Expression of the immediate-early gene-encoded protein Egr-1 (zif268) during in vitro classical conditioning

Expression of the immediate-early genes (IEGs) has been shown to be induced by activity-dependent synaptic plasticity or behavioral training and is thought to play an important role in long-term memory. In the present study, we examined the induction and expression of the IEG-encoded protein Egr-1 during an in vitro neural correlate of eyeblink classical conditioning. The results showed that Egr-1 protein expression as determined by immunocytochemistry and Western blot analysis rapidly increased during the early stages of conditioning and remained elevated during the later stages. Further, expression of Egr-1 protein required NMDA receptor activation as it was blocked by bath application of AP-5. These findings suggest that the IEG-encoded proteins such as Egr-1 are activated during relatively simple forms of learning in vertebrates. In this case, Egr-1 may have a functional role in the acquisition phase of conditioning as well as in maintaining expression of conditioned responses.     

Antagonism of lateral amygdala alpha1-adrenergic receptors facilitates fear conditioning and long-term potentiation

Norepinephrine receptors have been studied in emotion, memory, and attention. However, the role of alpha1-adrenergic receptors in fear conditioning, a major model of emotional learning, is poorly understood. We examined the effect of terazosin, an alpha1-adrenergic receptor antagonist, on cued fear conditioning. Systemic or intra-lateral amygdala terazosin delivered before conditioning enhanced short- and long-term memory. Terazosin delivered after conditioning did not affect consolidation. In vitro, terazosin impaired lateral amygdala inhibitory postsynaptic currents leading to facilitation of excitatory postsynaptic currents and long-term potentiation. Since alpha1 blockers are prescribed for hypertension and post-traumatic stress disorder, these results may have important clinical implications.Although norepinephrine (NE) has been widely studied as an important modulator of memory and emotion, comparatively little is known about the role of NE in amygdala-dependent Pavlovian fear conditioning, a major model for understanding the neural basis of fear learning and memory. In fear conditioning, an emotionally neutral conditioned stimulus (CS; i.e., tone) is temporally paired with an aversive unconditioned stimulus (US; i.e., footshock). After very few pairings, a lasting, robust CS–US association is acquired, and the CS elicits stereotypical defensive responses, including behavioral freezing (Blanchard and Blanchard 1969; Bolles and Fanselow 1980).The lateral nucleus of the amygdala (LA) is a key structure underlying fear conditioning (LeDoux 2000). Convergence of CS and US information in LA is believed to play an important role in initiating synaptic plasticity. Long-term potentiation (LTP)-like changes in LA CS processing are critical for fear memory storage (LeDoux 2000; Blair et al. 2001; Maren 2001; Walker and Davis 2002). LA receives auditory CS inputs from the thalamus and cortex and connects directly and indirectly with the central nucleus of the amygdala to control expression of Pavlovian fear responses.Of the noradrenergic receptor subtypes, alpha1 receptors have received the least attention in fear conditioning. LA receives NE-containing inputs from the locus coeruleus that fire tonically and phasically in response to aversive stimuli like footshock (Pitkänen 2000; Tanaka et al. 2000; Aston-Jones and Cohen 2005). Alpha1-adrenergic receptors are expressed in LA, most likely on both excitatory and inhibitory neurons (Jones et al. 1985; Domyancic and Morilak 1997). Alpha1 receptor activation stimulates GABA-mediated miniature inhibitory postsynaptic currents in LA (Braga et al. 2004), suggesting that alpha1 receptors contribute to inhibition in fear conditioning pathways. Several elegant experiments recently demonstrated that LA inhibition gates synaptic plasticity necessary for fear conditioning, and this inhibitory gate can be influenced by neuromodulators including NE (Stutzmann and LeDoux 1999; Shumyatsky et al. 2002; Bissière et al. 2003; Shaban et al. 2006; Shin et al. 2006; Tully et al. 2007). However, the role of alpha1 receptor activity in gating amygdala LTP and fear learning has never been examined.We hypothesized that alpha1 blockers would facilitate fear learning, possibly by impairing LA inhibition and facilitating LA LTP. To test this hypothesis, we injected rats with terazosin, a selective alpha1-adrenergic receptor antagonist, systemically or directly into LA before or after fear conditioning. We examined in vitro the effect of terazosin on LA pyramidal cell inhibitory postsynaptic currents (IPSCs) and excitatory postsynaptic currents (EPSCs) in response to fiber stimulation of the thalamic CS input pathway to LA, as well as the effect of terazosin on LA LTP in this same pathway. We found that intra-LA terazosin facilitated fear conditioning when injected before but not after training. We also found that terazosin impaired IPSCs in LA pyramidal cells, leading to facilitated EPSCs and LTP.Behavioral experiments were conducted on adult, male Sprague–Dawley rats (Hilltop Laboratory Animals) weighing approximately 300 g upon arrival. Rats were individually housed, maintained on a 12/12 h light/dark schedule, and allowed free access to food and water. Testing was conducted during the light phase. All procedures and experiments were approved by NYU''s Animal Care and Use Committee.For systemic injections, terazosin (20 mg/kg; Sigma) was dissolved in saline and injected intraperitoneally (i.p.) 30 min prior to conditioning in 1 mL/kg volume. For bilateral infusions, terazosin (125 ng/0.25 µL) was dissolved in aCSF and infused into the LA at 0.1 µL/min 30 min prior to or immediately after fear conditioning. Bilateral guide cannulae (22 gauge; Plastics One) aimed at LA (−3.3 mm anterior, 5.2 mm lateral, −7 mm dorsal to bregma) were surgically implanted as previously described (Sotres-Bayon et al. 2009). Rats were given at least 7 d to recover from surgery before testing. For infusions, dummy cannulae were removed, and infusion cannulae (28 gauge, +1 mm beyond guides) were inserted into guides. Infusion cannulae were connected to a 1.0 μL Hamilton syringe via polyethylene tubing. Infusion rate was controlled by a pump (PHD22/2000; Harvard Apparatus), and infusion cannulae were left in place for an additional 60 sec to allow diffusion of the solution away from the cannula tip, then dummy cannulae were replaced. Upon completion of the experiment, rats were euthanized, brains removed, and cannulae placements verified histologically as previously described (Sotres-Bayon et al. 2009).Two contexts (A and B) were used for all testing as previously described (Schiller et al. 2008). The grid floors in Context B were covered with black Plexiglas inserts to reduce generalization. No odors were used and chambers were cleaned between sessions. CSs were 30 sec, 5 kHz, 80 dB tones, and USs were 1 sec, 0.8 mA scrambled electric footshocks. Experiments consisted of two phases separated by 48 h: (1) fear conditioning in Context A and (2) long-term memory (LTM) test in Context B. On Day 1, rats were placed in Context A, allowed 5 min to acclimate, and then received three CS–US pairings separated by variable 5 min ITIs. On Day 3, rats were placed in Context B and allowed 5 min to acclimate before receiving one CS-alone presentation.The index of fear in behavioral experiments was “freezing,” the absence of all non-respiratory movement (Blanchard and Blanchard 1971; Fanselow 1980). Following testing, freezing was manually scored from DVDs by a scorer blind to group specification. Graphs represent group means ± SEM. Statistical analysis was conducted with GraphPad Prism.Whole-cell electrophysiological recordings were obtained from LA pyramidal cells using in vitro coronal slices from rats aged P21–P30 d as described in Cunha et al. (2010). Terazosin was bath-applied for 10 min to achieve stable responses before testing. The cells were voltage-clamped using an Axopatch 200B amplifier at −35 mV for recording EPSCs and IPSCs. Synaptic responses were evoked with sharpened tungsten bipolar stimulating electrodes. Internal capsule fibers containing thalamic afferents were stimulated for paired-pulse facilitation (PPF) (ISI = 50 msec; 0.1 Hz) using a photoelectric stimulus isolation unit with a constant current output. Cells were rejected if access resistance (8–26 MΩ) changed more than 15%. Signals were filtered at 2 kHz and digitized (Digidata 1440 A; Axon Instruments), and peak amplitude, 10%–90% rise time, and IPSC decay time constants were analyzed offline using pCLAMP10.2 software (Axon Instruments).Brain slices for LTP experiments were prepared from rats aged 3–5 wk as in Johnson et al. (2008) and maintained on an interface chamber at 31°C. Glass recording electrodes (filled with aCSF, 5 MΩ resistance) were guided to LA neurons. Bipolar stainless steel stimulating electrodes (75 kΩ) were positioned medial to LA in internal capsule fibers. Orthodromic synaptic potentials were evoked via an isolated current generator (Digitimer; 100 μsec pulses of 0.3–0.7 mA). Evoked field potentials were recorded with an Axoclamp 2B amplifier and Axon WCP software (Axon Instruments). Data were analyzed offline using WCP PeakFit (Axon Instruments). LTP was measured as a change in evoked field potential amplitude.Baseline responses were monitored at 0.05 Hz for 30 min with a stimulus intensity of 40%–50% of maximum fEPSP before LTP induction. Terazosin (10 µM) was superfused for 15 min, and then LTP was elicited by three tetanus trains (100 Hz × 1 sec, 3 min ITI) with the same intensity and pulse duration as the baseline stimuli. In one experiment, picrotoxin (PTX; 75 µM) was present in the perfusion solution to block fast GABAergic signaling.     

Spared anterograde memory for shock-probe fear conditioning after inactivation of the amygdala

Previous studies have shown that amygdala lesions impair avoidance of an electrified probe. This finding has been interpreted as indicating that amygdala lesions reduce fear. It is unclear, however, whether amygdala-lesioned rats learn that the probe is associated with shock. If the lesions prevent the formation of this association, then pretraining reversible inactivation of the amygdala should impair both acquisition and retention performance. To test this hypothesis, the amygdala was inactivated (tetrodotoxin; TTX; 1 ng/side) before a shock-probe acquisition session, and retention was tested 4 d later. The data indicated that, compared with rats infused with vehicle, rats infused with TTX received more shocks during the acquisition session, but more importantly, were not impaired on the retention test. In Experiment 2, we assessed whether the spared memory on the retention test was caused by overtraining during acquisition. We used the same procedure as in Experiment 1, with the exception that the number of shocks the rats received during the acquisition session was limited to four. Again the data indicated that amygdala inactivation did not impair performance on the retention test. These results indicate that amygdala inactivation does not prevent the formation of an association between the shock and the probe and that shock-probe deficits during acquisition likely reflect the amygdala's involvement in other processes.     

Synaptic plasticity in the lateral amygdala: a cellular hypothesis of fear conditioning

Fear conditioning is a form of associative learning in which subjects come to express defense responses to a neutral conditioned stimulus (CS) that is paired with an aversive unconditioned stimulus (US). Considerable evidence suggests that critical neural changes mediating the CS-US association occur in the lateral nucleus of the amygdala (LA). Further, recent studies show that associative long-term potentiation (LTP) occurs in pathways that transmit the CS to LA, and that drugs that interfere with this LTP also disrupt behavioral fear conditioning when infused into the LA, suggesting that associative LTP in LA might be a mechanism for storing memories of the CS-US association. Here, we develop a detailed cellular hypothesis to explain how neural responses to the CS and US in LA could induce LTP-like changes that store memories during fear conditioning. Specifically, we propose that the CS evokes EPSPs at sensory input synapses onto LA pyramidal neurons, and that the US strongly depolarizes these same LA neurons. This depolarization, in turn, causes calcium influx through NMDA receptors (NMDARs) and also causes the LA neuron to fire action potentials. The action potentials then back-propagate into the dendrites, where they collide with CS-evoked EPSPs, resulting in calcium entry through voltage-gated calcium channels (VGCCs). Although calcium entry through NMDARs is sufficient to induce synaptic changes that support short-term fear memory, calcium entry through both NMDARs and VGCCs is required to initiate the molecular processes that consolidate synaptic changes into a long-term memory.     

Auditory specific fear conditioning results in increased levels of synaptophysin in the basolateral amygdala

Auditory fear conditioning is one of the most well characterized models used in studies of learning and memory. In order to ensure the animals have been conditioned to fear the auditory stimulus, animals are generally tested for their response to this stimulus in a different context to that used for training. For this reason it is often unclear how much contextual fear conditioning the animals also acquire when they are trained. In this study, we have established a protocol for fear conditioning in mice which is explicit for auditory cues; mice trained using this protocol, show a very low fear response to contextual cues encountered during training. We have undertaken analysis to look for potential brain changes associated with this model by measuring levels of the synaptic vesicle protein, synaptophysin, in the basolateral nuclei of the amygdala following auditory fear conditioning. Our results show levels of synaptophysin were significantly higher in mice which learnt to associate the auditory stimulus with fear, in comparison to all non-learning control animals. These findings support the idea that synaptic plasticity associated with formation of fear conditioning to a single specific conditioned stimulus occurs within the basolateral nuclei of the amygdala. Furthermore, our results demonstrate the usefulness of this model in looking for changes in the brain specific for a defined learning event.     

Calcitonin gene-related peptide released within the amygdala is involved in Pavlovian auditory fear conditioning

The effects of CGRP and the CGRP receptor antagonist hCGRP(8-37) injected into the amygdala on both the acquisition and expression of fear behavior to a discrete auditory conditional stimulus (CS) and the training context were assessed. In Experiment 1, pretraining injections of CGRP but not hCGRP(8-37) produced fear-like behavior before any aversive stimuli were presented. While both compounds attenuated freezing to the contextual CS on the test day, neither affected learning about the auditory CS. In Experiment 2, pretesting injections of hCGRP(8-37) (0.63 mM) selectively attenuated freezing to the auditory CS but left freezing to the contextual CS intact. These data suggest that CGRP in the amygdala may selectively contribute to the expression of learning about auditory stimuli during fear conditioning.     

Influence of chronic corticosterone and glucocorticoid receptor antagonism in the amygdala on fear conditioning

Glucocorticoid receptor activation within the basolateral amygdala (BLA) during fear conditioning may mediate enhancement in rats chronically exposed to stress levels of corticosterone. Male Sprague-Dawley rats received corticosterone (400 microg/ml) in their drinking water (days 1-21), a manipulation that was previously shown to cause hippocampal CA3 dendritic retraction. Subsequently, rats were adapted to the fear conditioning chamber (day 22), then trained (day 23), and tested for conditioned fear to context and tone (day 25). Training consisted of two tone (20s) and footshock (500 ms, 0.25 mA) pairings. In Experiment 1, muscimol (4.4 nmol/0.5 microl/side), a GABAergic agonist, was microinfused to temporarily inactivate the BLA during training. Rats given chronic corticosterone showed enhanced freezing to context, but not tone, compared to vehicle-supplemented rats. Moreover, BLA inactivation impaired contextual and tone conditioning, regardless of corticosterone treatment. In Experiment 2, RU486 (0, 0.3, and 3.0 ng/0.2 microl/side) was infused on training day to antagonize glucocorticoid receptors in the BLA. Corticosterone treatment enhanced fear conditioning to context and tone when analyzed together, but not separately. Moreover, RU486 (3.0 ng/side) selectively exacerbated freezing to context in chronic corticosterone-exposed rats only, but failed to alter tone conditioning. Serum corticosterone levels were negatively correlated with contextual, not tone, conditioning. Altogether, these suggest that chronic corticosterone influences fear conditioning differently than chronic stress as shown previously. Moreover, chronic exposure to corticosteroids alters BLA functioning in a non-linear fashion and that contextual conditioning is influenced more than tone conditioning by chronic corticosterone and BLA glucocorticoid receptor stimulation.     

Differential fear conditioning induces reciprocal changes in the sensory responses of lateral amygdala neurons to the CS(+) and CS(-)

In classical fear conditioning, a neutral sensory stimulus (CS) acquires the ability to elicit fear responses after pairing to a noxious unconditioned stimulus (US). As amygdala lesions prevent the acquisition of fear responses and the lateral amygdaloid (LA) nucleus is the main input station of the amygdala for auditory afferents, the effect of auditory fear conditioning on the sensory responsiveness of LA neurons has been examined. Although conditioning was shown to increase CS-evoked LA responses, the specificity of the changes in responsiveness was not tested. Because conditioning might induce nonspecific increases in LA responses to auditory afferents, we re-examined this issue in conscious, head-restrained cats using a differential conditioning paradigm where only one of two tones (CS(+) but not CS(-)) was paired to the US. Differential conditioning increased unit and field responses to the CS(+), whereas responses to the CS(-) decreased. Such changes have never been observed in the amygdala except in cases where the CS(-) had been paired to the US before and fear responses not extinguished. This suggests that fear conditioning is not only accompanied by potentiation of amygdalopetal pathways conveying the CS(+) but also by the depression of sensory inputs unpaired to noxious stimuli.     

Glutamate receptor antagonist infusions into the basolateral and medial amygdala reveal differential contributions to olfactory vs. context fear conditioning and expression

The basolateral amygdala's involvement in fear acquisition and expression to visual and auditory stimuli is well known. The involvement of the basolateral and other amygdala areas in fear acquisition and expression to stimuli of other modalities is less certain. We evaluated the contribution of the basolateral and medial amygdala to olfactory and to context fear and fear conditioning by infusing into these areas the NMDA receptor antagonist AP5, the AMPA/kainate receptor antagonist NBQX, or vehicle prior to either odor-shock pairings or fear-potentiated startle testing. Pre-training AP5 infusions into the basolateral amygdala disrupted fear conditioning to the odor but not the context conditioned stimulus (CS). Pre-test NBQX infusions disrupted fear-potentiated startle to the odor but not context CS. Neither compound blocked fear conditioning when infused into the medial amygdala prior to training, but pre-test NBQX infusions did block fear-potentiated startle. The results confirm and extend recent findings suggesting a role for the basolateral amygdala in olfactory fear and fear conditioning, reveal an unexpected dissociation of the basolateral amygdala's involvement in discrete cue versus context fear and fear conditioning, and implicate for the first time the medial amygdala in fear-potentiated startle.     

Norepinephrine infused into the basolateral amygdala posttraining enhances retention in a spatial water maze task

Recent evidence indicates that the amygdala plays a role in modulating memory processes in other brain regions. For example, posttraining intra-amygdala infusions of amphetamine enhanced memory in both spatial and cued training water maze tasks; these tasks are known to depend on the integrity of the hippocampus and caudate nucleus, respectively. To determine whether this modulation is dependent on noradrenergic activation within a subregion of the amygdala (the basolateral nucleus), the present study examined the effects of posttraining microinfusions (0.2 microl) of norepinephrine or propranolol into the basolateral amygdala immediately following training in a spatial version of the water maze task. Rats received a four-trial training session on each of 2 consecutive days. On the third day, rats were given a 60-s probe test in the absence of a platform. Retention latencies obtained on the second training day revealed that norepinephrine dose-dependently enhanced retention for the location of the hidden platform. In contrast, propranolol significantly impaired retention. Probe trial analysis revealed that rats treated with 0.25 microg norepinephrine demonstrated a selective spatial bias for the training platform location relative to all other groups. These findings are consistent with others and support the view that the basolateral amygdala has a role in modulating memory storage by interacting with other brain regions.     

Early growth response gene 1 (Egr-1) is required for new and reactivated fear memories in the lateral amygdala

The immediate-early gene early growth response gene-1 (EGR-1, zif-268) has been extensively studied in synaptic plasticity and memory formation in a variety of memory systems. However, a convincing role for EGR-1 in amygdala-dependent memory consolidation processes has yet to emerge. In the present study, we have examined the role of EGR-1 in the consolidation and reconsolidation of amygdala-dependent auditory Pavlovian fear conditioning. In our first series of experiments, we show that EGR-1 is regulated following auditory fear conditioning in the lateral nucleus of the amygdala (LA). Next, we use antisense oligodeoxynucleotide (ODN) knockdown of EGR-1 in the LA to show that training-induced expression of EGR-1 is required for memory consolidation of auditory fear conditioning; that is, long-term memory (LTM) is significantly impaired while acquisition and short-term memory (STM) are intact. In a second set of experiments, we show that EGR-1 is regulated in the LA by retrieval of an auditory fear memory. We then show that retrieval-induced expression of EGR-1 in the LA is required for memory reconsolidation of auditory fear conditioning; that is, post-retrieval (PR)-LTM is significantly impaired while memory retrieval and PR-STM are intact. Additional experiments show these effects to be restricted to the LA, to be temporally graded, and unlikely to be due to nonspecific toxicity within the LA. Collectively, our findings strongly implicate a role for EGR-1 in both the initial consolidation and in the reconsolidation of auditory fear memories in the LA.     

Differential roles of the basolateral amygdala and nucleus basalis magnocellularis during post-reactivation contextual fear conditioning reconsolidation in rats

The roles of the basolateral amygdala and nucleus basalis magnocellularis in fear conditioning reconsolidation were investigated by means of tetrodotoxin bilateral inactivation performed 96 h after conditioning, immediately after reactivation training. Footshocks of 1.2 mA intensity were employed to induce the generalization phenomenon. Basolateral amygdala inactivation disrupts the contextual fear response and its generalization but not acoustic CS trace retention, when measured 72 and 96 h after tetrodotoxin administration. Nucleus basalis magnocellularis functional inactivation does not affect memory post-reactivation phase of any of the three conditioned fear responses. The present findings show a differential role of the two structures in fear memory reconsolidation and can be a starting point for future investigation of the neural circuits subserving generalization.     

Basolateral amygdala inactivation impairs learned (but not innate) fear response in rats

Numerous studies have suggested that the amygdala is involved in the formation of aversive memories, but the possibility that this structure is merely related to any kind of fear sensation or response could not be ruled out in previous studies. The present study investigated the effects of bilateral inactivation of the amygdaloid complex in rats tested in the plus-maze discriminative avoidance task. This task concomitantly evaluates aversive memory (by discrimination of the two enclosed arms) and innate fear (by open-arm exploration). Wistar rats (3-5 months-old) were implanted with bilateral guide cannulae into basolateral amygdala. After surgery, all subjects were given 1 week to recover before behavioral experiments. Afterwards, in experiment 1, 15 min prior to training, 0.5 μl of saline or muscimol (1 mg/ml) was infused in each side via microinjection needles. In experiment 2 the animals received injections immediately after the training session and in experiment 3 rats were injected prior to testing session (24 h after training). The main results showed that (1) pre-training muscimol prevented memory retention (evaluated by aversive arm exploration in the test session), but did not alter innate fear (evaluated by percent time in open arms); (2) post-training muscimol impaired consolidation, inducing increased percent in aversive arm exploration in the test session and (3) pre-testing muscimol did not affect retrieval (evaluated by aversive enclosed arm exploration in the test session). The results suggest that amygdala inactivation specifically modulated the learning of the aversive task, excluding a possible secondary effect of amygdala inactivation on general fear responses. Additionally, our data corroborate the hypothesis that basolateral amygdala is not the specific site of storage of aversive memories, since retention of the previously learned task was not affected by pre-testing inactivation.     

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.     

Contextual,but not auditory,fear conditioning is disrupted by neurotoxic selective lesion of the basal nucleus of amygdala in rats

The basolateral amygdala complex (BLA) is involved in acquisition of contextual and auditory fear conditioning. However, the BLA is not a single structure but comprises a group of nuclei, including the lateral (LA), basal (BA) and accessory basal (AB) nuclei. While it is consensual that the LA is critical for auditory fear conditioning, there is controversy on the participation of the BA in fear conditioning. Hodological and neurophysiological findings suggest that each of these nuclei processes distinct information in parallel; the BA would deal with polymodal or contextual representations, and the LA would process unimodal or elemental representations. Thus, it seems plausible to hypothesize that the BA is required for contextual, but not auditory, fear conditioning. This hypothesis was evaluated in Wistar rats submitted to multiple-site ibotenate-induced damage restricted to the BA and then exposed to a concurrent contextual and auditory fear conditioning training followed by separated contextual and auditory conditioning testing. Differing from electrolytic lesion and lidocaine inactivation, this surgical approach does not disturb fibers of passage originating in other brain areas, restricting damage to the aimed nucleus. Relative to the sham-operated controls, rats with selective damage to the BA exhibited disruption of performance in the contextual, but not the auditory, component of the task. Thus, while the BA seems required for contextual fear conditioning, it is not critical for both an auditory-US association, nor for the expression of the freezing response.     

Microinfusion of the D1 receptor antagonist, SCH23390 into the IL but not the BLA impairs consolidation of extinction of auditory fear conditioning

In auditory fear conditioning, repeated presentation of the tone in the absence of the shock leads to extinction of the acquired fear response. Both the medial prefrontal cortex (mPFC) and the basolateral amygdala (BLA) are involved in extinction. Here we examined this involvement by antagonizing D1 receptors in both regions, in the rat. We microinfused the D1 receptor antagonist, SCH23390, into the infra-limbic part of the mPFC (IL) or BLA at different time points. SCH23390 mircoinfused into the IL either before extinction acquisition or following short extinction training resulted in impairment of extinction consolidation. Microinfusion of SCH23390 into the BLA, prior to acquisition of extinction caused impairment in acquisition of extinction without affecting extinction consolidation. This is supported by the results showing that microinfusion of SCH23390 into the BLA following a short-training session did not affect consolidation. These results further strengthen the role of mPFC in consolidation of extinction while highlighting the role of the D1 receptors in this process.     

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.