

Fendt and Fanselow, 1999). On the other hand, the AHi is a prominent member of the corticomedial nuclear complex located in the caudal ventromedial angle of the amygdaloid body immediately caudal to the posterior part of the medial amygdala (Paxinos, 1995). The AHi forms a shallow, inverted basket-like formation around the dorsal and caudomedial aspect of the Co and Me (Paxinos, 1995). On its lateral aspect, the AHi merges with the BM and successively with the BL (Paxinos, 1995). The AHi is readily distinguished from the neighboring amygdaloid nuclei by its larger, more deeply staining and tightly packed cells (Paxinos, 1995). The AHi has both afferent and efferent connections with the Co, Me, BL, and Ce, efferent to the BM and afferent to the La (Canteras et al., 1992). Thus, the AHi has a close connection with these nuclei in the amygdala. Taken together with the results from previous studies, the present findings suggest that the AHi is likely to be involved in auditory fear conditioning. As shown in experiment 1, the freezing behaviors of AHi-lesioned rats were enhanced in cases of auditory fear conditioning. The hippocampus is thought to be involved in classical conditioning mainly when contextual information is utilized or when the conditioned stimulus is contextual (Jarrard, 1993; Holland and Bouton, 1999; Fanselow, 2000; Maren and Holt, 2000). However, dorsally, the AHi is in successive topographic relationship with the field CA1 (Paxinos, 1995). In cholinesterase preparations, the AHi shows moderate activity, i.e. similar to the reaction observed in the neighboring Ammon’s horn (Paxinos, 1995). Moreover, the AHi has both afferent and efferent connections with the ventral parts of the hippocampal field CA1. Therefore, these findings suggest that the AHi is also potentially involved in contextual conditioning. As shown in the results of experiment 2, the freezing behaviors of the AHi-lesioned rats were enhanced in the contextual conditioning experiments. Thus, the present study indicates that the AHi is potentially involved in both auditory and contextual fear conditioning. As shown in experiments 1 and 2, pretraining AHi lesions enhanced the duration of freezing behavior. However, it was not clear which points along the process from learning to expression of conditioning were modified by the lesions. Therefore, we performed a study in which both sides of the AHi were lesioned during the post-training period. However, as shown in experiments 3 and 4, the freezing behavior of post-training AHi-lesioned rats did not change compared with that of sham rats. We may consider memory formation as a model for the conditioning process, i.e. such a model would include the four following aspects, namely, the acquisition of conditioned memory during training, the memory consolidation during the period from training to the test session, and the memory retrieval and expression as a freezing behavior in the test session. The results in the post-training AHi lesions studies show that the lesions have no influence on the stages occurring after consolidation. Therefore, the present results suggest that the AHi plays a significant role in acquisition, but has no effect on other stages of the conditioning process.

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In this study, we found that the number of c-Fos and Egr-1 immunoreactive-positive cells in the medial prefrontal cortex (mPFC), amygdala, and periaqueductal gray (PAG) changed after the test session. The AHi has mutual connections with the other nuclei of the amygdala, and it shares the highest number of fiber connections with the Me and Co (Canteras et al., 1992). Furthermore, the Me and Co have efferent fibers to the BL, whereas the Co can undergo plastic changes in the context of fear processing. Animals that received lesions of the La or Ce, or of the entire amygdala, were dramatically impaired in the acquisition of conditioned freezing behavior in response to auditory fear conditioning, whereas little effect was observed in animals that had received lesions in the Me or Co (Nader et al., 2001). If anything, the Me and Co might have been related to the novelty (Radulovic et al., 1998). Recent chemical blockade experiments have indicated that the main role of the VLPAG in fear-conditioned response is to impose the immobility necessary for the expression of the freezing component. In the dorsal portion of the PAG (DLPAG and DMPAG)-lesioned group, there was no significant difference in the amount of freezing and in the blood pressure response (Leman et al., 2003). Thus, the DLPAG and DMPAG appear to play only a small role in the expression of the fear response. On the other hand, it has been suggested that the ventral portions of the PAG form an output nucleus responsible for mediating the specific conditioned response of freezing and this area is not thought to be involved in processes such as learning and memory (LeDoux, 1995). However, it should be noted that under some conditions, lesions of the dorsal portion of the PAG have been demonstrated as having an effect on learning (De Oca et al., 1998). The AHi is thus known to have the highest number of fiber connections with the Me and Co, but the Me, Co, DMPAG, and DLPAG might not be involved in the expressions observed in the test session. In this study, it was suggested that freezing behavior was not necessarily affected by changes in the expression of IEGs in the Me, Co, DMPAG, or DLPAG in experiments 3 and 4. In the prefrontal cortex, the AHi sends afferent fibers to the IL (Canteras et al., 1992). In addition, the IL is included in the mPFC with the dorsal anterior cingulate, PrL, frontal area 2, and the dorsal peduncular area. Inhibition as well as stimulation of dopamine receptors in the test session in the mPFC reduced conditioned fear (Pezze et al., 2003). Increased fear reactivity following lesions of the mPFC has also been reported (Morgan et al., 1993; Morgan and LeDoux, 1995). However, decreased fear reactivity (Frysztak and Neafsey, 1991), as well as no change (Gewirtz et al., 1997) in fear reactivity following lesioning of the mPFC have also been reported. For example, another group reported that mPFC lesions increased freezing behavior in response to contextual cues, and they also found reduced freezing behaviors among rats in response to discrete cues (Lacroix et al., 2000). Further studies will be needed in order to clarify the mechanisms of these re-

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sponses. However, it is possible that the mPFC is important for the retrieval and/or expression of conditioned fear. Contextual and auditory conditioned stimuli converge on La neurons, where these neurons come into association with unconditioned stimuli. Indirect projections from the La to the Ce via the BL and BM mediate the expression of conditioned stimuli– unconditioned stimuli associations (Goosens and Maren, 2001). In this study, it was suggested that the changes in freezing behaviors were related to changes in the expression of IEGs in the IL, BM, and LPAG; moreover the results of experiments 1 and 2 indicated that the observed changes in freezing behaviors were not associated with either the Me or the Co. As was observed in the BM, the number of Egr-1 immunoreactive-positive cells increased in both experiments, and it is therefore possible that the activation of neurons with high basal levels of expression might have been related to the memory retrieval or expression as a freezing behavior during the test session in this study.

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(Accepted 21 November 2003)