Anterograde amnesia for Pavlovian fear conditioning and the role of one-trial overshadowing: effects of preconditioning exposures to morphine in the rat

Four experiments studied anterograde deficits in Pavlovian fear conditioning following prolonged exposure to the mu-opioid receptor agonist morphine. Injections of morphine produced temporally graded anterograde amnesia characterized by deficits in contextual and conditioned-stimulus (CS) conditioning 1 or 7 days and selective impairment in CS conditioning 21 days after last injection. This anterograde deficit in conditioning did not recover across a retention interval, was absent when rats were tested immediately after conditioning, and required the presence of an auditory CS. These results suggest that anterograde deficits in Pavlovian fear conditioning emerged from differences in susceptibility to 1-trial overshadowing of context by CS.     

Temporally graded,context-specific retrograde amnesia and its alleviation by context preexposure: effects of postconditioning exposures to morphine in the rat

Five experiments studied retrograde impairments in Pavlovian fear conditioning following prolonged exposure to the opioid receptor agonist morphine. Injections of morphine commencing 1-7 days but not 14 days after conditioning produced amnesia for that conditioning episode. This amnesia was (a) selective such that morphine impaired freezing to the conditioning context but not to the auditory conditioned stimulus, (b) independent of the interval between the last injection of morphine and test, and (c) accompanied by a failure of contextual discrimination. Context preexposure protected context conditioning and discrimination from the amnestic effects of morphine. These results show that retrograde deficits in contextual fear conditioning are mediated by failures to consolidate a contextual representation.     

The L-Type voltage-gated calcium channel Cav1.3 mediates consolidation, but not extinction, of contextually conditioned fear in mice

Using pharmacological techniques, it has been demonstrated that both consolidation and extinction of Pavlovian fear conditioning are dependent to some extent upon L-type voltage-gated calcium channels (LVGCCs). Although these studies have successfully implicated LVGCCs in Pavlovian fear conditioning, they do not provide information about the specific LVGCC isoform involved. Both of the major LVGCC subtypes found in the brain (Cav1.2 and Cav1.3) are targets of the pharmacological manipulations used in earlier work. In this study, we used mice in which the gene for the pore-forming subunit (alpha1D) Cav1.3 was deleted (Cav1.3 knockout mice) to elucidate its contribution to consolidation and extinction of conditioned fear. We find that Cav1.3 knockout mice exhibit significant impairments in consolidation of contextual fear conditioning. However, once sufficiently overtrained, the Cav1.3 knockout mice exhibit rates of extinction that are identical to that observed in wild-type mice. We also find that Cav1.3 knockout mice perform as well as wild-type mice on the hidden platform version of the Morris water maze, suggesting that the consolidation deficit in conditioned fear observed in the Cav1.3 knockout mice is not likely the result of an inability to encode the context, but may reflect an inability to make the association between the context and the unconditioned stimulus.     

Trace fear conditioning is enhanced in mice lacking the delta subunit of the GABAA receptor

The delta subunit of the GABA(A) receptor (GABA(A)R) is highly expressed in the dentate gyrus of the hippocampus. Genetic deletion of this subunit reduces synaptic and extrasynaptic inhibition and decreases sensitivity to neurosteroids. This paper examines the effect of these changes on hippocampus-dependent trace fear conditioning. Compared to controls, delta knockout mice exhibited enhanced acquisition of tone and context fear. Hippocampus-independent delay conditioning was normal in these animals. These results suggest that reduced inhibition in the dentate gyrus facilitates the acquisition of trace fear conditioning. However, the enhancement in trace conditioning was only observed in female knockout mice. The sex-specificity of this effect may be a result of neuroactive steroids. These compounds vary during the estrus cycle, can increase GABAergic inhibition, and have been shown to impair hippocampus-dependent learning. We propose that activation of GABA(A)Rs by neuroactive steroids inhibits learning processes in the hippocampus. Knockouts are immune to this effect because of the reduced neurosteroid sensitivity that accompanies deletion of the delta subunit. Relationships between neurosteroids, hippocampal excitability, and memory are discussed.     

Activation of nociceptin opioid peptide (NOP) receptor impairs contextual fear learning in mice through glutamatergic mechanisms

The present study investigated whether the selective nociceptin opioid peptide (NOP) receptor agonist, Ro64-6198, impairs acquisition of fear conditioning through glutamatergic mechanisms. Systemic administration of Ro64-6198 (0.3 and 1 mg/kg) or the non-competitive NMDA receptor antagonist, MK-801 (0.03 and 0.1 mg/kg) prior to conditioning severely impaired contextual but not cued fear learning in C57BL/6N mice. When administered together at sub-effective doses, Ro64-6198 (0.5 mg/kg) and MK-801 (0.05 mg/kg), synergistically impaired contextual fear learning, but left cued fear learning intact. We next used the immediate shock deficit paradigm (ISD) to examine the effects of Ro64-6198 and MK-801 on contextual memory formation in the absence of the foot-shock. As expected, naive mice that were shocked briefly after being placed in the training chamber displayed no contextual fear conditioning. This learning deficit was elevated by prior exposure of mice to the training context. Furthermore, administration of Ro64-6198 and MK-801, either separately at amnesic doses (1 mg/kg and 0.1 mg/kg, respectively) or concomitantly at sub-effective doses (0.5 mg/kg and 0.05 mg/kg, respectively) significantly reduced the facilitating effects of context preexposure. These findings demonstrate the existence of functional antagonism between NOP and NMDA receptors that predominantly contributes to modulation of conditioned fear learning which involves spatial-processing demands.     

Opioid receptors mediate direct predictive fear learning: evidence from one-trial blocking

Pavlovian fear learning depends on predictive error, so that fear learning occurs when the actual outcome of a conditioning trial exceeds the expected outcome. Previous research has shown that opioid receptors, including μ-opioid receptors in the ventrolateral quadrant of the midbrain periaqueductal gray (vlPAG), mediate such predictive fear learning. Four experiments reported here used a within-subject one-trial blocking design to study whether opioid receptors mediate a direct or indirect action of predictive error on Pavlovian association formation. In Stage I, rats were trained to fear conditioned stimulus (CS) A by pairing it with shock. In Stage II, CSA and CSB were co-presented once and co-terminated with shock. Two novel stimuli, CSC and CSD, were also co-presented once and co-terminated with shock in Stage II. The results showed one-trial blocking of fear learning (Experiment 1) as well as one-trial unblocking of fear learning when Stage II training employed a higher intensity footshock than was used in Stage I (Experiment 2). Systemic administrations of the opioid receptor antagonist naloxone (Experiment 3) or intra-vlPAG administrations of the selective μ-opioid receptor antagonist CTAP (Experiment 4) prior to Stage II training prevented one-trial blocking. These results show that opioid receptors mediate the direct actions of predictive error on Pavlovian association formation.     

Beta2 subunit containing acetylcholine receptors mediate nicotine withdrawal deficits in the acquisition of contextual fear conditioning

Acute nicotine enhances contextual fear conditioning, whereas withdrawal from chronic nicotine produces impairments. However, the nicotinic acetylcholine receptors (nAChR) that are involved in nicotine withdrawal deficits in contextual fear conditioning are unknown. The present study used genetic and pharmacological techniques to investigate the nAChR subtype(s) involved in the effects of nicotine withdrawal on contextual fear conditioning. beta2 or alpha 7 nAChR subunit knockout (KO) and corresponding wild-type (WT) mice were withdrawn from 12 days of chronic nicotine treatment (6.3mg/kg/day), and trained with 2 conditioned stimulus (CS; 85 dB white noise)--unconditioned stimulus (US; 0.57 mA footshock) pairings on day 13. On day 14, mice were tested for contextual and cued freezing. beta2 KO mice did not show nicotine withdrawal-related deficits in contextual fear conditioning, in contrast to WT mice and alpha 7 KO mice. A follow-up study investigated if nicotine withdrawal disrupts acquisition or recall of contextual fear conditioning. The high affinity nAChR antagonist dihydro-beta-erythroidine (DH beta E; 3mg/kg) was administered prior to training or testing to precipitate withdrawal in chronic nicotine-treated C57BL/6 mice. Deficits in contextual fear conditioning were observed in chronic nicotine-treated mice when DH beta E was administered prior to training, but not when administered at testing. These results indicate that beta2-containing nAChRs, such as the alpha 4 beta 2 receptor, mediate nicotine withdrawal deficits in contextual fear conditioning. In addition, nicotine withdrawal selectively affects acquisition but not recall or expression of the learned response.     

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.     

Contextual-specificity of short-delay extinction in humans: renewal of fear-potentiated startle in a virtual environment

A recent fear-potentiated startle study in rodents suggested that extinction was not context dependent when extinction was conducted after a short delay following acquisition, suggesting that extinction can lead to erasure of fear learning in some circumstances. The main objective of this study was to attempt to replicate these findings in humans by examining the context specificity of short-delay extinction in an ABA renewal procedure using virtual reality environments. A second objective was to examine whether renewal, if any, would be influenced by context conditioning. Subjects underwent differential aversive conditioning in virtual context A, which was immediately followed by extinction in virtual context B. Extinction was followed by tests of renewal in context A and B, with the order counterbalanced across subjects. Results showed that extinction was context dependent. Evidence for renewal was established using fear-potentiated startle as well as skin conductance and fear ratings. In addition, although contextual anxiety was greater in the acquisition context than in the extinction context during renewal, as assessed with startle, context conditioning did not influence the renewal effect. These data do not support the view that extinction conducted shortly after acquisition is context independent. Hence, they do not provide evidence that extinction can lead to erasure of a fear memory established via Pavlovian conditioning.     

Trace and contextual fear conditioning is enhanced in mice lacking the α4 subunit of the GABAA receptor

The GABA_AR α4 subunit is highly expressed in the dentate gyrus region of the hippocampus at predominantly extra synaptic locations where, along with the GABA_AR δ subunit, it forms GABA_A receptors that mediate a tonic inhibitory current. The present study was designed to test hippocampus-dependent and hippocampus-independent learning and memory in GABA_AR α4 subunit-deficient mice using trace and delay fear conditioning, respectively. Mice were of a mixed C57Bl/6J X 129S1/X1 genetic background from α4 heterozygous breeding pairs. The α4-knockout mice showed enhanced trace and contextual fear conditioning consistent with an enhancement of hippocampus-dependent learning and memory. These enhancements were sex-dependent, similar to previous studies in GABA_AR δ knockout mice, but differences were present in both males and females. The convergent findings between α4 and δ knockout mice suggests that tonic inhibition mediated by α4βδ GABA_A receptors negatively modulates learning and memory processes and provides further evidence that tonic inhibition makes important functional contributions to learning and behavior.     

Associative regulation of Pavlovian fear conditioning: unconditional stimulus intensity, incentive shifts, and latent inhibition.

Conditional stimuli (CS) associated with painful unconditional stimuli (US) produce a naloxone-reversible analgesia. The analgesia serves as a negative-feedback regulation of fear conditioning that can account for the impact of US intensity and CS predictiveness on Pavlovian fear conditioning. In Experiment 1 training under naloxone produced learning curves that approached the same high asymptote despite US intensity. Shifting drug treatment during acquisition had effects that paralleled US intensity shifts. In Experiment 3 naloxone reversed Hall-Pearce (1979) negative transfer using a contextual CS, indicating that conditional analgesia acquired during the CS-weak-footshock phase retards acquisition in the CS-strong-footshock phase. Experiment 5 used a tone CS in both a latent-inhibition and a negative-transfer procedure. Only negative transfer was blocked by naloxone. Therefore, negative transfer but not latent inhibition is mediated by a reduction of US processing.     

Aversive conditioning in the rat: effects of a benzodiazepine and of an opioid agonist and antagonist on conditioned hypoalgesia and fear.

Hypoalgesia and fear co-occurred in rats trained on a heated floor and tested for their latencies to paw lick on that floor and to step down onto a nonheated floor. These responses were extinguished, suggesting a mediation by aversive conditioning processes. A benzodiazepine impaired the acquisition of aversive conditioning, but it did not attenuate the expression of conditioned hypoalgesia. The opioid agonist morphine also impaired acquisition across a range of drug doses and variations in hypoalgesic tolerance, whereas the opioid antagonist naloxone facilitated acquisition. The results are discussed in terms of the perceptual-defensive-recuperative (Fanselow, 1986) and working memory (Grau, 1987) models of the mechanisms for the co-occurrence of conditioned hypoalgesia and fear.     

Dissociated roles for the lateral and medial septum in elemental and contextual fear conditioning

Extensive evidence indicates that the septum plays a predominant role in fear learning, yet the direction of this control is still a matter of debate. Increasing data suggest that the medial (MS) and lateral septum (LS) would be differentially required in fear conditioning depending on whether a discrete conditional stimulus (CS) predicts, or not, the occurrence of an aversive unconditional stimulus (US). Here, using a tone CS-US pairing (predictive discrete CS, context in background) or unpairing (context in foreground) conditioning procedure, we show, in mice, that pretraining inactivation of the LS totally disrupted tone fear conditioning, which, otherwise, was spared by inactivation of the MS. Inactivating the LS also reduced foreground contextual fear conditioning, while sparing the higher level of conditioned freezing to the foreground (CS-US unpairing) than to the background context (CS-US pairing). In contrast, inactivation of the MS totally abolished this training-dependent level of contextual freezing. Interestingly, inactivation of the MS enhanced background contextual conditioning under the pairing condition, whereas it reduced foreground contextual conditioning under the unpairing condition. Hence, the present findings reveal a functional dissociation between the LS and the MS in Pavlovian fear conditioning depending on the predictive value of the discrete CS. While the requirement of the LS is crucial for the appropriate processing of the tone CS-US association, the MS is crucial for an appropriate processing of contextual cues as foreground or background information.     

The central nucleus of the amygdala is essential for acquiring and expressing conditional fear after overtraining

The basolateral complex of the amygdala (BLA) is critical for the acquisition and expression of Pavlovian fear conditioning in rats. Nonetheless, rats with neurotoxic BLA lesions can acquire conditional fear after overtraining (75 trials). The capacity of rats with BLA lesions to acquire fear memory may be mediated by the central nucleus of the amygdala (CEA). To examine this issue, we examined the influence of neurotoxic CEA lesions or reversible inactivation of the CEA on the acquisition and expression of conditional freezing after overtraining in rats. Rats with pretraining CEA lesions (whether alone or in combination with BLA lesions) did not acquire conditional freezing to either the conditioning context or an auditory conditional stimulus after extensive overtraining. Similarly, post-training lesions of the CEA or BLA prevented the expression of overtrained fear. Lastly, muscimol infusions into the CEA prevented both the acquisition and the expression of overtrained fear, demonstrating that the effects of CEA lesions are not likely due to the destruction of en passant axons. These results suggest that the CEA is essential for conditional freezing after Pavlovian fear conditioning. Moreover, overtraining may engage a compensatory fear conditioning circuit involving the CEA in animals with damage to the BLA.     

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.     

Cannabinoid CB1 receptor deficiency increases contextual fear memory under highly aversive conditions and long-term potentiation in vivo

The cannabinoid receptor type 1 (CB1) is abundantly expressed in the central nervous system where it negatively controls the release of several neurotransmitters. CB1 activity plays a crucial role in learning and memory and in synaptic plasticity. In the present study, the role of CB1 was investigated in three different hippocampus-dependent memory tasks and in in vivo hippocampal synaptic plasticity in knockout (CB1-ko) and wildtype mice. There was no difference in short-term and long-term social and object recognition memory between CB1-ko and wildtype mice. In contrast, in background contextual fear conditioning CB1-ko mice showed enhanced freezing levels in the conditioning context and increased generalised contextual fear after a high-intensity conditioning foot shock of 1.5 mA, but not after 0.7 mA. In in vivo field potential recordings in the dentate gyrus, CB1-ko mice displayed a decreased paired-pulse facilitation of the populations spikes, suggesting an altered inhibitory synaptic drive onto hippocampal granule cells. Furthermore, CB1-ko mice displayed significantly higher levels of in vivo long-term potentiation (LTP) in the dentate gyrus. In conclusion, CB1 deficiency leads to enhanced contextual fear memory and altered synaptic plasticity in the hippocampus, supporting the key role of endocannabinoid signalling in learning and memory, in particular following highly aversive encounters.     

Electrolytic lesions of the dorsal hippocampus disrupt renewal of conditional fear after extinction

There is a growing body of evidence that the hippocampus is critical for context-dependent memory retrieval. In the present study, we used Pavlovian fear conditioning in rats to examine the role of the dorsal hippocampus (DH) in the context-specific expression of fear memory after extinction (i.e., renewal). Pre-training electrolytic lesions of the DH blunted the expression of conditional freezing to an auditory conditional stimulus (CS), but did not affect the acquisition of extinction to that CS. In contrast, DH lesions impaired the context-specific expression of extinction, eliminating the renewal of fear normally observed to a CS presented outside of the extinction context. Post-extinction DH lesions also eliminated the context dependence of fear extinction. These results are consistent with those using pharmacological inactivation of the DH and suggest that the DH is required for using contextual stimuli to regulate the expression of fear to a Pavlovian CS after extinction.     

An immediate-shock freezing deficit with discrete cues: a possible role for unconditioned stimulus processing mechanisms.

Five experiments with C57BL/6 mice (Mus musculus) investigated whether failures in shock processing might contribute to deficits in freezing that occur after an animal receives a shock immediately on exposure to a conditioning context. Experiment 1 found that more contextual freezing resulted from delayed shocks than from immediate shocks across 4 shock intensities. Experiment 2 extended the immediate-shock freezing deficit to discrete stimuli. Experiment 3 found that preexposure to the to-be-conditioned cue did not facilitate immediate cued conditioning. Experiment 4 found that context preexposure enhanced context-evoked fear after an immediate shock. Experiment 5 found that context preexposure also enhanced immediate cued conditioning. These findings are problematic for current theories of the immediate-shock freezing deficit that focus exclusively on processing of the conditioned stimulus, and they suggest that failures in shock processing may contribute to the deficit.     

Gadd45b knockout mice exhibit selective deficits in hippocampus-dependent long-term memory

Growth arrest and DNA damage-inducible β (Gadd45b) has been shown to be involved in DNA demethylation and may be important for cognitive processes. Gadd45b is abnormally expressed in subjects with autism and psychosis, two disorders associated with cognitive deficits. Furthermore, several high-throughput screens have identified Gadd45b as a candidate plasticity-related gene. However, a direct demonstration of a link between Gadd45b and memory has not been established. The current studies first determined whether expression of the Gadd45 family of genes was affected by contextual fear conditioning. Gadd45b, and to a lesser extent Gadd45g, were up-regulated in the hippocampus following contextual fear conditioning, whereas Gadd45a was not. Next, Gadd45b knockout mice were tested for contextual and cued fear conditioning. Gadd45b knockout mice exhibited a significant deficit in long-term contextual fear conditioning; however, they displayed normal levels of short-term contextual fear conditioning. No differences between Gadd45b knockout and wild-type mice were observed in cued fear conditioning. Because cued fear conditioning is hippocampus independent, while contextual fear conditioning is hippocampus dependent, the current studies suggest that Gadd45b may be important for long-term hippocampus-dependent memory storage. Therefore, Gadd45b may be a novel therapeutic target for the cognitive deficits associated with many neurodevelopmental, neurological, and psychiatric disorders.     

Nicotine Produces a Within-Subject Enhancement of Contextual Fear Conditioning in C57BL/6 Mice Independent of Sex

Nicotine enhances learning including contextual fear conditioning. The present study extends previous work on nicotine and conditioned fear to examine the nature of nicotine's enhancement of contextual fear conditioning and sex differences in contextual fear conditioning in C57BL/6 mice using a within-subjects design. Mice were trained by pairing of an auditory stimulus of 80 dB, 6 cps train of broad-band clicks conditioned stimulus (CS) with a 2 sec., 0.35 mA shock unconditioned stimulus (US). Twenty-four hours later mice were tested for freezing in the original context, and one hour later mice were retested in the same context. A 0.5 mg/kg dose of nicotine was given either for three conditions: (1) before training, testing, and retesting; (2) before training and retesting; and (3) before retesting only. The use of a within-subjects design allowed for testing if nicotine would produce state-dependent deficits in contextual fear conditioning. Nicotine did enhance contextual fear conditioning in the groups that received nicotine for both training and testing. Nicotine, however, did not alter freezing when given on training but not testing or testing but not training. No sex differences, however, existed for conditioning or for nicotine's effects on conditioning. These results suggest that nicotine enhanced acquisition and retrieval processes but did not produce state-dependent deficits when administered just for training or just for testing.