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Although sex differences have been reported in hippocampal-dependent learning and memory, including contextual fear memories, the underlying molecular mechanisms contributing to such differences are not well understood. The present study examined the extent to which sex differences in contextual fear conditioning are related to differential activation of the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK), a protein kinase critically involved in memory formation. We first show that male rats exhibit more long-term retention of contextual fear conditioning than female rats. During a tone test, females spent more time freezing than males, although both sexes exhibited robust retention of auditory fear learning. Using Western blot analysis, we then show that phosphorylated ERK levels in ventral, but not dorsal, hippocampus are higher in males than females, relative to same-sex controls, 60 minutes after fear conditioning. Post-conditioning increases in ERK activation were observed in the amygdala in both males and females, suggesting a selective effect of sex on hippocampal ERK activation. Together, these findings suggest that differential activation of the ERK signal transduction pathway in male and female rats, particularly in the ventral hippocampus, is associated with sex differences in contextual fear.

Pavlovian fear conditioning has emerged as a leading behavioral paradigm for studying the neurobiological basis of learning and memory. Although considerable progress has been made in understanding the neural substrates of fear conditioning at the systems level, until recently little has been learned about the underlying cellular and molecular mechanisms. The success of systems-level work aimed at defining the neuroanatomical pathways underlying fear conditioning, combined with the knowledge accumulated by studies of long-term potentiation (LTP), has recently given way to new insights into the cellular and molecular mechanisms that underlie acquisition and consolidation of fear memories. Collectively, these findings suggest that fear memory consolidation in the amygdala shares essential biochemical features with LTP, and hold promise for understanding the relationship between memory consolidation and synaptic plasticity in the mammalian brain.

The lateral nucleus of the amygdala (LA) is an essential component of the neural circuitry underlying Pavlovian fear conditioning. Although blockade of NMDA receptors in LA and adjacent areas before training disrupts the acquisition of fear conditioning, blockade before testing also often disrupts the expression of fear responses. With this pattern of results, it is not possible to distinguish a contribution of NMDA receptors to plasticity from a role in synaptic transmission. In past studies, NMDA blockade has been achieved using the antagonist d,l-2-amino-5-phosphovalerate, which blocks the entire heteromeric receptor complex. The present experiments examined the effects of selective blockade of the NR2B subunit of the NMDA receptor in LA using the selective antagonist ifenprodil. Systemic injections of ifenprodil before training led to a dose-dependent impairment in the acquisition of auditory and contextual fear conditioning, whereas injections before testing had no effect. Intra-amygdala infusions of ifenprodil mirrored these results and, in addition, showed that the effects are attributable to a disruption of fear learning rather than a disruption of memory consolidation. NMDA receptors in LA are thus involved in fear conditioning, and the NR2B subunit appears to make unique contributions to the underlying plasticity.

'Consolidation' has been used to describe distinct but related processes. In considering the implications of our recent findings on the lability of reactivated fear memories, we view consolidation and reconsolidation in terms of molecular events taking place within neurons as opposed to interactions between brain regions. Our findings open up a new dimension in the study of memory consolidation. We argue that consolidation is not a one-time event, but instead is reiterated with subsequent activation of the memories.

Although much has been learned about the neurobiological mechanisms underlying Pavlovian fear conditioning at the systems and cellular levels, relatively little is known about the molecular mechanisms underlying fear memory consolidation. The present experiments evaluated the role of the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) signaling cascade in the amygdala during Pavlovian fear conditioning. We first show that ERK/MAPK is transiently activated-phosphorylated in the amygdala, specifically the lateral nucleus (LA), at 60 min, but not 15, 30, or 180 min, after conditioning, and that this activation is attributable to paired presentations of tone and shock rather than to nonassociative auditory stimulation, foot shock sensitization, or unpaired tone-shock presentations. We next show that infusions of U0126, an inhibitor of ERK/MAPK activation, aimed at the LA, dose-dependently impair long-term memory of Pavlovian fear conditioning but leaves short-term memory intact. Finally, we show that bath application of U0126 impairs long-term potentiation in the LA in vitro. Collectively, these results demonstrate that ERK/MAPK activation is necessary for both memory consolidation of Pavlovian fear conditioning and synaptic plasticity in the amygdala.

Previous studies have shown that long-term potentiation (LTP) can be induced in the lateral nucleus of the amygdala (LA) after stimulation of central auditory pathways and that auditory fear conditioning modifies neural activity in the LA in a manner similar to LTP. The present experiments examined whether intra-LA administration of inhibitors of protein synthesis or protein kinase A (PKA) activity, treatments that block LTP in hippocampus, interfere with memory consolidation of fear conditioning. In the first series of experiments, rats received a single conditioning trial followed immediately by intra-LA infusions of anisomycin (a protein synthesis inhibitor) or Rp-cAMPS (an inhibitor of PKA activity) and were tested 24 hr later. Results indicated that immediate post-training infusion of either drug dose-dependently impaired fear memory retention, whereas infusions 6 hr after conditioning had no effect. Additional experiments showed that anisomycin and Rp-cAMPS interfered with long-term memory (LTM), but not short-term memory (STM), of fear and that the effect on LTM was specific to memory consolidation processes rather than to deficits in sensory or performance processes. Findings suggest that the LA is essential for memory consolidation of auditory fear conditioning and that this process is PKA and protein-synthesis dependent.

Although the lateral and basal nuclei of the amygdala are believed to be essential for the acquisition of Pavlovian fear conditioning, studies using post-training manipulations of the amygdala in the inhibitory avoidance learning paradigm have recently called this view into question. We used the GABA(A) agonist muscimol to functionally inactivate these nuclei immediately after single-trial Pavlovian fear conditioning or single-trial inhibitory avoidance learning. Immediate post-training infusions of muscimol had no effect on Pavlovian conditioning but produced a dose-dependent effect on inhibitory avoidance. However, pre-training infusions dose-dependently disrupted Pavlovian conditioning. These findings indicate that the amygdala plays an essential role in the acquisition of Pavlovian fear conditioning and contributes to the modulation of memory consolidation of inhibitory avoidance but not of Pavlovian fear conditioning.

'New' memories are initially labile and sensitive to disruption before being consolidated into stable long-term memories. Much evidence indicates that this consolidation involves the synthesis of new proteins in neurons. The lateral and basal nuclei of the amygdala (LBA) are believed to be a site of memory storage in fear learning. Infusion of the protein synthesis inhibitor anisomycin into the LBA shortly after training prevents consolidation of fear memories. Here we show that consolidated fear memories, when reactivated during retrieval, return to a labile state in which infusion of anisomycin shortly after memory reactivation produces amnesia on later tests, regardless of whether reactivation was performed 1 or 14 days after conditioning. The same treatment with anisomycin, in the absence of memory reactivation, left memory intact. Consistent with a time-limited role for protein synthesis production in consolidation, delay of the infusion until six hours after memory reactivation produced no amnesia. Our data show that consolidated fear memories, when reactivated, return to a labile state that requires de novo protein synthesis for reconsolidation. These findings are not predicted by traditional theories of memory consolidation.

The induction of c-Fos-like immunoreactivity (c-FLI) in the nucleus of the solitary tract (NTS) has been shown to be correlated with behavioral expression of a conditioned taste aversion (CTA). However, because this cellular response is also dependent on an intact amygdala, it may represent the activation of a stress-related autonomic response. The present experiments addressed this possibility by evaluating the correlation between c-FLI in the intermediate division of the NTS (iNTS) and 2 measures of conditioned fear: freezing and changes in mean arterial pressure (MAP) and heart rate (HR). Exposure to the taste conditioned stimulus (CS) resulted in a marked induction of c-FLI in the iNTS, whereas exposure to a fear CS did not. Further, exposure to a taste CS did not selectively lead to increases in MAP or HR. Results suggest that induction of c-FLI in the iNTS may reflect the activation of a cell population whose function is unique to the CTA paradigm.

Two competing theories predict different effects on memory consolidation when the amygdala is inactivated after fear conditioning. One theory, based on studies using inhibitory avoidance training, proposes that the amygdala modulates the strength of fear learning, and post-training amygdala manipulations interfere with memory consolidation. The other, based on studies using Pavlovian fear conditioning, hypothesizes that fear learning occurs in the amygdala, and post-training manipulations after acquisition will not affect memory consolidation. We infused the GABAA agonist muscimol (4.4 nmol/side) or vehicle into lateral and basal amygdala (LBA) of rats either before or immediately after tone-foot shock Pavlovian fear conditioning. Pre-training infusions eliminated acquisition, whereas post-training infusions had no effect. These findings indicate that synaptic activity in LBA is necessary during learning, but that amygdala inactivation directly after training does not affect memory consolidation. Results suggest that essential aspects of plasticity underlying auditory fear conditioning take place within LBA during learning.

The induction of c-Fos-like immunoreactivity (c-FLI) in the intermediate division of the nucleus of the solitary tract (iNTS) has been shown to be a cellular correlate of the behavioral expression of a conditioned taste aversion (CTA). To further define neuroanatomical structures and pathways that contribute to this cellular response and to CTA learning in general, electrolytic lesions of insular (gustatory) cortex (IC) were combined with immunostaining for c-FLI. Rats were given either unilateral or bilateral electrolytic lesions of insular cortex or 'sham' operations. Following surgery, 'paired' animals were given a single conditioning trial consisting of intraoral infusion of 5-ml 0.15% sodium-saccharin followed by injection with LiCl (0.15 M, 20 ml/kg, i.p.) while 'unpaired' controls received a non-contingent saccharin-LiCl presentation. Rats with bilateral lesions showed no behavioral evidence of having acquired a CTA. Increases in c-FLI in iNTS were evident, but reduced, relative to 'sham' animals. Rats with unilateral-lesions displayed a CTA by rejecting the saccharin, although increases in c-FLI on the side of the iNTS ipsilateral to the lesion were reduced relative to that seen in 'sham' animals. A comparison of these results with those obtained after amygdala lesions supports the conclusion that amygdala and insular cortex are necessary, but not sufficient, for the behavioral expression of a CTA., (Copyright 1998 Elsevier Science B.V. All rights reserved.)

Sodium (Na) ingestion in rats depleted of Na is a strong, motivated behavior that is enhanced further when depleted rats are sham drinking. Dopamine plays a critical role in motivation, including reward associated with consumption of palatable tastes. The present studies assessed the role of dopamine in real and sham drinking of NaCl solutions after Na depletion with the diuretic furosemide (10 mg/kg). Dopamine (D2) receptor antagonists were evaluated (Haloperidol [0.1 mg/kg] and raclopride [0.2 mg/kg]), for their effects on sham and real drinking of 0.3 M NaCl. Sham drinking was markedly reduced by both antagonists whereas real drinking was unaffected. These effects did not appear to be due to malaise or suppression of motor behavior because drug-treated animals were able to increase ingestion substantially when offered less concentrated NaCl (0.1 M). These results suggest that the positive motivating properties of NaCl stimulation in depleted, sham-drinking rats are mediated by central D2 receptors.

The induction of c-Fos-like immunoreactivity (c-FLI) in the intermediate division of the nucleus of the solitary tract (iNTS) has been shown to be a reliable cellular correlate of the acquisition and/or behavioral expression of a conditioned taste aversion (CTA). To begin to define neuroanatomical structures and pathways that contribute to this cellular response and to CTA learning in general, electrolytic lesions of the amygdala were combined with immunostaining for c-FLI. Rats were given either unilateral or bilateral electrolytic lesions of the amygdala or "sham' operations. Following surgery "paired' animals were given a single conditioning trial consisting of intraoral infusion of 5 ml 0.15% sodium-saccharin followed by injection with LiCl (0.15 M, 20 ml/kg, i.p.) while "unpaired' controls received a non-contingent saccharin-LiCl presentation. When tested, unilateral-lesioned rats displayed a CTA by rejecting the saccharin, but increases in c-FLI were evident only on the side of the iNTS contralateral to the lesion. Rats with bilateral lesions showed no evidence of having acquired a CTA and no increase in c-FLI in iNTS relative to unpaired controls. These findings support involvement of amygdala in CTA learning and suggest that a lateralized connection between amygdala and iNTS is necessary for the conditioned c-FLI which is induced by exposure to a conditioned aversive taste.

Conditioned taste aversion (CTA) learning is a robust form of classical conditioning in which animals rapidly associate a flavor with aversive internal symptoms. The present study assessed CTA learning in transgenic mice deficient in a specific nonreceptor tyrosine kinase (the fyn mutant). Fyn mutants show impaired long-term potentiation and marked deficits in acquisition of spatial learning tasks. To assess whether they are also impaired in CTA learning, fyn mutant and wild-type mice received 2 conditioning trials consisting of access to a flavored solution followed by administration of LiCl. Fyn mutant mice acquired significant CTAs following a single conditioning trial and these aversions were comparable to those seen in wild-type mice. These results indicate that the fyn mutation does not interfere with the acquisition of CTAs and hence that this mutation is not associated with a global learning deficit.

A conditioned taste aversion (CTA) is a form of classical conditioning in which animals avoid a taste (conditioned stimulus; CS) which has been previously paired with a treatment (unconditioned stimulus; US) that produces transient illness. Recently, a reliable cellular correlate of the behavioral expression of a CTA was identified using c-Fos immunostaining as a marker of neuronal activation. Exposure to a saccharin solution (CS) which had previously been paired with lithium chloride (LiCl; US) induced significant c-Fos-like immunoreactivity (c-FLI) in the intermediate zone of the nucleus of the solitary tract (NTS), a response that was quite similar to that displayed following administration of LiCl alone. The present studies employed a variant of the chronic decerebrate rat preparation to explore whether circuitry intrinsic to the brainstem is sufficient for the induction of c-FLI in both as an unconditioned response to the LiCl and as a conditioned response to the saccharin. Using chronic hemidecerebrate rats, which have a unilateral brain transection at the level of the superior colliculus, we found that the unconditioned c-FLI to LiCI was unaltered by the transection, while the conditioned expression of c-FLI to the CS taste was evident only on the side of the NTS which retained neural connections with the forebrain. These findings strongly implicate forebrain input in this cellular correlate of CTA learning and also indicate that the pathways mediating the response to the US (LiCl) and the CS (saccharin) differ.

Temporal parameters of taste aversion learning are known to differ markedly from other learning paradigms in that acquisition occurs despite lengthy delays between exposure to conditioned (CS) and unconditioned stimulus (US). Far less consideration has been paid to very brief CS-US intervals and the possibility that this learning may also be distinguished by an ineffectiveness of close temporal contiguity between CS and US. The effectiveness of a very brief CS-US interval (10 s) was compared with that of 2 lengthier intervals (15 and 30 min). Temporal control of CS delivery (0.15% saccharin solution) into the oral cavity and US delivery (7.5 mg/kg apomorphine hydrochloride) into circulation involved infusion pumps and indwelling catheters. Using a 1-trial learning paradigm, CS-US delays of 15 and 30 min led to significant aversions whereas the 10-s CS-US interval did not, suggesting that close temporal contiguity between CS and US is neither necessary nor sufficient for conditioned taste aversion acquisition.

Amphetamine and lithium chloride (LiCl) are both effective unconditioned stimuli (USs) in the establishment of conditioned taste aversions (CTA) in the rat. However, the mechanism of action of these drugs is quite different with the area postrema and related emetic circuitry critical to the response to LiCl but not amphetamine. c-Fos immunohistochemistry was used to define brain regions activated during drug administration and during expression of a CTA using either amphetamine or LiCl as the US drug. Administration of LiCl induced dense c-Fos-like immunoreactivity (c-FLI) in the nucleus of the solitary tract (NTS) while amphetamine induced only light staining in this area. A conditioned stimulus (CS) saccharin solution paired with amphetamine, however, was associated with c-FLI in NTS in a pattern quite similar to that seen to a LiCl-paired CS. This suggests that the pattern of c-Fos expression to a taste CS after conditioning is characteristic of aversion conditioning, in general, and appears not to represent a matching of the conditioned response to specific unconditioned effects of the drug. To examine this conditioned response further, c-FLI to the aversive saccharin CS was compared to the response to quinine hydrochloride, which is innately aversive. Although behaviorally the animals' ingestive responses were quite similar, the saccharin CS induced significant elevations of c-FLI in NTS whereas the quinine did not. Thus, a taste which had become aversive by virtue of conditioning induced c-FLI expression in NTS while a taste which was inherently aversive did not.