A temporally distinct role for group I and group II metabotropic glutamate receptors in object recognition memory

Recognition memory, involving the ability to discriminate between a novel and familiar object, depends on the integrity of the perirhinal cortex (PRH). Glutamate, the main excitatory neurotransmitter in the cortex, is essential for many types of memory processes. Of the subtypes of glutamate receptor, metabotropic receptors (mGluRs) have received less study than NMDA receptors; thus, the reported experiments examined the role of mGluRs in familiarity discrimination in the rat PRH. Experiments 1 and 2 assessed the effects of systemic administration of MPEP, a group I mGluR (specifically mGluR5) antagonist, and/or LY341495, a group II mGluR antagonist, on a spontaneous object novelty preference task. Simultaneous antagonism of both group I and II mGluRs impaired familiarity discrimination following a 24-h but not a 15-min delay, while antagonism of either mGluR subtype alone had no effect at either delay. The impairment was in acquisition, as in Experiment 3 coadministration of MPEP and LY341495 did not affect recognition memory performance when administered either after the sample phase or prior to test. The impairment in long-term recognition memory was mediated by mGluRs in the PRH, as localized intracortical antagonism of group I and II mGluRs also produced a deficit (Experiment 4). No evidence was found for an involvement of group III mGluRs in the acquisition of long-term familiarity discrimination (Experiment 5). These findings establish that glutamatergic neurotransmission in the PRH via group I and II mGluRs is crucial for the acquisition, but not for the consolidation or retrieval of long-term object recognition memory.     

Timing of fear expression in trace and delay conditioning measured by fear-potentiated startle in rats

In two experiments, the time course of the expression of fear in trace (hippocampus-dependent) versus delay (hippocampus-independent) conditioning was characterized with a high degree of temporal specificity using fear-potentiated startle. In experiment 1, groups of rats were given delay fear conditioning or trace fear conditioning with a 3- or 12-sec trace interval between conditioned stimulus (CS) offset and unconditioned stimulus (US) onset. During test, the delay group showed fear-potentiated startle in the presence of the CS but not after its offset, whereas the trace groups showed fear-potentiated startle both during the CS and after its offset. Experiment 2 compared the time course of fear expression after trace conditioning with the time course in two delay conditioning groups: one matched to the trace conditioning group with respect to CS duration, and the other with respect to ISI. In all groups, fear was expressed until the scheduled occurrence of the US and returned to baseline rapidly thereafter. Thus, in both trace and delay fear conditioning, ISI is a critical determinant of the time course of fear expression. These results are informative as to the possible role of neural structures, such as the hippocampus, in memory processes related to temporal information.     

The role of metabotropic glutamate receptors and cortical adaptation in habituation of odor-guided behavior

Decreases in behavioral investigation of novel stimuli over time may be mediated by a variety of factors including changes in attention, internal state, and motivation. Sensory cortical adaptation, a decrease in sensory cortical responsiveness over prolonged stimulation, may also play a role. In olfaction, metabotropic glutamate receptors on cortical afferent pre-synaptic terminals have been shown to underlie both cortical sensory adaptation and habituation of odor-evoked reflexes. The present experiment examined whether blockade of sensory cortical adaptation through bilateral infusion of the group III metabotropic glutamate receptor antagonist cyclopropyl-4-phosphonophenylglycine (CPPG) into the anterior piriform cortex could reduce habituation of a more complex odor-driven behavior such as investigation of a scented object or a conspecific. The results demonstrate that time spent investigating a scented jar, or a conspecific, decreases over the course of a continuous 10 minute trial. Acute infusion of CPPG bilaterally into the anterior piriform cortex significantly enhanced the time spent investigating the scented jar compared to investigation time in control rats, without affecting overall behavioral activity levels. Infusions into the brain outside of the piriform cortex were without effect. CPPG infusion into the piriform cortex also produced an enhancement of time spent investigating a conspecific, although this effect was not significant.     

Differential roles of hippocampal metabotropic glutamate receptors 1 and 5 in inhibitory avoidance learning

Group I metabotropic glutamate receptors (mGlu1 and 5) have been implicated in synaptic plasticity and learning and memory. However, much of our understanding of how these receptors in different brain regions contribute to distinct memory stages in different learning tasks remains incomplete. The present study investigated the effects of the mGlu5 receptor antagonist, 2-methyl-6-(phenylethynyl)-pyridine (MPEP), and mGlu1 receptor antagonist, (S)-(+)-alpha-amino-4-carboxy-2-methylbenzene-acetic acid (LY 367385) in the dorsal hippocampus on the consolidation and extinction of memory for inhibitory avoidance learning. Male, Sprague-Dawley rats were trained in a single-trial step-down inhibitory avoidance task. MPEP, LY 367385 or saline were infused bilaterally into the CA1 region immediately after training or immediately after the first retention test which was given 24h after training. Rats receiving MPEP (1.5 or 5.0 microg/side) or LY 367385 (0.7 or 2.0 microg/side) infusion exhibited a dose-dependent decrease in retention when tested 24h later. MPEP was ineffective while LY 367385 significantly attenuated extinction when injected after the first retention test using an extinction procedure. These findings indicate a selective participation of hippocampal group I mGlu receptors in memory processing in this task.     

Generalization of conditioned fear-potentiated startle in humans: experimental validation and clinical relevance

Though generalization of conditioned fear has been implicated as a central feature of pathological anxiety, surprisingly little is known about the psychobiology of this learning phenomenon in humans. Whereas animal work has frequently applied methods to examine generalization gradients to study the gradual weakening of the conditioned-fear response as the test stimulus increasingly differs from the conditioned stimulus (CS), to our knowledge no psychobiological studies of such gradients have been conducted in humans over the last 40 years. The current effort validates an updated generalization paradigm incorporating more recent methods for the objective measurement of anxiety (fear-potentiated startle). The paradigm employs 10, quasi-randomly presented, rings of gradually increasing size with extremes serving as CS+ and CS−. The eight rings of intermediary size serve as generalization stimuli (GSs) and create a continuum-of-similarity from CS+ to CS−. Both startle data and online self-report ratings demonstrate continuous decreases in generalization as the presented stimulus becomes less similar to the CS+. The current paradigm represents an updated and efficacious tool with which to study fear generalization—a central, yet understudied conditioning-correlate of pathologic anxiety.     

Activation of cannabinoid CB1 receptors in the central amygdala impairs inhibitory avoidance memory consolidation via NMDA receptors

In the present study, we investigated the influence of bilateral intra-central amygdala (intra-CeA) microinjections of N-methyl-d-aspartate (NMDA) receptor agents on amnesia induced by a cannabinoid CB1 receptor agonist, arachydonilcyclopropylamide (ACPA). This study used a step-through inhibitory (passive) avoidance task to assess memory in adult male Wistar rats. The results showed that intra-CeA administration of ACPA (2 ng/rat) immediately after training decreased inhibitory avoidance (IA) memory consolidation as evidenced by a decrease in step-through latency on the test day, which was suggestive of drug-induced amnesia. Post-training intra-CeA microinjections of NMDA (0.0001, 0.001 and 0.01 μg/rat) did not affect IA memory consolidation. However co-administration of NMDA with ACPA (2 ng/rat) prevented the impairment of IA memory consolidation that was induced by ACPA. Although post-training intra-CeA administration of the NMDA receptor antagonist, d-(−)-2-amino-5-phosphonopentanoic acid (d-AP5; 0.01, 0.05 and 0.1 μg/rat) alone had no effect, its co-administration with an ineffective dose of ACPA (1 ng/rat) impaired IA memory consolidation. Post-training intra-CeA microinjection of an ineffective dose of d-AP5 (0.01 μg/rat) prevented an NMDA response to the impaired effect of ACPA. These results suggest that amnesia induced by intra-CeA administration of ACPA is at least partly mediated through an NMDA receptor mechanism in the Ce-A.     

Modulation of working, short- and long-term memory by nicotinic receptors in the basolateral amygdala in rats

Male Wistar rats were exposed to one-trial step-down inhibitory avoidance training using a 0.5 mA footshock. Through bilaterally implanted indwelling cannulae, they received bilateral 0.5 microL infusions of saline, mecamylamine (1.0 or 10.0 microg/side), or nicotine (0.6 or 3.0 microg/side) into the basolateral complex of the amygdaloid nucleus (BLA). Infusions were either 10 min before training (Experiment 1) or 4 min after training (Experiment 2). In Experiment 1, the animals were tested three times: first for working memory (WM) 5 s after training, then for short-term memory (STM) 90 min later, and finally for long-term memory (LTM) 24 h later. Mecamylamine depressed and nicotine enhanced WM, STM, and LTM. In Experiment 2, the treatments were given after WM was presumably over. Again, mecamylamine inhibited and nicotine enhanced STM and LTM. The results indicate that nAChRs in BLA participate in the regulation of WM formation and STM and LTM acquisition and consolidation.     

Neural substrates of individual differences in human fear learning: Evidence from concurrent fMRI, fear-potentiated startle, and US-expectancy data

To provide insight into individual differences in fear learning, we examined the emotional and cognitive expressions of discriminative fear conditioning in direct relation to its neural substrates. Contrary to previous behavioral-neural (fMRI) research on fear learning--in which the emotional expression of fear was generally indexed by skin conductance--we used fear-potentiated startle, a more reliable and specific index of fear. While we obtained concurrent fear-potentiated startle, neuroimaging (fMRI), and US-expectancy data, healthy participants underwent a fear-conditioning paradigm in which one of two conditioned stimuli (CS(+) but not CS(-)) was paired with a shock (unconditioned stimulus [US]). Fear learning was evident from the differential expressions of fear (CS(+) > CS(-)) at both the behavioral level (startle potentiation and US expectancy) and the neural level (in amygdala, anterior cingulate cortex, hippocampus, and insula). We examined individual differences in discriminative fear conditioning by classifying participants (as conditionable vs. unconditionable) according to whether they showed successful differential startle potentiation. This revealed that the individual differences in the emotional expression of discriminative fear learning (startle potentiation) were reflected in differential amygdala activation, regardless of the cognitive expression of fear learning (CS-US contingency or hippocampal activation). Our study provides the first evidence for the potential of examining startle potentiation in concurrent fMRI research on fear learning.     

Post-acquisition release of glutamate and norepinephrine in the amygdala is involved in taste-aversion memory consolidation

Amygdala activity mediates the acquisition and consolidation of emotional experiences; we have recently shown that post-acquisition reactivation of this structure is necessary for the long-term storage of conditioned taste aversion (CTA). However, the specific neurotransmitters involved in such reactivation are not known. The aim of the present study was to investigate extracellular changes of glutamate, norepinephrine, and dopamine within the rat amygdala using in vivo microdialysis during the acquisition and 1-h post-acquisition of CTA paradigm. Microdialysis monitoring showed a significant norepinephrine increase related to novel taste exposure and a glutamate increase after gastric malaise induction by i.p. LiCl administration. Interestingly, we found a spontaneous concomitant increase of glutamate and norepinephrine, but not dopamine, 45 min after conditioning, suggesting the presence of aversive learning-dependent post-acquisition signals in the amygdala. These signals seem to be involved in CTA consolidation process, since post-trial blockade of N-methyl-D-aspartate or β-adrenergic receptors impaired long- but not short-term memory. These data suggest that CTA long-term storage involves post-acquisition release of glutamate and norepinephrine in the amygdala.     

Exposure to a retrieval cue in rats induces changes in regional brain glucose metabolism in the amygdala and other related brain structures

Pre-test exposure to training-related cues is known to improve subsequent retention performance. To identify brain regions engaged in processes promoted by retrieval cues, a brain imaging approach using the [6-14C]glucose autoradiographic technique was used. Sprague-Dawley rats trained in a brightness discrimination avoidance task were submitted to different cueing conditions after a 1- or a 21-day training-to-test interval (TTI). Animals were either non-cued, cued with a box, or cued with a box and the light that served as a discriminative stimulus. Effects of the different cueing conditions on retention performance or on metabolic activity in 58 different brain regions were investigated. Rats cued with the light exhibited a subsequent improvement of their retention performance relative to controls, when tested at the 1- but not 21-days TTI, confirming our previous results. At the 1-day retention interval, a comparison between rats cued with the box and rats cued with the box and the light showed that the light cue significantly increased glucose uptake in a neuronal network composed of the lateral, basal, and central nuclei of the amygdala, the anterior and suprachiasmatic hypothalamic nuclei, the nucleus accumbens, the medial septum, and the insular cortex. In contrast, at the 21-day retention interval, both groups demonstrated similar cerebral metabolic activity. The present results indicate that exposure to a light cue increased metabolic activity in the previously mentioned brain structures only when the light acted as an effective retrieval cue, suggesting an involvement of this network in the processes triggered by a retrieval cue. Arguments are provided supporting the notion that the amygdala may play a key role in these processes. Whether the amygdala is a part of a neural network involved in retrieval processes or in neuromodulating systems that favour the efficacy of retrieval processes is also discussed.     

Nitric oxide synthesis in the basolateral complex of the amygdala is required for the consolidation and expression of fear potentiated startle but not shock sensitization of the acoustic startle

Nitric oxide (NO) is synthesized as a result of N-methyl-d-asparate (NMDA) receptor activation, it acts as an retrograde neurotransmitter freely diffusing across cell membranes interacting with its targets in a non-synaptic manner. Consequently, NO has been described as an extension of NMDA receptor activation. The targets of NO include cellular components within the basolateral complex of the amygdala (BLA) that are necessary for the consolidation of conditioned fear as well as targets that can significantly modulate neurotransmission associated with its expression. Given that both are NMDA receptor associated processes, this implies that NO may be an important intermediary of NMDA receptor activation and both fear memory consolidation and expression. The current study sought to examine this using visual fear conditioning and fear potentiated startle. Three experiments were conducted, rats received intra-BLA microinfusions of the global nitric oxide synthase inhibitor l-NAME either prior to fear conditioning, or expression of learned fear. Furthermore, NO's ability to modulate a NMDA receptor independent fear process was assessed by microinfusing l-NAME into the BLA prior to examination of the shock sensitization of the acoustic startle affect. The results indicated that NO was, indeed, required for both the consolidation and expression of learned fear, whereas it was not required for NMDA independent shock enhanced startle responding. This study illustrates that NO plays a pivotal role in the examined NMDA associated fear processes.     

Chronic nicotine exposure induces a long-lasting and pathway-specific facilitation of LTP in the amygdala

Nicotine, in the form of tobacco, is the most commonly used drug of abuse. In addition to its rewarding properties, nicotine also affects many cognitive and emotional processes that involve several brain regions, including hippocampus and amygdala. Long-term changes in synaptic strength in these brain regions after drug exposure may be importantly correlated with behavioral changes induced by nicotine. Here, we study the effect of chronic oral administration of nicotine on the long-term synaptic potentiation in the amygdala, a key structure for emotional memory. We find that oral administration of nicotine for 7 d produces a significant enhancement of LTP in the amygdala. This facilitation is pathway specific: Nicotine selectively facilitates LTP in the cortical-lateral amygdala pathway, but not the thalamic-lateral and the lateral-basolateral synaptic pathway. The synaptic facilitation induced by a 7-d exposure to nicotine is long-lasting, it persists for 72 h after cessation of nicotine but decays 8 d after its cessation. In contrast, a shorter exposure of nicotine (24 h) induces only a short-lasting facilitation of synaptic plasticity that dissipates 24 and 72 h after cessation of nicotine. The facilitation of LTP in the amygdala after exposure to nicotine is mediated by removal of GABAergic inhibition, is dependent on the activation NMDA receptors, and can be prevented by blocking either α7 or β2 nACh receptors. Our results indicate that chronic exposure to nicotine can promote the induction of long-lasting modifications of synapses in a specific pathway in the amygdala.These changes in synaptic plasticity may contribute to the complex neural adaptations and behaviors caused by nicotine.     

Amygdala infusions of an NR2B-selective or an NR2A-preferring NMDA receptor antagonist differentially influence fear conditioning and expression in the fear-potentiated startle test

Within the amygdala, most N-methyl-D-aspartic acid (NMDA) receptors consist of NR1 subunits in combination with either NR2A or NR2B subunits. Because the particular subunit composition greatly influences the receptors' properties, we investigated the contribution of both subtypes to fear conditioning and expression. To do so, we infused the NR1/NR2B receptor antagonist CP101,606 (0.5, 1.5, or 4.5 microg/amygdala) or the NR1/NR2A-preferring antagonist NVP-AAM077 (0.075, 0.25, 0.75, or 2.5 microg/amygdala) into the amygdala prior to either fear conditioning (i.e., light-shock pairings) or fear-potentiated startle testing. CP101,606 nonmonotonically disrupted fear conditioning but did not disrupt fear expression. NVP-AAM077 dose-dependently disrupted fear conditioning as well as fear expression. The results suggest that amygdala NR1/NR2B receptors play a special role in fear memory formation, whereas NR1/NR2A receptors participate more generally in synaptic transmission.     

Cannabinoid receptors activation and glucocorticoid receptors deactivation in the amygdala prevent the stress-induced enhancement of a negative learning experience

The enhancement of emotional memory is clearly important as emotional stimuli are generally more significant than neutral stimuli for surviving and reproduction purposes. Yet, the enhancement of a negative emotional memory following exposure to stress may result in dysfunctional or intrusive memory that underlies several psychiatric disorders. Here we examined the effects of stress exposure on a negative emotional learning experience as measured by a decrease in the magnitude of the expected quantity of reinforcements in an alley maze. In contrast to other fear-related negative experiences, reward reduction is more associated with frustration and is assessed by measuring the latency to run the length of the alley to consume the reduced quantity of reward. We also examined whether the cannabinoid receptors agonist WIN55,212-2 (5 μg/side) and the glucocorticoid receptors (GRs) antagonist RU-486 (10 ng/side) administered into the rat basolateral amygdala (BLA) could prevent the stress-induced enhancement. We found that intra-BLA RU-486 or WIN55,212 before stress exposure prevented the stress-induced enhancement of memory consolidation for reduction in reward magnitude. These findings suggest that cannabinoid receptors and GRs in the BLA are important modulators of stress-induced enhancement of emotional memory.     

The effect of amygdala lesions on conditional and unconditional vocalizations in rats

Electrolytic lesions centered on the amygdaloid central nucleus (ACe) resulted in the inability of rats to acquire a Pavlovian conditional vocalization response. Conditioning consisted of pairing a light conditional stimulus with a tailshock unconditional stimulus (US). The thresholds of three unconditional responses (URs) to tailshock were assessed prior to conditioning. These URs are organized at spinal (spinal motor reflexes), medullary (vocalizations during shock), and forebrain (vocalization afterdischarges, VADs) levels of the neuraxis. Compared to sham-lesioned controls, rats with amygdala lesions exhibited a selective elevation in the threshold of VADs. During conditioning the amplitude and duration of VADs were selectively reduced in amygdala-lesioned rats. These findings support earlier observations of that elicitation of VADs by tailshock correlates with the capacity of this US to support fear conditioning. The ACe may be involved in both associative and non-associative aspects of fear conditioning, but for progress in our understanding it is essential to evaluate its role in the generation of conditioning relevant URs.     

The medial amygdala: Serotonergic inhibition of shock-lnduced aggression and pain sensitivity in rats

Two aspects of the amygdaloid complex (corticomedial and basolateral) were examined with reference to serotonergic inhibition of shock-induced aggression. Fighting was significantly depressed by serotonergic stimulation (5-HT, 10 μg bilateral) in the corticomedial amygdala while serotonergic blockade (methysergide, 5 μg bilateral) in this region increased levels of fighting. No consistent effects were obtained with serotonergic manipulation of the basolateral amygdala. Further investigation revealed that the state of serotonergic activity in medial amygdaloid sites was associated with concomitant alterations in the animals' sensitivity to footshock. Results are discussed in relation to a) a general inhibitory role of serotonin in behavioural mechanisms and b) a dopaminergic-serotonergic balance for behavioural arousal involving medial amygdaloid nuclei.     

Activation of the cannabinoid receptor type 1 decreases glutamatergic and GABAergic synaptic transmission in the lateral amygdala of the mouse

The endogenous cannabinoid system has been shown recently to play a crucial role in the extinction of aversive memories. As the amygdala is presumably involved in this process, we investigated the effects of the cannabinoid receptor agonist WIN 55,212-2 (WIN-2) on synaptic transmission in the lateral amygdala (LA) of wild-type and cannabinoid receptor type 1 (CB1)-deficient mice. Extracellular field potential recordings and patch-clamp experiments were performed in an in vitro slice preparation. We found that WIN-2 reduces basal synaptic transmission and pharmacologically isolated AMPA receptor- and GABA_A receptor-mediated postsynaptic currents in wild-type, but not in CB1-deficient mice. These results indicate that, in the LA, cannabinoids modulate both excitatory and inhibitory synaptic transmission via CB1. WIN-2-induced changes of paired-pulse ratio and of spontaneous and miniature postsynaptic currents suggest a presynaptic site of action. Inhibition of G_i/o proteins and blockade of voltage-dependent and G protein-gated inwardly rectifying K⁺ channels inhibited WIN-2 action on basal synaptic transmission. In contrast, modulation of the adenylyl cyclase-protein kinase A pathway, and blockade of presynaptic N- and P/Q- or of postsynaptic L- and R/T-type voltage-gated Ca²⁺ channels did not affect WIN-2 effects. Our results indicate that the mechanisms underlying cannabinoid action in the LA partly resemble those observed in the nucleus accumbens and differ from those described for the hippocampus.     

Effect of prior exposure to a lithium- and an amphetamine-paired flavor on the acoustic startle response in rats

The experiments reported here evaluated the hypothesis that an amphetamine-paired flavor elicits conditioned fear-arousal, whereas a lithium-paired flavor elicits conditioned nausea-disgust by examining the effect of prior flavor exposure on an acoustic startle reaction (ASR). Exposure to a lithium-paired flavor by intraoral infusion, either immediately prior to a startle session (Experiment 1) or during a startle session (Experiments 2 and 3), resulted in a blunted ASR. In contrast, intraoral infusion of an amphetamine-paired flavor resulted in a potentiated ASR. The blunted ASR produced by exposure to a lithium-paired flavor dramatically reversed to a potentiated ASR when rats were pretreated with the antiemetic drug ondansetron prior to the saccharin-lithium pairing (Experiment 3). The findings shed light on a mechanism by which rewarding drugs produce conditioned taste avoidance in rats.