Activation of basolateral amygdala corticotropin-releasing factor 1 receptors modulates the consolidation of contextual fear

The basolateral amygdala complex (BLA) and central amygdala nucleus (CeA) are involved in fear and anxiety. In addition, the BLA contains a high density of corticotropin-releasing factor 1 (CRF₁) receptors in comparison to the CeA. However, the role of BLA CRF₁ receptors in contextual fear conditioning is poorly understood. In the present study, we first demonstrated in rats that oral administration of DMP696, the selective CRF₁ receptor antagonist, had no significant effects on the acquisition of contextual fear but produced a subsequent impairment in contextual freezing suggesting a role of CRF₁ receptors in the fear memory consolidation process. In addition, oral administration of DMP696 significantly reduced phosphorylation of cyclic AMP response element-binding protein (pCREB) in the lateral and basolateral amygdala nuclei, but not in the CeA, during the post-fear conditioning period. We then demonstrated that bilateral microinjections of DMP696 into the BLA produced no significant effects on the acquisition of conditioned fear but reduced contextual freezing in a subsequent drug-free conditioned fear test. Importantly, bilateral microinjections of DMP696 into the BLA at 5 min or 3 h, but not 9 h, after exposure to contextual fear conditioning was also effective in reducing contextual freezing in the conditioned fear test. Finally, microinfusions of either DMP696 into the CeA or a specific corticotropin-releasing factor 2 receptor antagonist in the BLA were shown to have no major effects on disrupting either contextual fear conditioning or performance of contextual freezing in the drug-free conditioned fear test. Collectively, results implicate a role of BLA CRF₁ receptors in activating the fear memory consolidation process, which may involve BLA pCREB-induced synaptic plasticity.     

Basolateral amygdala NMDA receptors are selectively involved in the acquisition of taste-potentiated odor aversion in the rat

In the taste-potentiated odor aversion (TPOA) paradigm, animals acquire a strong aversion to an odor that is followed by delayed intoxication only if a gustatory stimulus is presented with the odor during conditioning. Although previous work has shown that N-methyl-D-aspartate (NMDA) receptors in the basolateral nucleus of the amygdala (BLA) play a role in the acquisition of TPOA, the present study aimed at describing the process in which NMDA receptors in the BLA are involved during acquisition of TPOA. Male Long-Evans rats received intra-BLA infusions of the competitive NMDA receptor antagonist D,L-2-2-amino-5-phosphonovalerate (D-APV; 0.05 and 0.50 microg) immediately before or after the odor-taste conditioned stimulus (CS) presentation, or immediately before the test. Results showed that D-APV impaired acquisition of TPOA when infused before, but not after, the CS presentation, but did not affect retrieval. These results suggest that NMDA receptors of the BLA are involved in the formation of potentiation--by taste--of the olfactory memory trace, but not in the maintenance of this process.     

The role of dorsal hippocampus and basolateral amygdala NMDA receptors in the acquisition and retrieval of context and contextual fear memories

The authors used 3-phase context preexposure facilitation methodology to study the contribution of N-methyl-D-aspartate (NMDA) receptors in dorsal hippocampus (DH) and the basal lateral region of the amygdala (BLA) to (a) acquisition of the context memory, (b) retrieval of the context memory, (c) acquisition of context-shock association, and (d) retrieval of the context-shock association. The NMDA receptor antagonist D-2-amino-5 phosphonopentanoic acid (D-AP5) was injected into either the DH or BLA prior to (a) the context preexposure phase, (b) the immediate shock phase, or (c) the test for contextual fear. Antagonizing NMDA receptors in the DH impaired the acquisition of the context memory but did not affect its retrieval or retrieval of the fear memory. Antagonizing NMDA receptors with D-AP5 in the BLA impaired acquisition of the context-shock association but had no effect on the expression of fear. However, both DL-AP5 and L-AP5 reduced the expression of fear when they were injected into the amygdala prior to testing for contextual fear.     

Amygdalar NMDA receptors control the expression of associative reflex facilitation and three other conditional responses

Four conditional responses (CRs) were measured in rats implanted with bilateral cannulas in the basolateral nuclear complex of the amygdala (BLA). During retention testing in either the original training context or a shifted context, BLA was infused with artificial cerebral spinal fluid (ACSF) or ACSF containing an N-methyl-D-aspartate receptor antagonist (APV). Regardless of the testing context, APV infusion into BLA completely blocked the expression of conditional eyeblink facilitation and significantly attenuated the expression of conditional freezing, ultrasonic vocalization, and defecation. Discriminant analysis found eyeblink facilitation to be comparable to freezing in predicting group membership (APV vs. ACSF) and both to be better predictors than the other two CRs. The APV effect did not depend on the exact cannula positions within BLA.     

N-methyl-D-aspartate receptor antagonist APV blocks acquisition but not expression of fear conditioning.

The role of N-methyl-D-aspartate (NMDA) receptors in Pavlovian fear conditioning was examined using the NMDA antagonist DL-2-amino-5-phosphonovaleric acid (APV). Either APV (5 micrograms/rat) or saline was administered before the training phase, the testing phase, or both. APV completely blocked acquisition but not expression of fear conditioning. The L enantiomer of APV did not affect the acquisition of conditional fear. To separate encoding from consolidation processes, APV was administered either before or immediately after the footshock unconditional stimulus (US) during the training phase. The results indicate that APV must be present during the US to produce its effects on fear conditioning. The behavioral effect of the drug is not due to analgesic action because APV did not alter pain sensitivity. The data suggest that NMDA receptors are critical for the acquisition but not expression of fear conditioning. These effects on fear conditioning are parallel to the in vitro effects of APV on the acquisition but not expression of long-term potentiation (LTP) and suggest that endogenously generated NMDA-dependent LTP participates in the neural plasticity underlying fear conditioning.     

Selective impairment of long-term but not short-term conditional fear by the N-methyl-D-aspartate antagonist APV.

Previous research has indicated that the competitive N-methyl-D-aspartate (NMDA) antagonist APV (DL-2-amino-5-phosphonovalerate) prevents the Pavlovian conditioning of fear to contextual stimuli when tested 24 hr, but not immediately, after training. The present study investigated this differential time-dependent effect of APV on fear conditioning. Rats were given either APV or saline and presented with 3 footshocks in a distinctive chamber. Promptly after the shock, rats that had received APV exhibited a species-typical fear response-freezing. However, the freezing lasted for only a short period of time (less than 3 min) compared with that of controls. An immediate-shock procedure showed that freezing was entirely a conditional response to the chamber. In addition, the results of a savings test suggest that APV impairs storage rather than retrieval processes. These results indicate that there are two temporally distinct associative fear processes, a short-term NMDA-independent conditional fear and a long-term NMDA-dependent conditional fear.     

Effects of pre- or post-training entorhinal cortex AP5 injection on fear conditioning

Fear conditioning is one of the most studied paradigms to assess the neural basis of emotional memory. The circuitry involves NMDA receptor activation in the amygdala and, in the case of contextual conditioning, in the hippocampus. Entorhinal cortex is one of the major input/output structures to the hippocampus and also projects to the amygdala, both through glutamatergic transmission. Other learning tasks involving hippocampus and amygdala, such as inhibitory avoidance, require entorhinal cortex during acquisition and consolidation. However, the involvement of NMDA receptors mediated transmission in entorhinal cortex in fear conditioning acquisition and consolidation is not clear. To investigate that issue, rats were trained in fear conditioning to both contextual and tone conditioned stimulus. Immediately before, immediately, 30 or 90 min after training they received NMDA antagonist AP5 or saline injections bilaterally in the entorhinal cortex (AP-6.8 mm, L +/-5.0 mm DV-6.8 mm). Contextual fear conditioning was measured 24 h after training, and tone fear conditioning 48 h after training. AP5 injections selectively impaired contextual fear conditioning only when injected pre-training. Post-training injections had no effect. These findings suggest that entorhinal cortex NMDA receptors are necessary for acquisition, but not for consolidation, of contextual fear conditioning. On the other hand, both acquisition and consolidation of tone fear conditioning seem to be independent of NMDA receptors in the entorhinal cortex.     

Extended fear conditioning reveals a role for both N-methyl-D-aspartic acid and non-N-methyl-D-aspartic acid receptors in the amygdala in the acquisition of conditioned fear

Pavlovian conditioning is a useful tool for elucidating the neural mechanisms involved with learning and memory, especially in regard to the stimuli associated with aversive events. The amygdala has been repeatedly implicated as playing a significant role in the acquisition and expression of fear. If the amygdala is critical for the acquisition of fear, then it should contribute to this processes regardless of the parameters used to induce or evaluate conditioned fear. A series of experiments using reversible inactivation techniques evaluated the role of the amygdala in the acquisition of conditioned fear when training was conducted over several days in rats. Fear-potentiated startle was used to evaluate the acquisition of conditioned fear. Pretraining infusions of N-methyl-d-aspartic acid (NMDA) or non-NMDA receptor antagonists alone into the amygdala interfered with the acquisition of fear early in training, but not later. Pretraining infusions of a cocktail consisting of both an NMDA and non-NMDA antagonist interfered with the acquisition of conditioned fear across all days of training. Taken together these results suggest the amygdala may potentially be critical for the acquisition of conditioned fear regardless of the parameters utilized.     

Involvement of NMDA receptors within the amygdala in short- versus long-term memory for fear conditioning as assessed with fear-potentiated startle

Pretraining intra-amygdala infusions of the NMDA receptor antagonist. D,L-AP5, block fear-potentiated startle in rats tested 24+ hr after training. This may reflect a failure of either acquisition or retention. To evaluate these alternatives, rats were tested for fear-potentiated startle during fear conditioning (30 light-shock pairings [0.6 mA shock]), as well as 1-30 min and 48 hr after fear conditioning. Amygdala lesions abolished fear-potentiated startle at all train-test intervals. Intra-amygdala AP5 infusions (25 nmol/side) abolished fear-potentiated startle during the long-term test and had partial effects at shorter train-test intervals. When the level of fear-potentiated startle during the short-term test was lowered to that of the 48-hr test (i.e., by training rats with a lower, 0.3 mA footshock), AP5 abolished fear-potentiated startle at each timepoint. Thus, amygdala NMDA receptors appear to participate in the initial acquisition of fear memories.     

Intrahippocampal infusions of a metabotropic glutamate receptor antagonist block the memory of context-specific but not tone-specific conditioned fear

The role of metabotropic glutamate receptors (mGluRs) in the acquisition of learning and memory using fear conditioning as a behavioral model was examined. The mGluR antagonist (R, S)-alpha-methyl-4-carboxyphenylglycine (MCPG) was infused into the hippocampus 30 min before fear conditioning, and freezing was measured during both acquisition and retention tests. The results show that pretraining antagonism of MCPG-sensitive mGluRs in the hippocampus impaired context-specific memory for an aversive event during testing. The memory for tone-specific fear, however, remained intact despite pretraining infusion of MCPG. Treating rats with MCPG did not affect context- or tone-specific fear during acquisition. Results suggest that mGluR activation may play an important role in hippocampally mediated memory consolidation.     

The amygdala modulates hippocampus-dependent context memory formation and stores cue-shock associations

Preexposing rats to the context facilitates subsequent contextual fear conditioning. This effect depends on the hippocampus (J. W. Rudy, R. M. Barrientos, & R. C. O'Reilly, 2002). The authors report that inactivating the basolateral region of the amygdala (BLA) by injecting muscimol, a GABAA agonist, before or after preexposure reduced this effect. In contrast, bilateral injections of anisomycin, a protein synthesis inhibitor, into BLA did not impair the consolidation of the context memory. However, when injected after fear conditioning, anisomycin impaired consolidation of both contextual and auditory-cue fear conditioning. Results are consistent with 2 ideas about the amygdala's contribution to memory: (a) It modulates memory formation in other regions of the brain, and (b) it is a storage site for cue-shock associations.     

Functional interaction between entorhinal cortex and basolateral amygdala during trace conditioning of odor aversion in the rat

In rats, conditioned odor aversion (COA) occurs only if the time interval separating the odor from the subsequent intoxication is very short suggesting that the memory trace of the odor is subject to rapid decay. Recent results from our laboratory have found that lesion of the entorhinal cortex (EC), and activation of the basolateral nucleus of the amygdala (BLA) rendered COA tolerant to long interstimulus interval. The present study examined whether the odor memory trace depends on the interaction between the EC and the BLA. Rats lesioned in the EC received infusions of muscimol (a GABA(A) receptor agonist) into the BLA immediately after the odor presentation during acquisition of COA. Injection of muscimol into BLA prevented tolerance of COA to long interstimulus interval induced by EC lesions. This suggests that EC modulates the short-term memory trace of the odor by controlling the GABAergic activity of the BLA during acquisition of COA.     

Differential involvement of medial prefrontal cortex and basolateral amygdala extracellular signal-regulated kinase in extinction of conditioned taste aversion is dependent on different intervals of extinction following conditioning

Extinction reflects a decrease in the conditioned response (CR) following non-reinforcement of a conditioned stimulus. Behavioral evidence indicates that extinction involves an inhibitory learning mechanism in which the extinguished CR reappears with presentation of an unconditioned stimulus. However, recent studies on fear conditioning suggest that extinction erases the original conditioning if the time interval between fear acquisition and extinction is short. The present study examined the effects of different intervals between acquisition and extinction of the original memory in conditioned taste aversion (CTA). Male Long-Evans rats acquired CTA by associating a 0.2% sucrose solution with malaise induced by i.p. injection of 4 ml/kg 0.15 M LiCl. Two different time intervals, 5 and 24 h, between CTA acquisition and extinction were used. Five or 24 h after CTA acquisition, extinction trials were performed, in which a bottle containing 20 ml of a 0.2% sucrose solution was provided for 10 min without subsequent LiCl injection. If sucrose consumption during the extinction trials was greater than the average water consumption, then rats were considered to have reached CTA extinction. Rats subjected to extinction trials lasting 24 h, but not 5 h, after acquisition re-exhibited the extinguished CR following injection of 0.15 M LiCl alone 7 days after acquisition. Extracellular signal-regulated kinase (ERK) in the medial prefrontal cortex (mPFC) and basolateral nucleus of the amygdala (BLA) was examined by Western blot after the first extinction trial. ERK activation in the mPFC was induced after the extinction trial beginning 5 h after acquisition, whereas the extinction trial performed 24 h after acquisition induced ERK activation in the BLA. These data suggest that the original conditioning can be inhibited or retained by CTA extinction depending on the time interval between acquisition and extinction and that the ERK transduction pathway in the mPFC and BLA is differentially involved in these processes.     

Effects of muscarinic receptor antagonism in the basolateral amygdala on two-way active avoidance

The aim of the present study was to investigate whether the blockade of muscarinic receptors (mRs) in the basolateral amygdala (BLA), which receives important cholinergic inputs related to avoidance learning, affects the consolidation of two-way active avoidance (TWAA). In Experiment 1, adult male Wistar rats were bilaterally infused with scopolamine (SCOP, 20 μg/site) or PBS (VEH) in the BLA immediately after a single 30-trial acquisition session. Twenty-four hours later, avoidance retention was tested in an identical session. Results indicated that scopolamine in the BLA did not affect TWAA performance measured by the number of avoidance responses. Experiment 2 was conducted to test whether such a negative outcome might be due to the occurrence of overtraining during acquisition, which may indeed have a protective effect against scopolamine-induced memory deficits. In this experiment, rats were infused with scopolamine in the BLA immediately after a brief 10-trial acquisition session and tested 24 h later in a 30-trial retention session. The SCOP group showed significantly more avoidances and inter-trial crossings in the retention session than the VEH rats. Together, these results reveal that mRs blockade in the BLA does not disrupt TWAA consolidation and may even enhance avoidance performance when infused after a low number of acquisition trials. Performance factors, such as locomotor activity in the shuttle-box, may account, at least in part, for the facilitative effects of muscarinic antagonism in the BLA.     

NMDA receptor modulation of incidental learning in Pavlovian context conditioning

Rats exposed to a footshock show conditional fear when reexposed to the shock context. Immediate presentation of shock after placement in the context significantly reduces this fear. Preexposure to the context in the absence of shock, coupled with a minimum preshock interval during training, overcomes this immediate shock deficit. Because rats learn about the context during preexposure and express that learning after being reinforced, the context preexposure effect is an aversive analogue of latent learning. The authors examined the effect of the N-methyl-D-aspartate (NMDA) receptor antagonist D,L-2-amino-5-phosphovalerate (APV) on the facilitatory effect of context preexposure. Rats were preexposed to a chamber after APV administration. The next day they were placed in the same chamber without drug and received shock 35 s later. APV blocked the facilitatory effect of preexposure. Therefore NMDA receptors are important for contextual latent learning.     

Predator odor-induced conditioned fear involves the basolateral and medial amygdala

The basolateral (BLA) and medial nucleus (MeA) of the amygdala participate in the modulation of unconditioned fear induced by predator odor. However, the specific role of these amygdalar nuclei in predator odor-induced fear memory is not known. Therefore, fiber-sparing lesions or temporary inactivation of the BLA or MeA were made either prior to or after exposure to cat odor, and conditioned contextual fear behavior was examined the next day. BLA and MeA lesions produced significant reductions in cat odor-induced unconditioned and conditioned fear-related behavior. In addition, temporary pharmacological neural inactivation methods occurring after exposure to cat odor revealed subtle behavioral alterations indicative of a role of the BLA in fear memory consolidation but not memory retrieval. In contrast, the MeA appears to play a specific role in retrieval but not consolidation. Results show that the BLA participates in the conditioned and unconditioned cat odor stimulus association that underlies fear memory, underscore a novel role of the MeA in predator odor contextual conditioning, and demonstrate different roles of the BLA and MeA in modulating consolidation and retrieval of predator odor fear memory.     

Amygdala, hippocampus, and unconditioned fear

Embedded within contemporary views of emotional learning is a well-founded agreement that the amygdala plays a pivotal role in the formation and consolidation of aversive memories formed during fear conditioning. However, it is important to determine whether observed deficits are reflective of a memory impairment or whether they are simply attributable to a deficit in the performance of unconditioned fear responses such as freezing. Within the neurobiology of learning and memory literature, there is an ongoing debate concerning the potential role of the amygdala in the performance of unconditioned fear responses. A view put forth by Vazdarjanova and McGaugh (1998) suggests that the amygdala is not required for the formation and consolidation of the aversive memories formed during fear conditioning, but is essential in the performance of unconditioned fear responses. Data provided by Maren (1999) counter this view by positing that the amygdala is not required for the performance of fear responses, but its role is of a mnemonic nature in the conditioning of fear to neutral cues. To clarify the amygdala's participation in these two processes, a useful approach would involve a situation where animals with amygdala damage were examined for their unconditioned fear responses in reaction to footshock as well as the conditioning of these reactions to previously neutral cues paired with the aversive event. We have previously reported that rats with amygdala or hippocampal damage are impaired in discriminative fear conditioning to context. In the present experiment, we report the initial unconditioned fear responses to footshock by these same animals as well as the conditioned responses during testing. In both groups, the fear responses assessed (freezing, urination, defecation, and locomotion) were not impaired and did not differ from those expressed by the sham animals. The impairment of discriminative fear conditioning to context, in combination with the present experiment, represents a dissociation where damage to specific memory structures (amygdala or hippocampus) debilitates the mnemonic processes involved in fear conditioning, but not the performance of the fear responses per se.     

Contribution of the amygdala to learning and performance of conditional fear.

The amygdaloid complex appears to be an essential component in the neural systems mediating some forms of aversive Pavlovian conditioning. The relative contribution of this structure to acquisition and performance during fear conditioning was assessed by making temporary lesions in the amygdala prior to training or retention testing in a single-trial Pavlovian conditioning preparation. Microinjection of lidocaine HCl (5.0%, 1.0 microliters) into the amygdala prior to the presentation of a CS signalling footshock resulted in a significant attenuation of the performance of conditional fear, as indexed by the amount of time rats spent engaged in defensive freezing behavior during the retention session. However, similar treatment with lidocaine prior to the training session, during which the CS and UCS were paired, resulted in only a weak reduction in subsequent responding. Thus, while both acquisition- and performance-related processes take place within the amygdala, it appears that the latter are more sensitive to disruption using the present procedures. These results are discussed in terms of the general role played by this structure in aversive learning and motivational processes.     

The ventral hippocampus and fear conditioning in rats. Different anterograde amnesias of fear after tetrodotoxin inactivation and infusion of the GABA(A) agonist muscimol

Studies on the involvement of the rat hippocampus in classical fear conditioning have focused mainly on the dorsal hippocampus and conditioning to a context. However, the ventral hippocampus has intimate connections with the amygdala and the nucleus accumbens, which are involved in classical fear conditioning to explicit and contextual cues. Consistently, a few recent lesion studies have indicated a role for the ventral hippocampus in classical fear conditioning to explicit and contextual cues. The present study examined whether neuronal activity within the ventral hippocampus is important for the formation of fear memory to explicit and contextual cues by classical fear conditioning. Tetrodotoxin (TTX; 10 ng/side), which completely blocks neuronal activity, or muscimol (1 microg/side), which increases GABA(A) receptor-mediated inhibition, were bilaterally infused into the ventral hippocampus of Wistar rats before the conditioning session of a classical fear-conditioning experiment. Conditioning to a tone and the context were assessed using freezing as a measure of conditioned fear. TTX blocked fear conditioning to both tone and context. Muscimol only blocked fear conditioning to the context. The data of the present study indicate that activity of neurons in the ventral hippocampus is necessary for the formation of fear memory to both explicit and contextual cues and that neurons in the ventral hippocampus that bear the GABA(A) receptor are important for the formation of fear conditioning to a context. In addition, both bilateral muscimol (0.5 microg/side and 1 microg/side) and TTX (5 ng/side and 10 ng/side) infusion into the ventral hippocampus dose-dependently decreased locomotor activity in an open-field experiment.     

Hippocampus and Pavlovian fear conditioning in rats: muscimol infusions into the ventral, but not dorsal, hippocampus impair the acquisition of conditional freezing to an auditory conditional stimulus

The authors compared the effects of pharmacological inactivation of the dorsal hippocampus (DH) or ventral hippocampus (VH) on Pavlovian fear conditioning in rats. Freezing behavior served as the measure of fear. Pretraining infusions of muscimol, a GABAA receptor agonist, into the VH disrupted auditory, but not contextual, fear conditioning; DH infusions did not affect fear conditioning. Pretesting inactivation of the VH or DH did not affect the expression of conditional freezing. Pretraining electrolytic lesions of the VH reproduced the effects of muscimol infusions, whereas posttraining VH lesions disrupted both auditory and contextual freezing. Hence, neurons in the VH are importantly involved in the acquisition of auditory fear conditioning and the expression of auditory and contextual fear under some conditions.