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.     

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.     

NMDA receptor-dependent processes in the medial prefrontal cortex are important for acquisition and the early stage of consolidation during trace, but not delay eyeblink conditioning

Permanent lesions in the medial prefrontal cortex (mPFC) affect acquisition of conditioned responses (CRs) during trace eyeblink conditioning and retention of remotely acquired CRs. To clarify further roles of the mPFC in this type of learning, we investigated the participation of the mPFC in mnemonic processes both during and after daily conditioning using local microinfusion of the GABA(A) receptor agonist muscimol or the NMDA receptor antagonist APV into the rat mPFC. Muscimol infusions into the mPFC before daily conditioning significantly retarded CR acquisition and reduced CR expression if applied after sufficient learning. APV infusion also impaired acquisition of CRs, but not expression of well-learned CRs. When infusions were made immediately after daily conditioning, acquisition of the CR was partially impaired in both the muscimol and APV infusion groups. In contrast, rats that received muscimol infusions 3 h after daily conditioning exhibited improvement in their CR performance comparable to that of the control group. Both the pre- and post-conditioning infusion of muscimol had no effect on acquisition in the delay paradigm. These results suggest that the mPFC participates in both acquisition of a CR and the early stage of consolidation of memory in trace, but not delay eyeblink conditioning by NMDA receptor-mediated operations.     

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.     

Hippocampal regulation of context-dependent neuronal activity in the lateral amygdala

Pavlovian fear conditioning is a robust and enduring form of emotional learning that provides an ideal model system for studying contextual regulation of memory retrieval. After extinction the expression of fear conditional responses (CRs) is context-specific: A conditional stimulus (CS) elicits greater conditional responding outside compared with inside the extinction context. Dorsal hippocampal inactivation with muscimol attenuates context-specific CR expression. We have previously shown that CS-elicited spike firing in the lateral nucleus of the amygdala is context-specific after extinction. The present study examines whether dorsal hippocampal inactivation with muscimol disrupts context-specific firing in the lateral amygdala. We conditioned rats to two separate auditory CSs and then extinguished each CS in separate and distinct contexts. Thereafter, single-unit activity and conditional freezing were tested to one CS in both extinction contexts after saline or muscimol infusion into the dorsal hippocampus. After saline infusion, rats froze more to the CS when it was presented outside of its extinction context, but froze equally in both contexts after muscimol infusion. In parallel with the behavior, lateral nucleus neurons exhibited context-dependent firing to extinguished CSs, and hippocampal inactivation disrupted this activity pattern. These data reveal a novel role for the hippocampus in regulating the context-specific firing of lateral amygdala neurons after fear memory extinction.     

Distinct contributions of the basolateral amygdala and the medial prefrontal cortex to learning and relearning extinction of context conditioned fear

We studied the roles of the basolateral amygdala (BLA) and the medial prefrontal cortex (mPFC) in learning and relearning to inhibit context conditioned fear (freezing) in extinction. In Experiment 1, pre-extinction BLA infusion of the NMDA receptor (NMDAr) antagonist, ifenprodil, impaired the development and retention of inhibition but post-extinction infusion spared retention. Pre-extinction infusion of the GABA(A) agonist, muscimol, depressed freezing and impaired retention as did post-extinction infusion. In Experiment 2, pre-extinction mPFC infusion of ifenprodil spared the development of inhibition whereas muscimol depressed freezing. Both impaired retention when infused pre- or post-extinction. Thus, the development of inhibition involves NMDAr activation in the BLA, whereas its consolidation involves both NMDAr activation in the mPFC and NMDAr-independent mechanisms in the BLA. In Experiment 3, BLA infusion of ifenprodil impaired relearning and retention of inhibition when infused before but did not impair retention when infused after re-extinction. BLA infusion of muscimol depressed freezing but did not impair retention when infused before or after re-extinction. In Experiment 4, mPFC infusion of ifenprodil impaired relearning when infused before re-extinction, whereas muscimol depressed responses. Both drugs impaired retention when infused into the mPFC before or after re-extinction. Thus, relearning to inhibit fear responses involves NMDAr activation in both the BLA and mPFC and consolidation of the inhibitory memory involves NMDAr activation in the mPFC. However, relearning and consolidation occur in the absence of neuronal activity within the BLA. We propose that NMDAr in the mPFC supports relearning inhibition when the BLA is inactivated.     

Contextual Fear after Signalled versus Unsignalled Shocks: Effect of Extent of Prior Experience with Context and Signal

In the first two experiments, rats were differentially familiarized with a discrete stimulus and/ or a context prior to receiving either shocks signalled by that stimulus or unsignalled shocks in that context. As indexed by freezing, in none of the pre-exposure conditions did the signalled-shock rats consistently acquire less contextual fear than the unsignalled-shock animals. Both pre-exposure to the stimulus and relatively short pre-exposure to the future conditioning context resulted in more contextual fear in the signalled-shock than in the unsignalled-shock subjects. In a third experiment, freezing in the target conditioning context was especially enhanced in rats that had been familiarized with a stimulus, conditioned with the stimulus as a signal for shock, and subsequently further conditioned to the stimulus in a different, non-target context. The level of freezing to the stimulus in a neutral test context was positively related to the level of freezing in the target conditioning context in all experiments. These results were discussed in terms of context-shock, stimulus-shock, and context-stimulus associations.     

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.     

Scopolamine Selectively Disrupts the Acquisition of Contextual Fear Conditioning in Rats

Muscarinic cholinergic antagonism produces learning and memory deficits in a variety of hippocampal-dependent tasks. Hippocampal lesions produce both acquisition deficits and retrograde amnesia for contextual fear conditioning, but do not impact fear conditioning to discrete cues. In order to examine the effects of muscarinic antagonism in this paradigm, rats were given scopolamine (1 mg/kg) either before or for 3 days after a Pavlovian fear-conditioning session in which tones were paired with aversive footshocks. Fear to the context and the tone was assessed by measuring freezing in separate tests. It was found that pretraining, but not posttraining, scopolamine severely impaired contextual fear conditioning; tone conditioning was not affected under either condition (cf., Young, Bohenek, & Fanselow,Neurobiology of Learning and Memory,63,174–180, 1995).     

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.     

Complex effects of NMDA receptor antagonist APV in the basolateral amygdala on acquisition of two-way avoidance reaction and long-term fear memory

Although much has been learned about the role of the amygdala in Pavlovian fear conditioning, relatively little is known about an involvement of this structure in more complex aversive learning, such as acquisition of an active avoidance reaction. In the present study, rats with a pretraining injection of the N-methyl-D-aspartate (NMDA) receptor antagonist, 2-amino-5-phosphonopentanoic acid (APV), into the basolateral amygdala (BLA) were found to be impaired in two-way active avoidance learning. During multitrial training in a shuttle box, the APV-injected rats were not different from the controls in sensitivity to shock or in acquisition of freezing to contextual cues. However, APV injection led to impaired retention of contextual fear when tested 48 h later, along with an attenuation of c-Fos expression in the amygdala. These results are consistent with the role of NMDA receptors of the BLA in long-term memory of fear, previously documented in Pavlovian conditioning paradigms. The APV-induced impairment in the active avoidance learning coincided with deficits in directionality of the escape reaction and in attention to conditioned stimuli. These data indicate that normal functioning of NMDA receptors in the basolateral amygdala is required during acquisition of adaptive instrumental responses in a shuttle box but is not necessary for acquisition of short-term contextual fear in this situation.     

Effects of the selective M1 muscarinic receptor antagonist dicyclomine on emotional memory

The nonselective muscarinic antagonist scopolamine is known to impair the acquisition of some learning tasks such as inhibitory avoidance. There has been recent research into the effects of this drug in contextual fear conditioning and tone fear conditioning paradigms. The purpose of the present study was to assess the role of the selective M1 muscarinic antagonist dicyclomine in these paradigms and in the inhibitory avoidance test. Rats were administered different doses of dicyclomine or saline 30 min before acquisition training. The animals were tested 24 hr later, and it was observed that 16 mg/kg of dicyclomine impaired both contextual fear conditioning and inhibitory avoidance. However, dicyclomine (up to 64 mg/kg) did not affect tone fear conditioning. These results suggest that the selective M1 muscarinic antagonist dicyclomine differentially affects aversively motivated tasks known to be dependent on hippocampal integrity (such as contextual fear conditioning and inhibitory avoidance) but does not affect similar hippocampus-independent tasks.     

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.     

Enhancement of extinction memory consolidation: the role of the noradrenergic and GABAergic systems within the basolateral amygdala

Evidence from previous studies indicates that the noradrenergic and GABAergic influences within the basolateral amygdala (BLA) modulate the consolidation of memory for fear conditioning. The present experiments investigated whether the same modulatory influences are involved in regulating the extinction of fear-based learning. To investigate this issue, male Sprague Dawley rats implanted with unilateral or bilateral cannula aimed at the BLA were trained on a contextual fear conditioning (CFC) task and 24 and 48 h later were given extinction training. Immediately following each extinction session they received intra-BLA infusions of the GABAergic antagonist bicuculline (50 ng), the beta-adrenocepter antagonist propranolol (500 ng), bicuculline with propranolol, norepinephrine (NE) (0.3, 1.0, and 3.0 microg), the GABAergic agonist muscimol (125 ng), NE with muscimol or a control solution. To investigate the involvement of the dorsal hippocampus (DH) as a possible target of BLA activation during extinction, other animals were given infusions of muscimol (500 ng) via an ipsilateral cannula implanted in the DH. Bilateral BLA infusions of bicuculline significantly enhanced extinction, as did infusions into the right, but not left BLA. Propranolol infused into the right BLA together with bicuculline blocked the bicuculline-induced memory enhancement. Norepinephrine infused into the right BLA also enhanced extinction, and this effect was not blocked by co-infusions of muscimol. Additionally, muscimol infused into the DH did not attenuate the memory enhancing effects of norepinephrine infused into the BLA. These findings provide evidence that, as with original CFC learning, noradrenergic activation within the BLA modulates the consolidation of CFC extinction. The findings also suggest that the BLA influence on extinction is not mediated by an interaction with the dorsal hippocampus.     

Nicotine Produces a Within-Subject Enhancement of Contextual Fear Conditioning in C57BL/6 Mice Independent of Sex

Nicotine enhances learning including contextual fear conditioning. The present study extends previous work on nicotine and conditioned fear to examine the nature of nicotine's enhancement of contextual fear conditioning and sex differences in contextual fear conditioning in C57BL/6 mice using a within-subjects design. Mice were trained by pairing of an auditory stimulus of 80 dB, 6 cps train of broad-band clicks conditioned stimulus (CS) with a 2 sec., 0.35 mA shock unconditioned stimulus (US). Twenty-four hours later mice were tested for freezing in the original context, and one hour later mice were retested in the same context. A 0.5 mg/kg dose of nicotine was given either for three conditions: (1) before training, testing, and retesting; (2) before training and retesting; and (3) before retesting only. The use of a within-subjects design allowed for testing if nicotine would produce state-dependent deficits in contextual fear conditioning. Nicotine did enhance contextual fear conditioning in the groups that received nicotine for both training and testing. Nicotine, however, did not alter freezing when given on training but not testing or testing but not training. No sex differences, however, existed for conditioning or for nicotine's effects on conditioning. These results suggest that nicotine enhanced acquisition and retrieval processes but did not produce state-dependent deficits when administered just for training or just for testing.     

Nicotine enhances contextual fear conditioning in C57BL/6J mice at 1 and 7 days post-training

Nicotine has been demonstrated to enhance learning processes. The present experiments extend these results to examine the effects of nicotine on acquisition and consolidation of contextual and cued fear conditioning, and the duration of nicotine's enhancement of conditioned fear. C57BL/6 mice were trained with two pairings of an auditory CS and a foot shock US. Multiple doses of nicotine were given before or immediately after training and on testing day (0.0, 0.050, 0.125, 0.250, and 0.375 mg/kg, i.p). Freezing to both the context and auditory CS was measured 24h after training and again 1 week after training. Mice did not receive nicotine for the 1-week retest. Nicotine (0.125 and 0.250 mg/kg) given on both training and testing days enhanced freezing to the context at 24h. In addition, elevated freezing to the context was seen 1 week post-training in mice previously treated with 0.125 and 0.250 mg/kg nicotine. Thus, nicotine-treated mice did show elevated levels of freezing when retested 1 week later, even though no nicotine was administered at the 1-week retest. Mice that received nicotine on training day or testing day only and mice that received nicotine with mecamylamine, a nicotinic receptor antagonist, were not different from saline-treated mice. In addition, post-training administration of nicotine did not enhance fear conditioning. The present results indicate that nicotine enhancement of contextual fear conditioning depends on administration of nicotine on training and test days but results in a long-lasting enhancement of memories of contextual fear conditioning that remains in the absence of nicotine.     

Nicotine produces a within-subject enhancement of contextual fear conditioning in C57BL/6 mice independent of sex

Nicotine enhances learning including contextual fear conditioning. The present study extends previous work on nicotine and conditioned fear to examine the nature of nicotine’s enhancement of contextual fear conditioning and sex differences in contextual fear conditioning in C57BL/6 mice using a within-subjects design. Mice were trained by pairing of an auditory stimulus of 80 dB, 6 cps train of broad-band clicks conditioned stimulus (CS) with a 2 sec., 0.35 mA shock unconditioned stimulus (US). Twenty-four hours later mice were tested for freezing in the original context, and one hour later mice were retested in the same context. A 0.5 mg/kg dose of nicotine was given either for three conditions: (1) before training, testing, and retesting; (2) before training and retesting; and (3) before retesting only. The use of a within-subjects design allowed for testing if nicotine would produce state-dependent deficits in contextual fear conditioning. Nicotine did enhance contextual fear conditioning in the groups that received nicotine for both training and testing. Nicotine, however, did not alter freezing when given on training but not testing or testing but not training. No sex differences, however, existed for conditioning or for nicotine’s effects on conditioning. These results suggest that nicotine enhanced acquisition and retrieval processes but did not produc state-dependent deficits when administered just for training or just for testing.     

Temporally graded,context-specific retrograde amnesia and its alleviation by context preexposure: effects of postconditioning exposures to morphine in the rat

Five experiments studied retrograde impairments in Pavlovian fear conditioning following prolonged exposure to the opioid receptor agonist morphine. Injections of morphine commencing 1-7 days but not 14 days after conditioning produced amnesia for that conditioning episode. This amnesia was (a) selective such that morphine impaired freezing to the conditioning context but not to the auditory conditioned stimulus, (b) independent of the interval between the last injection of morphine and test, and (c) accompanied by a failure of contextual discrimination. Context preexposure protected context conditioning and discrimination from the amnestic effects of morphine. These results show that retrograde deficits in contextual fear conditioning are mediated by failures to consolidate a contextual representation.     

The memory-impairing effects of septal GABA receptor activation involve GABAergic septo-hippocampal projection neurons

Septal infusions of the gamma-aminobutyric acid (GABA)(A) agonist muscimol impair memory, and the effect likely involves the hippocampus. GABA(A) receptors are present on the perikarya of cholinergic and GABAergic septo-hippocampal (SH) projections. The current experiments determined whether GABAergic SH projections are involved in the memory-impairing effects of septal GABA(A) receptor activation. Experiment 1 tested whether combining septal co-infusions of subeffective doses of muscimol with scopolamine, a drug that selectively influences GABA SH projections, would produce memory deficits. Experiment 2 tested whether hippocampal infusions of a GABA(A) receptor antagonist would block the effects of septal muscimol infusions. Fifteen minutes prior to assessing spontaneous alternation (SA) or training in a multiple trial inhibitory avoidance (CMIA) task, male Sprague-Dawley rats were given septal infusions of vehicle, muscimol, scopolamine, or co-infusions of muscimol with scopolamine, or septal infusions of vehicle or muscimol combined with hippocampal infusions of vehicle or bicuculline. Septal co-infusions of muscimol with scopolamine significantly impaired SA and CMIA. Hippocampal bicuculline infusions blocked deficits produced by septal muscimol infusions in SA and attenuated deficits produced in CMIA. Combined, these findings suggest that GABAergic SH projections are involved in the memory-impairing effects of septal GABA receptor activation.     

Pre-training prevents context fear conditioning deficits produced by hippocampal NMDA receptor blockade

These experiments explore parallels between the neurobiological substrates of spatial and context learning. Male Long-Evans rats were employed in a context fear conditioning protocol that involved sequential acquisition and testing in two distinct contexts. Rats received three unsignaled footshocks in one context and were tested for context fear, measured as freezing, the next day. Four days later, the procedure was repeated in a different, distinct context. Rats received a hippocampal infusion of either the NMDA receptor antagonist 5-amino-phosphonovaleric acid (APV, 10 microg) or vehicle prior to training in each context. NMDA receptor blockade was effective in impairing context learning only in the first context. Context fear acquisition was not impaired by APV in the second context, indicating that pre-training (in the first context) mitigated the effects of APV. These data agree with those seen previously in the water maze, where pre-training prevented learning deficits produced by NMDA receptor blockade. The data thus suggest that the neurobiological substrates of context learning and place learning overlap.