Context preexposure prevents forgetting of a contextual fear memory: implication for regional changes in brain activation patterns associated with recent and remote memory tests

Contextual fear conditioning was maintained over a 15-day retention interval suggesting no forgetting of the conditioning experience. However, a more subtle generalization test revealed that, as the retention interval increased, rats showed enhanced generalized fear to an altered context. Preexposure to the training context prior to conditioning, however, prevented this enhanced generalized fear from developing. These results support the hypothesis that the memory representation of the context degrades as the memory ages and is responsible for enhanced generalization. The implications of these results for systems consolidation versus forgetting interpretations of regional changes in neural activation patterns that occur as memories age are discussed.     

Critical role of the 65-kDa isoform of glutamic acid decarboxylase in consolidation and generalization of Pavlovian fear memory

Evidence suggests that plasticity of the amygdalar and hippocampal GABAergic system is critical for fear memory formation. In this study we investigated in wild-type and genetically manipulated mice the role of the activity-dependent 65-kDa isozyme of glutamic acid decarboxylase (GAD65) in the consolidation and generalization of conditioned fear. First, we demonstrate a transient reduction of GAD65 gene expression in the dorsal hippocampus (6 h post training) and in the basolateral complex of the amygdala (24 h post training) during distinct phases of fear memory consolidation. Second, we show that targeted ablation of the GAD65 gene in Gad65(-/-) mice results in a pronounced context-independent, intramodal generalization of auditory fear memory during long-term (24 h or 14 d) but not short-term (30 min) memory retrieval. The temporal specificity of both gene regulation and memory deficits in Gad65 mutant mice suggests that GAD65-mediated GABA synthesis is critical for the consolidation of stimulus-specific fear memory. This function appears to involve a modulation of neural activity patterns in the amygdalo-hippocampal pathway as indicated by a reduction in theta frequency synchronization between the amygdala and hippocampus of Gad65(-/-) mice during the expression of generalized fear memory.     

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

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

Effects of Age on the Generalization and Incubation of Memory in the F344 Rat

Freezing (immobility) in the presence of aversive stimuli is a species-specific behavior that is used as an operational measure of fear. Conditioning of this response to discrete sensory stimuli and environmental context cues has been used as a tool to study the neuropsychology of memory dynamics and their development over the lifespan. Three age groups of F344 rats (3, 9, and 27 month) received tone–foot shock pairing (or tone only) in a distinctive chamber on two consecutive days. Separate subgroups of rats from each age group were then tested, at retention intervals of 1, 20, 40, or 60 days, for context-mediated fear in the environment in which they were trained, for generalization of the fear response to a novel chamber, and for fear of the tone. Beginning at day 20, the 27-month-old rats exhibited less freezing behavior than did younger rats when tested in the conditioning context. This age difference was a result of freezing behavior becoming progressively stronger with time in the two younger age groups, a phenomenon that has been referred to as memory incubation. Incubation of the contextual fear response was not detected in the old rats. In a novel context, all age groups exhibited significantly more freezing than did control animals. There was also pronounced incubation of this generalized freezing response, and the extent of incubation declined significantly with age. In the novel context, the freezing response to the tone was robust in all age groups and increased over time, in constant proportion to the degree of freezing elicited by the novel context itself, prior to tone onset. The fact that old animals are known to be relatively selectively impaired in forms of memory that depend on a functional hippocampus suggests a possible explanation for the reduced incubation effects seen in old rats; however, whether the increased expression of fear over time is mediated by a hippocampal-dependent memory consolidation process or whether it reflects a generalized increase in the gain of the circuitry mediating the fear response itself, remains to be determined.     

Glucocorticoid involvement in memory formation in a rat model for traumatic memory

Contextual fear conditioning under training conditions involving high stressor intensities has been proposed as an animal model for traumatic memories. The strength of memory for this task has been related to the intensity of the conditioning stressor and post-training corticosterone values. However, administration of a glucocorticoid receptor (GR) antagonist only attenuated memory for this task in rats conditioned at a moderate shock intensity (0.4 mA), but failed to influence conditioning in rats trained at a high shock intensity (1 mA). Here, we further questioned whether interfering with glucocorticoid action at the time of training might be effective in influencing contextual fear conditioning in rats trained under different shock intensities. Rats were subcutaneously injected with the glucocorticoid synthesis inhibitor metyrapone (50, 100 mg/kg) 90 min before being trained in the contextual fear conditioning task, at either 0.4 or 1 mA shock intensities. The results showed that metyrapone, in a dose-dependent manner: (i) attenuated long-term expression of contextual fear conditioning, both in 0.4- and 1 mA-trained rats; and (ii) efficiently prevented increased plasma corticosterone concentration. In addition to further supporting a facilitating role of glucocorticoids in memory consolidation, these findings suggest a critical involvement of these hormones in the formation of traumatic memories.     

Generalization of contextual fear as a function of familiarity: the role of within- and between-context associations

Three experiments in rats investigated the generalization of conditioned fear from one context (B) to both a preexposed context (A) and a novel context (C). In each experiment, when the conditioning context (B) had been preexposed, there was greater generalization to context A than to context C; but when B was novel at the outset of conditioning this difference between A and C was not observed. The implications of these results for associative treatments of the development of contextual memories are evaluated.     

Effects of post-training hippocampal injections of midazolam on fear conditioning

Benzodiazepines have been useful tools for investigating mechanisms underlying learning and memory. The present set of experiments investigates the role of hippocampal GABA(A)/benzodiazepine receptors in memory consolidation using Pavlovian fear conditioning. Rats were prepared with cannulae aimed at the dorsal hippocampus and trained with a series of white noise-shock pairings. In the first experiment, animals received intrahippocampal infusion of midazolam or vehicle immediately or 3 h after training. Then, 24 h later, freezing to the training context and the white noise were measured independently. Results show infusion of midazolam immediately, but not 3 h, after training selectively attenuates contextual fear conditioning. In the second experiment, animals received intrahippocampal infusions of an antisense oligodeoxynucleotide (ODN) targeting the alpha5 subunit of the GABA(A) receptor or a missense control for several days prior to training and testing. Immediately after training, animals received an infusion of either midazolam or vehicle. Western blots conducted after testing showed a significant decrease in alpha5-containing GABA(A) receptor protein. This reduction did not alter the effectiveness of midazolam immediately after training at impairing context fear memory. Therefore, alpha5-containing GABA(A) receptors may not contribute to the effects of midazolam on context fear conditioning when given immediately post-training.     

Cholinergic modulation of the hippocampus during encoding and retrieval of tone/shock-induced fear conditioning

We investigated the role of acetylcholine (ACh) during encoding and retrieval of tone/shock-induced fear conditioning with the aim of testing Hasselmo's cholinergic modulation model of encoding and retrieval using a task sensitive to hippocampal disruption. Lesions of the hippocampus impair acquisition and retention of contextual conditioning with no effect on tone conditioning. Cholinergic antagonists also impair acquisition of contextual conditioning. Saline, scopolamine, or physostigmine was administered directly into the CA3 subregion of the hippocampus 10 min before rats were trained on a tone/shock-induced fear conditioning paradigm. Freezing behavior was used as the measure of learning. The scopolamine group froze significantly less during acquisition to the context relative to controls. The scopolamine group also froze less to the context test administered 24 h posttraining. A finer analysis of the data revealed that scopolamine disrupted encoding but not retrieval. The physostigmine group initially froze less during acquisition to the context, although this was not significantly different from controls. During the context test, the physostigmine group froze less initially but quickly matched the freezing levels of controls. A finer analysis of the data indicated that physostigmine disrupted retrieval but not encoding. These results suggest that increased ACh levels are necessary for encoding new spatial contexts, whereas decreased ACh levels are necessary for retrieving previously learned spatial contexts.     

Infusion of lidocaine into the dorsal hippocampus before or after the shock training phase impaired conditioned freezing in a two-phase training task of contextual fear conditioning

Learning in a contextual fear conditioning task involves forming a context representation and associating it with a shock. The dorsal hippocampus (DH) is implicated in representing the context, but whether it also has a role in associating the context and shock is unclear. To address this issue, male Wistar rats were trained on the task by a two-phase training paradigm, in which rats learned the context representation on day 1 and then reactivated it to associate with the shock on day 2; conditioned freezing was tested on day 3. Lidocaine was infused into the DH at various times in each of the two training sessions. Results showed that intra-DH infusion of lidocaine shortly before or after the context training session on day 1 impaired conditioned freezing, attesting to the DH involvement in context representation. Intra-DH infusion of lidocaine shortly before or after the shock training session on day 2 also impaired conditioned freezing. This deficit was reproduced by infusing lidocaine or APV (alpha-amino-5-phosphonovaleric acid) into the DH after activation of the context memory but before shock administration. The deficit was not due to drug-induced state-dependency, decreased shock sensitivity or reconsolidation failure of the contextual memory. These results suggest that in contextual fear conditioning integrity of the DH is required for memory processing of not only context representation but also context-shock association.     

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

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

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 24h later. Immediately after reactivation, rats were injected with corticosterone (1, 3 or 10mg/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 3mg/kg. Propranolol (5mg/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.     

Repeatedly reactivated memories become more resistant to hippocampal damage

We examined whether repeated reactivations of a context memory would prevent the typical amnesic effects of post-training damage to the hippocampus (HPC). Rats were given a single contextual fear-conditioning session followed by 10 reactivations, involving a brief return to the conditioning context (no shock). Subsequently, the rats received sham or complete lesions of the HPC. When tested for retention, the HPC rats that experienced the reactivations froze significantly more than nonreactivation HPC rats and did not significantly differ from their respective control group. These findings suggest that memory reactivations contribute to long-term memories becoming independent of the HPC.     

Hippocampal regulation of context-dependent neuronal activity in the lateral amygdala

Pavlovian fear conditioning is a robust and enduring form of emotional learning that provides an ideal model system for studying contextual regulation of memory retrieval. After extinction the expression of fear conditional responses (CRs) is context-specific: A conditional stimulus (CS) elicits greater conditional responding outside compared with inside the extinction context. Dorsal hippocampal inactivation with muscimol attenuates context-specific CR expression. We have previously shown that CS-elicited spike firing in the lateral nucleus of the amygdala is context-specific after extinction. The present study examines whether dorsal hippocampal inactivation with muscimol disrupts context-specific firing in the lateral amygdala. We conditioned rats to two separate auditory CSs and then extinguished each CS in separate and distinct contexts. Thereafter, single-unit activity and conditional freezing were tested to one CS in both extinction contexts after saline or muscimol infusion into the dorsal hippocampus. After saline infusion, rats froze more to the CS when it was presented outside of its extinction context, but froze equally in both contexts after muscimol infusion. In parallel with the behavior, lateral nucleus neurons exhibited context-dependent firing to extinguished CSs, and hippocampal inactivation disrupted this activity pattern. These data reveal a novel role for the hippocampus in regulating the context-specific firing of lateral amygdala neurons after fear memory extinction.     

Body temperature as a conditional response measure for pavlovian fear conditioning

On six days rats were exposed to each of two contexts. They received an electric shock in one context and nothing in the other. Rats were tested later in each environment without shock. The rats froze and defecated more often in the shock-paired environment; they also exhibited a significantly larger elevation in rectal temperature in that environment. The rats discriminated between each context, and we suggest that the elevation in temperature is the consequence of associative learning. Thus, body temperature can be used as a conditional response measure in Pavlovian fear conditioning experiments that use footshock as the unconditional stimulus.     

Conditional forebrain deletion of the L-type calcium channel Ca V 1.2 disrupts remote spatial memories in mice

To determine whether L-type voltage-gated calcium channels (L-VGCCs) are required for remote memory consolidation, we generated conditional knockout mice in which the L-VGCC isoform Ca(V)1.2 was postnatally deleted in the hippocampus and cortex. In the Morris water maze, both Ca(V)1.2 conditional knockout mice (Ca(V)1.2(cKO)) and control littermates displayed a marked decrease in escape latencies and performed equally well on probe trials administered during training. In distinct contrast to their performance during training, Ca(V)1.2(cKO) mice exhibited significant impairments in spatial memory when examined 30 d after training, suggesting that Ca(V)1.2 plays a critical role in consolidation of remote spatial memories.     

Intrahippocampal scopolamine impairs both acquisition and consolidation of contextual fear conditioning

Lesions of the dorsal hippocampus have been shown to disrupt both the acquisition and the consolidation of memories associated with contextual fear (fear of the place of conditioning), but do not affect fear conditioning to discrete cues (e.g., a tone). Blockade of central muscarinic cholinergic receptor activation results in selective acquisition deficits of contextual fear conditioning, but reportedly has little effect on consolidation. Here we show for the first time that direct infusion of the muscarinic cholinergic receptor antagonist, scopolamine, into the dorsal hippocampus produces a dose-dependent deficit in both acquisition and consolidation of contextual fear conditioning, while having no impact on simple tone conditioning.     

Both exposure to a novel context and associative learning induce an upregulation of AKAP150 protein in mouse hippocampus

A-kinase anchoring protein 150 (AKAP150) is a multi-enzyme signaling complex that coordinates the action of PKA, PKC, and PP2B at neuronal membranes and synapses. We measured levels of AKAP150 protein in the hippocampus 6h after training mice in a contextual fear conditioning paradigm. In contextual fear conditioning mice learn to associate a context with a footshock presentation. Mice were divided in four experimental groups with different training protocols: naive, no footshock exposure, immediate footshock exposure, and footshock 3min after exposure to the context. We found that AKAP150 protein levels were increased upon exposing mice to the novel context independent of the training protocol. However, when the animals were habituated to the experimental context, only mice that learned to associate the context with the footshock showed an upregulation of AKAP150. We suggest that upregulated levels of AKAP150 contribute to processing the exposure to a novel context and associative learning.     

Post-training unilateral amygdala lesions selectively impair contextual fear memories

The basolateral amygdala (BLA) and the dorsal hippocampus (dHPC) are both structures with key roles in contextual fear conditioning. During fear conditioning, it is postulated that contextual representations of the environment are formed in the hippocampus, which are then associated with foot shock in the amygdala. However, it is not known to what extent a functional connection between these two structures is required. This study investigated the effect on contextual and cued fear conditioning of disconnecting the BLA and dHPC, using asymmetrically placed, excitotoxic unilateral lesions. Post-training lesions selectively impaired contextual, but not cued, fear, while pretraining lesions resulted in a similar but nonsignificant pattern of results. This effect was unexpectedly observed in both the contralateral disconnection group and the anticipated ipsilateral control, which prompted further examination of individual unilateral lesions of BLA and dHPC. Post-training unilateral dHPC lesions had no effect on contextual fear memories while bilateral dHPC lesions and unilateral BLA lesions resulted in a near total abolition of contextual fear but not cued conditioned fear. Again, pretraining unilateral BLA lesions resulted in a strong but nonsignificant trend to the impairment of contextual fear. Furthermore, an analysis of context test-induced Fos protein expression in the BLA contralateral to the lesion site revealed no differences between post-training SHAM and unilateral BLA lesioned animals. Therefore, post-training unilateral lesions of the BLA are sufficient to severely impair contextual, but not cued, fear memories.     

睡眠对恐惧学习的影响及其认知神经机制

睡眠问题可能会诱发恐惧相关情绪障碍(焦虑、创伤性应激障碍、恐怖症等),研究睡眠影响恐惧学习的认知神经机制,有助于增强对恐惧相关情绪障碍的预测、诊断和治疗。以往研究表明睡眠剥夺影响恐惧习得和消退主要是通过抑制vmPFC活动,阻碍其与杏仁核的功能连接,从而导致恐惧习得增强或是消退学习受损。进一步研究发现睡眠不同阶段对恐惧学习相关脑区有独特的影响:剥夺(缺乏)快速眼动睡眠会抑制vmPFC活动、增强杏仁核、海马激活,导致恐惧习得增强,消退学习受损,此外边缘皮层的功能连接减少破坏了记忆巩固(恐惧记忆和消退记忆);而慢波睡眠主要与海马变化有关,慢波睡眠期间进行目标记忆重激活可促进恐惧消退学习。未来研究需要增加睡眠影响恐惧泛化的神经机制研究、及昼夜节律中断对恐惧消退的影响,以及关注动物睡眠研究向人类睡眠研究转化中存在的问题。