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.     

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.     

A different recruitment of the lateral and basolateral amygdala promotes contextual or elemental conditioned association in Pavlovian fear conditioning

Convergent data suggest dissociated roles for the lateral (LA) and basolateral (BLA) amygdaloid nuclei in fear conditioning, depending on whether a discrete conditioned stimulus (CS)-unconditional stimulus (US) or context-US association is considered. Here, we show that pretraining inactivation of the BLA selectively impaired conditioning to context. In contrast, inactivation of the LA disrupted conditioning to the discrete tone CS, but also either impaired or enhanced contextual conditioning, depending on whether the context was in the foreground or in the background. Hence, these findings refine the current model of the amygdala function in emotional learning by showing that the BLA and the LA not only differentially contribute to elemental and context-US association, but also promote, through their interaction, the most relevant of these two associations.     

The contextual malleability of approach-avoidance training effects: approaching or avoiding fear conditioned stimuli modulates effects of approach-avoidance training

Previous research showed that the repeated approaching of one stimulus and avoiding of another stimulus typically leads to more positive evaluations of the former stimuli. In the current study, we examined whether approach and avoidance training (AAT) effects on evaluations of neutral stimuli can be modulated by introducing a regularity between the approach-avoidance actions and a positive or negative (feared) stimulus. In an AAT task, participants repeatedly approached one neutral non-word and avoided another neutral non-word. Half of the participants also approached a negative fear-conditioned stimulus (CS+) and avoided a conditioned safe stimulus (CS?). The other half of the participants avoided the CS+ and approached the CS?. Whereas participants in the avoid CS+ condition exhibited a typical AAT effect, participants in the approach CS+ condition exhibited a reversed AAT effect (i.e. they evaluated the approached neutral non-word as more negative than the avoided non-word). These findings provide evidence for the malleability of the AAT effect when strongly valenced stimuli are approached or avoided. We discuss the practical and theoretical implications of our findings.     

Administration of corticosterone after memory reactivation disrupts subsequent retrieval of a contextual conditioned fear memory: dependence upon training intensity

Reactivation of stabilized memories returns them to a labile state and causes them to undergo extinction or reconsolidation processes. Although it is well established that administration of glucocorticoids after training enhance consolidation of contextual fear memories, but their effects on post-retrieval processes are not known. In this study, we first asked whether administration of corticosterone after memory reactivation would modulate subsequent expression of memory in rats. Additionally, we examined whether this modulatory action would depend upon the strength of the memory. We also tested the effect of propranolol after memory reactivation. Adult male Wistar rats were trained in a fear conditioning system using moderate (0.4 mA) or high shock (1.5 mA) intensities. For reactivation, rats were returned to the chamber for 90 s 24h later. Immediately after reactivation, rats were injected with corticosterone (1, 3 or 10mg/kg) or vehicle. One, 7 and 14 days after memory reactivation, rats were returned to the context for 5 min, and freezing behavior was scored. The findings indicated that corticosterone when injected after memory reactivation had no significant effect on recall of a moderate memory, but it impaired recall of a strong memory at a dose of 3mg/kg. Propranolol (5mg/kg) given after the reactivation treatment produced a modest impairment that persisted over three test sessions. Further, the results showed that corticosterone, but not propranolol deficit was reversed by a reminder shock. These findings provide evidence that administration of glucocorticoids following memory reactivation reduces subsequent retrieval of strong, but not moderate, contextual conditioned fear memory likely via acceleration of memory extinction. On the other hand, propranolol-induced amnesia may result from blockade of reconsolidation process. Further studies are needed to determine the underlying mechanisms.     

Lesions of the dorsal subiculum and the dorsal hippocampus impaired pattern separation in a task using distinct and overlapping visual stimuli

The contribution of the dorsal subiculum (DS) and of the dorsal hippocampus (DH) to memory for distinct and overlapping visual stimuli was examined. Rats with selective lesions of the DS or the DH were compared to sham-operated rats on a delayed matching-to-place task guided by distal visual cues in a modified radial-arm maze. Overlapping distal visual cues could be perceived from three arm entrances (adjacent arms) and a unique set of distal cues were more likely to be seen from the other two arm entrances (distinct arms). Rats with DS lesions were impaired on trials with baited adjacent arms, but not on trials with baited distinct arms. Rats with DH lesions were impaired on both types of trials. These results suggest that the DS and the DH are necessary for pattern separation and that they may have different contributions to memory.     

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.     

Early extinction after fear conditioning yields a context-independent and short-term suppression of conditional freezing in rats

Extinction of Pavlovian fear conditioning in rats is a useful model for therapeutic interventions in humans with anxiety disorders. Recently, we found that delivering extinction trials soon (15 min) after fear conditioning yields a short-term suppression of fear, but little long-term extinction. Here, we explored the possible mechanisms underlying this deficit by assessing the suppression of fear to a CS immediately after extinction training (Experiment 1) and the context specificity of fear after both immediate and delayed extinction training (Experiment 2). We also examined the time course of the immediate extinction deficit (Experiment 3). Our results indicate that immediate extinction produces a short-lived and context-independent suppression of conditional freezing. Deficits in long-term extinction were apparent even when the extinction trials were given up to 6 h after conditioning. Moreover, this deficit was not due to different retention intervals that might have influenced the degree of spontaneous recovery after immediate and delayed extinction (Experiment 4). These results suggest that fear suppression under immediate extinction may be due to a short-term, context-independent habituation process, rather than extinction per se. Long-term extinction memory only develops when extinction training occurs at least six hours after conditioning.Pavlovian fear conditioning and extinction are important behavioral models for studying the brain mechanisms underlying the acquisition, storage, retrieval, and suppression of traumatic fear (LeDoux 2000; Maren 2001, 2005; Kim and Jung 2005). In this procedure, an emotionally neutral stimulus, such as a tone, is paired with an aversive stimulus (US), such as an electric foot shock. After a few tone–foot shock pairings, the previous neutral tone becomes a potent conditioned stimulus (CS) and acquires the ability to elicit fear responses, such as freezing (CR). However, with repeated presentations of the CS-alone, the previously acquired CR gradually subsides, a process called extinction (Davis et al. 2003; Maren and Quirk 2004; Kim and Jung 2005; Myers and Davis 2007). The behavioral processes and the underlying neural mechanisms of extinction have attracted extensive attention in contemporary research of learning and memory (Bouton et al. 2006). Indeed, it has been suggested that failure to extinguish fear may contribute to post-traumatic stress disorder (PTSD) (Bouton et al. 2001; Rothbaum and Davis 2003). To avoid the possible long-term consequences and costs of PTSD or other anxiety disorders, clinical interventions are essential. While early interventions may manage the stress response to trauma, their efficacy has been challenged, because the acute intense stress of the traumatic experience might actually exacerbate relapse of fear (McNally 2003; Rothbaum and Davis 2003; Gray and Litz 2005). Thus, it is essential to learn when these interventions generate the best long-term extinction of fear responses.In a recent study, we demonstrated that delivering extinction trials shortly after fear conditioning yields poor long-term fear reduction (Maren and Chang 2006; but, see Myers et al. 2006). We observed that conditional freezing decreased during extinction training, but recovered completely 24 h later. This was true even when we gave 225 massed extinction trials 15 min after fear conditioning. However, in these experiments the within-session decrease in fear in rats that underwent extinction was similar to that in rats that were not exposed to extinction trials. Thus, it is unclear to what extent the short-term fear suppression we observed was due to a loss of fear to the context, the auditory CS, or both. It is also not clear whether fear suppression was due to extinction or, alternatively, another learning process such as habituation.To examine these issues further, in the present study we first assessed fear suppression to the auditory CS after immediate extinction by probing CS fear 15 min after extinction training. In a second experiment, we examined whether short-term fear suppression to the CS is renewed outside of the extinction context, as context specificity is one of the hallmarks of extinction (Bouton 2002; Ji and Maren 2007). In the third and fourth experiments, we examined the temporal delay necessary between conditioning and extinction to yield long-term suppression of fear. In our previous work (Maren and Chang 2006), all phases of training were conducted in the same context. Therefore, fear to the context decreased conditional freezing to the tone, particularly when extinction occurred shortly after conditioning, a time at which sensitized context fear was high. In an effort to isolate fear to the tone CS during extinction, we conducted extinction and test sessions in a context that was different from the conditioning context (i.e., an ABB procedure, where each letter denotes the context used for conditioning, extinction, and test, respectively). Our results reveal that delivering CS-alone trials shortly after fear conditioning produces a short-lived and context-independent suppression of freezing. This fear suppression may be due to a short-term, context-independent habituation process, rather than extinction. Furthermore, poor long-term extinction occurs even when the extinction trials were administered up to 6 h after conditioning.     

Lesions of the ventral hippocampus, but not the dorsal hippocampus, impair conditioned fear expression and inhibitory avoidance on the elevated T-maze

The hippocampus has been suggested to be involved in spatial (or configural) memory and also in the inhibition of certain response or goal alternatives. An increasing number of anatomical, physiological, and behavioral studies indicate that the hippocampus is functionally heterogeneous along the dorsal-ventral axis. Identification of distinct behavioral roles for the dorsal (DH) and ventral (VH) hippocampus may resolve differences between the various theoretical accounts of hippocampal function. The present study examined the effects of electrolytic lesions restricted to the DH or VH on fear-conditioned freezing, passive avoidance on the elevated T-maze (ETM) test of anxiety, and general activity in male Sprague-Dawley (Charles-River derived) albino rats. We found that rats with lesions of the VH, but not DH showed reduced freezing to both context and tone conditioned stimuli (CS). Rats with VH lesions also showed a reduced latency to emerge from the enclosed arm on trials 2 and 3 of the ETM (indicating reduced anxious behavior), while having no effect on the latency to escape from the open arms on trial 4. There were no differences in activity between the groups. These results indicate that the VH and DH are differentially involved in passive avoidance on the ETM and conditioned freezing to context and tone CS. We suggest that the VH may be specifically involved in modulating goal-oriented, defensive behavior expression through hypothalamic and amygdaloid connections.     

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.     

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.     

Lesion of the ventral periaqueductal gray reduces conditioned fear but does not change freezing induced by stimulation of the dorsal periaqueductal gray

Previously-reported evidence showed that freezing to a context previously associated with footshock is impaired by lesion of the ventral periaqueductal gray (vPAG). It has also been shown that stepwise increase in the intensity of the electrical stimulation of the dorsal periaqueductal gray (dPAG) produces alertness, then freezing, and finally escape. These aversive responses are mimicked by microinjections of GABA receptor antagonists, such as bicuculline, or blockers of the glutamic acid decarboxylase (GAD), such as semicarbazide, into the dPAG. In this work, we examined whether the expression of these defensive responses could be the result of activation of ventral portion of the periaqueductal gray. Sham- or vPAG electrolytic–lesioned rats were implanted with an electrode in the dPAG for the determination of the thresholds of freezing and escape responses. The vPAG electrolytic lesions were behaviorally verified through a context-conditioned fear paradigm. Results indicated that lesion of the vPAG disrupted conditioned freezing response to contextual cues associated with footshocks but did not change the dPAG electrical stimulation for freezing and escape responses. In a second experiment, lesion of the vPAG also did not change the amount of freezing and escape behavior produced by microinjections of semicarbazide into the dPAG. These findings indicate that freezing and escape defensive responses induced by dPAG stimulation do not depend on the integrity of the vPAG. A discussion on different neural circuitries that might underlie different inhibitory and active defensive behavioral patterns that animals display during threatening situations is presented.     

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.     

Latent growth curve analysis of fear during a speech task before and after treatment for social phobia

Models of social phobia highlight the importance of anticipatory anxiety in the experience of fear during a social situation. Anticipatory anxiety has been shown to be highly correlated with performance anxiety for a variety of social situations. A few studies show that average ratings of anxiety during the anticipation and performance phases of a social situation decline following treatment. Evidence also suggests that the point of confrontation with the feared stimulus is the peak level of fear. No study to date has evaluated the pattern of anxious responding across the anticipation, confrontation, and performance phases before and after treatment, which is the focus of the current study. Socially phobic individuals (N = 51) completed a behavioral avoidance task before and after two types of manualized cognitive behavioral therapy, and gave ratings of fear during the anticipation and performance phases.Results from latent growth curve analysis were the same for the two treatments and suggested that before treatment, anxiety sharply increased during the anticipation phase, was highly elevated at the confrontation, and gradually increased during the performance phase. After treatment, anxiety increased during the anticipation phase, although at a much slower rate than at pretreatment, peaked at confrontation, and declined during the performance phase. The findings suggest that anticipatory experiences are critical to the experience of fear for public speaking and should be incorporated into exposures.     

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.     

Unconditioned freezing is enhanced in an appetitive context: implications for the contextual dependency of unconditioned fear

It has been well established that expression of conditioned fear is context independent, but the context dependency of unconditioned fear expression has rarely been explored. A recent study reported that unconditioned freezing in rats is enhanced in a familiar context, which suggests that unconditioned fear expression can be modulated by contextual processing. In order to further explore this possibility we examined unconditioned freezing in novel, familiar, and appetitive contexts; and attempted to identify brain regions critical for context-related changes in unconditioned freezing by measuring c-Fos mRNA levels in emotional circuits. Unconditioned freezing was enhanced in the appetitive context, and this enhancement was accompanied by increased c-Fos mRNA expression in the medial amygdala and hippocampus, but attenuated expression in the medial prefrontal cortex. In the appetitive context, expectation of a reward coupled with detection of threat may have enhanced unconditioned fear expression, which suggests that unconditioned fear expression can be modulated by contextual factors. Context-related expectancy mismatch may explain the enhancement of unconditioned fear expression seen in this study and warrants further examination.     

Glucose injections into the dorsal hippocampus or dorsolateral striatum of rats prior to T-maze training: modulation of learning rates and strategy selection

The present experiments examined the effects of injecting glucose into the dorsal hippocampus or dorsolateral striatum on learning rates and on strategy selection in rats trained on a T-maze that can be solved by using either a hippocampus-sensitive place or striatum-sensitive response strategy. Percentage strategy selection on a probe trial (P(crit)) administered after rats achieved criterion (nine of 10 correct choices) varied by group. All groups predominately exhibited a response strategy on a probe trial administered after overtraining, i.e., after 90 trials. In experiment 1, rats that received intrahippocampal glucose injections showed enhanced acquisition of the T-maze and showed increased use of response solutions at P(crit) compared with that of unimplanted and artificial cerebral spinal fluid (aCSF)-treated groups. These findings suggest that glucose enhanced hippocampal functions to accelerate the rate of learning and the early adoption of a response strategy. In experiment 2, rats that received intrastriatal glucose injections exhibited place solutions early in training and reached criterion more slowly than did aCSF controls, with learning rates comparable to those of unoperated and operated-uninjected controls. Relative to unoperated, operated-uninjected and glucose-injected rats, rats that received intrastriatal aCSF injections showed enhanced acquisition of the T-maze and increased use of response solutions at P(crit). The unexpected enhanced acquisition seen after striatal aCSF injections suggests at least two possible interpretations: (1) aCSF impaired striatal function, thereby releasing competition with the hippocampus and ceding control over learning to the hippocampus during early training trials; and (2) aCSF enhanced striatal functioning to facilitate striatal-sensitive learning. With either interpretation, the results indicate that intrastriatal glucose injections compensated for the aCSF-induced effect. Finally, enhanced acquisition regardless of treatment was accompanied by rapid adoption of a response solution for the T-maze.     

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.