Functional anatomy of neural circuits regulating fear and extinction.

The memory of fear extinction is context dependent: fear that is suppressed in one context readily renews in another. Understanding of the underlying neuronal circuits is, therefore, of considerable clinical relevance for anxiety disorders. Prefrontal cortical and hippocampal inputs to the amygdala have recently been shown to regulate the retrieval of fear memories, but the cellular organization of these projections remains unclear. By using anterograde tracing in a transgenic rat in which neurons express a dendritically-targeted PSD-95:Venus fusion protein under the control of a c-fos promoter, we found that, during the retrieval of extinction memory, the dominant input to active neurons in the lateral amygdala was from the infralimbic cortex, whereas the retrieval of fear memory was associated with greater hippocampal and prelimbic inputs. This pattern of retrieval-related afferent input was absent in the central nucleus of the amygdala. Our data show functional anatomy of neural circuits regulating fear and extinction, providing a framework for therapeutic manipulations of these circuits.

Social modulation of learning in rats.

It is well known that emotions participate in the regulation of social behaviors and that the emotion displayed by a conspecific influences the behavior of other animals. In its simplest form, empathy can be characterized as the capacity to be affected by and/or share the emotional state of another. However, to date, relatively little is known about the mechanisms by which animals that are not in direct danger share emotions. In the present study, we used a model of between-subject transfer of fear to characterize the social interaction during which fear is transmitted, as well as the behavioral effects of socially transmitted fear. We found that (1) during social interaction with a recently fear-conditioned partner, observers and demonstrators exhibit social exploratory behaviors rather than aggressive behaviors; (2) learning and memory in a shock-motivated shuttle avoidance task are facilitated in rats that underwent a social interaction with a partner that had been fear conditioned; and (3) a brief social interaction with a recently fear-conditioned partner immediately before fear conditioning increases conditioned freezing measured on the next day. The observed effects were not due to a stress-induced increase in pain sensitivity or analgesia. Collectively, these data suggest that a brief social interaction with a cage mate that has undergone an aversive learning experience promotes aversive learning in an otherwise naïve animal. We argue that socially transferred fear is an adaptation that promotes defensive behavior to potentially dangerous situations in the environment.

Sex differences in social modulation of learning in rats.

In its simplest form, empathy can be characterized as the capacity to share the emotional experiences among individuals, a phenomenon known as emotional contagion. Recent research shows that emotional contagion and its adaptive role can be studied in rodents. However, it is not known whether sex differences observed in human empathy extend to its more primitive forms. In the present study, we used a rat model of emotional contagion to compare the behavioral consequences of social transfer of information about threat, and the subsequent neural activation patterns in male and female rats. We found that: (1) males and females display a similar behavioral pattern during the interaction with either a fear-conditioned or a control rat; (2) interaction with a fear-conditioned conspecific positively modulates two-way avoidance learning in male and diestral female rats but not in estral females; and (3) such interaction results in increased c-Fos expression in the central and lateral nuclei of the amygdala and the prelimbic and infralimbic cortex in males, whereas in females no such changes were observed. Collectively, our results point to the occurrence of sex and estrus cycle phase differences in susceptibility to emotional contagion and underlying neuronal activation in rodents.

Effects of stimulus modality on the shuttle activity in rats.

Shuttle activity during repeated presentation of irrelevant auditory (white band noise) and visual (darkness) stimuli was studied in 32 male Möll-Wistar rats. The subjects were randomly divided into two groups. In Group ND the auditory stimulus was used in the first habituation session, and the visual stimulus in the second habituation session. The opposite sequence of stimuli was applied in Group DN. House light was used as a background stimulus in both groups of subjects. The rate of crossing from one compartment to the other was markedly enhanced by noise, regardless of the position of the auditory stimulus in the session's sequence. A clear decrease of response rate was caused by noise termination, whereas neither darkness offset nor onset changed the relatively high and stable rate of responding. The results of this study provided convincing evidence of the energizing influence of the irrelevant auditory cue on the shuttle activity in rats. Moreover, these findings showed that a habituation procedure provides an efficient tool to analyze pure unconditional features of the to-be-conditioned stimuli, and to foresee several response modifications during further conditioning.

Effects of social rearing conditions on conditioned suppression in rats.

Twenty-two rats were reared in standard conditions during the first two months of their life. Then the animals were divided into two groups exposed to different rearing conditions. Twelve animals (Group SO) were housed socially, six animals per cage, and for three weeks they were subjected to sensory stimulation in an enriched environment. The other ten subjects were kept individually (Group IN); one rat per mesh cage, in conditions of relatively impoverished sensory stimulation. In both groups the training of the conditioned emotional response (CER) was performed when animals were three months old. In contrast to IN subjects, the rats subjected to permanent social contacts and reared in the enriched environment (Group SO) revealed almost equally low instrumental response rates in trials with the conditioned stimulus (CS) paired with nociceptive foot-shock (US), and in periods when no CS and/or US were applied. The results suggested that early exposure to an enriched environment caused a later decrease of the animals' capability to differentiate between the aversive CS and cues of the experimental context. This cognitive impairment was probably a secondary effect of fear generalized to the entire experimental situation.

Social modulation in extinction of aversive memories.

Return of fear after extinction is a considerable challenge for the efficacy of exposure-based therapies. Fear recovery is most often modeled in the laboratory by changing the experimental context and studied in isolated animals. Since social context is an important factor affecting behavior, the question arises how it influences the recovery of extinguished fear. Here we present two novel behavioral models that allow studying social modulation of extinction memory retrieval. We show that the presence of a fearful cage mate results in a robust renewal of freezing as well as avoidance responses that were previously successfully extinguished.

Acoustic startle and disruption of prepulse inhibition by dizocilpine in selectively bred mice.

In this study we examined the relationship between genetically produced differences in the magnitude of prepulse inhibition (PPI) of the acoustic startle response (ASR) and stress induced swim analgesia in genetically different strains of mice. Prepulse inhibition of the ASR and its changes due to dizocilpine (MK-801) injection were studied in 180 mice. The animals used in this study were obtained from our colony of 54 generation, Swiss-Webster mice selectively bred for high and low magnitude of analgesia. Three month old male mice of the high analgesia (HA) and the low analgesia (LA) lines, in addition to randomly bred controls (C) were used in the experiment. Thirty minutes before the ASR session the mice were injected intraperitoneally with saline or with 0.15, 0.25, 0.5 mg÷kg of dizocilpine maleate. Prepulses suppressed the acoustic startle response in all lines in a prepulse intensity-dependent manner, but only the differences between the weakest and the strongest prepulses appeared significant. Two-way ANCOVA performed separately for each line revealed a significant effect of dizocilpine and prepulse intensity. Only in the HA line, however, the disruption of PPI from the injection of dizocilpine was evidenced by significant treatment by prepulse interaction. That means the prepulses decreased ASR significantly less in dizocilpine- treated animals than in saline-treated animals. The results confirmed that the mouse lines manifesting differential ASR magnitudes along with different degrees of PPI sensitivity to dizocilpine, might be suitable for pharmacogenetic studies on the glutaminergic mechanism of the startle response.

Differential startle magnitude in mice selected for high and low swim analgesia is not related to difference in nociception.

The acoustic startle response (ASR) elicited by 110 dB 10-ms pulses was studied in relation to pain sensitivity in mouse lines selectively bred for high (HA) and for low (LA) swim analgesia. The magnitudes of ASR, similarly as hot-plate latencies, differed between the lines in the rank order HA is greater than unselected controls (C) greater than LA. The animals' nociception did not change after the ASR session consisting of a sequence of 20 acoustic stimuli. Morphine hydrochloride (5 and 10 mg per kg i.p.) increased hot-plate latencies in the order of HA greater than C greater than LA, and was not effective on ASR magnitude in HA as well as in C mice. In the LA line, 10 mg per kg of morphine slightly attenuated ASR, but caused only a little analgesia. We conclude that (1) the difference in ASR between the selected lines is inversely correlated with the difference in pain sensitivity; (2) the magnitude of ASR is not altered by morphine analgesia; (3) the procedure of ASR using brief acoustic pulses is not stressful enough to elicit a form of stress analgesia. The lack of a direct relationship between the readiness to startle and pain sensation may be beneficial for an animal's survival in dangerous situations. It is beneficial when the startle to a warning signal precedes defensive behaviors and it often must be effectuated in a state of decreased nociception.

Functional internal complexity of amygdala: focus on gene activity mapping after behavioral training and drugs of abuse.

The amygdala is a heterogeneous brain structure implicated in processing of emotions and storing the emotional aspects of memories. Gene activity markers such as c-Fos have been shown to reflect both neuronal activation and neuronal plasticity. Herein, we analyze the expression patterns of gene activity markers in the amygdala in response to either behavioral training or treatment with drugs of abuse and then we confront the results with data on other approaches to internal complexity of the amygdala. c-Fos has been the most often studied in the amygdala, showing specific expression patterns in response to various treatments, most probably reflecting functional specializations among amygdala subdivisions. In the basolateral amygdala, c-Fos expression appears to be consistent with the proposed role of this nucleus in a plasticity of the current stimulus-value associations. Within the medial part of the central amygdala, c-Fos correlates with acquisition of alimentary/gustatory behaviors. On the other hand, in the lateral subdivision of the central amygdala, c-Fos expression relates to attention and vigilance. In the medial amygdala, c-Fos appears to be evoked by emotional novelty of the experimental situation. The data on the other major subdivisions of the amygdala are scarce. In conclusion, the studies on the gene activity markers, confronted with other approaches involving neuroanatomy, physiology, and the lesion method, have revealed novel aspects of the amygdala, especially pointing to functional heterogeneity of this brain region that does not fit very well into contemporarily active debate on serial versus parallel information processing within the amygdala.

Between-subject transfer of emotional information evokes specific pattern of amygdala activation.

Emotional states displayed by an animal or a human can seriously affect behavior of their conspecifics. The amygdala plays a crucial role in the processing of emotions. In this study, we describe an experimental rat model of between-subject transfer of emotional information and its effects on activation of the amygdala. The rats were kept in pairs, and one animal (designated as "demonstrator") was treated to specific behavioral training of either foot-shock-reinforced context conditioning or just exposure to a novel context. We next examined the influence of the demonstrators on the exploratory behavior of their cagemates (called "observers") and the observers' performance of the acoustic startle response. We report that we can distinguish both groups of observers from the control animals (as shown by startle-response measure) and distinguish between observers (by means of indexing the exploration), with respect to whether they were paired with demonstrators treated to different experimental conditions. Furthermore, we show that the observers have most of their amygdala activated (as revealed by c-Fos mapping) to the same level as the demonstrators and, in the case of the central amygdala, to an even higher level. Moreover, the level of c-Fos expression in the observers reflected the specific behavioral treatment of the demonstrators with whom they were paired. Thus, in this study, we have shown that undefined emotional information transferred by a cohabitant rat can be evaluated and measured and that it evokes very strong and information-specific activation of the amygdala.

Differential involvement of the central amygdala in appetitive versus aversive learning.

Understanding the function of the distinct amygdaloid nuclei in learning comprises a major challenge. In the two studies described herein, we used c-Fos immunolabeling to compare the engagement of various nuclei of the amygdala in appetitive and aversive instrumental training procedures. In the first experiment, rats that had already acquired a bar-pressing response to a partial food reinforcement were further trained to learn that an acoustic stimulus signaled either continuous food reinforcement (appetitive training) or a footshock (aversive training). The first training session of the presentation of the acoustic stimulus resulted in significant increases of c-Fos immunolabeling throughout the amygdala; however, the pattern of activation of the nuclei of the amygdala differed according to the valence of motivation. The medial part of the central amygdala (CE) responded, surprisingly, to the appetitive conditioning selectively. The second experiment was designed to extend the aversive versus appetitive conditioning to mice, trained either for place preference or place avoidance in an automated learning system (INTELLICAGE). Again, much more intense c-Fos expression was observed in the medial part of the CE after the appetitive training as compared to the aversive training. These data, obtained in two species and by means of novel experimental approaches balancing appetitive versus aversive conditioning, support the hypothesis that the central nucleus of the amygdala is particularly involved in appetitively motivated learning processes.

Involvement of the lateral and dorsolateral amygdala in conditioned stimulus modality dependent two-way avoidance performance in rats.

Post-lesion learning and performance of shuttle-box avoidance and subsequent transfer to two warning signals (CSs) of different modality were investigated in 27 rats subjected either to a sham lesion (Group NORM), electrolytic injuries of the lateral amygdaloid nucleus (Group LAT), or combined lesions of the amygdalostriatal transition area and dorsolateral amygdala (Group D-LAT). All groups were divided into two subgroups according to warning signal sequences. In the first subgroup (D-DN-N sequence) the subjects were initially trained with the visual CS (darkness - D), then transferred to the more salient visual and auditory compound CS (darkness and noise - DN), and finally to the auditory CS alone (noise - N). The opposite arrangement of the CSs (N-ND-D sequence) was employed in the second subgroup. A small interference with shuttle-box learning, and no transfer deficit were seen in Group LAT, whereas D- LAT rats showed dramatically slow and inconsistent acquisition of avoidance responding followed by rapid weakening of performance during the training. In contrast to controls, in both lesioned groups avoidance and intertrial responding (ITR) were independent of the CS modality changes. The results indicate differential involvement of the lateral, and also of the dorsolateral amygdala, and amygdalostriatal transition area in CS processing, as well as in the mechanisms related to consolidation of the associations created during avoidance training.

Complex effects of NMDA receptor antagonist APV in the basolateral amygdala on acquisition of two-way avoidance reaction and long-term fear memory.

Although much has been learned about the role of the amygdala in Pavlovian fear conditioning, relatively little is known about an involvement of this structure in more complex aversive learning, such as acquisition of an active avoidance reaction. In the present study, rats with a pretraining injection of the N-methyl-D-aspartate (NMDA) receptor antagonist, 2-amino-5-phosphonopentanoic acid (APV), into the basolateral amygdala (BLA) were found to be impaired in two-way active avoidance learning. During multitrial training in a shuttle box, the APV-injected rats were not different from the controls in sensitivity to shock or in acquisition of freezing to contextual cues. However, APV injection led to impaired retention of contextual fear when tested 48 h later, along with an attenuation of c-Fos expression in the amygdala. These results are consistent with the role of NMDA receptors of the BLA in long-term memory of fear, previously documented in Pavlovian conditioning paradigms. The APV-induced impairment in the active avoidance learning coincided with deficits in directionality of the escape reaction and in attention to conditioned stimuli. These data indicate that normal functioning of NMDA receptors in the basolateral amygdala is required during acquisition of adaptive instrumental responses in a shuttle box but is not necessary for acquisition of short-term contextual fear in this situation.