Memory enhancement of classical fear conditioning by post-training injections of corticosterone in rats

There is extensive evidence that post-training administration of the adrenocortical hormone corticosterone facilitates memory consolidation processes in a variety of contextual and spatial-dependent learning situations. The present experiments examine whether corticosterone can modulate memory of auditory-cue classical fear conditioning, a learning task that is not contingent on contextual or spatial representations. Male Sprague-Dawley rats received three pairings of a single-frequency auditory stimulus and footshock, followed immediately by a post-training subcutaneous injection of either corticosterone (1.0 or 3.0mg/kg) or vehicle. Retention was tested 24h later in a novel test chamber and suppression of ongoing motor behavior served as the measure of conditioned fear. Corticosterone dose-dependently facilitated suppression of motor activity during the 10-s presentation of the auditory cue. As corticosterone administration did not alter responding after unpaired presentations of tone and shock, tone alone, shock alone or absence of tone/shock, the findings indicated that corticosterone selectively facilitated memory of the tone-shock association. Furthermore, injections of corticosterone given 3h after training did not alter motor activity during retention testing, demonstrating that corticosterone enhanced time-dependent memory consolidation processes. These findings provide evidence that corticosterone modulates the consolidation of memory for auditory-cue classical fear conditioning and are consistent with a wealth of data indicating that glucocorticoids can modulate a wide variety of emotionally influenced memories.     

Olfactory fear conditioning paradigm in rats: Effects of midazolam,propranolol or scopolamine

In rodents, fear conditioned responses are more pronounced toward olfactory stimulus, since olfaction is a dominant sense in these subjects. The present study was outlined to investigate if the association between coffee odor (CS1) and electrical footshock (US) would be an effective model for the study of fear-induced behavior and whether compounds used in humans for emotional-related disorders such as midazolam, propranolol, or scopolamine, applied during the different stages of fear conditioning (acquisition, consolidation and expression), affect the defensive responses to both, the olfactory CS1, and the context (CS2) where the CS1 had been presented (second order conditioning). The results revealed that five pairings between coffee odor (CS1) and electrical footshock (US) were able to elicit consistent defensive responses and a second order conditioning to the context (CS2). Midazolam (0.375–0.5 mg/kg; i.p.) treatment was able to interfere with the CS1–US association and with the consolidation of the aversive information. The propranolol (5–10 mg/kg; i.p.) treatment interfered with the CS1–US association, with the retention of fear memory and with the CS1–CS2 association. Propranolol also attenuated the expression of conditioned fear responses when applied before the CS1 test session. Scopolamine (0.6–1.2 mg/kg; i.p.) treatment impaired the acquisition of CS1–US and CS1–CS2 associations, and also disrupted the expression of conditioned fear responses when injected prior to the CS1 test session. These findings have pointed out the usefulness for the olfactory fear conditioning paradigm to investigate drug effects on the acquisition, consolidation and expression of fear conditioned responses.     

The benzodiazepine midazolam does not impair Pavlovian fear conditioning but regulates when and where fear is expressed.

Rats were injected with a benzodiazepine (midazolam) and shocked after presentation of an auditory conditioned stimulus (CS). They were then tested for fear reactions (freezing) to the CS in either the original context or a 2nd context after either a short (1-day) or long (21-day) retention interval. Rats tested in the original context froze less after 1 day than rats tested after that interval in the 2nd context or rats tested after 21 days. Moreover, rats tested after the long interval in the original context froze less than rats tested after that interval in the 2nd context. Therefore, midazolam does not impair the acquisition of conditioned fear but regulates when and where that fear is expressed. These effects of midazolam were interpreted as a contextually controlled deficit in the expression of conditioned fear that is similar to that associated with latent inhibition and extinction (M. E. Bouton, 1993).     

Amygdala and hippocampal activity during acquisition and extinction of human fear conditioning

Previous functional magnetic resonance imaging (fMRI) studies have characterized brain systems involved in conditional response acquisition during Pavlovian fear conditioning. However, the functional neuroanatomy underlying the extinction of human conditional fear remains largely undetermined. The present study used fMRI to examine brain activity during acquisition and extinction of fear conditioning. During the acquisition phase, participants were either exposed to light (CS) presentations that signaled a brief electrical stimulation (paired group) or received light presentations that did not serve as a warning signal (control group). During the extinction phase, half of the paired group subjects continued to receive the same treatment, whereas the remainder received light alone. Control subjects also received light alone during the extinction phase. Changes in metabolic activity within the amygdala and hippocampus support the involvement of these regions in each of the procedural phases of fear conditioning. Hippocampal activity developed during acquisition of the fear response. Amygdala activity increased whenever experimental contingencies were altered, suggesting that this region is involved in processing changes in environmental relationships. The present data show learning-related amygdala and hippocampal activity during human Pavlovian fear conditioning and suggest that the amygdala is particularly important for forming new associations as relationships between stimuli change.     

Pre-training prevents context fear conditioning deficits produced by hippocampal NMDA receptor blockade

These experiments explore parallels between the neurobiological substrates of spatial and context learning. Male Long-Evans rats were employed in a context fear conditioning protocol that involved sequential acquisition and testing in two distinct contexts. Rats received three unsignaled footshocks in one context and were tested for context fear, measured as freezing, the next day. Four days later, the procedure was repeated in a different, distinct context. Rats received a hippocampal infusion of either the NMDA receptor antagonist 5-amino-phosphonovaleric acid (APV, 10 microg) or vehicle prior to training in each context. NMDA receptor blockade was effective in impairing context learning only in the first context. Context fear acquisition was not impaired by APV in the second context, indicating that pre-training (in the first context) mitigated the effects of APV. These data agree with those seen previously in the water maze, where pre-training prevented learning deficits produced by NMDA receptor blockade. The data thus suggest that the neurobiological substrates of context learning and place learning overlap.     

Nicotine enhances contextual fear conditioning in C57BL/6J mice at 1 and 7 days post-training

Nicotine has been demonstrated to enhance learning processes. The present experiments extend these results to examine the effects of nicotine on acquisition and consolidation of contextual and cued fear conditioning, and the duration of nicotine's enhancement of conditioned fear. C57BL/6 mice were trained with two pairings of an auditory CS and a foot shock US. Multiple doses of nicotine were given before or immediately after training and on testing day (0.0, 0.050, 0.125, 0.250, and 0.375 mg/kg, i.p). Freezing to both the context and auditory CS was measured 24h after training and again 1 week after training. Mice did not receive nicotine for the 1-week retest. Nicotine (0.125 and 0.250 mg/kg) given on both training and testing days enhanced freezing to the context at 24h. In addition, elevated freezing to the context was seen 1 week post-training in mice previously treated with 0.125 and 0.250 mg/kg nicotine. Thus, nicotine-treated mice did show elevated levels of freezing when retested 1 week later, even though no nicotine was administered at the 1-week retest. Mice that received nicotine on training day or testing day only and mice that received nicotine with mecamylamine, a nicotinic receptor antagonist, were not different from saline-treated mice. In addition, post-training administration of nicotine did not enhance fear conditioning. The present results indicate that nicotine enhancement of contextual fear conditioning depends on administration of nicotine on training and test days but results in a long-lasting enhancement of memories of contextual fear conditioning that remains in the absence of nicotine.     

Effects of stress and sex on acquisition and consolidation of human fear conditioning

We examined the relationship between stress hormone (cortisol) release and acquisition and consolidation of conditioned fear learning in healthy adults. Participants underwent acquisition of differential fear conditioning, and consolidation was assessed in a 24-h delayed extinction test. The acquisition phase was immediately followed by an 11-min psychosocial stress period (arithmetic test combined with a public speech). Salivary cortisol was sampled at various time points before and after acquisition and retention of fear conditioning. Results showed two effects of endogenous cortisol. Post-acquisition cortisol correlated with fear acquisition in male but not female participants. In addition, post-acquisition cortisol correlated with consolidation of fear but only in those participants with high cortisol levels. We conclude that in the short term, a robust and sexually dimorphic relationship exists between fear learning and stress hormone levels. For those participants whose fear learning is accompanied by high stress hormone levels, a long-term relationship exists between cortisol release and memory consolidation. These short-term and long-term effects may relate to the differential involvement of mineralocorticoid and glucocorticoid receptor subtypes, respectively. The findings have implications for understanding the role of stress, sex, and hormones in different stages of fear learning and memory.     

Effects of ventral and dorsal CA1 subregional lesions on trace fear conditioning

Recent lines of research have focused on dissociating function between the dorsal and ventral hippocampus along space and anxiety dimensions. In the dorsal hippocampus, the CA1 subregion has been implicated in the acquisition of contextual fear as well as in the trace interval in trace fear conditioning. The present study was designed to test the relative contributions of dorsal (dCA1) and ventral CA1 (vCA1) in trace fear conditioning. Long-Evans rats received ibotenate lesions of the ventral CA1 (n=7), dorsal CA1 (n=9), or vehicle control lesions (n=8) prior to trace fear conditioning acquisition. Results suggest dCA1 and vCA1 groups show no significant deficits during acquisition when compared to control groups. dCA1 and vCA1 both show deficits in the retention of contextual fear when tested 24 h post-acquisition (P<.05 and P<.01, respectively), and vCA1 was impaired relative to dCA1 (P<.05). This is suggestive of a graded involvement in contextual retention between the dorsal and ventral aspects of CA1. dCA1 showed no deficit for retention of conditioned fear to the tone or the trace when tested 48 h post-acquisition, whereas vCA1 did show a significant deficit for the trace interval and a slight, non-significant reduction in freezing to the tone, when compared to the control group (p<.05). Overall the data are suggestive of a graded involvement in retention of fear conditioning between the dorsal and ventral aspects of CA1, but it is likely that vCA1 may be critically involved in retention of trace fear conditioning.     

Learning strategies during fear conditioning

This paper describes a model of fear learning, in which subjects have an option of behavioral responses to impending social defeat. The model generates two types of learning: social avoidance and classical conditioning, dependent upon (1) escape from or (2) social subordination to an aggressor. We hypothesized that social stress provides the impetus as well as the necessary information to stimulate dichotomous goal-oriented learning. Specialized tanks were constructed to subject rainbow trout to a conditioning paradigm where the conditioned stimulus (CS) is cessation of tank water flow (water off) and the unconditioned stimulus (US) is social aggression from a larger conspecific. Following seven daily CS/US pairings, approximately half of the test fish learned to consistently escape the aggression to a neutral chamber through a small escape hole available only during the interaction. The learning curve for escaping fish was dramatic, with an 1100% improvement in escape time over 7 days. Fish that did not escape exhibited a 400% increase in plasma cortisol and altered brain monoamine response to presentation of the CS alone. Elevated plasma cortisol levels represent classical fear conditioning in non-escaping fish, while a lack of fear conditioning and a decreased latency to escape over the training period in escapers indicates learned escape.     

Naloxone and Pavlovian fear conditioning

Previous research has shown that pretreating rats with the opiate antagonist naloxone increases the freezing that follows painful electric shock. Three experiments, using freezing behavior as a dependent variable, were carried out to determine whether the drug might cause this effect by enhancing fear conditioning. Two of the studies employed a differential context fear-conditioning paradigm. Naloxone did not affect freezing behavior during the preshock adaptation period. In Experiment 1, naloxone was found to increase resistance to extinction in the S+ context. In Experiment 2, naloxone was found to increase freezing in the S+ context. This effect was dependent upon administering naloxone during training but not dependent on administering it during testing. The third study employed a generalization paradigm. It was found that naloxone's effect on postshock freezing was dependent on the place of testing; as the contextual cues of the test chamber were changed from those of the conditioning chamber, the effect of naloxone on freezing was reduced. The results of these experiments lend strong support to the hypothesis that naloxone increases freezing by enhancing the conditioning of fear to contextual stimuli associated with shock.     

Unblocking in Pavlovian fear conditioning

Six experiments used rats to study blocking and unblocking of fear learning. An excitatory stimulus (A) blocked fear learning to a neutral stimulus (B). Unblocking of B occurred if the AB compound signaled an increase in unconditioned stimulus (US) intensity or number. Assessments of associative change during blocking showed that more was learned about B than A. Such assessments during unblocking revealed that more was learned about B than A following an increase in US intensity but not US number. These US manipulations had no differential effects on single-cue learning. The results show that variations in US intensity or number produce unblocking of fear learning, but for each there is a different profile of associative change and a potentially different mechanism.     

Effects of recent exposure to a conditioned stimulus on extinction of Pavlovian fear conditioning

In six experiments we studied the effects of a single re-exposure to a conditioned stimulus (CS; "retrieval trial") prior to extinction training (extinction-reconsolidation boundary) on the development of and recovery from fear extinction. A single retrieval trial prior to extinction training significantly augmented the renewal and reinstatement of extinguished responding. Augmentation of recovery was not observed if the retrieval and extinction training occurred in different contexts. These results contrast with those reported in earlier papers by Monfils and coworkers in rats and by Schiller and coworkers in humans. We suggest that these contrasting results could depend on the contrasting influences of either: (1) occasion-setting contextual associations vs. direct context-CS associations formed as a consequence of the retrieval trial or (2) discrimination vs. generalization between the circumstances of conditioning and extinction.     

Coantagonism of glutamate receptors and nicotinic acetylcholinergic receptors disrupts fear conditioning and latent inhibition of fear conditioning

The present study investigated the hypothesis that both nicotinic acetylcholinergic receptors (nAChRs) and glutamate receptors (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors (AMPARs) and N-methyl-d-aspartate glutamate receptors (NMDARs)) are involved in fear conditioning, and may modulate similar processes. The effects of the nAChR antagonist mecamylamine administered alone, the AMPAR antagonist NBQX administered alone, and the NMDAR antagonist MK-801 administered alone on cued fear conditioning, contextual fear conditioning, and latent inhibition of cued fear conditioning were examined. In addition, the effects of coadministration of either mecamylamine and NBQX or mecamylamine and MK-801 on these behaviors were examined. Consistent with previous studies, neither mecamylamine nor NBQX administered alone disrupted any of the tasks. However, coadministration of mecamylamine and NBQX disrupted both contextual fear conditioning and latent inhibition of cued fear conditioning. In addition, coadministration of mecamylamine with a dose of MK-801 subthreshold for disrupting either task disrupted both contextual fear conditioning and latent inhibition of cued fear conditioning. Coadministration of mecamylamine and NBQX, and coadministration of mecamylamine with a dose of MK-801 subthreshold for disrupting fear conditioning had little effect on cued fear conditioning. These results suggest that nAChRs and glutamate receptors may support similar processes mediating acquisition of contextual fear conditioning and latent inhibition of fear conditioning.     

Effects of hippocampal injections of a novel ligand selective for the alpha 5 beta 2 gamma 2 subunits of the GABA/benzodiazepine receptor on Pavlovian conditioning

Benzodiazepine pharmacology has led to greater insight into the neural mechanisms underlying learning and anxiety. The synthesis of new compounds capable of modulating responses produced by these receptors has been made possible by the development of an isoform model of the GABA(A)/benzodiazepine receptor complex. In the current experiment, rats were pretreated with several concentrations of the novel ligand RY024 (an alpha 5 beta 2 gamma 2 -selective benzodiazepine receptor inverse agonist) in the hippocampus and were trained in a Pavlovian fear conditioning paradigm. RY024 independently produced fear-related behavior prior to training and, at the highest concentration, decreased the strength of conditioning observed 24 h after training. These data provide further evidence for the involvement of hippocampal GABA(A)/benzodiazepine receptors in learning and anxiety.     

Asymmetrical stimulus generalization following differential fear conditioning

Rodent ultrasonic vocalizations (USVs) are ethologically critical social signals. Rats emit 22 kHz USVs and 50 kHz USVs, respectively, in conjunction with negative and positive affective states. Little is known about what controls emotional reactivity to these social signals. Using male Sprague–Dawley rats, we examined unconditional and conditional freezing behavior in response to the following auditory stimuli: three 22 kHz USVs, a discontinuous tone whose frequency and on–off pattern matched one of the USVs, a continuous tone with the same or lower frequencies, a 4 kHz discontinuous tone with an on–off pattern matched to one of the USVs, and a 50 kHz USV. There were no differences among these stimuli in terms of the unconditional elicitation of freezing behavior. Thus, the stimuli were equally neutral before conditioning. During differential fear conditioning, one of these stimuli (the CS⁺) always co-terminated with a footshock unconditional stimulus (US) and another stimulus (the CS⁻) was explicitly unpaired with the US. There were no significant differences among these cues in CS⁺-elicited freezing behavior. Thus, the stimuli were equally salient or effective as cues in supporting fear conditioning. When the CS⁺ was a 22 kHz USV or a similar stimulus, rats discriminated based on the principal frequency and/or the temporal pattern of the stimulus. However, when these same stimuli served as the CS⁻, discrimination failed due to generalization from the CS⁺. Thus, the stimuli differed markedly in the specificity of conditioning. This strikingly asymmetrical stimulus generalization is a novel bias in discrimination.