Temporary inactivation of dorsal hippocampus attenuates explicitly nonspatial, unimodal, contextual fear conditioning

The current study examined the effects of temporary inactivation of the DH on freezing, rearing, ambulating, grooming, and whisking behavior in an explicitly nonspatial contextual fear conditioning paradigm in which olfactory stimuli served as temporally and spatially diffuse contexts. Prior either to training, testing, or both, male Sprague–Dawley rats received bilateral microinfusions of saline or the GABA_A agonist muscimol into the DH. Results indicate that temporary inactivation of DH produced both anterograde and retrograde deficits in contextually conditioned freezing, while sparing the acquisition and expression of freezing to a discrete auditory or olfactory CS. These data suggest that there is a decidedly nonspatial component to the role of DH in contextual conditioning, and that olfactory contextual conditioning is a fruitful means of further exploring this function.     

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.     

Sleep deprivation selectively impairs memory consolidation for contextual fear conditioning

Many behavioral and electrophysiological studies in animals and humans have suggested that sleep and circadian rhythms influence memory consolidation. In rodents, hippocampus-dependent memory may be particularly sensitive to sleep deprivation after training, as spatial memory in the Morris water maze is impaired by rapid eye movement sleep deprivation following training. Spatial learning in the Morris water maze, however, requires multiple training trials and performance, as measured by time to reach the hidden platform is influenced by not only spatial learning but also procedural learning. To determine if sleep is important for the consolidation of a single-trial, hippocampus-dependent task, we sleep deprived animals for 0–5 and 5–10 h after training for contextual and cued fear conditioning. We found that sleep deprivation from 0–5 h after training for this task impaired memory consolidation for contextual fear conditioning whereas sleep deprivation from 5–10 h after training had no effect. Sleep deprivation at either time point had no effect on cued fear conditioning, a hippocampus-independent task. Previous studies have determined that memory consolidation for fear conditioning is impaired when protein kinase A and protein synthesis inhibitors are administered at the same time as when sleep deprivation is effective, suggesting that sleep deprivation may act by modifying these molecular mechanisms of memory storage.     

Footshock intensity and generalization in contextual and auditory-cued fear conditioning in the rat

The relationship between US (footshock) intensity and the two conditioned freezing responses (to acoustic CS and to "context") was investigated in fear conditioning. Administered footshock intensity was 0.00, 0.15, 0.30, 0.60, 0.90, and 1.20 mA to six different groups of 70-day-old male Albino Wistar rats. To measure contextual freezing, the animals were again placed inside the conditioning apparatus without acoustic CS and US presentation. To measure acoustic CS freezing, the animals were placed in a totally different apparatus and only the acoustic CS was presented. The 0.15 mA footshock intensity was not sufficient to condition the animals, in fact no freezing was exhibited as in the non-shocked control group. The 0.30 mA footshock intensity was sufficient only to condition the animals to the acoustic CS, whereas the 0.60 mA was sufficient to condition the animals both to acoustic CS and to context. Footshock intensities (0.90 and 1.20 mA) did not elicit any significant increase in conditioned freezing for either acoustic CS or context but at the highest one the generalization phenomenon appeared (freezing in the different context before presentation of acoustic CS). Acoustic CS freezing to all over-threshold intensities was longer than that to context. In conclusion, freezing responses to acoustic CS and context after increasing footshock intensities follow distinct patterns, and intermediate footshock intensities (0.60 and 0.90 mA) appear to be the most useful for eliciting conditioned freezing responses to acoustic CS and to context without inducing a generalized fear status contamination.     

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.     

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.     

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.     

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.     

Transgenic inhibition of neuronal calcineurin activity in the forebrain facilitates fear conditioning, but inhibits the extinction of contextual fear memories

It is unclear whether protein phosphatases, which counteract the actions of protein kinases, play a beneficial role in the formation and extinction of previously acquired fear memories. In this study, we investigated the role of the calcium/calmodulin dependent phosphatase 2B, also known as calcineurin (CaN) in the formation of contextual fear memory and extinction of previously acquired contextual fear. We used a temporally regulated transgenic approach, that allowed us to selectively inhibit neuronal CaN activity in the forebrain either during conditioning or only during extinction training leaving the conditioning undisturbed. Reducing CaN activity through the expression of a CaN inhibitor facilitated contextual fear conditioning, while it impaired the extinction of previously formed contextual fear memory. These findings give the first genetic evidence that neuronal CaN plays an opposite role in the formation of contextual fear memories and the extinction of previously formed contextual fear memories.     

Role of muscarinic M1 receptors in inhibitory avoidance and contextual fear conditioning

The objective of the present study was to observe the effects of pre-training or post-training administration of dicyclomine, a M1 muscarinic antagonist, on inhibitory avoidance (IA) and contextual fear conditioning (CFC) and to investigate if the effects observed with the pre-training administration of dicyclomine are state-dependent. For each behavioral procedure (IA and CFC) groups of Wistar male rats were treated with saline or dicyclomine either 30 min before training (pre-training), immediately after training or 30 min before training/30 min before test (pre-training/pre-test). The animals were tested 24 h after training. The acquisition of IA and CFC was impaired by pre-training administration of dicyclomine. The consolidation of both tasks was not affected by dicyclomine given immediately after training. Pre-training/pre-test administration of dicyclomine impaired both tasks, an effect similar to that observed in the group which only received pre-training administration. Pre-test treatment induced dissociation between both tasks, impairing CFC retrieval, without interfering with the animals avoidance response. These results show that the dicyclomine did not affect IA and CFC consolidation, suggesting specific involvement of M1 muscarinic receptor only in acquisition these tasks, and these effects was not state-dependent. However, it is possible that the retrieval of these tasks may be mediated, at least in part, by different neurochemical mechanisms and may be dissociated by dicyclomine.     

Pentylenetetrazole as an unconditioned stimulus for olfactory and contextual fear conditioning in rats

The association of five footshocks with a neutral odor is able to establish an olfactory fear conditioning in rats. The present study sought to investigate whether the systemic administration of pentylenetetrazole (PTZ; 3.75–15 mg/kg) would turn the coffee odor in a conditioned stimulus in the fear conditioning paradigm. The results showed that rats started to display risk assessment and avoidance after PTZ (15 mg/kg)–coffee odor pairing. When three mild footshocks (0.4 mA for 2 s) were delivered during this pairing, the conditioned response exhibited was greater than before. In both cases, however, pretreatment with the benzodiazepine midazolam (MDZ. 0.5 mg/kg i.p.) fully counteracted the expression of these defensive behaviors. Moreover, after being paired with 15 mg/kg of PTZ alone or combined with footshocks, the coffee odor was able to promote a new fear conditioning related to the context where it was re-exposed. The present findings point out the usefulness of PTZ as an unconditioned stimulus to promote fear conditioning to olfactory and contextual cues in rats.     

The development of cued versus contextual conditioning in a predictable and an unpredictable human fear conditioning preparation

In this human fear conditioning study, the online development of conditioned US-expectancy to discrete cues and background contexts was measured in two groups. In the paired group (n=30), the CS was systematically followed by an aversive shock (US). In the unpaired group (n=30), CS and US were presented explicitly unpaired. Using US-expectancy ratings, we replicated the basic finding already illustrated in humans with startle modulation. In the paired group, the CS elicited more US-expectancy than the context, whereas in the unpaired group, the context elicited more US-expectancy than the CS. Interestingly, we also observed a trial-by-trial development of conditioning to the context in the unpaired group as indicated by a significant linear trend. This gradual development and the evidence for the role of US-expectancy in contextual fear add to the idea that cued and contextual fear rely on the same basic associative processes.     

Transswitching and contextual conditioning

An empirical evaluation of temporal aspects of contextual conditioning was conducted in relation to Asratyan’s (1965) theory of transswitching and to an alternative explanation that was partly stimulated by the Rescorla-Wagner model (Rescorla and Wagner 1972). On the basis of a human electrodermal conditioning preparation suggested by Kimmel and Ray (1978), five groups with 12 subjects each were run. The results indicated that the basic phenomena of transswitching are robust and therefore could be replicated; but the Asratyan theory was rejected. All the results supported an alternative explanation: in contextual conditioning, duration of contextual stimuli is less important than order. Phasic switching is due to simultaneous occurrence of stimuli (differential compound conditioning) and is therefore compatible with the Rescorla-Wagner model. Tonic switching is due to signals that occur before a marked sequence of conditioning trials, in part a challenge to the Rescorla-Wagner model. Long delays between critical events can perhaps be compensated for by mediating memory processes.     

Trace and contextual fear conditioning require neural activity and NMDA receptor-dependent transmission in the medial prefrontal cortex

The contribution of the medial prefrontal cortex (mPFC) to the formation of memory is a subject of considerable recent interest. Notably, the mechanisms supporting memory acquisition in this structure are poorly understood. The mPFC has been implicated in the acquisition of trace fear conditioning, a task that requires the association of a conditional stimulus (CS) and an aversive unconditional stimulus (UCS) across a temporal gap. In both rat and human subjects, frontal regions show increased activity during the trace interval separating the CS and UCS. We investigated the contribution of prefrontal neural activity in the rat to the acquisition of trace fear conditioning using microinfusions of the γ-aminobutyric acid type A (GABA_A) receptor agonist muscimol. We also investigated the role of prefrontal N-methyl-d-aspartate (NMDA) receptor-mediated signaling in trace fear conditioning using the NMDA receptor antagonist 2-amino-5-phosphonovaleric acid (APV). Temporary inactivation of prefrontal activity with muscimol or blockade of NMDA receptor-dependent transmission in mPFC impaired the acquisition of trace, but not delay, conditional fear responses. Simultaneously acquired contextual fear responses were also impaired in drug-treated rats exposed to trace or delay, but not unpaired, training protocols. Our results support the idea that synaptic plasticity within the mPFC is critical for the long-term storage of memory in trace fear conditioning.The prefrontal cortex participates in a wide range of complex cognitive functions including working memory, attention, and behavioral inhibition (Fuster 2001). In recent years, the known functions of the prefrontal cortex have been extended to include a role in long-term memory encoding and retrieval (Blumenfeld and Ranganath 2006; Jung et al. 2008). The prefrontal cortex may be involved in the acquisition, expression, extinction, and systems consolidation of memory (Frankland et al. 2004; Santini et al. 2004; Takehara-Nishiuchi et al. 2005; Corcoran and Quirk 2007; Jung et al. 2008). Of these processes, the mechanisms supporting the acquisition of memory may be the least understood. Recently, the medial prefrontal cortex (mPFC) has been shown to be important for trace fear conditioning (Runyan et al. 2004; Gilmartin and McEchron 2005), which provides a powerful model system for studying the neurobiological basis of prefrontal contributions to memory. Trace fear conditioning is a variant of standard “delay” fear conditioning in which a neutral conditional stimulus (CS) is paired with an aversive unconditional stimulus (UCS). Trace conditioning differs from delay conditioning by the addition of a stimulus-free “trace” interval of several seconds separating the CS and UCS. Learning the CS–UCS association across this interval requires forebrain structures such as the hippocampus and mPFC. Importantly, the mPFC and hippocampus are only necessary for learning when a trace interval separates the stimuli (Solomon et al. 1986; Kronforst-Collins and Disterhoft 1998; McEchron et al. 1998; Takehara-Nishiuchi et al. 2005). This forebrain dependence has led to the hypothesis that neural activity in these structures is necessary to bridge the CS–UCS temporal gap. In support of this hypothesis, single neurons recorded from the prelimbic area of the rat mPFC exhibit sustained increases in firing during the CS and trace interval in trace fear conditioning (Baeg et al. 2001; Gilmartin and McEchron 2005). Similar sustained responses are not observed following the CS in delay conditioned animals or unpaired control animals. This pattern of activity is consistent with a working memory or “bridging” role for mPFC in trace fear conditioning, but it is not clear whether this activity is actually necessary for learning. We address this issue here using the γ-aminobutyric acid type A (GABA_A) receptor agonist muscimol to temporarily inactivate cellular activity in the prelimbic mPFC during the acquisition of trace fear conditioning.The contribution of mPFC to the long-term storage (i.e., 24 h or more) of trace fear conditioning, as opposed to a strictly working memory role (i.e., seconds to minutes), is a matter of some debate. Recent reports suggest that intact prefrontal activity at the time of testing is required for the recall of trace fear conditioning 2 d after training (Blum et al. 2006a), while mPFC lesions performed 1 d after training fail to disrupt the memory (Quinn et al. 2008). The findings from the former study may reflect a role for prelimbic mPFC in the expression of conditional fear rather than memory storage per se (Corcoran and Quirk 2007). However, blockade of the intracellular mitogen-activated protein kinase (MAPK) cascade during training impairs the subsequent retention of trace fear conditioning 48 h later (Runyan et al. 2004). Activation of the MAPK signaling cascade can result in the synthesis of proteins necessary for synaptic strengthening, providing a potential mechanism by which mPFC may participate in memory storage. To better understand the nature of the prefrontal contribution to long-term memory, more information is needed about fundamental plasticity mechanisms in this structure. Dependence on N-methyl-d-aspartate receptors (NMDAR) is a key feature of many forms of long-term memory, both in vitro and in vivo. The induction of long-term potentiation (LTP) in the hippocampus, a cellular model of long-term plasticity and information storage, requires NMDAR activation (Reymann et al. 1989). Genetic knockdown or pharmacological blockade of NMDAR-mediated neurotransmission in the hippocampus impairs several forms of hippocampus-dependent memory, including trace fear conditioning (Tonegawa et al. 1996; Huerta et al. 2000; Quinn et al. 2005), but it is unknown if activation of these receptors is necessary in the mPFC for the acquisition of trace fear conditioning. Data from in vivo electrophysiology studies have shown that stimulation of ventral hippocampal inputs to prelimbic neurons in mPFC produces LTP, and the induction of prefrontal LTP depends upon functional NMDARs (Laroche et al. 1990; Jay et al. 1995). If the role of mPFC in trace fear conditioning goes beyond simply maintaining CS information in working memory, then activation of NMDAR may be critical to memory formation. We test this hypothesis by reversibly blocking NMDAR neurotransmission with 2-amino-5-phosphonovaleric acid (APV) during training to examine the role of prefrontal NMDAR to the acquisition of trace fear conditioning.Another important question is whether mPFC contributes to the formation of contextual fear memories. Fear to the training context is acquired simultaneously with fear to the auditory CS in both trace and delay fear conditioning. Conflicting reports in the literature suggest the role of mPFC in contextual fear conditioning is unclear. Damage to ventral areas of mPFC prior to delay fear conditioning has failed to impair context fear acquisition (Morgan et al. 1993). Prefrontal lesions incorporating dorsal mPFC have in some cases been reported to augment fear responses to the context (Morgan and LeDoux 1995), while blockade of NMDAR transmission has impaired contextual fear conditioning (Zhao et al. 2005). Post-training lesions of mPFC impair context fear retention (Quinn et al. 2008) in trace and delay conditioning. Contextual fear responses were assessed in this study to determine the contribution of neuronal activity and NMDAR-mediated signaling in mPFC to the acquisition of contextual fear conditioning.     

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.     

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.