Hitting Ras where it counts: Ras antagonism in the basolateral amygdala inhibits long-term fear memory

Several studies have implicated the Ras/mitogen-activated protein kinase (MAPK) pathway in Pavlovian fear conditioning. RasGRF1 knockout mice show significant deficits in acquisition of long-term fear memories and long-term potentaition (LTP) in the basolateral amygdala (BLA). MAPK kinase inhibition also impairs fear conditioning and amygdaloid LTP. However, there is no direct evidence to date for the involvement of Ras itself in fear conditioning. To address this issue, we examined the effects of intra-amygdala infusions of the selective Ras antagonist farnesylthiosalicylic acid (FTS) on the acquisition and expression of conditional freezing in rats. Micro-infusions of FTS into the BLA prior to contextual fear conditioning significantly impaired acquisition of long-term contextual fear memory in a dose-dependent manner. Post-training FTS infusions had no effect on acquisition of long-term fear memory. The effects of FTS on fear conditioning were specific for the BLA. Finally, intra-amygdala infusions of FTS inhibited MAPK activation in BLA. Collectively, these results provide further evidence for the involvement of amygdaloid Ras in the acquisition of long-term conditional fear memory.     

Protein synthesis inhibition in the basolateral amygdala following retrieval does not impair expression of morphine-associated conditioned place preference

Conditioned place preference is an animal model used to evaluate the affective properties of natural rewards and drugs of abuse. This animal model is a kind of classical conditioning that depends on learning and memory. The basolateral amygdala (BLA) plays an important role in the consolidation and extinction of memory for this task. However, there is a lack of evidence demonstrating protein synthesis dependent reconsolidation following retrieval in conditioned animals. In other words, is it possible to observe morphine-associated place preference if recall of this preference is disrupted? Accordingly, we investigated this hypothesis by BLA infusion of protein synthesis inhibitor, anisomycin, immediately after retrieval (test) in conditioned place preference paradigm. In the first experiment, the conditioned animals were exposed to the two sides of the apparatus for 15 min in a drug-free state during retrieval. In the second experiment, the animals received an injection of morphine (7.5 mg/kg, i.p.) and immediately after, they were exposed to the two sides of the apparatus for 15 min. Finally in the third experiment, after habituation and training in the conditioned place preference task, the animals received an injection of the unconditioned stimulus (morphine, i.p.; 7.5 mg/kg) followed by confinement for 10 min in the morphine-paired compartment (conditioned stimulus) during memory retrieval. For the three experiments the animals were subsequently exposed in a free-drug state to the two sides of the apparatus for the retest. Our results show that the protein synthesis inhibition in all of these experimental designs had no effect on conditioned place preference memory under conditions that would initiate reconsolidation, suggesting that if reconsolidation of a conditioned place preference task exists it is not mediated by protein synthesis in basolateral amygdala. The effect of anisomycin on consolidation of contextual fear conditioning was also investigated as a positive control to assure that the negative results were not due to methodological problems. Using the same dose of anisomycin (62.5 microg/1 microl) in morphine-associated place preference procedures, we have found that this anisomycin dose blocks the consolidation of contextual fear memory, ruling out the possibility that these negative results can be attributed to methodological problem of some sort.     

Protein synthesis in the amygdala, but not the auditory thalamus, is required for consolidation of Pavlovian fear conditioning in rats

The amygdala is an essential neural substrate for Pavlovian fear conditioning. Nevertheless, long-term synaptic plasticity in amygdaloid afferents, such as the auditory thalamus, may contribute to the formation of fear memories. We therefore compared the influence of protein synthesis inhibition in the amygdala and the auditory thalamus on the consolidation of Pavlovian fear conditioning in Long-Evans rats. Rats received three tone-footshock trials in a novel conditioning chamber. Immediately after fear conditioning, rats were infused intra-cranially with the protein synthesis inhibitor, anisomycin. Conditional fear to the tone and conditioning context was assessed by measuring freezing behaviour in separate retention tests conducted at least 24 h following conditioning. Post-training infusion of anisomycin into the amygdala impaired conditional freezing to both the auditory and contextual stimuli associated with footshock. In contrast, intra-thalamic infusions of anisomycin or a broad-spectrum protein kinase inhibitor [1-(5'-isoquinolinesulphonyl)-2-methylpiperazine, H7] did not affect conditional freezing during the retention tests. Pre-training intra-thalamic infusion of the NMDA receptor antagonist 2-amino-5-phosphonopentanoic acid (APV), which blocks synaptic transmission in the auditory thalamus, produced a selective deficit in the acquisition of auditory fear conditioning. Autoradiographic assays of cerebral [14C]-leucine incorporation revealed similar levels of protein synthesis inhibition in the amygdala and thalamus following intra-cranial anisomycin infusions. These results reveal that the establishment of long-term fear memories requires protein synthesis in the amygdala, but not the thalamus, after auditory fear conditioning. Forms of synaptic plasticity that depend on protein synthesis, such as long-term potentiation, are likely candidates for the encoding and long-term storage of fear memories in the amygdala.     

Trace fear conditioning depends on NMDA receptor activation and protein synthesis within the dorsal hippocampus of mice

Various lesion studies demonstrated that trace but not delay fear conditioning requires an intact hippocampal formation. Our present study examined the role of NMDA receptor activation and protein synthesis within the dorsal hippocampus for acquisition of fear memories following trace (5-s trace) and delay conditioning. To this end male C57BL/6JOlaHsd mice were chronically implanted with guide cannulae targeting the dorsal hippocampus. Fifteen minutes before conditioning mice received a bilateral intrahippocampal injection of either the NMDA receptor antagonist AP5 (0.5 or 1 microg per 0.5 microl per side) or of anisomycin, an inhibitor of protein synthesis (62.5 microg per 0.5 microl per side). Control mice were treated with vehicle (Ringer's solution). Blocking NMDA receptors before trace but not delay conditioning dose-dependently attenuated the freezing response to the tone as assessed 24 h after conditioning. The same findings were obtained after blocking protein synthesis within the dorsal hippocampus. These data indicate that the hippocampus shows synaptic plasticity during trace conditioning that requires an activation of NMDA receptors and protein synthesis as prerequisites for the acquisition of fear memory.     

Theta resynchronization during reconsolidation of remote contextual fear memory

We have recently demonstrated high theta-phase synchronization between the lateral amygdala and CA1 area of the hippocampus during retrieval of long-term (1 day) fear memory, and not during short-term (2 h) or remote memory retrieval (30 days). These results indicated that the amygdalo-hippocampal interaction reflects a dynamic change of ensemble activities related to various stages of fear memory storage. In this study, we investigated theta activity during the reconsolidation of a remote contextual fear memory by re-exposing animals to the shock context 30 days after training. Consistent with our previous results, high theta synchronization was no longer apparent during re-exposure to the shock context, but was significantly higher 1 day after context re-exposure. These data indicate that the reconsolidation of remote contextual fear memory includes changes in ensemble activities between the lateral amygdala and CA1.     

The role of NMDA glutamate receptors,PKA, MAPK,and CAMKII in the hippocampus in extinction of conditioned fear

Pavlovian conditioning involves the association of initially neutral conditioned stimuli (CS) with unconditioned stimuli (US) that elicit a response. In contextual fear conditioning in rodents, the CS is the context of a training apparatus and the US is a foot shock. Retrieval of memory of the training is tested by presenting the CS alone. But a retrieval test also initiates extinction of the conditioned response. That is, presentation of the CS alone results in new learning, i.e., the CS no longer predicts the US. Here we report that extinction is triggered by two hippocampal signaling pathways underlying retrieval (the cAMP-dependent protein kinase and the mitogen-activated protein kinase pathways) and two other mechanisms that become activated at the same time and are not necessary for retrieval (N-methyl-D-aspartate glutamatergic receptors and the calcium/calmodulin-dependent protein kinase II signaling pathway). Thus, the molecular mechanisms underlying acquisition and/or consolidation of the memory for extinction are similar to those described for the acquisition and/or consolidation of the original contextual fear.     

Selective excitotoxic lesions of the hippocampus and basolateral amygdala have dissociable effects on appetitive cue and place conditioning based on path integration in a novel Y-maze procedure

The hippocampus and amygdala are thought to be functionally distinct components of different learning and memory systems. This functional dissociation has been particularly apparent in pavlovian fear conditioning, where the integrity of the hippocampus is necessary for contextual conditioning, and of the amygdala for discrete cue conditioning. Their respective roles in appetitive conditioning, however, remain equivocal mainly due to the lack of agreement concerning the operational definition of a 'context'. The present study used a novel procedure to measure appetitive conditioning to spatial context or to a discrete cue. Following selective excitotoxic lesions of the hippocampus (HPC) or basolateral amygdala (BLA), rats were initially trained to acquire discrete CS-sucrose conditioning in a Y-maze apparatus with three topographically identical chambers, the chambers discriminated only on the basis of path integration. The same group of animals then underwent 'place/contextual conditioning' where the CS presented in a chamber assigned as the positive chamber was paired with sucrose, but the same CS presented in either of the other two chambers was not. Thus, spatial context was the only cue that the animal could use to retrieve the value of the CS. HPC lesions impaired the acquisition of conditioned place preference but facilitated the acquisition of cue conditioning, while BLA lesions had the opposite effect, retarding the acquisition of cue conditioning but leaving the acquisition of conditioned place preference intact. Here we provide strong support for the notion that the HPC and BLA subserve complementary and competing roles in appetitive cue and contextual conditioning.     

Basolateral amygdala involvement in memory reconsolidation processes that facilitate drug context-induced cocaine seeking

Understanding the neurobiological underpinnings of putative memory stabilization processes that maintain context–response–cocaine associations in long‐term memory and underlie contextual control over addictive behavior is of great interest from an addiction treatment perspective. Using an instrumental animal model of contextual drug relapse we show that the protein synthesis inhibitor anisomycin, administered into the basolateral amygdala (BLA) immediately after limited (15‐ or 60‐min) re‐exposure to a previously cocaine‐paired context, subsequently disrupted the ability of the previously cocaine‐paired context to reinstate extinguished cocaine‐seeking behavior relative to vehicle. Consistent with a BLA‐mediated memory reconsolidation deficit, a similar impairment in cocaine‐seeking behavior was not observed in (i) ‘no‐reactivation’ control groups that received anisomycin into the BLA after (re)exposure to either a novel unpaired or an extinction‐paired context or in (ii) a neuroanatomical control group that received anisomycin into the posterior caudate–putamen, dorsally adjacent to the BLA, after re‐exposure to the cocaine‐paired context. Furthermore, anisomycin administered into the BLA after brief (5‐min) or extensive (120‐min) re‐exposure to the cocaine‐paired context (which was sufficient to extinguish cocaine‐seeking behavior in a vehicle control group) also failed to alter responding. Together, these findings suggest that re‐exposure to a cocaine‐paired context in the absence of cocaine reinforcement is sufficient to trigger memory reconsolidation processes that support future drug‐seeking behavior. The presence and duration of drug‐related memory reactivation critically influences, and anisomycin‐sensitive mechanisms in the BLA selectively control, this phenomenon. These findings support the feasibility of novel pharmacotherapeutic approaches that selectively inhibit the reconsolidation of cocaine‐related memories in order to prevent drug relapse.     

Extinction of auditory fear conditioning requires MAPK/ERK activation in the basolateral amygdala

Whereas the neuronal substrates underlying the acquisition of auditory fear conditioning have been widely studied, the substrates and mechanisms mediating the acquisition of fear extinction remain largely elusive. Previous reports indicate that consolidation of fear extinction depends on the mitogen-activated protein kinase/extracellular-signal regulated kinase (MAPK/ERK) signalling pathway and on protein synthesis in the medial prefrontal cortex (mPFC). Based on experiments using the fear-potentiated startle paradigm suggesting a role for neuronal plasticity in the basolateral amygdala (BLA) during fear extinction, we directly addressed whether MAPK/ERK signalling in the basolateral amygdala is necessary for the acquisition of fear extinction using conditioned freezing as a read-out. First, we investigated the regional and temporal pattern of MAPK/ERK activation in the BLA following extinction learning in C57Bl/6J mice. Our results indicate that acquisition of extinction is associated with an increase of phosphorylated MAPK/ERK in the BLA. Moreover, we found that inhibition of the MAPK/ERK signalling pathway by intrabasolateral amygdala infusion of the MEK inhibitor, U0126, completely blocks acquisition of extinction. Thus, our results indicate that the MAPK/ERK signalling pathway is required for extinction of auditory fear conditioning in the BLA, and support a role for neuronal plasticity in the BLA during the acquisition of fear extinction.     

CA3 NMDA receptors are crucial for rapid and automatic representation of context memory

It is argued that the hippocampus contributes to acquisition of context-specific memory although neural mechanisms have not been clarified. To evaluate the role of CA3 in context-specific memory, we developed one-trial context discrimination tasks to test acquisition and retrieval of contextual memory in CA3 pyramidal cell-restricted N-methyl-d-aspartate (NMDA) receptor knockout mice. Mutants were unable to discriminate conditioned and no-shock contexts 3 h after one-trial avoidance training. These phenotypes were not evident 24 h after one-trial training or 3 h after multi-trial training. Following one-trial contextual fear conditioning, mutants showed a selective deficit in context discrimination during a retention test 3 h after acquisition, although overall freezing levels were similar to those of the control mice. As in the avoidance task, this context discrimination impairment was not observed 24 h after initial conditioning. Interestingly, extending the post-shock period to 3 min during the one-trial fear conditioning task eliminated the discrimination deficit observed at the 3 h retention interval. These results suggest that: (i) impaired rapid context discrimination during the recall test is dependent on the duration of post-shock period during conditioning; (ii) CA3 NMDA receptors are critically involved in rapid and automatic formation of a unified context memory representation from the sensory information; (iii) CA3 NMDA receptors support contextual pattern separation; (iv) fear memory to foot-shock is acquired without CA3 NMDA receptors. It appears that rapid and automatic context memory representations from one-time experience are mediated, at least in part, by CA3 NMDA receptors.     

Rac1 Signaling in Amygdala Astrocytes Regulates Fear Memory Acquisition and Retrieval

The importance of astrocytes in behavior control is increasingly appreciated, but little is known about the effects of their dynamic activity in regulating learning and memory. In the present study, we constructed AAVs of photoactivatable and photoinactivatable Ras-related C3 botulinum toxin substrate 1 (Rac1) under the mGFAP promoter, which enabled the manipulation of Rac1 activity in astrocytes by optical stimulation in free-moving mice. We found that both up-regulation and down-regulation of astrocytic Rac1 activity in the basolateral amygdala (BLA) attenuated memory acquisition in a fear conditioning mouse model. Meanwhile, neuronal activation in the BLA induced by memory acquisition was inhibited under both the up- and down-regulation of astrocytic Rac1 activity during training. In terms of the impact on fear memory retrieval, we found both up- and down-regulation of BLA astrocytic Rac1 activity impaired memory retrieval of fear conditioning and memory retrieval-induced neuronal activation. Notably, the effect of astrocytic Rac1 on memory retrieval was reversible. Our results demonstrate that the normal activity of astrocytic Rac1 is necessary for the activation of neurons and memory formation. Both activation and inactivation of astrocytic Rac1 activity in the BLA reduced the excitability of neurons, and thereby impaired fear memory acquisition and retrieval.     

Memory for context is impaired by injecting anisomycin into dorsal hippocampus following context exploration

Pre-exposure to the context facilitates the small amount of contextual fear conditioning that is normally produced by immediate shock. This context pre-exposure facilitation effect provides a convenient way to study the rat's learning about context. We recently reported that anterograde damage to dorsal hippocampus prevents this facilitation. The present experiments strengthen this conclusion by showing that the protein synthesis inhibitor, anisomycin, injected bilaterally into the dorsal hippocampus following context pre-exposure also significantly reduces the facilitation effect. The same treatment given immediately after immediate shock, however, had no effect on facilitation. These results support theories that assume that, (a) contextual fear involves two processes, acquiring and storing a conjunctive representation of a context and associating that representation with fear; and (b) the hippocampus contributes to contextual fear by participating in the storage of the memory representation of the context.     

Latent inhibition of cued fear conditioning: an NMDA receptor-dependent process that can be established in the presence of anisomycin

Much of the research examining the biological basis for long-term memories has focused on mechanisms that support the formation of conditioned associations. Less information is available on biological mechanisms which underlie processes that modify the strength of conditioned associations. Latent inhibition is a phenomenon by which pre-exposure to a to-be-conditioned stimulus (CS) weakens subsequent conditioning of that CS to an unconditioned stimulus (US). Here we report that latent inhibition of cued fear conditioning is dependent on NMDA receptor activation. MK-801 (1 mg/kg), an NMDA receptor antagonist, abolished latent inhibition of cued fear conditioning. This dose of MK-801 administered before training did not disrupt cued fear conditioning. Conversely, anisomycin (150 mg/kg), a protein synthesis inhibitor, had no effect on latent inhibition of cued fear conditioning when administered 20 min before, immediately after, or 2, 4, 6, or 8 h after CS pre-exposure. Furthermore, continuous anisomycin administration (50 mg/kg, administered every 2 h for 6 h starting 20 min prior to pre-exposure) did not disrupt latent inhibition of cued fear conditioning. In addition, anisomycin had no effect on a long-lasting version of latent inhibition of cued fear conditioning that was maintained over a 7-day interval. Anisomycin administered before training, however, disrupted learning of the CS-US association. These findings suggest that latent inhibition of cued fear conditioning is a long-lasting NMDA receptor-dependent process that can develop during the inhibition of protein synthesis.     

Inhibition of hippocampal protein synthesis following recall disrupts expression of episodic-like memory in trace conditioning

Transition of short-term to long-term memory is referred to as consolidation and the process is dependent on protein synthesis. Recently, several studies have shown that expression of consolidated memory for simple forms of learning tasks (e.g., delay conditioning, contextual fear, inhibitory avoidance) becomes vulnerable to disruption by inhibition of protein synthesis when administered shortly after recall. In the present study, we address whether recall-induced dependence on protein synthesis is a fundamental property that can be applied to a form of memory requiring attentional awareness or is specific to memories for simple forms of conditioning. Trace fear conditioning is a form of learning that requires an active memory trace to associate a conditioned stimulus (CS) with an unconditioned stimulus (US) separated by time. Our data demonstrate that whether a CS-alone recall trial in a novel context acts as an extinction or reactivation trial depends on the strength of the original memory. Inhibition of protein synthesis following the recall trial in animals receiving one trace conditioning training session (that gives rise to weak memory) resulted in enhanced CS-elicited freezing compared with vehicle control, as a result of impaired extinction memory, but had no effect on contextual memory. However, inhibition of hippocampal protein synthesis following the recall trial in animals receiving two trace conditioning training sessions (that gives rise to stronger memory) resulted in impaired retention of both trace CS-US associative and contextual memory despite that the context-US association was not directly reactivated. This provides evidence that, for a robust memory, the CS-alone recall trial results in the reactivation of an episodic-like memory, including trace CS- and contextual-memory, and that hippocampal information storage for the memory as a whole is returned to a labile state requiring de novo protein synthesis. This and other studies are consistent with the role of the hippocampus in coordinating episodic memory retrieval.     

Histamine H3 receptor-mediated impairment of contextual fear conditioning and in-vivo inhibition of cholinergic transmission in the rat basolateral amygdala

We investigated the effects of agents acting at histamine receptors on both, spontaneous release of ACh from the basolateral amygdala (BLA) of freely moving rats, and fear conditioning. Extensive evidence suggests that the effects of histamine on cognition might be explained by the modulation of cholinergic systems. Using the microdialysis technique in freely moving rats, we demonstrated that perfusion of the BLA with histaminergic compounds modulates the spontaneous release of ACh. The addition of 100 mm KCl to the perfusion medium strongly stimulated ACh release, whereas, 0.5 microm tetrodotoxin (TTX) inhibited spontaneous ACh release by more than 50%. Histaminergic H3 antagonists (ciproxifan, clobenpropit and thioperamide), directly administered to the BLA, decreased ACh spontaneous release, an effect fully antagonized by the simultaneous perfusion of the BLA with cimetidine, an H2 antagonist. Local administration of cimetidine alone increased ACh spontaneous release slightly, but significantly. Conversely, the administration of H1 antagonists failed to alter ACh spontaneous release. Rats receiving intra-BLA, bilateral injections of the H3 antagonists at doses similar to those inhibiting ACh spontaneous release, immediately after contextual fear conditioning, showed memory consolidation impairment of contextual fear conditioning. Post-training, bilateral injections of 50 microg scopolamine also had an adverse effect on memory retention. These observations provide the first evidence that histamine receptors are involved in the modulation of cholinergic tone in the amygdala and in the consolidation of fear conditioning.     

Facilitating influence of stress on the consolidation of fear memory induced by a weak training: Reversal by midazolam pretreatment

It is well known that an emotionally arousing experience usually results in a robust and persistent memory trace. The present study explored the potential mechanisms involved in the influence of stress on the consolidation of a contextual fear memory in animals subjected to a weak fear training protocol, and whether pretreatment with intra-basolateral amygdala or systemic administration of midazolam (MDZ) prevents the potential stress-induced influence on fear memory formation. A previous restraint session facilitated fear retention, this effect was not due to a sensitized effect of restraint on the footshock experience. MDZ, both systemically or intra-basolateral amygdala infusion prior to the restraint, attenuated the stress-induced promoting influence on fear memory formation. In addition, stress exposure activated the ERK1/2 pathway in basolateral amygdala (BLA) after the weak training procedure but not after the immediate footshock protocol. Similar to our behavioral findings, MDZ attenuated stress-induced elevation of phospho-ERK2 (p-ERK2) in BLA following the acquisition session. Given that the activation of ERK1/2 pathway is essential for associative learning, we propose that stress-induced facilitation of p-ERK2 in BLA is an important mechanism for the promoting influence of stress on the consolidation of contextual fear memory.     

Contextual fear conditioning is associated with an increase of acetylcholine release in the hippocampus of rat

The effects of contextual fear conditioning on the release of acetylcholine (ACh) in the hippocampus of freely moving rats was assessed using microdialysis. Measures were carried out during both acquisition and retention testing (re-exposure to the conditioning chamber) and compared between animals that either received foot-shocks as unconditioned stimulus (conditioned group) or no foot-shocks (control group) during acquisition. Results showed that during acquisition, hippocampal ACh extracellular level was increased with respect to baseline but that this increase was of similar magnitude in both groups. By contrast, re-exposure to the conditioning chamber the day after (retention testing) produced a significantly greater increase in ACh extracellular level in the conditioned (that, otherwise, displayed conditioned freezing behavior to contextual cues), than in the control group (which displayed virtually no freezing). This enhanced hippocampal ACh release seems to result from the greater hippocampal processing of contextual stimuli in conditioned animals with respect to controls.     

Paradoxical facilitatory effect of fornix lesions on acquisition of contextual fear conditioning in mice

The present study examined the effect of fornix lesions on freezing behavior elicited by contextual and phasic conditioned stimuli. Male mice of the C57Bl/6 strain received electrolytic lesions of the fornix. Ten days following the lesion, they were submitted to acquisition of one-trial classical fear conditioning involving the pairing of an auditory conditioned stimulus (CS) with a footshock unconditioned stimuli (US). Analysis of conditioned fear responses showed that fornix lesions enhanced the freezing response elicited by exposure to the conditioning chamber 24 h after a single tone-shock pairing. In contrast, the two groups did not differ on their fear responses during the auditory cue test. Analysis of the time-course of freezing behavior during re-exposure to the conditioning chamber suggests, however, that the observed fornix lesion-induced facilitation of freezing to the conditioning chamber is more likely due to a facilitation of the processing of a simple (unimodal) rather than polymodal (contextual) CS-US association.