Amygdala, hippocampus and discriminative fear conditioning to context

Amygdala, hippocampus and discriminative fear conditioning to context

Behavioural Brain Research 108 (2000) 1 – 19 www.elsevier.com/locate/bbr Research report Amygdala, hippocampus and discriminative fear conditioning ...

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Behavioural Brain Research 108 (2000) 1 – 19 www.elsevier.com/locate/bbr

Research report

Amygdala, hippocampus and discriminative fear conditioning to context Elena A. Antoniadis *, Robert J. McDonald Department of Psychology, Uni6ersity of Toronto, 100 St. George St., Toronto, Ont., Canada M5S 3G3 Received 20 December 1998; received in revised form 3 May 1999; accepted 21 July 1999

Abstract Various measures of fear have been shown to condition to a fearful context with different acquisition rates (Antoniadis EA, McDonald RJ. Fear conditioning to context expressed by multiple measures of fear in the rat, Behav Brain Res 1999;101(1):1–14). Freezing, locomotion, urination and preference are ‘fast’ measures of fear in that they discriminatively condition to context after a single training session, while ultrasonic vocalizations and defecation are ‘slow’ measures of fear given that they condition following three training sessions. In the present experiment we sought to assess the contribution of the amygdala and the hippocampus in this form of learning. Existing views differ on the degree of involvement of each memory structure. This discord probably emerges from the common use of non-discriminative paradigms and the assessment of a single measure of fear. With the use of a discriminative paradigm and the assessment of multiple measures of fear, results indicate that the amygdala is a memory structure that selectively mediates the conditioning of heart rate, and the hippocampus selectively mediates the conditioning of defecation and body temperature. The conditioning of preference, locomotion, freezing and ultrasonic vocalizations, necessitate the participation of both memory structures while the conditioning of urination does not seem to require the participation of either the hippocampus or the amygdala. The proposed view ascribes an equal role in fear conditioning to both the amygdala and the hippocampus. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Rat; Fear; Context conditioning; Memory systems

1. Introduction Precise information about how the nervous system acquires and stores information and then uses this information to guide behavior is essential to our understanding of both normal and abnormal manifestations of behavior. Recently, there has been a resurgence of interest in how environmental context influences learning and memory. The types of influence contextual stimuli have on behaviour and the neural substrates subserving this type of learning is fundamental to our understanding of the complexities of conditioned behaviours in the mammal. Accordingly, embedded within contemporary surveys of learning is a broadly acknowledged account of * Corresponding author. Tel.: +1-416-978-1708; fax: + 1-416-9784811. E-mail address: [email protected] (E.A. Antoniadis)

the importance that context has on the regulation of learning and performance. The impact of contextual information has also been widely recognized in human behaviour in which context is considered as a behaviour-shaping force that directs, constrains, and regulates human actions [46]. Environmental context conditioning is the acquisition and retention of the significance (positive or negative) of a situation or environmental context. Aversive context conditioning is usually measured by the degree to which an involuntary response (such as freezing) can be elicited by a previously neutral context that was paired with an aversive stimulus (shock). Unfortunately, the precise circumstances in which learning and memory systems in the medial temporal lobe are essential for fear conditioning to context are not well understood. There is, however, significant agreement about the identity of neural circuits in the hypothalamus and brainstem that underlie the basic

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unconditioned responses exhibited in animals and humans immediately following an aversive event. In nonhuman animals, these unconditioned responses may include changes in heart rate, respiration, body temperature, vocalizations, freezing, defecation, and urination. These unconditioned responses occur when animals are exposed to aversive stimuli like predators, footshock, and loud noises. However, there is substantial discord in the literature concerning the essential components of the forebrain that underlie the complex cognitive processes important for the impressive ability that mammals have for learning and remembering important environmental contexts. Some researchers suggest that the hippocampus is critical for stimulus selection in fear conditioning to context [52]. Others support a synergistic interaction between the amygdala and the hippocampus in this form of learning in that the hippocampal representation of the context must output through the amygdala for the context-shock association to occur [4,22,27,36]. Another view suggests that the amygdala and the hippocampus independently access unconditioned fear responses and the participation of each system is determined by the complexity of the environmental context [15,45,47]. Finally, others ascribe a limited role to the hippocampus, suggesting than an intact hippocampus is only required when there is a competition for associative strength between the static context and a phasic cue [35,37]. The main reason, we believe, for this discord concerning the role of various medial temporal lobe structures in context conditioning is that virtually all of these experiments use non-discriminative procedures which render the obtained results very difficult to interpret because of various confounds associated with this procedure. Another problem with these experiments is that although in the normal animal fear is expressed in a variety of autonomic and behavioral responses, most of these experiments only measure one response measure of fear. Related to this problem is the fact that most of the experiments described above use different response measures, making direct comparisons of the disparate data difficult. Given this lack of agreement on the respective roles of the amygdala and hippocampus in fear conditioning to context, a re-assessment of their participation is needed. Using a discriminative fear conditioning to context paradigm and assessment of eight different response measures of fear, Antoniadis and McDonald [1] found that there are different learning rate parameters for the discriminative development of these responses. A subset of these fear responses including freezing, urination, locomotion, and preference showed discriminative conditioning after a single training session. The other fear responses including heart rate, ultrasonic vocalizations, and defecation required three training sessions for discriminative conditioning to

emerge. The demonstration of different learning rate parameters for subsets of fear responses suggests that different learning systems and mechanisms may contribute to the acquisition and expression of different conditioned fear responses. The brain lesion technique can serve as a powerful tool for assessing the participation of different fear responses in a discriminative fear conditioning to context paradigm so in the present experiment we tested the effects of neurotoxic lesions to the amygdala or hippocampus on acquired fear to environmental cues using a discriminative procedure and assessment of eight different response measures of fear [1].

2. Method

2.1. Subjects Twenty-four male Long-Evans rats were used. The animals were individually housed in single Plexiglas cages (24 cm long× 22 cm wide×20 cm high), and were maintained on a 12-h light, 12-h dark cycle. The rats weighed approximately 300–325 g at arrival, and were given free access to food and water.

2.2. Apparatus A white square prism (41 cm long × 41 cm wide ×29 cm high) served as one context, and a black triangle prism (61 cm long × 61 cm wide × 30 cm high) served as the other context. Isoamyl acetate served as the olfactory cue in the black triangle prism and eucalyptus served as the olfactory cue in the white square. An alley (16.5 cm long× 11 cm wide× 11 cm high) connected the two chambers. The flooring of all chambers consisted of grids, but a Plexiglas platform was placed on top of the grids of the chamber that served as the unpaired context, while the grids served as the tactile cues in the chamber that served as the paired context. The chambers were placed on a Plexiglas table 100 cm above the floor. A mirror (91 cm long × 61 cm wide), inclined by 45° provided a full view of the chambers. A camera placed 2 feet in front of the mirror allowed the experimenter to video tape ongoing behaviour throughout all phases of the experiment. The training phase of the experiment was conducted in two different rooms within two different laboratories. Animals experienced the paired chamber in the shock room, different from the one where they experienced the unpaired chamber (safe room). The entire apparatus including the chambers, the computer, the shock generator, the video camera, the mirror and the Dataquest equipment were transported back and forth on a trolley. In order to assess conditioning to the chamber and not any fear acquired by the room, all testing occurred in the safe

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room. As such, greater fear in the paired context expressed by one or many of the measures assessed can only be attributed to the aversive properties acquired by the paired context.

2.3. Surgery 2.3.1. Lesions All rats undergoing surgery were first injected with 0.2 mg of atropine to facilitate respiration and were subsequently anaesthetized with sodium pentobarbital (65 mg/kg i.p.). The rats were assigned randomly to one of the three treatment groups: amygdala damage, hippocampus damage and sham lesion. Eight animals were assigned to each group. Bilateral neurotoxic lesions were made with the use of the Paxinos and Watson atlas [34] to locate all coordinates. Lesions were stereotaxically placed and the coordinates were measured in relation to bregma and the skull surface. Neurotoxic lesions of the hippocampus were made with injections of NMDA infused through 30 gauge stainless steel cannulae over 3 min. The injection coordinates were: 3.1 mm posterior, 1 mm lateral and 3.6 mm ventral, 3.1 mm posterior, 2.0 mm lateral and 3.6 mm ventral, 4.1 mm posterior, 2.0 mm lateral and 4.0 mm ventral, 4.1 mm posterior, 3.5 mm lateral and 4.0 mm ventral, 5.0 mm posterior, 3.0 mm lateral and 4.1 mm ventral, 5.0 mm posterior, 5.2 mm lateral and 5.0 mm ventral, 5.0 mm posterior, 5.2 mm lateral and 7.3 mm ventral, 5.8 mm posterior, 4.4 mm lateral and 4.4 mm ventral, 5.8 mm posterior, 5.1 mm lateral and 6.2 mm ventral 5.8 mm posterior, 5.1 mm lateral and 7.5 mm ventral. The total volume injected in the hippocampus was 2.10 ml. The amygdala was also damaged by NMDA infusion. The coordinates were: 2.3 mm posterior, 4.8 mm lateral, and 9.4 mm ventral, 3.3 mm posterior, 4.6 mm lateral, and 9.4 mm ventral. The total volume injected in the amygdala was 2.4 ml. Prior to NMDA injections all animals received 1 ml of Valium to prevent the occurrence of any seizures. Upon completion, the incision was closed using stainless steel wound clips. Hibotaene was applied upon the incision site. Following the lesion, animals were allowed to recover for 7 days before the transmitter implantation was to begin. 2.3.2. Implant surgery Rats were first injected with 0.2 mg of atropine to facilitate respiration, and were subsequently anaesthetized with sodium pentobarbital (65 mg/kg i.p.). Biocompatible and hermetically sealed transmitters (model TA10 CA F40, Data Sciences, St Paul, MN) were implanted into the peritoneal cavity. Following implantation of the transmitter, animals were allowed to recover for 7 days.

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2.3.3. Histology Subsequent to the completion of behavioural testing, all animals were anesthetized with somnotol and perfused cardially with 0.9% saline and with 4% paraformaldehyde. Brains were removed and stored in 20% sucrose, 4% paraformaldehyde overnight and cut at − 17°C with a cryostat. Forty-micrometer coronal sections were cut from tissue of the hippocampus and amygdala lesioned groups. These sections were mounted on gelatin-coated slides and stained with cresyl violet. Lesions were then examined under a light microscope and reconstructed on rat brain atlas sections from Paxinos and Watson [34]. 2.4. Data collection 2.4.1. Heart rate and body temperature Implanted transmitters produced a temperature and electrocardiogram (ECG) signal (10 mV peak-to-peak biopotential) that was telemetrically transmitted via radio-frequency signals to a receiver (model RLA1020, Data Sciences) placed close to the cage. The receiver converted these signals into a form accessible to the Dataquest PC-based data acquisition system which processed the data into digital form. ECG and body temperature were sampled for 5 s every 10 s, and heart rate was extracted from the ECG. 2.4.2. Ultrasonic 6ocalizations A bat detector (Mini-3 bat detector, Hockley, Birmingham) equipped with a high frequency microphone served as the ultrasonic signal device and transduced ultrasounds into the audible range. This bat detector, placed non-intrusively underneath the chamber grids, was set at 22 kHz because of previously reported findings that rats emit vocalizations in the 20–28 kHz range in reaction to noxious stimuli such as footshock [9]. The bat detector was in turn connected to a CTR102 tape recorder. All vocalizations were recorded and kept as part of the permanent data set. Headphones were connected to the bat detector, allowing the experimenter to audit and count the number of occurrences during the experiment. 2.4.3. Freezing Freezing was defined as a total absence of body or head movement except that associated with breathing. Video tapes containing all the test sessions were scored by the experimenter at the end of the experiment, and this measure of fear was quantified in amount of time spent freezing in seconds. 2.4.4. Locomotion Equi distant lines (2 inches) perpendicular to the chamber grids were drawn on the Plexiglas table and were used to quantify locomotor behaviour. The num-

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ber of times that the animal crossed any of the lines served as an index of the amount of locomotion occurring in the paired and unpaired context. This measure was also assessed by re-scoring the video tapes.

2.4.5. Urination During the testing sessions the experimenter counted the number of emissions by each animal in each context, and confirmed the initial assessment by scoring the video tapes. 2.4.6. Defecation During the testing sessions the experimenter counted the number of feces produced by each animal in each chamber. This initial assessment was confirmed by scoring the video tapes. 2.4.7. Preference test Time spent in each chamber was assessed by the experimenter during the testing phase and confirmed during the video scoring. Dwell time was recorded when both forepaws were past the threshold of the doorway into one of the chambers. Dwell time was not recorded when both forepaws were past the threshold of the doorway into the alley. We have an a-priori interest in the emergence and development of the behavioural and physiological measures described above. 2.5. Procedure The entire experiment consisted of four phases: (1) baseline recordings; (2) pre-exposure; (3) training; (4) testing.

2.5.1. Baseline The first phase consisted of baseline recordings which lasted 4 days. Rats’ home cages were individually placed on top of the receiver (model RLA1020) while ECG and body temperature were recorded for a total period of 2 min. This procedure was used to get an assessment of baseline heart rate and body temperature. For the next 3 min, rats were individually handled by the experimenter in an attempt to render the room and the experimenter neutral. On each of the 4 days, rats had two baseline sessions, one in each room. The order in which the animals experienced these two rooms during the baseline procedures was counterbalanced. One of the rooms was designated the shock room because all of the shocks occurred there. The other room was designated the safe room because no shocks ever occurred there during conditioning. 2.5.2. Pre-exposure This phase consisted of pre-exposure to the entire

apparatus for two consecutive days (10 min/day). This pre-exposure occurred in the safe room for both days. Animals were placed in the middle alley and were given free access to the experimental apparatus for a total period of 10 min each day.

2.5.3. Training During the training phase of the experiment, each daily trial lasted 5 min. On day one of each training session, half the animals were confined in their assigned paired context and received one set of three footshocks (1 mA) in the shock room at the 2-, 3-, and 4-min mark of the training session. The other half of the rats were confined in their assigned unpaired context and received no foot shock in the safe room. The order in which the animals experienced each context was counterbalanced so that half the animals were confined in the ‘paired’ context on day one for 5 min of each session and confined in the ‘unpaired’ context on day 2 for 5 min. The chamber that served as the paired context was also counterbalanced so that half the animals experienced the aversive event in the black triangle and the other half experienced the aversive event in the white square. Animals always experienced their paired context in the shock room and their unpaired context in the safe room. 2.5.4. Testing As an attempt to capture the amount of fear conditioned to the chamber as opposed to any fear that may have generalized to the room, all testing took place in the safe room. A testing session lasted 2 days. Half of the animals were confined individually in the paired context on day one of the session, and the other half were individually confined in the paired context on day 2 of the session. The conditioning and testing sequence was repeated three times. Each test lasted 20 min. The results of our previous study [1] suggested that body temperature was not a measure of conditioned fear, given that this response did not condition differentially in the paired than in the unpaired context as did the other measures assessed. We speculated for the present study that either an additional conditioning session or an expanded testing window size of 20 min for each session may be required for a difference in body temperature to emerge between the paired and unpaired context. 2.5.5. Preference test For the last phase of the experiment, animals were given free access to the entire apparatus by being individually placed in the alley. Animals were expected to spend more time in the unpaired context and express an active avoidance of the paired context.

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3. Results

3.1. Histology Fig. 1 shows reconstructions of the smallest (open circles) and the largest (shaded areas) of the amygdala. As can be seen, large portions of both the anterior and posterior amygdala were damaged. Neurotoxic lesions of the amygdala produced extensive cell loss and gliosis in the central, basolateral, and lateral nuclei of the

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amygdala. One of the rats in the amygdala lesion group also sustained damage to the piriform cortex unilaterally. Based on the lesion sites, three rats were removed from the group. One rat sustained unilateral damage to the dorsal amygdala which extended to the caudate putamen. The second rat sustained posterior hippocampal damage unilaterally, and the third rat had unilateral damage to the amygdala. Fig. 2 shows reconstructions of the smallest (open circles) and the largest (shaded area) lesions of the

Fig. 1. Drawings of neurotoxic lesions of the amygdala. The shaded areas represent the maximum extent of all lesions and the open circles represent the minimum extent of all lesions for all rats.

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Fig. 2. Drawings of neurotoxic lesions of the hippocampus. The open circles represent the maximum extent of all lesions and the shaded areas represent the minimum extent of all lesions for all rats.

Response

Group

Session

Location

Group×Session

Group×Location

Session×Location

Group×Session ×Location

Freezing

F(2,21) =0.87, P\0.05 Heart rate: last 10 min F(2,21)=1.35, P\0.05 Body temp.: first 10 F(2,21)= 4.20, min PB0.05* Body temp.: last 10 F(2,21)= 7.04, min PB0.05* Defecation F(2,21)=1.53, P\0.05 Urination F(2,21)= 1.34, P\0.05 Locomotion F(2,21)= 3.05, P\0.05 Ultrasonic vocalizaF(2,21)=3.26, tions P\0.05 Pre-exposure F(2,21)= 0.53, P\0.05 Preference F(2,21)=2.02, P\0.05 * Statistically significant.

F(2,42) = 0.46, P\0.05 F(2,42) = 4.32, PB0.05* F(2,42) =0.19, P\0.05 F(2,42) = 1.27, P\0.05 F(2,42) = 9.18, PB0.05* F(2,42) =3.34, PB0.05* F(2,42) =0.41, P\0.05 F(2,42) = 0.67, P\0.05

F(1,21) = 15.43, PB0.05* F(1,21) =32.56, PB0.05* F(1,21) = 12.28, PB0.05* F(1,21) = 0.35, P\0.05 F(1,21) =19.51, PB0.05* F(1,21) = 33.39, PB0.05* F(1,21) = 28.70, PB0.05* F(1,21) =5.68, PB0.05* F(1,21) =0.01, P\0.05 F(1,21) =0.06, P\0.05

F(4,42) =1.38, P\0.05 F(4,42) =4.00, PB0.05* F(4,42) = 0.60, P\0.05 F(4,42) = 1.32, P\0.05 F(4,42) =0.26, P\0.05 F(4,42) = 1.42, P\0.05 F(4,42) = 3.31, PB0.05* F(4,42) =1.61, P\0.05

F(2,21) =7.35, PB0.05* F(2,21) = 4.12, PB0.05* F(2,21) = 1.03, P\0.05 F(2,21) =1.45, P\0.05 F(2,21) = 0.02, P\0.05 F(2,21) = 0.19, P\0.05 F(2,21) = 4.51, PB0.05* F(2,21) = 3.20, P\0.05 F(2,21) = 0.44, P\0.05 F(2,21) = 1.41, P\0.05

F(2,42)=0.34, P\0.05 F(2,42) =0.79, P\0.05 F(2,42) = 2.42, P\0.05 F(2,42)=3.65, PB0.05* F(2,42) =1.72, P\0.05 F(2,42) = 0.09, P\0.05 F(2,42) =2.06, P\0.05 F(2,42) =0.26, P\0.05

F(4,42) =0.96, P\0.05 F(4,42) = 0.87, P\0.05 F(4,42) =1.24, P\0.05 F(4,42) =0.21, P\0.05 F(4,42) =1.44, P\0.05 F(4,42) =0.79, P\0.05 F(4,42) =0.95, P\0.05 F(4,42) =1.34, P\0.05

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Table 1 Statistical analyses of main effects and interactions

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Sham Session 1 Freezing Heart rate: last 10 min Body temp.: first 10 min Body temp.: last 10 min Defecation Urination Locomotion Ultrasonic vocalizations Pre-exposure Preference

Amygdala Session 2

F(1,21)=5.93, F(1,21)= 5.93, PB0.05* PB0.05* F(1,21)= 14.37, F(1,21)= 27.69, PB0.05* PB0.05* F(1,21)=1.79, F(1,21)=2.64, P\0.05 P\0.05 F(1,21)= 0.50, F(1,21)= 0.23, P\0.05 P\0.05 F(1,21)= 1.55, F(1,21)= 0.16, P\0.05 P\0.05 F(1,21)=6.58, F(1,21)= 6.58, PB0.05* PB0.05* F(1,21) =9.50, F(1,21)= 19.20, PB0.05* PB0.05* F(1,21)= 1.43, F(1,21)=11.43, P\0.05 PB0.05* F(1,7)=0.44, P\0.05 F(1,7)=8.07, PB0.05*

* Statistically significant.

Hippocampus

Session 3

Session 1

Session 2

F(1,21) =41.45, PB0.05* F(1,21) =6.67, PB0.05* F(1,21) = 21.11, PB0.05* F(1,21) =5.99, PB0.05* F(1,21) =16.24, PB0.05* F(1,21) =8.40, PB0.05* F(1,21) = 21.21, PB0.05* F(1,21) = 7.54, PB0.05*

F(1,21) = 0.012, F(1,21) =0.46, P\0.05 P\0.05 F(1,21) =0.98, F(1,21) = 0.56, P\0.05 P\0.05 F(1,21) = 0.26, F(1,21) =0.21, P\0.05 P\0.05 F(1,21) = 0.98, F(1,21) = 0.61, P\0.05 P\0.05 F(1,21) = 0.55, F(1,21) =4.00, P\0.05 P\0.05 F(1,21) = 1.64, F(1,21) =2.74, P\0.05 P\0.05 F(1,21) = 1.40, F(1,21) =0.31, P\0.05 P\0.05 F(1,21) =2.21, F(1,21) = 0.25, P\0.05 P\0.05 F(1,7)= 0.002, P\0.05 F(1,7)= 0.00004, P\0.05

Session 3

Session 1

Session 2

F(1,21) = 2.60, P\0.05 F(1,21) = 0.69, P\0.05 F(1,21) = 14.30, PB0.05* F(1,21) = 1.35, P\0.05 F(1,21) = 4.94, PB0.05* F(1,21) = 5.83, PB0.05* F(1,21) =3.13, P\0.05 F(1,21) = 0.04, P\0.05

F(1,21) =0.04, F(1,21) = 1.84, P\0.05 P\0.05 F(1,21)= 7.20, F(1,21) =4.49, PB0.05* PB0.05* F(1,21) =0.52, F(1,21) = 1.44, P\0.05 P\0.05 F(1,21) =0.43, F(1,21) =0.22, P\0.05 P\0.05 F(1,21) =2.23, F(1,21) =4.00, P\0.05 P\0.05 F(1,21)= 0.41, F(1,21) =7.03, P\0.05 PB0.05* F(1,21)= 6.01, F(1,21) = 0.86, PB0.05* P\0.05 F(1,21) = 0.00, F(1,21) =0.00, P\0.05 P\0.05 F(1,7) = 0.43, P\0.05 F(1,7) = 0.90, P\0.05

Session 3 F(1,21) =1.55, P\0.05 F(1,21) = 6.67, PB0.05* F(1,21) =0.45, P\0.05 F(1,21) =0.05, P\0.05 F(1,21) =2.78, P\0.05 F(1,21) =5.83, PB0.05* F(1,21) =0.079, P\0.05 F(1,21) = 0.00, P\0.05

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Table 2 Statistical analyses of planned comparisons for each group at each session for all the responses

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hippocampus. The animals included in this group had large lesions of both dorsal and ventral portions of the hippocampus. Neurotoxic lesions of the hippocampus produced substantial cell loss and gliosis in the hippocampus. All of the lesions in the hippocampal group were virtually identical in extent of damage except for one animal that had some bilateral sparing of the ventral portions of the hippocampus. All the statistical analyses of the main effects: group, location and session, as well as the interactions for all the responses are found in Table 1. The planned comparisons for each group at each session are found in Table 2, and the planned comparisons for all the measures between all the groups are found in Table 3.

graph shows that the sham group (n= 8) spent more time freezing in the paired than in the unpaired context at test session 1 [F(1,21)= 5.93, P B 0.05], test session 2 [F(1,21)= 5.93, PB 0.05], and test session 3 [F(1,21)= 41.45, PB 0.05]. The amygdala (n= 8) and hippocampal (n= 8) lesioned animals showed no notable difference in their freezing behavior across all testing sessions. These findings suggest that both the amygdala and the hippocampus lesioned groups were unable to show discriminative freezing regardless of the amount of training, while the sham group showed higher freezing in the paired context starting at test session 1 and throughout test session 2 and 3.

3.2. Summary of results

3.2.2. Heart rate: last 10 min As previously observed [1], discriminative heart rate conditioning only emerges during the last 10 min of the test session in normals. In the present experiment the same findings emerged in controls, and therefore the first 10 min data for heart rate is not analyzed in the

3.2.1. Freezing Fig. 3 shows the mean amount of time the rats in the three different groups spent freezing in the paired and unpaired contexts during the three testing sessions. The

Table 3 Statistical analyses of planned comparisons between the groups for all the responses

Freezing Heart rate: last 10 min Body temp.: first 10 min Body temp.: last 10 min Defecation Urination Locomotion Ultrasonic vocalizations

Amygdala versus Sham

Hippocampus versus Sham

Amygdala versus Hippocampus

F(1,21)= 9.02, F(1,21)= 8.21, F(1,21)= 1.06, F(1,21)= 1.95, F(1,21)= 0.05, F(1,21)= 0.36, F(1,21)= 7.26, F(1,21)= 3.40,

F(1,21) =12.72, PB0.05* F(1,21) =1.59, P\0.05 F(1,21) =1.91, P\0.05 F(1,21) =2.39, P\0.05 F(1,21) =0.01, P\0.05 F(1,21) =0.20, P\0.05 F(1,21) =6.24, PB0.05* F(1,21) =5.58, PB0.05*

F(1,21) =0.31, P\0.05 F(1,21) =2.57, P\0.05 F(1,21) =0.12, P\0.05 F(1,21) =0.02, P\0.05 F(1,21) =0.008, P\0.05 F(1,21) =0.02, P\0.05 F(1,21) =0.03, P\0.05 F(1,21) =0.33, P\0.05

PB0.05* PB0.05* P\0.05 P\0.05 P\0.05 P\0.05 PB0.05* P\0.05

* Statistically significant.

Fig. 3. Mean amount of time (s) spent freezing in the paired and unpaired chambers during test sessions 1, 2 and 3 by rats with amygdala or hippocampus lesions and the sham animals.

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Fig. 4. Mean heart rate (BPM: beats/min) in the paired and unpaired context during the last 10 min of sessions 1, 2 and 3 for rats with amygdala or hippocampus lesions and the sham animals.

sham group or either of the lesioned groups. Fig. 4 shows the mean heart rate during the last 10 min of each session for the various groups. As can be seen in the graph, mean heart rate was higher in the paired versus the unpaired context for the sham group and hippocampus lesioned group across all sessions, while the amygdala lesioned group showed similar heart rate in both chambers. In the sham group, discriminatively higher heart rate in the paired context emerged at test session 1 [F(1,21)= 14.37, PB 0.05], and remained significantly higher at test session 2 [F(1,21) = 27.69, PB 0.05], and test session 3 [F(1,21) =6.67, P B 0.05]. For the hippocampus lesioned group, heart rate was significantly higher in the paired context at test session 1 [F(1,21)= 7.20, PB 0.05], test session 2 [F(1,21) = 4.49, P B 0.05], and test session 3 [F(1,21) = 6.67, P B 0.05]. For the amygdala lesioned group heart rate did not differ between the two chambers throughout the entire experiment. These findings suggest that the sham and hippocampus lesioned group were able to show discriminatively higher heart rate during the last 10 min block of each session in the paired chamber, while the amygdala lesioned group showed an impairment in this measure of conditioned fear to context.

3.2.3. Body temperature: first 10 min Fig. 5A shows the mean body temperature during the first 10 min of each session for the three groups. As can be seen in the graph, for the amygdala lesioned and sham group, body temperature was similar in both chambers during the first two sessions, and became significantly higher in the unpaired context during test

session 3 for both the sham [F(1,21)= 21.11, P B 0.05], and the amygdala lesioned group [F(1,21)= 14.30, PB 0.05]. Body temperature in the hippocampus lesioned group was similar in both chambers during the entire experiment. These results suggest that body temperature is a measure of fear that can condition to a context in a discriminative manner because it is higher in the unpaired chamber during the first 10-min block of the third session in the sham group. These data also illustrate that the amygdala lesioned group is able to exhibit the same discrimination as the sham lesioned group, while the hippocampus lesioned group demonstrated no such difference, as body temperature was similar in both chambers during the entire experiment.

3.2.4. Body temperature: last 10 min Fig. 5B shows the mean body temperature for the three groups in the paired and unpaired contexts during the last 10 min of each session. As can be seen in the graph, body temperature in the sham group is higher in the unpaired context across all testing sessions, while the amygdala and hippocampus lesioned groups showed no such discrimination. In the sham group, body temperature between the two chambers was not significantly different at test session 1 [F(1,21)=0.50, P\ 0.05], test session 2 [F(1,21)=0.23, P\ 0.05], but became significantly different at test session 3 [F(1,21)=5.99, PB 0.05]. In the amygdala and hippocampus lesioned group, the difference in body temperature between the two chambers did not differ significantly across all testing sessions. These results suggest that the sham group showed discriminative

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Fig. 7. Mean number of emissions of urine in the paired and unpaired context during test sessions 1, 2 and 3 by rats with amygdala or hippocampus lesions and the sham animals.

Fig. 5. (A) Mean body temperature (°C) in the paired and unpaired context during the first 10 min of session 1, 2 and 3 for rats with amygdala or hippocampus lesions and the sham animals. (B) Mean body temperature (°C) in the paired and unpaired context during the last 10 min of sessions 1, 2 and 3 for rats with amygdala or hippocampus lesions and the sham animals.

Fig. 6. Mean amount of fecal matter (number of feces) emitted in the paired and unpaired context during test sessions 1, 2 and 3 by rats with amygdala or hippocampus lesions and the sham animals.

conditioned body temperature during the last 10-min block of session 3, and that rats with amygdala and hippocampus lesions were impaired at this discriminative ability. Comparisons between the first 10 min and the last 10 min in body temperature for the sham group revealed a significant difference between the two time points [F(1,14)= 24.84, PB0.05], indicating that overall body temperature was higher during the last half of each testing session. Body temperature was also significantly higher during the last half of every session in the amygdala group [F(1,14)=53.04, PB 0.05], and the hippocampus lesioned group [F(1,14)= 10.63, PB 0.05].

3.2.5. Defecation Fig. 6 shows the mean amount of feces emitted in each chamber by the three groups throughout the three testing sessions. For the sham group, the level of defecation was similar in the paired and in the unpaired context at test session 1 [F(1,21)= 1.55, P\ 0.05], and session 2 [F(1,21)= 0.16, P\ 0.05], but became considerably higher in the paired context at test session 3 [F(1,21)= 16.24, PB 0.05]. The level of defecation for the amygdala lesioned group between the two chambers was similar in test session 1 and 2, but became notably higher in the paired chamber at test session 3 [F(1,21)=4.94, PB 0.05]. The hippocampus lesioned group demonstrated no such discrimination, as level of defecation was similar across both contexts during the three testing sessions. These results suggest that conditioned defecation is a measure of fear that was impaired in animals with hippocampus lesions but remained intact in sham and amygdala lesioned animals. 3.2.6. Urination Fig. 7 shows the mean level of urination for the three groups across the three test sessions. For the sham

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group, urination was consistently greater in the paired context at test session 1 [F(1,21) =6.58, P B 0.05], test session 2 [F(1,21)=6.58, P B0.05], and test session 3 [F(1,21)=8.40, PB 0.05]. For the amygdala lesioned group, level of urination only became higher in the paired context at test session 3 [F(1,21) = 5.83, PB 0.05], while the hippocampus lesioned group demonstrated this discrimination at test session 2 [F(1,21)= 7.03, PB 0.05] and session 3 [F(1,21) 5.83, P B 0.05]. These results suggest that conditioned urination is not impaired following hippocampal or amygdala lesions, and suggests that another memory system may participate in the conditioning of this fear response.

3.2.7. Locomotion Fig. 8 shows the level of locomotion in both chambers for the three groups. As can be seen there was more locomotion in the unpaired than in the paired chamber in the sham group across test session 1 [F(1,21)=9.50, PB 0.05], test session 2 [F(1,21)= 19.20, PB0.05], and test session 3 [F(1,21)=21.21, PB 0.05]. For the amygdala lesioned group the level of locomotion between the two contexts did not differ significantly across all sessions. The hippocampus lesioned group showed a higher level of locomotion in the paired chamber at test session 1 [F(1,21)=6.01, PB 0.05], but this extinguished at session 2 [F(1,21)= 0.86, P\ 0.05], and test session 3 [F(1,21)= 0.079, P\ 0.05]. These results suggest that amygdala lesioned animals are impaired at expressing a higher level of locomotion in the unpaired chamber across the entire experiment, and that their hippocampal counterparts only demonstrate such ability in the first session. 3.2.8. Ultrasonic 6ocalizations Fig. 9 shows the number of emissions produced in both chambers across the three testing sessions for all groups. As shown in the graph, the sham group emitted considerably more vocalizations in the paired chamber at test session 2 [F(1,21)=11.43, PB0.05], and test session 3 [F(1,21)= 7.54, PB 0.05]. Both the amygdala and hippocampus lesioned group did not show any difference in their amount of vocalizations within the two chambers. These results suggest that conditioned vocalizations in a discriminative manner is a measure of fear that is impaired by either amygdala or hippocampus lesions.

Fig. 8. Mean level of locomotion (number of crossings) in the paired and unpaired context during test sessions 1, 2 and 3 for rats with amygdala or hippocampus lesions and the sham animals.

Fig. 9. Mean amount of ultrasonic vocalizations emitted in the paired and unpaired context during test sessions 1, 2 and 3 by rats with amygdala or hippocampus lesions and the sham animals.

3.2.9. Pre-exposure Fig. 10 (A) shows the mean amount of time that the three groups spent in each chamber during the pre-exposure phase. The graph indicates that all groups showed no difference in dwell time between the black triangle prism and white square prism. A closer examination indicated that the time spent in the paired and unpaired chambers did not differ in the sham group [F(1,7)= 0.44, P\ 0.05], in the amygdala lesioned group [F(1,7)= 0.002, P\ 0.05], and in the hippocampus lesioned group [F(1,7)= 0.43, P\0.05]. These results suggest that the groups have no initial preference for any one of the chambers, and that this is an unbiased procedure [10]. 3.2.10. Preference test Fig. 10 (B) shows the mean amount of time that the three groups spent in the paired and unpaired chambers during the preference test. The graph illustrates that the sham group spent more time in the unpaired chamber while the amygdala and hippocampus lesioned groups spent an equal amount of time in both chambers. A

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Fig. 10. (A). Mean amount of time (s) spent in the black triangle and the white square prism during the pre-exposure phase by rats with amygdala or hippocampus lesions and the sham animals. (B) Mean amount of time (s) spent in the paired and unpaired context during the preference test by rats with amygdala or hippocampus lesions and the sham animals.

closer examination revealed that the sham group spent significantly more time in the unpaired chamber during the preference test [F(1,7) =8.07, P B 0.05], but that the amygdala [F(1,7)=0.00004, P \ 0.05], and the hippocampus lesioned groups [F(1,7) = 0.90, P \0.05], did not show a preference for the paired chamber. That there is no significant difference in dwell time for both the amygdala and hippocampus lesioned groups reveals an impairment on this conditioned measure of fear.

4. Discussion The responses examined in the present study occur in fearful situations and are ultimately controlled by motoneurons in the spinal cord and brain stem. These fear responses have been found to be modulated by forebrain structures that are now known to form the socalled emotional motor system [5,17]. Forebrain structures do not project directly to autonomic or somatic motoneurons in the spinal cord or brain stem, but rather, these sites modulate behaviours such as urination, vocalizations, cardiovascular changes and defensive postures, via premotor interneurons in the caudal brain stem that act as a relay [6]. The measures of fear assessed in the present experiment and their development across the three testing sessions have been examined in relation to the neural circuits that exist between the spinal cord, brain stem and the learning and memory structures of interest: the hippocampus and the amygdala. This depiction of the connections between each memory structure and the sites that are involved in the unconditioned emergence of the fear responses, may enable us to understand how each mem-

ory structure participates in fear conditioning to context. For our assessment, figures illustrating the neural circuits that might enable each forebrain system to influence the conditioning of the fear response(s) are depicted. Fig. 11 (A) shows the synergistic interactions between the amygdala and hippocampus and the possible eventual influence of freezing, locomotion, ultrasonic vocalizations and preference. Fig. 11 (B) shows the connections that exist between the amygdala and brainstem structures that potentially mediate the emergence of conditioned heart rate. Fig. 11 (C) shows the connections that exist between the hippocampus and subcortical sites that may influence the fear conditioning to context of certain fear responses including body temperature and defecation. We will describe the possible anatomical circuits for the conditioning of defecation as mediated by the hippocampus, and the conditioning of heart rate as mediated by the amygdala. This represents a unique dissociation between the two memory systems in that amygdala lesions impair conditioned heart rate but not defecation, and hippocampal lesions impair conditioned defecation but not heart rate.

4.1. Anatomy 4.1.1. Heart rate Amygdala damage: discrimination never appears. Hippocampus damage: discrimination appears in session 1. The effect of amygdala ablation was the suppression of normally elevated heart rate in the paired context. These data suggest that some component of the amygdala has projections to areas of the central nervous

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system mediating cardiovascular activity and that through these projections the amygdala comes to control these homeostatic mechanisms during emotional activity and emotional learning. The medulla includes the nucleus of the solitary tract (NTS), and the dorsal motor nucleus (DVN), which are sensory and motor subdivisions that subserve cardiovascular regulation in the rabbit, cat, rat and monkey [18,19]. The nucleus of the solitary tract (NTS) is a visceroceptive cell group and is located in the dorsal medulla. The immediately adjacent dorsal motor nucleus of the vagus nerve is one of the main origins of preganglionic parasympathetic fibers, and it controls cardiovascular and other visceromotor functions [3,12,20,32]. Converging evidence suggests that the central nucleus of the amygdala has direct projections to the nucleus of the solitary tract (NTS), and the subjacent dorsal motor nucleus (DVN), which may therefore influence cardiovascular and other autonomic responses. The idea that the ACE exerts control over cardiovascular activity has been heavily sustained by staining techniques, demonstrating that the central

nucleus projects to the nucleus of the solitary tract and the dorsal motor nucleus within the vagus nerve, in the rabbit [43], and in the rat [44,49]. Interestingly, the heaviest subcortical afferentiation of the NTS originates from the central nucleus of the amygdala [50]. van der Kooy et al. [50] suggested that forebrain structures like the ACE, the medial lateral prefrontal cortex, the bed nucleus of the stria terminalis to name a few, may form a visceral forebrain which exerts influences on cardiovascular regulation during emotional activity. This suggestion has been previously supported by experiments reporting the interference of conditioned heart rate to a single CS following the ablation of the amygdaloid complex, supporting that the amygdala is a critical link in the neural circuit underlying aversive emotional conditioning [21,25,26,38]. Major projections from ACE descend via the lateral hypothalamic area to reach the ventrolateral medulla, the NTS and the pariaqueductal gray matter (PAG) [23,40,51]. The anatomical findings stated above strongly suggest that the memory system that includes the amygdala comes to

Fig. 11. (A) Proposed neural circuit depicting the synergistic interaction between the hippocampus and the amygdala and the possible mediation of ultrasonic vocalizations, locomotion and freezing. (B) Proposed neural circuit involved in the control of conditioned heart rate by the amygdala. (C) Proposed neural circuitry for the influence of body temperature and defecation by the hippocampus.

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control the conditioning of heart rate to a fearful context via its anatomical relation to structures that subserve cardiovascular changes. Ablation of this memory system would result in a deficit in the conditioning of this fear response to a fearful context, as observed in the present study.

4.1.2. Defecation Amygdala damage: discrimination appears at test session 3. Hippocampus damage: discrimination never appears. The motoneurons controlling the anal sphincter are located in the dorsomedial nucleus of the ventral horn at the level of L5 and L6 in the rat [31,42]. This cell group is separate from the cell group controlling the urethral sphicter, which is located in the dorsolateral nucleus of the ventral horn. In the cat, dog, monkey and man, the motoneurons innervating the anal and urethral sphincter are located in the same cell group called the nucleus of ONUF [24,33,39,41,48]. The ventral horn of the spinal cord that contains the cell group controlling the anal sphincter, receives most of its projections from caudal portions of the ventromedial medulla, including the nuclei raphe pallidus and obscurus [16]. The participation of the raphe nucleus has recently been supported in the conditioning of defecation to a fearful context. Lesions of the median raphe nucleus resulted in an impairment of conditioned defecation [2], while the electrical stimulation of this site caused behavioural and autonomic manifestations of fear that included defecation [14]. The pathway between the hippocampus and the raphe nucleus [7,8,13] may be responsible for the modulation of defecation in a fearful environment. Amygdala lesions do not interfere with the acquisition of conditioned defecation [47,50], but hippocampal lesions do interfere with the conditioning of this fear response [47]. Accordingly, in the present study, animals with hippocampus lesions were impaired at this form of autonomic conditioning to context, while their amygdala counterparts demonstrated normal conditioning. 4.2. Learning rate parameters It has been previously reported that control animals express discriminative fear conditioning to context in the measures of fear assessed, and that subsets of these measures have different learning rate parameters [1]. The sham lesioned animals of the present study showed the same pattern in the development of their fear responses. From our previous study, body temperature did not seem to be a measure of conditioned fear to context. However, we noted that across the three testing sessions, the difference in body temperature between the paired and unpaired chamber was increasing from the first to the third session [1]. We tentatively

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concluded that if this is a measure of fear that can condition to a fearful context, it may have a slower learning rate parameter, and it may therefore require either additional conditioning, or an extended testing window. Accordingly, in the present experiment we opted for the latter option and extended our testing window to 20 min per session. With this arrangement, body temperature emerged as a conditioned measure of fear, as it was significantly higher in the unpaired context during the entire length of session 3. As such we qualify it as a slow measure of conditioned fear. We have previously reported that freezing, urination and locomotion are rapid measures of conditioned fear, as they emerge in a discriminative manner during the first 10-min block of the first and each subsequent testing session. We also classified heart rate, defecation, and ultrasonic vocalizations as slow measures of conditioned fear as they only surfaced in a discriminative way during the last 10-min block of the third session [1]. In the present experiment, we report that freezing, urination, locomotion, defecation, and ultrasonic vocalizations have the same learning rate parameter as previously shown. Heart rate however, showed a different pattern under the present circumstances, as it emerged in a discriminative manner during the last 10 min of every testing session. As such, conditioned heart rate does not fit the criterion of a purely ‘fast’ measure of conditioned fear as it does not appear during the first 10 min of the first and any subsequent testing session. Similarly, it cannot be classified as a strictly ‘slow’ measure of fear as it appears starting at session 1. A strict ‘slow’ measure of fear requires more than one testing session. Under the present circumstances, heart rate does not fit any of the classifications that we have previously defined for conditioned measures of fear, which prompts us to expand our spectrum and include a definition for measures of fear that are neither ‘slow’ nor ‘fast’, but whose temporal emergence is more susceptible to the interaction between the testing session and window size. The present experiment allowed us to expand our classifications and include a categorization for such measures of fear that we will refer to as a ‘intermediate’ measures of fear.

4.3. Current theories In the present experiment, we attempted to understand the participation of the hippocampus and amygdala in fear conditioning to environmental cues, by using a recently developed discriminative fear conditioning to context paradigm [1]. The paradigm involved multiple training and testing sessions and the assessment of multiple fear responses which included: preference, freezing, heart rate, ultrasonic vocalizations, defecation, body temperature, urination and locomotion. The results of the present experiment clearly re-

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vealed that the amygdala is a learning and memory structure that participates in fear conditioning to context and contributes exclusively to the emergence of conditioned heart rate. The hippocampus is also a learning and memory structure that participates in fear conditioning to context and contributes exclusively to the emergence of conditioned defecation and body temperature. Interestingly, the amygdala and hippocampus are required for the conditioning of freezing, ultrasonic vocalizations, locomotion and preference. Conditioned urination emerged in both the hippocampus and amygdala lesioned groups, suggesting that this measure of fear is mediated by another memory system, and that neither system is essential for its conditioning. There are currently four predominant views that ascribe roles to the hippocampus and amygdala in fear conditioning to context. Our present results are inconsistent with these views on the degree of involvement of each memory structure. The first view of the role of the hippocampus in fear conditioning to context suggests that the hippocampus is critical for stimulus selection during learning [52]. Support for this view comes from an experiment using an Oddling–Smee procedure which manipulates the predictive value of a context by training different cues which were associated with footshock all the time, half the time, or none of the time. Conditioning to the background context was then tested and normal animals showed graded contextual conditioning based on the predictive value of the context during training. Rats with lesions of the hippocampus showed enhanced fear conditioning to context in all conditions. This pattern of data is taken to suggest that the hippocampal rats are enhanced at fear conditioning to context. The second view states that complex hippocampal representations of context must output through amygdala circuitry to be associated with the unconditioned response to the shock. This view represents an example of a synergistic interaction between the hippocampus and amygdala [27]. Support for this view comes from recent experiments that have shown that rats with damage to the amygdala are impaired at the acquisition and retention of conditioned fear to a single cue and to a context paired with a mild foot-shock [22,36]. Furthermore, rats with damage to the hippocampus were impaired in the acquisition of conditioned fear to context but not to a single cue paired with shock [4]. Others have suggested that both the hippocampus and amygdala can independently access unconditioned responses to the shock and that the nature of the representation needed to identify/recognize the environmental context dictates the necessity for hippocampal or amygdala circuitry. Support for this alternative view comes from experiments showing that rats with hippocampal damage, but not disruption of amygdala circuits, are impaired at the acquisition of conditioned fear to context [15,22,45,47].

The final view argues for a limited role of the hippocampus in fear conditioning to context. Phillips and Ledoux [37] suggest that an intact hippocampus is necessary for fear conditioning to context only when there is a competition for associative strength between the static context and a predictive phasic cue. Consistent with this view of hippocampal function, rats with hippocampal damage show impairments on fear conditioning to context when there was a cue/context competition but show normal acquisition during static context conditioning [35,37]. The results obtained with our discriminative procedure and the assessment of multiple measures of fear require us to propose a new view to clarify the participation of the hippocampus and amygdala in fear conditioning to context. The core of the proposed view ascribes an equal role in fear conditioning to context to both the amygdala and the hippocampus. This proposal stems from the present findings that the conditioning of heart rate is selectively mediated by a memory structure that involves the amygdala, while the conditioning of defecation and body temperature is exclusively mediated by a memory system that includes the hippocampus. However, our findings cannot solely be accounted for by a view espousing that the hippocampus and amygdala independently participate in fear conditioning to context, given that the conditioning of a subset of fear responses requires the participation of both memory structures. The discriminative conditioning of freezing, ultrasonic vocalizations, locomotion and preference seem to necessitate a synergistic interaction between the amygdala and hippocampus. The present data are taken to suggest that the conditioning of urination may be mediated by a memory system other than the hippocampus or the amygdala. As previously reported subsets of these fear responses in the normal rat show different acquisition rates [1]. Freezing, urination, locomotion and preference are ‘fast’ measures of fear in that they discriminatively condition to context following a single training session. Ultrasonic vocalizations and defecation are ‘slow’ measures of fear, given that they require multiple training sessions to condition to a fearful context. Heart rate is an ‘intermediate’ measure of fear given that it requires an expanded testing window of 20 min to emerge. Interestingly, conditioned defecation and conditioned body temperature, measures that are exclusively mediated by the hippocampus, are both ‘slow’ measures. Freezing, locomotion and preference are ‘fast’ measures of fear in that they discriminatively condition to context following a single training session. Interestingly, they all necessitate the participation of the hippocampus and the amygdala. Ultrasonic vocalizations is a ‘slow’ measure of fear which also requires the participation of both memory structures. Neither the hippocampus nor the amygdala seem to be essential for the conditioning

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of urination to a fearful context. Heart rate is an ‘intermediate’ measure of fear and it is exclusively modulated by the amygdala. Although there is no clear-cut separation between the measures mediated by the hippocampus and those mediated exclusively by the amygdala on the basis of acquisition rates, there is an apparent pattern. It seems that the hippocampus selectively modulates ‘slow’ measures of fear. The amygdala exclusively mediates the ‘intermediate’ measure of fear. Both memory structures interact synergistically to modulate fast measures of fear: locomotion, freezing and preference, and ultrasonic vocalizations, a ‘slow’ measure of fear.

4.4. What factors determine the participation of the amygdala and the hippocampus in fear conditioning to context? Three factors seem to determine which long-term memory system is activated during fear conditioning to context. The degree of similarity between the two contexts, determines whether the situation is ambiguous or non-ambiguous, and defines the information available to the animal. This in turn influences the necessity of different memory systems. Secondly, the output examined by the experimenter may be limiting in terms of all the possible outlets of fear expression and the mediation of these outlets by different memory systems. Finally the activation of specific memory systems in this form of learning is also influenced by the behaviors that the animal can engage in during training. For instance, if the animal is unrestrained, the ability to explore the surroundings may influence the degree to which contextual information is processed. The experiments that we report in this paper do not address the issue of representations formed by the two learning and memory systems. According to our findings, the degree of participation of each system is based on the selective mediation of fear responses by each system. However, the proposed view would make certain predictions if there were a greater cue ambiguity by increasing the similarity between the two chambers and rendering the discrimination more dependent on hippocampal processing. The hippocampus seems necessary when context discrimination has high associative interference from overlapping (common) cues found in both chambers, and creates two different ‘contextual’ representations of the chambers, which then become associated with the shock. Accordingly, hippocampal lesions have been reported to impair the freezing response in discriminative fear conditioning to context that involve a high cue ambiguity [11,29]. The importance of hippocampal processing in high cue ambiguity situations was highlighted by the findings of an operant conditioning experiment, where animals with hippocampal lesions were impaired in configural tasks

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like negative patterning and biconditional discrimination but not contextual-conditional discrimination [30]. Negative patterning has the highest within session ambiguity and contextual-conditional discrimination has the lowest. Similarly, in a radial arm maze set-up, the discrimination between two adjacent arms of a radial arm maze is high in cue ambiguity given the overlapping spatial elements. Accordingly, hippocampal lesions resulted in an impairment on this version of the place discrimination task but not a version which involved non-adjacent arms and was therefore lower in cue ambiguity [28]. The above stated results in combination with the present findings suggest that level of cue ambiguity, amount of training and the measures of fear assessed, are three important factors and that the interaction between these factors may have an influence in determining which learning and memory system participates in this form of conditioning. We would predict that if the present paradigm were highly ambiguous, damage to the hippocampus would result in the impairment of conditioned body temperature and defecation as well as the responses that require both memory systems. Overtraining would be required for the conditioning of heart rate given that the conditioning of this response would require the amygdala to detect some cue dimension on which the chambers differ, but the conditioned response mediated by the amygdala would eventually emerge. Accordingly, animals with damage to the amygdala would be impaired in the conditioning of heart rate and the responses that require both systems. With the contextual representation formed by the intact hippocampus, the conditioning of body temperature and defecation would not be impaired. The issue of amygdalar and hippocampal contextual representations is of importance and should be addressed with the purpose of assessing how each representation contributes in solving a highly ambiguous situation. In conclusion, although we admit that the view presented in this paper concerning the neural organization of fear conditioning to context is not elegant, it provides a lucid appraisal as to how each memory system participates in the conditioning of many measures of fear.

Acknowledgements This work was supported by an NSERC operating grant awarded to Dr R.J. McDonald. We thank Michael Child and Andrew Gristock for excellent animal care. We are grateful to Dr Stephan Brudzynski for insightful comments on the neural basis of ultrasonic vocalizations, and Nancy Hong for valuable advice on presentation of the anatomy section. We thank Eric Rieux for technical assistance, and James Dix for precious mechanical help.

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