Selective excitotoxic lesions of the hippocampus and basolateral amygdala have dissociable effects on appetitive cue and place conditioning based on path integration in a novel Y-maze procedure

The hippocampus and amygdala are thought to be functionally distinct components of different learning and memory systems. This functional dissociation has been particularly apparent in pavlovian fear conditioning, where the integrity of the hippocampus is necessary for contextual conditioning, and of the amygdala for discrete cue conditioning. Their respective roles in appetitive conditioning, however, remain equivocal mainly due to the lack of agreement concerning the operational definition of a 'context'. The present study used a novel procedure to measure appetitive conditioning to spatial context or to a discrete cue. Following selective excitotoxic lesions of the hippocampus (HPC) or basolateral amygdala (BLA), rats were initially trained to acquire discrete CS-sucrose conditioning in a Y-maze apparatus with three topographically identical chambers, the chambers discriminated only on the basis of path integration. The same group of animals then underwent 'place/contextual conditioning' where the CS presented in a chamber assigned as the positive chamber was paired with sucrose, but the same CS presented in either of the other two chambers was not. Thus, spatial context was the only cue that the animal could use to retrieve the value of the CS. HPC lesions impaired the acquisition of conditioned place preference but facilitated the acquisition of cue conditioning, while BLA lesions had the opposite effect, retarding the acquisition of cue conditioning but leaving the acquisition of conditioned place preference intact. Here we provide strong support for the notion that the HPC and BLA subserve complementary and competing roles in appetitive cue and contextual conditioning.     

Brain tissue oxygen amperometry in behaving rats demonstrates functional dissociation of dorsal and ventral hippocampus during spatial processing and anxiety

Traditionally, the function of the hippocampus (HPC) has been viewed in unitary terms, but there is growing evidence that the HPC is functionally differentiated along its septotemporal axis. Lesion studies in rodents and functional brain imaging in humans suggest a preferential role for the septal HPC in spatial learning and a preferential role for the temporal HPC in anxiety. To better enable cross-species comparison, we present an in vivo amperometric technique that measures changes in brain tissue oxygen at high temporal resolution in freely-moving rats. We recorded simultaneously from the dorsal (septal; dHPC) and ventral (temporal; vHPC) HPC during two anxiety tasks and two spatial tasks on the radial maze. We found a double-dissociation of function in the HPC, with increased vHPC signals during anxiety and increased dHPC signals during spatial processing. In addition, dHPC signals were modulated by spatial memory demands. These results add a new dimension to the growing consensus for a differentiation of HPC function, and highlight tissue oxygen amperometry as a valuable tool to aid translation between animal and human research.     

NMDA receptor antagonism in the basolateral but not central amygdala blocks the extinction of Pavlovian fear conditioning in rats

Glutamate receptors in the basolateral complex of the amygdala (BLA) are essential for the acquisition, expression and extinction of Pavlovian fear conditioning in rats. Recent work has revealed that glutamate receptors in the central nucleus of the amygdala (CEA) are also involved in the acquisition of conditional fear, but it is not known whether they play a role in fear extinction. Here we examine this issue by infusing glutamate receptor antagonists into the BLA or CEA prior to the extinction of fear to an auditory conditioned stimulus (CS) in rats. Infusion of the α‐amino‐3‐hydroxyl‐5‐methyl‐4‐isoxazole‐propionate (AMPA) receptor antagonist, 2,3‐dihydroxy‐6‐nitro‐7‐sulfamoyl‐benzo[f]quinoxaline‐2,3‐dione (NBQX), into either the CEA or BLA impaired the expression of conditioned freezing to the auditory CS, but did not impair the formation of a long‐term extinction memory to that CS. In contrast, infusion of the N‐methyl‐d ‐aspartate (NMDA) receptor antagonist, d,l ‐2‐amino‐5‐phosphonopentanoic acid (APV), into the amygdala, spared the expression of fear to the CS during extinction training, but impaired the acquisition of a long‐term extinction memory. Importantly, only APV infusions into the BLA impaired extinction memory. These results reveal that AMPA and NMDA receptors within the amygdala make dissociable contributions to the expression and extinction of conditioned fear, respectively. Moreover, they indicate that NMDA receptor‐dependent processes involved in extinction learning are localized to the BLA. Together with previous work, these results reveal that NMDA receptors in the CEA have a selective role acquisition of fear memory.     

Extinction of auditory fear conditioning requires MAPK/ERK activation in the basolateral amygdala

Whereas the neuronal substrates underlying the acquisition of auditory fear conditioning have been widely studied, the substrates and mechanisms mediating the acquisition of fear extinction remain largely elusive. Previous reports indicate that consolidation of fear extinction depends on the mitogen-activated protein kinase/extracellular-signal regulated kinase (MAPK/ERK) signalling pathway and on protein synthesis in the medial prefrontal cortex (mPFC). Based on experiments using the fear-potentiated startle paradigm suggesting a role for neuronal plasticity in the basolateral amygdala (BLA) during fear extinction, we directly addressed whether MAPK/ERK signalling in the basolateral amygdala is necessary for the acquisition of fear extinction using conditioned freezing as a read-out. First, we investigated the regional and temporal pattern of MAPK/ERK activation in the BLA following extinction learning in C57Bl/6J mice. Our results indicate that acquisition of extinction is associated with an increase of phosphorylated MAPK/ERK in the BLA. Moreover, we found that inhibition of the MAPK/ERK signalling pathway by intrabasolateral amygdala infusion of the MEK inhibitor, U0126, completely blocks acquisition of extinction. Thus, our results indicate that the MAPK/ERK signalling pathway is required for extinction of auditory fear conditioning in the BLA, and support a role for neuronal plasticity in the BLA during the acquisition of fear extinction.     

Lesions of basolateral amygdala impair extinction of CS motivational value,but not of explicit conditioned responses,in Pavlovian appetitive second-order conditioning

The basolateral amygdala (BLA) is important for the modification of the motivational significance of events through associative learning. In previous work, we found that BLA was critical for the acquisition of conditioned reinforcement value to a visual conditioned stimulus (CS) paired with food. Unlike normal rats, rats with neurotoxic lesions of the BLA failed to acquire Pavlovian second-order conditioning to an auditory stimulus paired with the first-order visual CS in the absence of food. In this experiment, we examined the role of BLA in the extinction of the previously acquired conditioned reinforcement value of a Pavlovian CS. Rats received first-order visual CS-food pairings prior to either BLA- or sham-lesions. Subsequent CS-alone extinction training reduced the ability of the visual CS to reinforce second-order conditioning of an auditory stimulus in the sham-lesioned rats, but not in the BLA-lesioned rats. Despite this persistence of the conditioned reinforcement value of the visual first-order CS in the BLA-lesioned rats, no effects of the lesions were observed on extinction of the explicit behavioural conditioned responses elicited by that CS.     

Conditioned suppression and freezing as measures of aversive Pavlovian conditioning: effects of discrete amygdala lesions and overtraining

Freezing and suppression are measures of conditioned fear that correlate in unlesioned animals. Both the basolateral (BLA) and central (CeN) nuclei of the amygdala are required for conditioned freezing, though there can be recovery with overtraining. The neuroanatomical substrates of conditioned suppression are less clear, with evidence both for a specific requirement of the CeN and for disruption by BLA lesions. The present study investigated the impact of selective excitotoxic lesions of the BLA and CeN upon the acquisition and expression of conditioned fear, measured by freezing and both on-baseline and off-baseline conditioned suppression in the same rats. BLA and CeN lesions both abolished all measures of conditioned fear after 9 trials of fear conditioning. However, when conditioning was extended to 33 trials, whereas rats with combined lesions of both the BLA and CeN continued to show no conditioned fear responses, there was a pattern of recovery observed after selective lesions. There was a partial recovery of freezing with both lesions, and full recovery of conditioned suppression, except for off-baseline suppression in CeN lesioned rats. These results indicate that with few conditioning trials, both the BLA and CeN are required in a serial manner for conditioned fear responses, but that overtraining can mitigate such impairments, likely involving parallel pathways in and through the amygdala.     

Medial prefrontal cortex activity during the extinction of conditioned fear: an investigation using functional near-infrared spectroscopy

The majority of fear conditioning studies in humans have focused on fear acquisition rather than fear extinction. For this reason only a few functional imaging studies on fear extinction are available. A large number of animal studies indicate the medial prefrontal cortex (mPFC) as neuronal substrate of extinction. We therefore determined mPFC contribution during extinction learning after a discriminative fear conditioning in 34 healthy human subjects by using functional near-infrared spectroscopy. During the extinction training, a previously conditioned neutral face (conditioned stimulus, CS+) no longer predicted an aversive scream (unconditioned stimulus, UCS). Considering differential valence and arousal ratings as well as skin conductance responses during the acquisition phase, we found a CS+ related increase in oxygenated haemoglobin concentration changes within the mPFC over the time course of extinction. Late CS+ trials further revealed higher activation than CS- trials in a cluster of probe set channels covering the mPFC. These results are in line with previous findings on extinction and further emphasize the mPFC as significant for associative learning processes. During extinction, the diminished fear association between a former CS+ and a UCS is inversely correlated with mPFC activity--a process presumably dysfunctional in anxiety disorders.     

Fear conditioning in adolescents with anxiety disorders: results from a novel experimental paradigm

OBJECTIVE: Considerable research examines fear conditioning in adult anxiety disorders but few studies examine youths. Adult data suggest that anxiety disorders involve elevated fear but intact differential conditioning. We used a novel paradigm to assess fear conditioning in pediatric anxiety patients. METHOD: Sixteen individuals with anxiety disorders and 38 healthy comparisons viewed two photographs of actresses displaying neutral expressions. One picture served as the conditioned stimulus (CS), paired with a fearful expression and a shrieking scream (CS+), whereas the other picture served as a CS unpaired with the aversive outcome (CS-). Conditioning was indexed by self-reported fear. Subjects participated in two visits involving conditioning and extinction trials. RESULTS: Both groups developed greater fear of the CS+ relative to CS-. Higher fear levels collapsed across each CS characterized anxious relative to healthy subjects, but no significant interaction between group and stimulus type emerged. Fear levels at visit 1 predicted avoidance of visit 2. Fear levels to both CS types showed stability even after extinction. CONCLUSIONS: Consistent with adult data, pediatric anxiety involves higher fear levels following conditioning but not greater differential conditioning. Extending these methods to neuroimaging studies may elucidate neural correlates of fear conditioning. Implications for exposure therapies are discussed.     

The role of the basolateral amygdala in stimulus-reward memory and extinction memory consolidation and in subsequent conditioned cued reinstatement of cocaine seeking

The consolidation of cue-cocaine associations and extinction learning (i.e. cue-no cocaine associations) into long-term memory probably regulates the long-lasting control of conditioned stimuli (CS) over cocaine-seeking behaviour, and the basolateral amygdala (BLA) may play a role in this phenomenon. To test this hypothesis, rats previously trained to self-administer cocaine underwent a single classical conditioning (CC) session, during which they received passive pairings of cocaine infusions and a novel light + tone stimulus complex. After additional self-administration sessions in the absence of CS presentation and subsequent extinction training sessions, the ability of the CS to reinstate cocaine-seeking on five test days was assessed. Rats received intra-BLA microinfusions of vehicle or the Na+-channel blocker tetrodotoxin (TTX) immediately after CC (consolidation of CS-cocaine associations) or immediately after reinstatement testing (consolidation of extinction learning). TTX administered immediately after CC attenuated subsequent CS-induced reinstatement. In contrast, TTX administered after the first reinstatement test impaired the extinction of cocaine-seeking behaviour during a second reinstatement test by disrupting extinction memory. Overall, these findings suggest that Na+ channel-mediated mechanisms within the BLA mediate the consolidation of both cocaine-stimulus association and extinction learning, two processes that have opposite effects on subsequent cue-induced cocaine-seeking behaviour.     

Fear extinction deficits following acute stress associate with increased spine density and dendritic retraction in basolateral amygdala neurons

Stress‐sensitive psychopathologies such as post‐traumatic stress disorder are characterized by deficits in fear extinction and dysfunction of corticolimbic circuits mediating extinction. Chronic stress facilitates fear conditioning, impairs extinction, and produces dendritic proliferation in the basolateral amygdala (BLA), a critical site of plasticity for extinction. Acute stress impairs extinction, alters plasticity in the medial prefrontal cortex‐to‐BLA circuit, and causes dendritic retraction in the medial prefrontal cortex. Here, we examined extinction learning and basolateral amygdala pyramidal neuron morphology in adult male rats following a single elevated platform stress. Acute stress impaired extinction acquisition and memory, and produced dendritic retraction and increased mushroom spine density in basolateral amygdala neurons in the right hemisphere. Unexpectedly, irrespective of stress, rats that underwent fear and extinction testing showed basolateral amygdala dendritic retraction and altered spine density relative to non‐conditioned rats, particularly in the left hemisphere. Thus, extinction deficits produced by acute stress are associated with increased spine density and dendritic retraction in basolateral amygdala pyramidal neurons. Furthermore, the finding that conditioning and extinction as such was sufficient to alter basolateral amygdala morphology and spine density illustrates the sensitivity of basolateral amygdala morphology to behavioral manipulation. These findings may have implications for elucidating the role of the amygdala in the pathophysiology of stress‐related disorders.     

Impaired acquisition of classically conditioned fear-potentiated startle reflexes in humans with focal bilateral basolateral amygdala damage

Based on studies in rodents, the basolateral amygdala (BLA) is considered a key site for experience-dependent neural plasticity underlying the acquisition of conditioned fear responses. In humans, very few studies exist of subjects with selective amygdala lesions and those studies have only implicated the amygdala more broadly leaving the role of amygdala sub-regions underexplored. We tested a rare sample of subjects (N = 4) with unprecedented focal bilateral BLA lesions due to a genetic condition called Urbach–Wiethe disease. In a classical delay fear conditioning experiment, these subjects showed impaired acquisition of conditioned fear relative to a group of matched control subjects (N = 10) as measured by fear-potentiation of the defensive eye-blink startle reflex. After the experiment, the BLA-damaged cases showed normal declarative memory of the conditioned association. Our findings provide new evidence that the human BLA is essential to drive fast classically conditioned defensive reflexes.     

Amygdala and nucleus accumbens involvement in appetitive extinction

Extinction of appetitive conditioning is regarded as an important model for the treatment of psychiatric disorders like addiction. However, very few studies have investigated its neural correlates. Therefore, we investigated neural correlates of appetitive extinction in a large human sample including all genders (N = 76, 40 females) to replicate and extend results from a previous study. During differential appetitive conditioning, one stimulus (CS+) was paired with the chance to win a monetary reward, whereas another stimulus (CS?) was not. During appetitive extinction on the next day, neither the CS+ nor the CS? were reinforced. After successful acquisition of appetitive conditioning, the extinction phase elicited significant reductions of valence and arousal ratings toward the CS+ and a significant reduction in skin conductance responses to the CS+ from early to late extinction. On a neural level, early extinction showed significant differential (CS+ ? CS?) activation in dACC and hippocampus, whereas involvement of the vACC and caudate nucleus did not replicate. The differential activation of amygdala and nucleus accumbens during late extinction was replicated, with the amygdala displaying significantly higher differential activation during the late phase of extinction as compared to the early phase of extinction. We show discernible signals for reward learning and extinction in subregions of amygdala and nucleus accumbens after extinction learning. This successful replication underlines the role of nucleus accumbens and amygdala in neural models of appetitive extinction in humans that was previously only based on animal findings.     

Flexible spatial learning requires both the dorsal and ventral hippocampus and their functional interactions with the prefrontal cortex

When faced with changing contingencies, animals can use memory to flexibly guide actions, engaging both frontal and temporal lobe brain structures. Damage to the hippocampus (HPC) impairs episodic memory, and damage to the prefrontal cortex (PFC) impairs cognitive flexibility, but the circuit mechanisms by which these areas support flexible memory processing remain unclear. The present study investigated these mechanisms by temporarily inactivating the medial PFC (mPFC), the dorsal HPC (dHPC), and the ventral HPC (vHPC), individually and in combination, as rats learned spatial discriminations and reversals in a plus maze. Bilateral inactivation of either the dHPC or vHPC profoundly impaired spatial learning and memory, whereas bilateral mPFC inactivation primarily impaired reversal versus discrimination learning. Inactivation of unilateral mPFC together with the contralateral dHPC or vHPC impaired spatial discrimination and reversal learning, whereas ipsilateral inactivation did not. Flexible spatial learning thus depends on both the dHPC and vHPC and their functional interactions with the mPFC.     

A thalamo-cortico-amygdala pathway mediates auditory fear conditioning in the intact brain

The neural substrates of fear conditioning in rats have been well characterized, with converging lines of evidence indicating that conditioned stimulus (CS) and unconditioned stimulus (US) information form a CS-US association in the amygdala. Auditory CS information can reach the amygdala via two routes: a direct thalamo-amygdala pathway, and an indirect thalamo-cortico-amygdala pathway. Although either pathway can fully support learning when the alternate pathway is disrupted, many studies to date have argued that the thalamo-amygdala pathway is the principal auditory CS pathway in intact brains. To test this hypothesis, we trained rats in auditory fear conditioning, and 24 h later lesioned either pathway, leaving the alternate pathway intact. Later, animals were tested for conditioned freezing to the auditory CS. We report that lesions of the thalamo-amygdala pathway produced severe but incomplete deficits in freezing during the tone retention test, while lesions of the thalamo-cortico-amygdala pathway completely abolished freezing during tone presentation. These results suggest that the thalamo-cortico-amygdala pathway is the principal auditory CS pathway when the brain is intact.     

Ventral,but not dorsal,hippocampus inactivation impairs reward memory expression and retrieval in contexts defined by proximal cues

The hippocampus (HPC) has been widely implicated in the contextual control of appetitive and aversive conditioning. However, whole hippocampal lesions do not invariably impair all forms of contextual processing, as in the case of complex biconditional context discrimination, leading to contention over the exact nature of the contribution of the HPC in contextual processing. Moreover, the increasingly well‐established functional dissociation between the dorsal (dHPC) and ventral (vHPC) subregions of the HPC has been largely overlooked in the existing literature on hippocampal‐based contextual memory processing in appetitively motivated tasks. Thus, the present study sought to investigate the individual roles of the dHPC and the vHPC in contextual biconditional discrimination (CBD) performance and memory retrieval. To this end, we examined the effects of transient post‐acquisition pharmacological inactivation (using a combination of GABA_A and GABA_B receptor agonists muscimol and baclofen) of functionally distinct subregions of the HPC (CA1/CA3 subfields of the dHPC and vHPC) on CBD memory retrieval. Additional behavioral assays including novelty preference, light‐dark box and locomotor activity test were also performed to confirm that the respective sites of inactivation were functionally silent. We observed robust deficits in CBD performance and memory retrieval following inactivation of the vHPC, but not the dHPC. Our data provides novel insight into the differential roles of the ventral and dorsal HPC in reward contextual processing, under conditions in which the context is defined by proximal cues.     

Auditory cortex lesions prevent the extinction of Pavlovian differential heart rate conditioning to tonal stimuli in rabbits

The present study examined the role of the auditory cortex in the extinction of differentially conditioned heart rate (HR) responses in rabbits. Lesions were placed bilaterally in wither the auditory cortex or the visual cortex. Three days after recovery from surgery, the auditory cortex lesion group and the visual cortex lesion control group were habituated to the tonal conditioned stimuli (CSs), and then given 2 days of Pavlovian differential conditioning (60 trials per day) in which one tone (CS+) was always paired with the unconditioned stimulus and another tone (CS−) was never paired with the uncondition stimulus. Animals that had demonstrated reliable differential conditioning (CS+ response at least 5 beats greater than the CS− response) were placed on an extinction schedule for 7 days. The extinction schedule was identical to the differential conditioning schedule with the exception that shock never followed the CS+. The results of the study indicate that auditory cortex lesions prevent the extinction of differential bradycardia conditioned responses (CRs) to tonal CSs. Whereas the bradycardia responses to the CS+ quickly extinguished in the group that had control lesions in the visual cortex, the auditory cortex lesion group continued to exhibit significantly larger bradycardiac HR CRs to the CS+ relative to the CS− during all 7 days of extinction. These results suggest that the animals in the auditory cortex lesioned group did not inhibit responses to a previously reinforced stimulus (i.e. CS+) as well as animals with control lesions in the visual cortex.     

Behavioral and neuropsychological foundations of olfactory fear conditioning

Pavlovian fear conditioning procedures have been a fruitful means of exploring the neural substrates of associative learning. There is now substantial evidence suggesting that many aspects of conditioned fear depend critically upon the integrity of the amygdala and the perirhinal cortex. Recent studies in our laboratory examining the contributions of these areas to olfactory and contextual fear conditioning are reviewed; collectively the results of these studies suggest that the amygdala participates critically in the acquisition and expression of fear conditioned to both an olfactory conditioned stimulus (CS) and to the training context, while the perirhinal cortex contributes to olfactory, but not contextual, fear conditioning. Moreover, it appears that perirhinal cortex may play a prominent role in recognition of the CS following conditioning. These results are discussed in light of the extent to which they replicate and extend previous research examining the contributions of these areas to fear conditioned to auditory and visual CSs.     

UPDATED META‐ANALYSIS OF CLASSICAL FEAR CONDITIONING IN THE ANXIETY DISORDERS

The aim of the current study was twofold: (1) to systematically examine differences in fear conditioning between anxiety patients and healthy controls using meta‐analytic methods, and (2) to examine the extent to which study characteristics may account for the variability in findings across studies. Forty‐four studies (published between 1920 and 2013) with data on 963 anxiety disordered patients and 1,222 control subjects were obtained through PubMed and PsycINFO, as well as from a previous meta‐analysis on fear conditioning (Lissek et al. 10 ). Results demonstrated robustly increased fear responses to conditioned safety cues (CS?) in anxiety patients compared to controls during acquisition. This effect may represent an impaired ability to inhibit fear in the presence of safety cues (CS?) and/or may signify an increased tendency in anxiety disordered patients to generalize fear responses to safe stimuli resembling the conditioned danger cue (CS+). In contrast, during extinction, patients show stronger fear responses to the CS+ and a trend toward increased discrimination learning (differentiation between the CS+ and CS?) compared to controls, indicating delayed and/or reduced extinction of fear in anxiety patients. Finally, none of the included study characteristics, such as the type of fear measure (subjective vs. psychophysiological index of fear), could account significantly for the variance in effect sizes across studies. Further research is needed to investigate the predictive value of fear extinction on treatment outcome, as extinction processes are thought to underlie the beneficial effects of exposure treatment in anxiety disorders.     

Internuclear synapse in the amygdala is not facilitated in fear conditioning

The amygdala is essential for fear learning and memory. Synaptic transmission is enhanced in two pathways in the amygdala in fear conditioning. In this study we examined whether lateral (LA) to basolateral (BLA) amygdala synapses are potentiated and participate in intra-amygdala plasticity during the maintenance of fear memory. Our data showed that synaptic strength from the LA (ventrolateral) to the BLA (parvicellular) pathway was not increased after fear conditioning and suggests that this pathway does not integrate information relevant to the coding of memories in auditory fear learning.     

Basolateral amygdala lesions disrupt latent inhibitionin rats

Latent inhibition (LI) refers to the retardation of acquisition of conditioned responding produced by repeated non-reinforced preexposure to the conditioned stimulus (CS) prior to its pairing with the unconditioned stimulus (US) during conditioning. LI has recently been shown to depend upon the integrity of temporal lobe structures, including regions of the hippocampal formation such as the entorhinal cortex. The present study investigated the effects of excitotoxic lesions of another temporal lobe structure, the basolateral nucleus of the amygdala (BLA), on LI. LI was studied in a within-subjects appetitive conditioning preparation in which an auditory CS was paired with food US. In this procedure, preexposure to the CS results in slower acquisition of magazine approach behaviour. Lesions of the BLA reduced the effect of preexposure, disrupting LI. This result suggests that the BLA can play a crucial role in LI. The possible involvement of interactions between BLA and entorhinal cortex in LI is discussed.