Consolidation Of Contextual Fear Memory Research Articles

Reactivating Memories for Consolidation

The consolidation of memory is thought to occur via a hippocampal-neocortical dialog involving reactivation of memory patterns in the hippocampus during sharp-wave ripples. In this issue of Neuron, Nakashiba et al. demonstrate that CA3 output is required for consolidation of contextual fear memory. They also show that lack of CA3 output results in a decrease in ripple-related reactivation, providing additional evidence for a role of ripple-related reactivation in the consolidation process.

Segregated Populations of Hippocampal Principal CA1 Neurons Mediating Conditioning and Extinction of Contextual Fear

Learning processes mediating conditioning and extinction of contextual fear require activation of several key signaling pathways in the hippocampus. Principal hippocampal CA1 neurons respond to fear conditioning by a coordinated activation of multiple protein kinases and immediate early genes, such as cFos, enabling rapid and lasting consolidation of contextual fear memory. The extracellular signal-regulated kinase (Erk) additionally acts as a central mediator of fear extinction. It is not known however, whether these molecular events take place in overlapping or nonoverlapping neuronal populations. By using mouse models of conditioning and extinction of fear, we set out to determine the time course of cFos and Erk activity, their cellular overlap, and regulation by afferent cholinergic input from the medial septum. Analyses of cFos(+) and pErk(+) cells by immunofluorescence revealed predominant nuclear activation of either protein during conditioning and extinction of fear, respectively. Transgenic cFos-LacZ mice were further used to label in vivo Fos(+) hippocampal cells during conditioning followed by pErk immunostaining after extinction. The results showed that these signaling molecules were activated in segregated populations of hippocampal principal neurons. Furthermore, immunotoxin-induced lesions of medial septal neurons, providing cholinergic input into the hippocampus, selectively abolished Erk activation and extinction of fear without affecting cFos responses and conditioning. These results demonstrate that extinction mechanisms based on Erk signaling involve a specific population of CA1 principal neurons distinctively regulated by afferent cholinergic input from the medial septum.

Post-training administration of corticosterone enhances consolidation of contextual fear memory and hippocampal long-term potentiation in rats

This study was designed to examine the effect of corticosterone on consolidation of contextual fear memory and hippocampal long-term potentiation (LTP) in rats. In Experiment 1, dose–response effects of corticosterone on consolidation of contextual fear memory were determined. Immediately after training in contextual fear conditioning task, rats received different doses of corticosterone. Testing 24 h later, it revealed that corticosterone enhanced memory consolidation in an inverted U shape as evidenced in increased freezing behavior of corticosterone-treated animals. The most effective dose was 3 mg/kg. In Experiment 2, LTP was examined in rats whose memory consolidation has been enhanced with corticosterone. The rats were trained as the above and received corticosterone (3 mg/kg) immediately after training. Immediately or up to one day after retention test, rats were anesthetized with urethane for LTP experiments. For LTP induction, three episodes of high frequency stimuli, 30 s apart, were delivered to the perforant path, each consisting of 10 stimuli at 250 Hz. LTP was assessed by measuring the increase in the initial slope of the population excitatory post-synaptic potentials and the amplitude of the population spikes. Data indicated that animals whose memory has been enhanced by corticosterone, also displayed enhanced hippocampal LTP. The above findings suggest that glucocorticoids may enhance contextual fear memory consolidation via enhancing hippocampal LTP.

Disruption of Fear Memory Through Dual-Hormone Gene Therapy

The basolateral complex of the amygdala (BLA) is uniquely affected by steroid hormones. Whereas glucocorticoids (GCs)--the adrenal hormones released during stress--increase the excitability of BLA neurons, estrogen decreases it. In this study, we used a vector designed to express a chimeric gene that contains the GC-binding domain of the GC receptor (GR) and the DNA binding domain of the estrogen receptor (ER) ("ER/GR") in infected neurons; as a result, it transduces GC signals into estrogenic ones. We microinfused ER/GR bilaterally into the BLA of rats to determine whether it would impair fear conditioning, a valuable BLA-dependent paradigm for studying the neural basis of emotional memory. Expression of ER/GR in the BLA caused robust expression of the transgene and a significant disruption of both auditory and contextual long-term fear memory consolidation, whereas fear learning and post-shock freezing remained intact. These data show that dual gene therapy with ER/GR might be a useful tool for understanding the role of steroid hormones in the storage of traumatic memories.

Upregulation of Calcium/Calmodulin-Dependent Protein Kinase IV Improves Memory Formation and Rescues Memory Loss with Aging

Previous studies have suggested that calcium/calmodulin-dependent protein kinase IV (CaMKIV) functions as a positive regulator for memory formation and that age-related memory deficits are the result of dysfunctional signaling pathways mediated by cAMP response element-binding protein (CREB), the downstream transcription factor of CaMKIV. Little is known, however, about the effects of increased CaMKIV levels on the ability to form memory in adult and aged stages. We generated a transgenic mouse overexpressing CaMKIV in the forebrain and showed that the upregulation of CaMKIV led to an increase in learning-induced CREB activity, increased learning-related hippocampal potentiation, and enhanced consolidation of contextual fear and social memories. Importantly, we also observed reduced hippocampal CaMKIV expression with aging and a correlation between CaMKIV expression level and memory performance in aged mice. Genetic overexpression of CaMKIV was able to rescue associated memory deficits in aged mice. Our findings suggest that the level of CaMKIV expression correlates positively with the ability to form long-term memory and implicate the decline of CaMKIV signaling mechanisms in age-related memory deficits.

Inhibition of PKA anchoring to A-kinase anchoring proteins impairs consolidation and facilitates extinction of contextual fear memories

Both genetic and pharmacological studies demonstrated that contextual fear conditioning is critically regulated by cyclic AMP-dependent protein kinase (PKA). Since PKA is a broad range protein kinase, a mechanism for confining its activity is required. It has been shown that intracellular spatial compartmentalization of PKA signaling is mediated by A-kinase anchoring proteins (AKAPs). Here, we investigated the role of PKA anchoring to AKAPs in different stages of the memory process (acquisition, consolidation, retrieval and extinction) using contextual fear conditioning, a hippocampus-dependent learning task. Mice were injected intracerebroventricularly or intrahippocampally with the membrane permeable PKA anchoring disrupting peptides St-Ht31 or St-superAKAP-IS at different time points during the memory process. Blocking PKA anchoring to AKAPs resulted in an impairment of fear memory consolidation. Moreover, disrupted PKA anchoring promoted contextual fear extinction in the mouse hippocampus. We conclude that the temporal and spatial compartmentalization of hippocampal PKA signaling pathways, as achieved by anchoring of PKA to AKAPs, is specifically instrumental in long-term contextual fear memory consolidation and extinction, but not in acquisition and retrieval.

Loss of Extinction in CA1-NMDA Receptor deficient Mice. A novel model for PTSD?

Hippocampal NMDA receptors are involved in contextual fear learning. Here we report that a gene deletion of the NR1 subunit of the NMDA receptor, localized to CA1 neurons of the dorsal hippocampus via AAV-Cre injection in fNR1 mice, results in a selective loss of fear memory extinction, but not its acquisition. Furthermore, CA1-NMDA receptor deficient mice show enhanced anxiety and hyperactivity after fear conditioning. These symptoms are typical of anxiety disorders. A focal NMDA receptor loss was induced in CA1 neurons of the hippocampus via bilateral stereotaxic AAV-Cre injections. We confirmed the loss of NMDA receptor function using whole-cell patch clamp recordings from CA1-pyramidal neurons. The area of gene loss was identified for all animals from in-situ hybridisations of NR1 mRNA. AAV-lacZ injected mice served as control group. AAV-Cre and AAV-lacZ injected mice were trained in a contextual fear conditioning paradigm with three footshocks of 0.5 mA each. No difference was observed in baseline freezing, nor acute fear after the shocks. Following re-exposure to the context 24h later, both groups showed consolidation of contextual fear memory. But extinguishing this memory for 9 days lead to a significant reduction in amount of freezing in CA1-NMDA receptor deficient mice. This behavior was context-specific. Two weeks after fear conditioning, the knock-out group also revealed enhanced anxiety in open field, dark/light box and elevated plus maze. Sensitized fear was observed 6 weeks after traumatizing. Our data suggest that NMDA receptors of CA1 neurons are crucial for extinction of fear memory. As extinction is impaired in PTSD patients, our CA1-NMDA receptor deficient mice might serve as a novel model for enhanced anxiety disorders and help finding new treatment strategies for PTSD.

Are β-adrenergic receptors in the hippocampal CA1 region required for retrieval of contextual fear memory?

It is well established that β-adrenoceptors (β-ARs) in the hippocampal CA1 region are involved in regulating synaptic plasticity and are essential for acquisition and consolidation of spatial memory and contextual fear memory. Previous studies reported that β-ARs in the CA1 region are also involved in memory retrieval. The present study re-examined the role of hippocampal β-ARs in retrieval of conditioned contextual fear. We bilaterally infused a high dose of the β-AR antagonist propranolol (15 μg in 1 μl saline) into the CA1 region 30 min before retention test and found that propranolol produced no deficit in retrieval of either 1-day or 7-day contextual fear. We then examined if β-AR stimulation would produce a beneficial effect. The β-AR agonist isoproterenol (10 μg in 1 μl saline) was infused into the CA1 region 30 min before retention test. Surprisingly, isoproterenol did not enhance but severely disrupted retrieval of 7-day contextual fear memory, with no impact on retrieval of 1-day contextual fear memory. The present study argues against the previous conclusion that β-ARs in the CA1 region play a role in memory retrieval. β-ARs in the CA1 region may be dispensable for retrieval of conditioned contextual fear.

Administration of corticosterone after memory reactivation disrupts subsequent retrieval of a contextual conditioned fear memory: Dependence upon training intensity

Reactivation of stabilized memories returns them to a labile state and causes them to undergo extinction or reconsolidation processes. Although it is well established that administration of glucocorticoids after training enhance consolidation of contextual fear memories, but their effects on post-retrieval processes are not known. In this study, we first asked whether administration of corticosterone after memory reactivation would modulate subsequent expression of memory in rats. Additionally, we examined whether this modulatory action would depend upon the strength of the memory. We also tested the effect of propranolol after memory reactivation. Adult male Wistar rats were trained in a fear conditioning system using moderate (0.4 mA) or high shock (1.5 mA) intensities. For reactivation, rats were returned to the chamber for 90 s 24 h later. Immediately after reactivation, rats were injected with corticosterone (1, 3 or 10 mg/kg) or vehicle. One, 7 and 14 days after memory reactivation, rats were returned to the context for 5 min, and freezing behavior was scored. The findings indicated that corticosterone when injected after memory reactivation had no significant effect on recall of a moderate memory, but it impaired recall of a strong memory at a dose of 3 mg/kg. Propranolol (5 mg/kg) given after the reactivation treatment produced a modest impairment that persisted over three test sessions. Further, the results showed that corticosterone, but not propranolol deficit was reversed by a reminder shock. These findings provide evidence that administration of glucocorticoids following memory reactivation reduces subsequent retrieval of strong, but not moderate, contextual conditioned fear memory likely via acceleration of memory extinction. On the other hand, propranolol-induced amnesia may result from blockade of reconsolidation process. Further studies are needed to determine the underlying mechanisms.

Biphasic ERK1/2 activation in both the hippocampus and amygdala may reveal a system consolidation of contextual fear memory

There is accumulating evidences to suggest that memory consolidation in some conditions involves two waves of neuronal plastic change. Using two fear conditioning procedures in male C57BL/6J mice, we have recently shown that consolidation of the foreground contextual fear memory required two waves of ERK1/2 activation in hippocampal CA1, while consolidation of cue conditioning was only associated with the early phase of activation. The present experiment further showed that this bi-phasic pattern of ERK1/2 activation was not restricted to hippocampal CA1, but could also be observed in other fear memory-related brain areas. The unpaired conditioning procedure (context in foreground) induced two waves of ERK1/2 activation in hippocampal CA1 and CA3, as well as in the LA and BLA nuclei of the amygdala. In contrast, the paired conditioning procedure (context in background) led to a transient early phase only in hippocampal CA1 and LA. In addition, ERK1/2 phosphorylation in the hippocampus was found to correlate with that in the amygdala nuclei specifically after the unpaired procedure. Taken together, our data suggest that the observed biphasic pattern of neuronal plastic events may reflect the interplay between hippocampal and amygdala activity-dependent plasticity critical for the system consolidation of contextual fear memory.

SNAP-25 in hippocampal CA3 region is required for long-term memory formation

SNAP-25 is a synaptosomal protein of 25 kDa, a key component of synaptic vesicle-docking/fusion machinery, and plays a critical role in exocytosis and neurotransmitter release. We previously reported that SNAP-25 in the hippocampal CA1 region is involved in consolidation of contextual fear memory and water-maze spatial memory (Hou et al. European J Neuroscience, 20: 1593–1603, 2004). SNAP-25 is expressed not only in the CA1 region, but also in the CA3 region, and the SNAP-25 mRNA level in the CA3 region is higher than in the CA1 region. Here, we provide evidence that SNAP-25 in the CA3 region is also involved in learning/memory. Intra-CA3 infusion of SNAP-25 antisense oligonucleotide impaired both long-term contextual fear memory and water-maze spatial memory, with short-term memory intact. Furthermore, the SNAP-25 antisense oligonucleotide suppressed the long-term potentiation (LTP) of field excitatory post-synaptic potential (fEPSP) in the mossy-fiber pathway (DG-CA3 pathway), with no effect on paired-pulse facilitation of the fEPSP. These results are consistent with the notion that SNAP-25 in the hippocampal CA3 region is required for long-term memory formation.

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 μg/1 μl) 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.

Foreground contextual fear memory consolidation requires two independent phases of hippocampal ERK/CREB activation

Fear conditioning is a popular model for investigating physiological and cellular mechanisms of memory formation. In this paradigm, a footshock is either systematically associated to a tone (paired conditioning) or is pseudorandomly distributed (unpaired conditioning). In the former procedure, the tone/shock association is acquired, whereas in the latter procedure, the context/shock association will prevail. Animals with chronically implanted recording electrodes show enhanced amplitude of the extracellularly recorded field EPSP in CA1 pyramidal cells for up to 24 h after unpaired, but not paired, fear conditioning. This is paralleled by a differential activation of the ERK/CREB pathway in CA1, which is monophasic in paired conditioning (0-15 min post-conditioning), but biphasic (0-1 h and 9-12 h post-conditioning) in unpaired conditioning as revealed by immunocytochemistry and Western blotting. Intrahippocampal injection of the MEK inhibitor U0126 prior to each phase prevents the activation of both ERK1/2 and CREB after unpaired conditioning. Block of any activation phase leads to memory impairment. We finally reveal that the biphasic activation of ERK/CREB activity is independently regulated, yet both phases are critically required for the consolidation of long-term memories following unpaired fear conditioning. These data provide compelling evidence that CA1 serves different forms of memory by expressing differential cellular mechanisms that are dependent on the training regime.

Encoding, consolidation, and retrieval of contextual memory: Differential involvement of dorsal CA3 and CA1 hippocampal subregions

Studies on human and animals shed light on the unique hippocampus contributions to relational memory. However, the particular role of each hippocampal subregion in memory processing is still not clear. Hippocampal computational models and theories have emphasized a unique function in memory for each hippocampal subregion, with the CA3 area acting as an autoassociative memory network and the CA1 area as a critical output structure. In order to understand the respective roles of the CA3- and CA1-hippocampal areas in the formation of contextual memory, we studied the effects of the reversible inactivation by lidocaine of the CA3 or CA1 areas of the dorsal hippocampus on acquisition, consolidation, and retrieval of a contextual fear conditioning. Whereas infusions of lidocaine never impaired elementary tone conditioning, their effects on contextual conditioning provided interesting clues about the role of these two hippocampal regions. They demonstrated first that the CA3 area is necessary for the rapid elaboration of a unified representation of the context. Secondly, they suggested that the CA1 area is rather involved in the consolidation process of contextual memory. Third, they showed that CA1 or CA3 inactivation during retention test has no effect on contextual fear retrieval when a recognition memory procedure is used. In conclusion, our findings point as evidence that CA1 and CA3 subregions of the dorsal hippocampus play important and different roles in the acquisition and consolidation of contextual fear memory, whereas they are not required for context recognition.

Deficit in long-term contextual fear memory induced by blockade of beta-adrenoceptors in hippocampal CA1 region.

The present study investigated the effects of intra-CA1 infusion of d,l-propranolol, the beta-adrenergic antagonist, on memory for contextual fear conditioning. d,l-Propranolol administered 5 min ('0 h') postconditioning impaired long-term but not short-term contextual fear memory, while it was ineffective when administered 6 h postconditioning, suggesting that there is a time window for beta-adrenoceptors to play a role. Thus, we conclude that beta-adrenoceptors in area CA1 are involved in regulating consolidation of contextual fear memory, with '0 h' but not 6 h post-training, a sensitive time point for the beta-adrenergic involvement.

Time-dependent involvement of PKA/PKC in contextual memory consolidation

Rats were implanted bilaterally with cannulae into the dorsal hippocampus and trained in a Pavlovian fear-conditioning paradigm. Four groups of rats were infused intra-cranially with 1-(5′-isoquinolinesulfonyl)-2-methylpiperazine (H7-dihydrochloride), a potent inhibitor of both protein kinase C (PKC) and cAMP-dependent protein kinase (PKA), at different time intervals in order to examine their involvement in the acquisition and consolidation of contextual fear memory. We demonstrate a significant consolidation deficit of long-term contextual fear-conditioning memory that is maximal when PKA and PKC are inhibited at 90 min post-training. These results suggest the existence of a critical time window, during which these enzymes must be activated for the consolidation of long-term memories.

Prolonged fear responses in mice lacking dopamine D 1 receptor

Dopamine is an important neurotransmitter involved in learning and memory including emotional memory. The involvement of dopamine in conditioned fear has been widely documented. However, little is known about the molecular mechanisms that underlie contextual fear conditioning and memory consolidation. To address this issue, we used dopamine D 1-deficient mice (D 1−/−) and their wild-type (D 1+/+) and heterozygote (D 1+/−) siblings to assess aversive learning and memory. We quantified two different aspects of fear responses to an environment where the mice have previously received unsignaled footshocks. Using one-trial step-through passive avoidance and conditioned freezing paradigms, mice were conditioned to receive mild inescapable footshocks then tested for acquisition, retention and extinction of conditioned fear responses 5 min after and up to 45–90 days post-training. No differences were observed among any of the genotypes in the acquisition of passive avoidance response or fear-induced freezing behavior. However, with extended testing, D 1−/− mice exhibited prolonged retention and delayed extinction of conditioned fear responses in both tasks, suggesting that D 1−/− mice are capable of acquiring aversive learning normally. These findings demonstrate that the dopamine D 1 receptor is not important for acquisition or consolidation of aversive learning and memory but has an important role in modulating the extinction of fear memory.

P-462 - Trimethyltin impairs acquisition and consolidation of contextual fear memory in rats

P-462 - Trimethyltin impairs acquisition and consolidation of contextual fear memory in rats

Auditory Thalamus, Dorsal Hippocampus, Basolateral Amygdala, and Perirhinal Cortex Role in the Consolidation of Conditioned Freezing to Context and to Acoustic Conditioned Stimulus in the Rat

On the basis of previous experimental evidence, it is known that the auditory thalamus (AT), the dorsal hippocampus (DH), the basolateral amygdala (BLA), and the perirhinal cortex (PC) are involved in the mnemonic processing of conditioned freezing. In particular, BLA and PC appear to be involved both in conditioned stimulus (CS) and context conditioned freezing. Through AT, the auditory CS is sent to other sites, whereas DH is involved in context conditioning. Nevertheless, the existing evidence does not make it possible to assess AT, DH, BLA, and PC involvement during the consolidation phase of conditioned freezing. To address this question, fully reversible tetrodotoxin (TTX) inactivation was performed on adult male Wistar rats having undergone CS and context fear training. Anesthetized animals were injected stereotaxically with TTX (either 5 or 10 ng in 0.5 or 1.0 microliter of saline, according to site dimensions) at increasing post-acquisition delays. Context and CS freezing durations were measured during retention testing, always performed 48 and 72 hr after TTX administration. The results showed that AT inactivation does not disrupt consolidation of either contextual or auditory fear memories. In contrast, inactivation of the other three structures disrupted consolidation. For the DH, this disruption was specific to contextual cues and only occurred when inactivation was performed early (up to 1.5 hr) after training. The BLA and PC were shown to be involved in the consolidation of both contextual and auditory fear. Their involvement persisted for longer periods of time (2d for BLA and 8 d for PC). These findings provide information to build a temporal profile for the post-training processing of fear memories in structures known to be important for this form of learning. The results are discussed in relation to previous studies on conditioned freezing and other aversive conditioned response neural correlates.