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.     

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.     

Deletion of the mu opioid receptor results in impaired acquisition of Pavlovian context fear

The mu opioid receptor may constitute a critical component of a negative feedback system that regulates Pavlovian fear conditioning. We investigated context fear conditioning acquisition and expression in mu opioid receptor knockout mice (on an inbred, C57 genetic background). We discovered that the mu receptor knockout results in an unexpected and significant deficit in context fear acquisition. Mice lacking the mu receptor showed normal fear acquisition when subjected to a 1-day fear conditioning protocol but evinced deficient fear learning when acquisition was conducted across 5 days. The knockout mice showed normal reactivity to footshock in both fear conditioning protocols. Finally, we confirmed the effectiveness of the receptor deletion in the C57 strain by subjecting the mice to a test of morphine analgesia in the hot-plate assay. As has been seen with mixed genetic background, the receptor deletion resulted in a complete lack of analgesic response to 10 mg/kg morphine. Surprisingly, mice with a single copy of the mu receptor gene (heterozygous knockouts) showed intact sensitivity to morphine but a significant deficit in Pavlovian fear conditioning. The results indicate that deletion of the mu receptor gene impairs fear conditioning and that the conditioning and analgesia effects of heterozygous deletion are dissociable. The conditioning deficit seen in this line of mice may be related to impairment in hippocampus function.     

c-Fos expression reveals aberrant neural network activity during cued fear conditioning in APPswe transgenic mice

The neural circuitry underlying emotional learning and memory is known to involve both the amygdala and hippocampus. Both of these structures undergo anatomical and functional changes during the course of Alzheimer's disease. The present study used expression of the immediate early gene c-Fos to examine the effect of amyloid-induced synaptic pathology on neural activity in the hippocampus and amygdala immediately following Pavlovian fear conditioning. Tg2576 mice underwent cued fear conditioning and the regional interdependencies of c-Fos expression in the hippocampus and the amygdala were assessed using structural equation modelling. Tg2576 mice displayed normal acquisition of conditioned freezing to a punctate auditory cue paired with shock. However, the analysis of c-Fos expression indicated abnormal regional activity in the hippocampal dentate gyrus of Tg2576 mice. Structural equation modelling also supported the view that activity within the amygdala was independent of hippocampal activity in Tg2576 mice (unlike control mice) and regional interaction between the dentate gyrus and CA3 region was disrupted. The results provide novel insight into the effects of excess amyloid production on brain region interdependencies underpinning emotional learning.     

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.     

Changing the rate and hippocampal dependence of trace eyeblink conditioning: slow learning enhances survival of new neurons

Trace eyeblink conditioning in which a conditioned stimulus and unconditioned stimulus are separated by a gap, is hippocampal dependent and can rescue new neurons in the adult dentate gyrus from death (e.g., Beylin et al., 2001; Gould et al., 1999). Tasks requiring more training trials for reliable expression of the conditioned response are most effective in enhancing survival of neurons (Waddell & Shors, 2008). To dissociate hippocampal dependence from acquisition rate, we facilitated hippocampal-dependent trace eyeblink conditioning in two ways: a shorter trace interval and signaling the intertrial interval with a post-US cue. Trace conditioning with a shorter trace interval (250ms) requires an intact hippocampus, and acquisition is faster relative to rats trained with a 500ms trace interval (e.g., Weiss et al., 1999). Using excitotoxic hippocampal lesions, we confirmed that eyeblink conditioning with the 250 or 500ms trace interval is hippocampal dependent. However, training with the post-US cue was not hippocampal dependent. The majority of lesion rats in this condition reached criterion of conditioned responding. To determine whether hippocampal dependence is sufficient to rescue adult-generated neurons in the dentate gyrus, rats were injected with BrdU and trained in one of the three trace eyeblink arrangements one week later. Of these training procedures, only the 500ms trace interval enhanced survival of new cells; acquisition of this task proceeded slowly relative to the 250ms and post-US cue conditions. These data demonstrate that rate of acquisition and not hippocampal dependence determines the impact of learning on adult neurogenesis.     

Trace fear conditioning is reduced in the aging rat

Auditory trace fear conditioning is a hippocampus-dependent learning task that requires animals to associate an auditory conditioned stimulus (CS) and a fear-producing shock-unconditioned stimulus (US) that are separated by an empty 20-s trace interval. Previous studies have shown that aging impairs learning performance on hippocampus-dependent tasks. This study measured heart rate (HR) and freezing fear responses to determine if aging impairs hippocampus-dependent auditory trace fear conditioning in freely moving rats. Aging and Young rats received one long-trace fear conditioning session (10 trials). Each trial consisted of a tone-CS (5 s) and a shock-US separated by an empty 20-s trace interval. The next day rats received CS-alone retention trials. Young rats showed significantly larger HR and freezing responses on the initial CS-alone retention trials compared to the Aging rats. A control group of aging rats received fear conditioning trials with a short 1-s trace interval separating the CS and US. The Aging Short-Trace Group showed HR and freezing responses on the initial CS alone retention trials that were similar to the Young Long-Trace Group, but greater than the Aging Long-Trace Group. A second aging control group received unpaired CSs and USs, and showed no HR or freezing responses on CS-alone retention trials. These data show that HR and freezing are effective measures for detecting aging-related deficits in trace fear conditioning.     

Differential contributions of dorsal vs. ventral hippocampus to auditory trace fear conditioning

The effect of excitotoxic lesions of dorsal vs. ventral hippocampus on the acquisition and expression of auditory trace fear conditioning was examined in two studies. In Experiment 1, animals received excitotoxic lesions of either the dorsal or ventral hippocampus or sham surgeries one week prior to conditioning, and were tested 24 h later. In Experiment 2, animals received excitotoxic lesions of either the dorsal or ventral hippocampus or sham surgeries 24 h after training, and were tested one week after surgery. Both pre- and post-training lesions of ventral hippocampus impaired the acquisition and expression, respectively, of auditory trace fear conditioning. Pre-training lesions of dorsal hippocampus had no effect on the acquisition of trace fear conditioning, while post-training lesions of dorsal hippocampus dramatically impaired expression during subsequent testing. Although in some cases animals with lesions of ventral hippocampus exhibited locomotor hyperactivity, it is unlikely that the pattern of observed deficits can be attributed to this effect. Collectively these data suggest that the dorsal and ventral hippocampus may contribute differentially to the mnemonic processes underlying fear trace conditioning.     

Simultaneous training on two hippocampus-dependent tasks facilitates acquisition of trace eyeblink conditioning

A common cellular alteration, reduced post-burst afterhyperpolarization (AHP) in CA1 neurons, is associated with acquisition of the hippocampus-dependent tasks trace eyeblink conditioning and the Morris water maze. As a similar increase in excitability is correlated with these two learning paradigms, we sought to determine the interactive behavioral effects of training animals on both tasks by using either a consecutive or simultaneous training design. In the consecutive design, animals were trained first on either the trace eyeblink conditioning task for six sessions, followed by training on the water maze task for six sessions, or vice versa. The simultaneous design consisted of six or 11 training days; animals received one session/day of both trace eyeblink conditioning and water maze training. Separate groups were used for consecutive and simultaneous training. Animals trained on both tasks simultaneously were significantly facilitated in their ability to acquire the trace eyeblink conditioning task; no effect of simultaneous training was seen on the water maze task. No effect was seen on acquisition for either task when using the consecutive training design. Taken together, these findings provide insight into how the hippocampus processes information when animals learn multiple hippocampus-dependent tasks.     

Overexpression of hAPPswe impairs rewarded alternation and contextual fear conditioning in a transgenic mouse model of Alzheimer's disease

One of the hallmarks of the pathology in Alzheimer's disease is the deposition of amyloid plaques throughout the brain, especially within the hippocampus and amygdala. Transgenic mice that overexpress the Swedish mutation of human amyloid precursor protein (hAPPswe; Tg2576) show age-dependent memory deficits in hippocampus-dependent learning tasks. However, the performance of aged Tg2576 mice in amygdala-dependent learning tasks has not been thoroughly assessed. We trained young (2–4 mo) and old (16–18 mo) Tg2576 and wild-type mice in a T-maze alternation task (hippocampus-dependent) and a Pavlovian fear-conditioning task (amygdala- and hippocampus-dependent). As previously reported, old Tg2576 mice showed impaired acquisition of rewarded alternation; none of these mice reached the criterion of at least five out of six correct responses over three consecutive days. In contrast, old Tg2576 mice showed normal levels of conditional freezing to an auditory conditional stimulus (CS) and acquired a contextual discrimination normally. However, when the salience of the fear-conditioning context was decreased, old (12–14 mo) Tg2576 mice were impaired at acquiring fear to the conditioning context, but not to the tone CS. Histological examination of a subset of the mice verified the existence of amyloid plaques in the cortex, hippocampus, and amygdala of old, but not young, Tg2576 mice. Hence, learning and memory deficits in old Tg2576 mice are limited to hippocampus-dependent tasks, despite widespread amyloid deposition in cortex, hippocampus, and amygdala.     

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.     

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.     

Translocation and activation of Rac in the hippocampus during associative contextual fear learning

Small G proteins including Rac are mediators of changes in neuronal morphology associated with synaptic plasticity. Previous studies in our laboratory showed that Rac is highly expressed in the adult mouse hippocampus, a brain area that exhibits robust synaptic plasticity and is crucial for the acquisition of memories. In this study, we investigated whether Rac was involved in NMDA receptor-dependent associative fear learning in the area CA1 of adult mouse hippocampus. We found that Rac translocation and activation was increased in the hippocampus following associative fear conditioning in mice, and that these increases are blocked by intraperitoneal injection of the NMDA receptor channel blocker MK801 at the acquisition stage. Our data indicate that NMDA receptor-dependent associative fear learning alters Rac localization and function in the mouse hippocampus.     

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.     

Histone acetylation rescues contextual fear conditioning in nNOS KO mice and accelerates extinction of cued fear conditioning in wild type mice

Epigenetic regulation of chromatin structure is an essential molecular mechanism that contributes to the formation of synaptic plasticity and long-term memory (LTM). An important regulatory process of chromatin structure is acetylation and deacetylation of histone proteins. Inhibition of histone deacetylase (HDAC) increases acetylation of histone proteins and facilitate learning and memory. Nitric oxide (NO) signaling pathway has a role in synaptic plasticity, LTM and regulation of histone acetylation. We have previously shown that NO signaling pathway is required for contextual fear conditioning. The present study investigated the effects of systemic administration of the HDAC inhibitor sodium butyrate (NaB) on fear conditioning in neuronal nitric oxide synthase (nNOS) knockout (KO) and wild type (WT) mice. The effect of single administration of NaB on total H3 and H4 histone acetylation in hippocampus and amygdala was also investigated. A single administration of NaB prior to fear conditioning (a) rescued contextual fear conditioning of nNOS KO mice and (b) had long-term (weeks) facilitatory effect on the extinction of cued fear memory of WT mice. The facilitatory effect of NaB on extinction of cued fear memory of WT mice was confirmed in a study whereupon NaB was administered during extinction. Results suggest that (a) the rescue of contextual fear conditioning in nNOS KO mice is associated with NaB-induced increase in H3 histone acetylation and (b) the accelerated extinction of cued fear memory in WT mice is associated with NaB-induced increase in H4 histone acetylation. Hence, a single administration of HDAC inhibitor may rescue NO-dependent cognitive deficits and afford a long-term accelerating effect on extinction of fear memory of WT mice.     

Effects of ventral and dorsal CA1 subregional lesions on trace fear conditioning

Recent lines of research have focused on dissociating function between the dorsal and ventral hippocampus along space and anxiety dimensions. In the dorsal hippocampus, the CA1 subregion has been implicated in the acquisition of contextual fear as well as in the trace interval in trace fear conditioning. The present study was designed to test the relative contributions of dorsal (dCA1) and ventral CA1 (vCA1) in trace fear conditioning. Long-Evans rats received ibotenate lesions of the ventral CA1 (n=7), dorsal CA1 (n=9), or vehicle control lesions (n=8) prior to trace fear conditioning acquisition. Results suggest dCA1 and vCA1 groups show no significant deficits during acquisition when compared to control groups. dCA1 and vCA1 both show deficits in the retention of contextual fear when tested 24 h post-acquisition (P<.05 and P<.01, respectively), and vCA1 was impaired relative to dCA1 (P<.05). This is suggestive of a graded involvement in contextual retention between the dorsal and ventral aspects of CA1. dCA1 showed no deficit for retention of conditioned fear to the tone or the trace when tested 48 h post-acquisition, whereas vCA1 did show a significant deficit for the trace interval and a slight, non-significant reduction in freezing to the tone, when compared to the control group (p<.05). Overall the data are suggestive of a graded involvement in retention of fear conditioning between the dorsal and ventral aspects of CA1, but it is likely that vCA1 may be critically involved in retention of trace fear conditioning.     

Differential regulation of glutamic acid decarboxylase gene expression after extinction of a recent memory vs. intermediate memory

Extinction reduces fear to stimuli that were once associated with an aversive event by no longer coupling the stimulus with the aversive event. Extinction learning is supported by a network comprising the amygdala, hippocampus, and prefrontal cortex. Previous studies implicate a critical role of GABA in extinction learning, specifically the GAD65 isoform of the GABA synthesizing enzyme glutamic acid decarboxylase (GAD). However, a detailed analysis of changes in gene expression of GAD in the subregions comprising the extinction network has not been undertaken. Here, we report changes in gene expression of the GAD65 and GAD67 isoforms of GAD, as measured by relative quantitative real-time RT-PCR, in subregions of the amygdala, hippocampus, and prefrontal cortex 24-26 h after extinction of a recent (1-d) or intermediate (14-d) fear memory. Our results show that extinction of a recent memory induces a down-regulation of Gad65 gene expression in the hippocampus (CA1, dentate gyrus) and an up-regulation of Gad67 gene expression in the infralimbic cortex. Extinguishing an intermediate memory increased Gad65 gene expression in the central amygdala. These results indicate a differential regulation of Gad gene expression after extinction of a recent memory vs. intermediate memory.     

长期双酚A暴露对成年小鼠恐惧记忆的影响

双酚A (bisphenol, BPA)是一种广泛应用于塑料制品的环境内分泌干扰物, 具有弱雌激素和抗雄激素活性, 与雌激素受体有一定的亲和力。本实验探讨长期BPA暴露对成年小鼠恐惧记忆的影响及其神经机制。将9周龄雄性小鼠暴露于BPA (0.4、4、40 mg/kg/d) 90 d, 建立小鼠亚慢性BPA暴露模型后, 进行场景性条件恐惧训练, 然后分别在电击后1 hr及24 hr检测小鼠的僵立时间, 同时在电击前、电击后1 hr及24 hr检测海马相关蛋白表达变化。结果表明, BPA (4、40 mg/kg/d)暴露延长小鼠场景性条件恐惧训练后1 hr及24 hr的僵立时间。Western blot蛋白检测结果显示, 行为训练前, BPA降低了小鼠NMDA受体NR1亚基表达水平, 上调组蛋白去乙酰化酶2表达。行为训练后1 hr及24 hr, BPA促进海马NMDA受体亚基NR1和组蛋白H3乙酰化表达, 抑制组蛋白去乙酰化酶2表达, 同时促进ERK1/2磷酸化水平。以上结果表明, 长期BPA暴露提高成年小鼠恐惧记忆获得的同时延长恐惧记忆的保持; 该作用可能通过激活海马的ERK1/2通路而改变核内组蛋白乙酰化和NMDA受体水平进行。     

Trace and contextual fear conditioning are impaired following unilateral microinjection of muscimol in the ventral hippocampus or amygdala, but not the medial prefrontal cortex

Trace fear conditioning, in which a brief empty "trace interval" occurs between presentation of the CS and UCS, differs from standard delay conditioning in that contributions from both the hippocampus and prelimbic medial prefrontal cortex (PL mPFC) are required to form a normal long term memory. Little is currently known about how the PL interacts with various temporal lobe structures to support learning across this temporal gap between stimuli. We temporarily inactivated PL along with either ventral hippocampus or amygdala in a disconnection design to determine if these structures functionally interact to acquire trace fear conditioning. Disconnection (contralateral injections) of the PL with either the ventral hippocampus or amygdala impaired trace fear conditioning; however, ipsilateral control rats were also impaired. Follow-up experiments examined the effects of unilateral inactivation of the PL, ventral hippocampus, or amygdala during conditioning. The results of this study demonstrate that unilateral inactivation of the ventral hippocampus or amygdala impairs memory, while bilateral inactivation of the PL is required to produce a deficit. Memory deficits after unilateral inactivation of the ventral hippocampus or amygdala prevent us from determining whether the mPFC functionally interacts with the medial temporal lobe using a disconnection approach. Nonetheless, our findings suggest that the trace fear network is more integrated than previously thought.     

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.