Continuous Multiple-trial Inhibitory Avoidance Research Articles

Hippocampal infusions of glucose reverse memory deficits produced by co-infusions of a GABA receptor agonist

Although septal infusions of glucose typically have positive effects on memory, we have shown repeatedly that this treatment exacerbates memory deficits produced by co-infusions of γ-aminobutyric acid (GABA) receptor agonists. The present experiments tested whether this negative interaction between glucose and GABA in the medial septum would be observed in the hippocampus, a brain region where glucose typically has positive effects on memory. Specifically, we determined whether hippocampal infusions of glucose would reverse or exacerbate memory deficits produced by hippocampal co-infusions of the GABA receptor agonist muscimol. Fifteen minutes prior to either assessing spontaneous alternation (SA) or continuous multiple trial inhibitory avoidance (CMIA) training, male Sprague–Dawley-derived rats were given bilateral hippocampal infusions of vehicle (phosphate-buffered saline [PBS], 1 μl/2 min), glucose (33 or 50 nmol), muscimol (0.3 or 0.4 μg, SA or 3 μg, CMIA) or muscimol and glucose combined in one solution. The results indicated that hippocampal infusions of muscimol alone decreased SA scores and CMIA retention latencies. More importantly, hippocampal infusions of glucose, at doses that had no effect when infused alone, attenuated (33 nmol) or reversed (50 nmol) the muscimol-induced memory deficits. Thus, although co-infusions of glucose with muscimol into the medial septum impair memory, the present findings show that an opposite effect is observed in the hippocampus. Collectively, these findings suggest that the memory-impairing interaction between glucose and GABA in the medial septum is not a general property of the brain, but rather is brain region-dependent.

Theophylline attenuates microwave-induced impairment of memory acquisition

Numerous studies have shown that acute microwave exposure causes cognitive deficits in animals, possibly via hyperthermia, but the biological effect of microwave exposure on memory processing is still unknown. The release of adenosine is demonstrated to be a general way for the cells to respond to metabolically stressful conditions such as hypoxia and ischemia. The present study aimed to examine whether adenosine mediates biological effects of microwave exposure on memory processing using a continuous multiple-trial inhibitory avoidance task. Results demonstrated that microwave exposure for 20 min before training impaired memory acquisition and retention performance in mice, assessed by the number of training trials and by latency to enter the dark compartment. The mice exposed to microwave radiation showed a dose-dependent hyperthermia. Moreover, the cell numbers of hippocampus were decreased in the mice receiving microwave exposure at an average power density of 50 mW/cm 2, indicating the anatomical correlation to hippocampal-amygdaloid structures corresponding with the memory disrupt of the mice. Administration of theophylline, a nonspecific adenosine receptor antagonist, 30 min before microwave exposure, completely antagonized the impairment of inhibitory avoidance acquisition but not retention. These results suggest that the adenosine regulation pathway was partially involved in microwave-induced impairment of inhibitory avoidance memory.

Hippocampal infusions of pyruvate reverse the memory-impairing effects of septal muscimol infusions

Hippocampal infusions of glucose reverse memory deficits in spontaneous alternation and in a continuous multiple trial inhibitory avoidance task. The current experiments tested whether glucose metabolism may participate in these effects of glucose. Specifically, these experiments determined whether the glycolytic metabolite pyruvate would mimic these effects of glucose. Male Sprague–Dawley rats were given septal infusions of vehicle or the gamma-aminobutyric acid (GABA) receptor agonist muscimol (0.15 nmol for spontaneous alternation or 5 nmol for continuous multiple trial inhibitory avoidance) combined with hippocampal infusions of vehicle or pyruvate (200 nmol) 15 min prior to assessing spontaneous alternation or training in a continuous multiple trial inhibitory avoidance task. The infusions of muscimol decreased percent alternation scores and continuous multiple trial inhibitory avoidance retention latencies tested 48 h after training. More importantly, hippocampal infusions of pyruvate reversed the deficits produced by septal infusions of muscimol on both tasks. These findings show for the first time that hippocampal infusions of pyruvate influence memory and suggest that glucose may affect memory via glycolytic metabolism.

The enhancing effects of hippocampal infusions of glucose are not restricted to spatial working memory

Extensive evidence shows that hippocampal infusions of glucose enhance spontaneous alternation (SA) performance or reverse deficits in this task. The current experiments determined whether the enhancing effects of hippocampal infusions of glucose are restricted to spatial working memory. Specifically we tested whether hippocampal infusions of glucose would reverse deficits in an emotional reference memory task (continuous multiple trial inhibitory avoidance [CMIA]) produced by septal infusions of the γ-aminobutyric acid agonist muscimol. Male Sprague–Dawley rats were given septal infusions of vehicle or muscimol (0.15 nmol: SA; 5 nmol: CMIA) combined with hippocampal infusions of vehicle or glucose (50 nmol) 15 min prior to assessing SA or CMIA training. CMIA retention was tested 48 h later. Muscimol infusions decreased percent alternation scores and avoidance retention latencies. Importantly, hippocampal infusions of glucose reversed the deficits produced by the septal muscimol infusions on both tasks. These findings show for the first time that hippocampal glucose infusions also influence emotional memory, indicating that the enhancing effects of glucose generalize to memory tasks that vary in motivational and cognitive demand.

Phthalic Acid Amygdalopetal Lesion of the Nucleus Basalis Magnocellularis Induces Reversible Memory Deficits in Rats

The basolateral amygdala (BLA) is extensively implicated in emotional learning and memory. The current study investigated the contribution of cholinergic afferents to the BLA from the nucleus basalis magnocellularis in influencing aversive learning and memory. Sprague-Dawley rats were given permanent unilateral phthalic acid (300 ng) lesions of the nucleus basalis magnocellularis and were chronically implanted with cannulas aimed at the ipsilateral BLA. Lesioned rats showed a pronounced inhibitory avoidance task retention deficit that was attenuated by acute posttraining infusions of the muscarinic cholinergic agonist oxotremorine (4 ng) or the indirect agonist physostigmine (1 μg) into the BLA. Continuous multiple-trial inhibitory avoidance training and testing revealed that lesioned rats have a mild acquisition deficit, requiring approximately 1 additional shock to reach the criterion, and a pronounced consolidation deficit as indicated by a shorter latency to enter the shock compartment on the retention test. Because lesioned rats did not differ from sham-operated controls in performance on a spatial water maze task or in shock sensitivity, it is not likely that the memory impairments produced by the phthalic acid lesions are due to any general sensory or motor deficits. These findings suggest that the dense cholinergic projection from the nucleus basalis magnocellularis to the BLA is involved in both the acquisition and the consolidation of the aversive inhibitory avoidance task.

Infusions of physostigmine into the hippocampus or the entorhinal cortex attenuate avoidance retention deficits produced by intra-septal infusions of the GABA agonist muscimol

Septal γ-aminobutyric acid (GABA) receptor activation is known to disrupt memory formation, although the mechanisms underlying this impairment remain unclear. The present study explored the possibility that high levels of septal GABA receptor activity might impair memory by down-regulating acetylcholine (ACh) function in archicortex and entorhinal cortex. To test this possibility, rats were trained on an avoidance task 15 min after receiving intra-septal infusions of vehicle or muscimol (5 nmol/0.5 μl) combined with unilateral intra-hippocampal (10 μl/1 μl) or intra-entorhinal cortex (1.875 μg/0.25 μl) infusions of vehicle or the acetylcholinesterase inhibitor physostigmine. We demonstrate that these infusions do not alter acquisition performance on a continuous multiple trial inhibitory avoidance task. However, intra-septal infusions of muscimol dramatically impair retention performance 48 h later. More importantly, infusions of physostigmine into the hippocampus or the entorhinal cortex, at doses that do not influence acquisition or retention performance when infused alone, attenuate the impairing effects of the muscimol infusions on retention. We suggest that high levels of septal GABA receptor activity might impair memory by down-regulating ACh levels in the hippocampal region, and that such memory impairments can be ameliorated by increasing ACh levels in the hippocampus or entorhinal cortex.

Glucocorticoid interactions with the β-adrenoceptor-cyclic AMO system in the basolateral amygdala in regulating memory consolidation

We previously reported finding differential effects of unilateral infusion of lidocaine into the right and left amygdalae prior to a retention test 24 h after one-trial inhibitory avoidance training. The present study was undertaken to determine whether the degree of training in an escape task influences the effects of unilateral inactivation of the amygdala with lidocaine prior to the retention test. Male Sprague-Dawley rats, implanted with bilateral cannulae aimed at the amygdaloid complex received 0 (no shock), 2, or 10 training trials in a footshock-motivated escape task (0.4 mA). Forty-eight h later, the rats received bilateral microinfusions into the amygdalae of either buffer, or lidocaine into one amygdala and buffer in the other, 5 min before they were tested for retention of the escape training using a continuous multiple trial inhibitory avoidance (CMIA) procedure (0.4 mA). Forty-eight h after CMIA training the animals were retested, this time without influence of drugs. As indicated by initial response latencies on the first retention test, unilateral inactivation of either the right or the left amygdala severely impaired retention performance for the escape training regardless of the number of prior escape training trials received. However, unilateral inactivation of either amygdala had no effect on acquisition of CMIA. Furthermore, unilateral inactivation of either amygdala during CMIA training did not affect retention tested 48 h later. These results suggest that the differential involvement of the right and left amygdalae may be limited to certain training conditions. Furthermore, these findings demonstrate opposite effects on 2 different measures of retention indicating that the method of testing retention affects the interpretation of the memory impairment induced by infusion of lidocaine into the amygdala.

(±)-α-Methyl-4-carboxyphenylglycine, a metabotropic glutamate receptor blocker, impairs retention of an inhibitory avoidance task in rats when infused into the basolateral nucleus of the amygdala

The amygdala is important for memory processes of emotionally motivated learning and the amygdala glutamatergic system may play a key role in this process. In this study we assessed the effect of the infusion of (±)-α-methyl-4-carboxyphenylglycine (MCPG), a metabotropic glutamate receptor (mGluR) antagonist, into the basolateral complex of the amygdala (BLA) on the learning and retention of an emotionally motivated task. Rats received either vehicle or three different doses of MCPG (0.2, or 1.0, or 5.0 μg/0.2μl/side, respectively) bilaterally into the BLA, 5 min before they were trained in a continuous multiple-trial inhibitory avoidance (CMIA) task. Response latencies during the training were recorded. Retention was assessed 8 days later. MCPG in the doses given did not significantly affect the acquisition of the CMIA task. However, MCPG at a dose of 5.0 μg/0.2 μl/side impaired the long-term retention test performance. Additionally, a nociception test indicated that dose of MCPG infused into the BLA did not affect the footshock sensitivity. Our results indicate that MCPG, when infused into the BLA of rats prior to the training, impaired long-term memory of aversive training without affecting acquisition.

Task-dependent effects of the antidepressant/antipanic drug phenelzine on memory.

Phenelzine (PLZ) is a non-selective monoamine oxidase (MAO) inhibitor commonly used to treat depression and panic disorder. In addition to increasing levels of biogenic amines in the brain, PLZ elevates brain levels of the amino acid gamma-aminobutyric acid (GABA; Baker et al. 1991; present study). Given the extensive evidence implicating biogenic amines and GABA in mnemonic processes, PLZ may affect learning and memory. To investigate this possibility, male Sprague-Dawley rats were given PLZ sulfate (15 or 30 mg/kg, based on free base weight) 2 h prior to training in a continuous multiple trial inhibitory avoidance (CMIA) and spatial water maze task. Retention was assessed 48 h later. The results indicated that PLZ enhanced CMIA and impaired water maze retention performance. Compared to control rats, rats given PLZ took significantly longer to re-enter the shock compartment and swam longer distances before reaching the escape platform on the retention tests. These effects of PLZ did not appear to be the result of PLZ-induced changes in acquisition or retrieval processes, activity levels, or footshock sensitivity. Combined, these findings indicate that PLZ influences memory in a task-dependent manner. These differential effects of PLZ may be the result of contrasting influences of GABA and biogenic amines on memory.

Opposite effects on two different measures of retention following unilateral inactivation of the amygdala

We previously reported finding differential effects of unilateral infusion of lidocaine into the right and left amygdalae prior to a retention test 24 h after one-trial inhibitory avoidance training. The present study was undertaken to determine whether the degree of training in an escape task influences the effects of unilateral inactivation of the amygdala with lidocaine prior to the retention test. Male Sprague-Dawley rats, implanted with bilateral cannulae aimed at the amygdaloid complex received 0 (no shock), 2, or 10 training trials in a footshock-motivated escape task (0.4 mA). Forty-eight h later, the rats received bilateral microinfusions into the amygdalae of either buffer, or lidocaine into one amygdala and buffer in the other, 5 min before they were tested for retention of the escape training using a continuous multiple trial inhibitory avoidance (CMIA) procedure (0.4 mA). Forty-eight h after CMIA training the animals were retested, this time without influence of drugs. As indicated by initial response latencies on the first retention test, unilateral inactivation of either the right or the left amygdala severely impaired retention performance for the escape training regardless of the number of prior escape training trials received. However, unilateral inactivation of either amygdala had no effect on acquisition of CMIA. Furthermore, unilateral inactivation of either amygdala during CMIA training did not affect retention tested 48 h later. These results suggest that the differential involvement of the right and left amygdalae may be limited to certain training conditions. Furthermore, these findings demonstrate opposite effects on 2 different measures of retention indicating that the method of testing retention affects the interpretation of the memory impairment induced by infusion of lidocaine into the amygdala.

Adrenocortical suppression blocks the memory-enhancing effects of amphetamine and epinephrine.

This study examined glucocorticoid-adrenergic interactions in modulating acquisition and memory storage for inhibitory avoidance training. Systemically (s.c.) administered amphetamine (1 mg/kg), but not epinephrine (0.1 mg/kg) or the peripherally acting amphetamine derivative 4-OH amphetamine (2 mg/kg), given to rats shortly before training facilitated acquisition performance in a continuous multiple-trial inhibitory avoidance (CMIA) task. Adrenocortical suppression with the 11beta-hydroxylase inhibitor metyrapone (50 mg/kg; s.c.), given to rats 90 min before training, did not block the effect of amphetamine and did not affect acquisition performance of otherwise untreated animals. Retention of CMIA and one-trial inhibitory avoidance was enhanced by either pre- or posttraining injections of amphetamine as well as 4-OH amphetamine and epinephrine. The finding that injections of amphetamine and epinephrine have comparable effects on memory is consistent with the view that amphetamine may modulate memory storage, at least in part, by inducing the release of epinephrine from the adrenal medulla. Metyrapone pretreatment blocked the memory-enhancing effects of amphetamine, 4-OH amphetamine, and epinephrine but did not affect retention performance of otherwise untreated animals. Posttraining injections of different doses of epinephrine (ranging from 0.0001 to 1.0 mg/kg) produced a dose-dependent memory enhancement for inhibitory avoidance training and metyrapone blocked the memory-enhancing effects of all these doses. These findings provide further evidence that the sympathoadrenal and adrenocortical systems are intimately coupled during processes of memory storage.

Footshock facilitates the expression of aversively motivated memory in rats given post-training amygdala basolateral complex lesions

We previously reported that increased training in an escape task partially attenuates the memory impairment produced by large amygdala lesions induced 1 week following training. The present study examined the effect of amount of preoperative training on the retention of rats with lesions restricted to the amygdala basolateral complex. Rats received 1 or 10 training trials in a footshock-motivated escape task and 1 week later sham lesions or neurotoxic lesions of the basolateral complex. Four days after recovery from the surgery they were tested for inhibitory avoidance retention and then 2 days later given continuous multiple trial inhibitory avoidance training (CMIA) in the same apparatus. The basolateral complex lesions significantly decreased the retention latencies of rats given 1 or 10 trials. However, following administration of footshock on the CMIA task, the performance of the lesioned rats reflected the degree of preoperative escape training. The basolateral complex lesions also increased open field locomotor activity, an effect that may have contributed to the shorter retention latencies in lesioned animals. These findings indicate that an intact amygdala basolateral complex is not critical for the retention of the escape training.

Muscimol injected into the right or left amygdaloid complex differentially affects retention performance following aversively motivated training

The effects of intra-amygdala infusion of the GABA A agonist, muscimol, prior to retention testing was examined. In Expt. 1, rats were trained in a one-trial inhibitory-avoidance task and given bilateral intra-amygdala infusions of vehicle or muscimol or simultaneous unilateral infusions of each, 5 min before the retention test 24 h after training. Expt. 2 used the same procedures as those in Expt. 1 but two retention measures were taken: initial step-through latency and the number of trials to reach criterion during continuous multiple-trial inhibitory-avoidance (CMIA) training. Groups given bilateral infusions of muscimol or unilateral infusion of muscimol into the right amygdala had significantly shorter latencies to enter the dark compartment than groups given bilateral infusions of vehicle or unilateral infusion of muscimol into the left amygdala. Bilateral muscimol infusions impaired acquisition of CMIA relative to bilateral vehicle infusions or unilateral muscimol infusion into the left amygdala. These results suggest differential involvement of the right and left amygdalae in memory.

Differential effects of pretraining inactivation of the right or left amygdala on retention of inhibitory avoidance training.

Rats with bilateral cannulas aimed at the amygdalae received bilateral infusions of either buffer or lidocaine hydrochloride, or unilateral infusions of each, 5 min before continuous multiple-trial inhibitory avoidance (CMIA) training. Retention was tested 48 hr later. Some of the rats were retrained at this time and tested again 48 hr later. Bilateral infusions of lidocaine prior to the initial training impaired acquisition, retention, and relearning of the CMIA task. Unilateral infusions of lidocaine into the right or left amygdala did not affect acquisition. Rats given lidocaine into the right amygdala were impaired on retention 48 hr later. The findings are consistent with others indicating involvement of the amygdala in acquisition and consolidation of aversively motivated learning and suggest possible differential involvement of the right and left amygdalae in memory consolidation.

Infusion of the GABAergic antagonist bicuculline into the medial septal area does not block the impairing effects of systemically administered midazolam on inhibitory avoidance retention

This experiment investigated the effect of intraseptal administration of the GABAergic antagonist bicuculline methiodide on benzodiazepine-induced amnesia. Male Sprague-Dawley rats were implanted with cannula aimed at the medial septal area and allowed to recover for 1 week. Ten minutes prior to training in a continuous multiple trial inhibitory avoidance task, buffer solution or bicuculline methiodide (56 or 100 pmol/0.5 microliter) was injected into the medial septal area. This infusion was immediately followed by systemic (ip) administration of saline or midazolam (1.5 or 3.0 mg/kg). In comparison with saline controls, animals given the higher dose of midazolam (3.0 mg/kg), required more trials to reach acquisition criterion (remaining in the starting chamber for 100 s). This midazolam-induced acquisition deficit was blocked by an intraseptal infusion of bicuculline methiodide (100 pmol). On a 48-h retention test the performance of animals given either dose of midazolam was significantly impaired relative to vehicle controls. Furthermore, although intraseptal infusion of bicuculline methiodide prior to systemic injection of midazolam blocked the midazolam-induced acquisition impairment, bicuculline did not block the midazolam-induced retention impairment. These results suggest that although the medial septal area may be involved in midazolam-induced acquisition deficits, this area is not a critical site of action for benzodiazepine-induced effects on inhibitory avoidance retention.

Memory of rats with amygdala lesions induced 30 days after footshock-motivated escape training reflects degree of original training.

This experiment was conducted to determine whether the amount of preoperative training influences the effects, on retention, of amygdala lesions induced 30 days after escape training. Rats received 1 or 10 footshock-motivated escape training trials; 30 days later, sham or neurotoxic amygdala lesions were induced. Results of an inhibitory avoidance test performed 4 days after surgery indicated that amygdala lesions impaired retention performance; however, increased preoperative training partially attenuated the retention deficit. Increased preoperative training also attenuated the impairing effects of the lesions on retention assessed in a continuous multiple-trial inhibitory avoidance task given 36 days after the original escape training. The finding that amygdala-lesioned rats remembered the escape training suggests that the amygdala is not a critical locus of the changes underlying the long-term retention of footshock-motivated escape training.

Bicuculline administered into the amygdala blocks benzodiazepine-induced amnesia

This experiment investigated the effect of intra-amygdala administration of the GABAergic antagonist bicuculline methiodide on benzodiazepine-induced amnesia. Male Sprague-Dawley rats were implanted bilaterally with cannulae aimed at the amygdala and allowed to recover for 1 week. Ten minutes before training in a continuous multiple trial inhibitory avoidance task a buffer solution or bicuculline methiodide (56 pmol/0.5 microliters) was injected bilaterally into the amygdala and this injection was immediately followed by a systemic injection of saline or midazolam (1.0 mg/kg). In comparison with saline controls, midazolam-treated animals required more trials to reach the acquisition criterion of remaining in the starting chamber for 100 s. The midazolam effect on acquisition was not attenuated by intra-amygdala infusion of bicuculline methiodide, suggesting that the midazolam-induced changes in acquisition behavior do not involve the amygdaloid GABAergic system. On a 48-h retention test the performance of the midazolam-treated animals was significantly poorer than that of the controls. However, the retention performance of animals given intra-amygdala injections of bicuculline methiodide prior to the systemic injection of midazolam was comparable to that of the saline controls. These results suggest that the amygdaloid GABAergic system mediates the impairing effects of midazolam on retention of inhibitory avoidance training.

Effects of intra-amygdala injections of NMDA receptor antagonists on acquisition and retention of inhibitory avoidance

These experiments examined the effects of intra-amygdala injections of NMDA receptor antagonists on the acquisition and retention of inhibitory avoidance. In Expt. I, rats received bilateral intra-amygdala injections of the NMDA antagonists d,l-AP5 (1–10 μg), d-AP5 (0.03–1 μg), CPP (0.125 or 0.375 μg), or MK-801 (0.2 or 0.5 μg) prior to training in a continuous multiple-trial inhibitory avoidance (CMIA) task. Acquisition of the task was not significantly affected by any of the drug injections. In contrast, all three competitive antagonists, d,l-AP5, d-AP5 and CPP, produced dose-dependent impairment of 48 h retention performance. Although the MK-801 injections did not significantly impair retention performance, the retention scores of the 0.5 μg MK-801 group were bimodally distributed, indicating retention impairment in a subgroup of the animals given that dose. Intra-amygdala injections of 3 or 10 μg d,l-AP5 did not affect footshock sensitivity (Expt. II) or locomotor activity (Expt. III) and their retention-impairing effects were not due to induction of state dependency (Expt. IV). The retention-impairing effects of intra-amygdala injections of NMDA antagonists were not due to diffusion of the drugs dorsally: injections of 1 μg d-AP5 into the striatal area directly above the amygdala impaired acquisition but not retention performance (Expt. V). The retention-impairing effects of 1 μg d-AP5 or 0.5 μg MK-801 were attenuated by giving additional training to the animals shortly after receiving intra-amygdala injections (Expt. VI). The implications of these findings for hypotheses concerning amygdala function in learning and memory are discussed.

Basolateral amygdala lesions block diazepam-induced anterograde amnesia in an inhibitory avoidance task.

This experiment examined the effects of diazepam (DZP) on acquisition and retention of an inhibitory avoidance response by rats with excitotoxic-induced lesions of central (CE), lateral (LAT), or basolateral (BL) amygdala nuclei. Sham-operated and lesioned rats received i.p. injections of DZP (2.0 mg per kg of body weight) 30 min before training in a continuous multiple-trial inhibitory avoidance task. Retention was tested 48 h later. Acquisition was not impaired by the lesions or the DZP. Retention was impaired in animals with CE and LAT lesions in comparison with sham-operated controls. DZP impaired retention in the sham-operated controls as well as CE- and LAT-lesioned animals but did not affect retention in animals with BL lesions. These findings indicate that the DZP-induced anterograde amnesia for inhibitory avoidance training is mediated through influences involving the BL amygdala nucleus.

Increased training in an aversively motivated task attenuates the memory-impairing effects of posttraining N-methyl-D-aspartate-induced amygdala lesions.

This study was designed to examine the effect of variations in the amount of preoperative training on the retention deficit produced by posttraining lesions of the amygdaloid complex (AC). Rats received 1, 10, or 20 training trials in a footshock-motivated escape task 7 days before receiving N-methyl-D-aspartate lesions of the AC. Inhibitory avoidance retention performance, which was measured 4 days postoperatively, indicated that increased training improved retention in AC-lesioned animals as well as in control animals. The retention performance of AC-lesioned animals was impaired when compared with that of controls; however, the impairment was partially attenuated by increased preoperative training. The finding that AC-lesioned animals displayed greater locomotor activity on the retention test compared with nonlesioned controls suggests that the increased activity may have contributed to the impaired inhibitory avoidance retention performance. Two days after the retention test, some of the AC-lesioned animals were subsequently trained on a continuous multiple-trial inhibitory avoidance response in the same apparatus. AC lesions did not block acquisition or retention of the task. These findings suggest that the amygdala may not be a critical site for the permanent changes mediating stimulus-affect associations based on extensive training.