The hippocampus and appetitive Pavlovian conditioning: effects of excitotoxic hippocampal lesions on conditioned locomotor activity and autoshaping

The hippocampus (HPC) is known to be critically involved in the formation of associations between contextual/spatial stimuli and behaviorally significant events, playing a pivotal role in learning and memory. However, increasing evidence indicates that the HPC is also essential for more basic motivational processes. The amygdala, by contrast, is important for learning about the motivational significance of discrete cues. This study investigated the effects of excitotoxic lesions of the rat HPC and the basolateral amygdala (BLA) on the acquisition of a number of appetitive behaviors known to be dependent on the formation of Pavlovian associations between a reward (food) and discrete stimuli or contexts: (1) conditioned/anticipatory locomotor activity to food delivered in a specific context and (2) autoshaping, where rats learn to show conditioned discriminated approach to a discrete visual CS+. While BLA lesions had minimal effects on conditioned locomotor activity, hippocampal lesions facilitated the development of both conditioned activity to food and autoshaping behavior, suggesting that hippocampal lesions may have increased the incentive motivational properties of food and associated conditioned stimuli, consistent with the hypothesis that the HPC is involved in inhibitory processes in appetitive conditioning.     

Dorsal hippocampal contributions to unimodal contextual conditioning

Although there is general consensus that the hippocampus is not critically involved in the acquisition of fear conditioned to an explicit conditioned stimulus (CS), the extent to which the hippocampus participates in contextual fear conditioning remains unclear. To further characterize the potential role of the hippocampus in contextual fear conditioning, the present experiments examined the effect of excitotoxic lesions of dorsal hippocampus on the acquisition of a novel contextual fear conditioning paradigm in which a unimodal (olfactory) cue served to disambiguate discrete "contexts" within a single behavioral training chamber. Selective lesions of dorsal hippocampus severely attenuated olfactory contextual conditioning without affecting conditioning to an explicit auditory or olfactory CS. Additional experiments indicate that these contextual conditioning deficits cannot be attributed to a lesion-induced decrement in olfactory perception, a preferential impairment of "weak" forms of conditioning, or hyperactivity. Thus, the hippocampus appears to contribute importantly to the acquisition of fear conditioned to explicitly nonspatial, unimodal, temporally, and spatially diffuse contextual stimuli.     

Hemodynamic responses in amygdala and hippocampus distinguish between aversive and neutral cues during Pavlovian fear conditioning in behaving rats

Lesion and electrophysiological studies in rodents have identified the amygdala and hippocampus (HPC) as key structures for Pavlovian fear conditioning, but human functional neuroimaging studies have not consistently found activation of these structures. This could be because hemodynamic responses cannot detect the sparse neuronal activity proposed to underlie conditioned fear. Alternatively, differences in experimental design or fear levels could account for the discrepant findings between rodents and humans. To help distinguish between these alternatives, we used tissue oxygen amperometry to record hemodynamic responses from the basolateral amygdala (BLA), dorsal HPC (dHPC) and ventral HPC (vHPC) in freely‐moving rats during the acquisition and extinction of conditioned fear. To enable specific comparison with human studies we used a discriminative paradigm, with one auditory cue [conditioned stimulus (CS)+] that was always followed by footshock, and another auditory cue (CS?) that was never followed by footshock. BLA tissue oxygen signals were significantly higher during CS+ than CS? trials during training and early extinction. In contrast, they were lower during CS+ than CS? trials by the end of extinction. dHPC and vHPC tissue oxygen signals were significantly lower during CS+ than CS? trials throughout extinction. Thus, hemodynamic signals in the amygdala and HPC can detect the different patterns of neuronal activity evoked by threatening vs. neutral stimuli during fear conditioning. Discrepant neuroimaging findings may be due to differences in experimental design and/or fear levels evoked in participants. Our methodology offers a way to improve translation between rodent models and human neuroimaging.     

Fear conditioning in C57/BL/6 and DBA/2 mice: variability in nucleus accumbens function according to the strain predisposition to show contextual- or cue-based responding

The contribution of the nucleus accumbens shell, the dorsal hippocampus, and the basolateral amygdala to contextual and explicit cue fear conditioning was assessed in C57BL/6 (C57) and DBA/2 (DBA) mice showing differences in processing contextual information associated with consistent but non-pathological variations in hippocampal functionality. Mice from both strains with bilateral ibotenic acid or sham lesions located in each area were introduced in a conditioning chamber and exposed twice to the pairing of a tone (2 x 8 s, 2000 Hz, 80 dB) with a shock (2 s, 0.7 mA). On the following day, mice were first exposed to the training context then to the tone in a different context. Freezing behaviour was scored in all situations. C57 showed more freezing to the context than to the tone whereas DBA showed more freezing to the tone than to the context. In C57, both nucleus accumbens and hippocampal lesions impaired acquisition of contextual fear conditioning but paradoxically improved acquisition of cue fear conditioning, whereas amygdala lesions disrupted performance in every task. In DBA, nucleus accumbens lesions, like amygdala lesions, impaired acquisition of both contextual and cue fear conditioning, whereas hippocampal lesions did not produce any effect. The parallelism between the effect of nucleus accumbens and hippocampus lesions in C57, and between the effect of nucleus accumbens and amygdala lesions in DBA points to a variability in nucleus accumbens function according to the strain specialization to develop context- or cue-based responding.     

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.     

Differential regulation of the expression of contextual freezing and fear-potentiated startle by 5-HT mechanisms of the median raphe nucleus

It has previously been shown that the median raphe nucleus (MR) is one of the main sources of projections to the septum and hippocampus. 5-HT projections from this nucleus to the hippocampus are implicated in the acquisition and expression of contextual fear (background stimuli), as assessed by freezing. It has also been reported that amygdala is involved in the acquisition of conditioned fear to foreground cues such as light, used as CS. As the MR projects to the hippocampus and amygdala, the role of this raphe nucleus in fear conditioning to contextual and classical fear conditioning remains to be elucidated. The present study examined the involvement of the MR serotonergic mechanisms in the expression of two distinct types of conditioned fear responses: contextual freezing and fear conditioning to explicit cue (light) measured in a fear-potentiated startle (FPS) procedure. Animals received MR electrolytic lesions of or microinjections of 8-OH-DPAT (8-hydroxy-2-(di-n-propylamino tetralin) (1 microg/0.2 microl) into the MR, 1 or 7 days after two consecutive training sessions in which they received 10 pairings of the CS (light, 4 s)-US (foot-shocks 0.6 mA, 1s) and were tested in a contextual fear paradigm and in a FPS procedure. The startle was clearly potentiated in the presence of light-CS in animals bearing lesions of or microinjected with 8-OH-DPAT into MR at 1 or 7 days post-training. However, animals bearing MR electrolytic lesions or microinjections of 8-OH-DPAT into the MR at 1 day, but not at 7 days post-training, showed a significant decrease in time spent in freezing than control ones. Thus, the memory for contextual conditioned fear seems to be formed during a time-window shorter than 1 week. As FPS may be produced in lesioned rats unable to freeze to fear contextual stimuli, dissociable systems seem to be recruited in each condition. Thus, the production of contextual freezing and fear-potentiated startle are conveyed by distinct 5-HT-mediated circuits of the MRN.     

Electrolytic lesions to nucleus accumbens core and shell have dissociable effects on conditioning to discrete and contextual cues in aversive and appetitive procedures respectively

The nucleus accumbens (n. acc.) has been implicated in conditioning to both discrete and contextual cues but its precise role is as yet controversial because conflicting patterns of effect have been reported. These inconsistencies may relate to the extent to which the lesions used encroach on different subfields of n. acc. and the use of different task variants. The present study compared the effects of selective lesions of shell and core subfields of nucleus accumbens (n. acc.) across aversive and appetitive trace conditioning variants.

In both experiments, an auditory stimulus was contiguous with footshock or food, or presented at a trace interval. A continuous flashing light in each case provided an experimental background stimulus. Conditioning to the cues provided by the experimental chambers was also assessed. Rats with electrolytic lesions to the n. acc. shell and core showed different patterns of effect in aversive (Experiment 1) and appetitive (Experiment 2) variants of this procedure. In Experiment 1, the core lesion reduced the difference between trace and contiguously conditioned groups, in responding to the discrete noise stimulus. However, neither lesion had any detectable effect on contextual conditioning. In Experiment 2, the shell lesion clearly increased contextual conditioning, selectively in the trace conditioned group, but neither lesion had any detectable effect on discrete cue conditioning.

Thus, whilst the shell and core lesions produced dissociable effects on discrete cue and contextual conditioning, the conclusions to be drawn depend on the procedural variant in use.     

Context conditioning and extinction in humans: differential contribution of the hippocampus, amygdala and prefrontal cortex

Functional magnetic resonance imaging was used to investigate the role of the hippocampus, amygdala and medial prefrontal cortex (mPFC) in a contextual conditioning and extinction paradigm provoking anxiety. Twenty-one healthy persons participated in a differential context conditioning procedure with two different background colours as contexts. During acquisition increased activity to the conditioned stimulus (CS+) relative to the CS− was found in the left hippocampus and anterior cingulate cortex (ACC). The amygdala, insula and inferior frontal cortex were differentially active during late acquisition. Extinction was accompanied by enhanced activation to CS+ vs. CS− in the dorsal anterior cingulate cortex (dACC). The results are in accordance with animal studies and provide evidence for the important role of the hippocampus in contextual learning in humans. Connectivity analyses revealed correlated activity between the left posterior hippocampus and dACC (BA32) during early acquisition and the dACC, left posterior hippocampus and right amygdala during extinction. These data are consistent with theoretical models that propose an inhibitory effect of the mPFC on the amygdala. The interaction of the mPFC with the hippocampus may reflect the context-specificity of extinction learning.     

Effect of mediodorsal thalamic nucleus lesion on contextual fear conditioning in rats

Much evidence from animal and clinical studies has shown that the mediodorsal nucleus of the thalamus (MD) is related to various types of memory, such as visual recognition, object-reward association, spatial working, and reference memory; however, few studies have investigated its role in emotion-related learning and memory processes. This study compared the effect of pre- and posttraining bilateral lesions of the mediodorsal thalamic nucleus with those of the amygdala on contextual conditioned fear. Both pre- and posttraining amygdala lesions almost eliminated conditioned freezing, and significantly blocked postshock freezing when behavioral tests were performed immediately after footshocks, reconfirming previous studies that the amygdala is implicated in the learning of Pavlovian conditioning. Both pre- and posttraining lesions of the mediodorsal nucleus of the thalamus significantly attenuated conditioned freezing but had no effect on postshock freezing. In contrast to lesions of the amygdala, those of the mediodorsal thalamic nucleus failed to alter the increased defecation induced by conditioned fear stress. Our results suggest that the mediodorsal nucleus of the thalamus has an important role in acquisition, consolidation or retrieval in Pavlovian contextual fear conditioning. Possible neural circuits, incorporating the amygdala, MD, and hippocampus, and the functional similarity of the MD and hippocampus in contextual fear conditioning, are also discussed.     

Hippocampal NMDA receptor blockade impairs CREB phosphorylation in amygdala after contextual fear conditioning

In contextual fear conditioning (CFC), hippocampus is thought to process environmental stimuli into a configural representation of the context and send it to amygdala nuclei, which current evidences point to be the site of CS‐US association and fear memory storage. If it is true, hippocampus should influence learning‐induced plasticity in the amygdala nuclei after CFC acquisition. To test this, we infused wistar rats with saline or AP5, a NMDA receptor antagonist, in the dorsal hippocampus just before a CFC session, in which they were conditioned to a single shock, exposed to the context with no shocks or received an immediate shock. The rats were perfused, their brains harvested and immunohistochemically stained for cAMP element binding protein (CREB) phosphorylation ratio (pCREB/CREB) in lateral (LA), basal (B) and central (CeA) amygdala nuclei. CFC showed a learning‐specific increase in pCREB ratio in B and CeA, in conditioned‐saline rats compared to context and immediate shocked ones. Further, conditioned rats that received AP5 showed a decrease in pCREB ratio in LA, B and CeA. Our results support the current ideas that the role of hippocampus in contextual fear conditioning occurs by sending contextual information to amygdala to serve as conditioned stimulus. © 2013 Wiley Periodicals, Inc.     

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.     

Contingent versus incidental context processing during conditioning: Dissociation after excitotoxic hippocampal plus dentate gyrus lesions

This experiment explored whether excitotoxic hippocampus plus dentate gyrus (HPC/DG) lesions in rats would dissociate the differential processing of contextual cues during the performance of learned associations when (1) their processing during training is incidental to successful learning or (2) the solution of a discrimination problem is contingent on their processing. A series of training stages were conducted, beginning with appetitive conditioning to two stimuli (X and Y), each of which was trained in one of two different contexts (operant chambers A and B) (i.e., AX+, BY+). Conditioning was indexed as appetitive responding. The animals were then trained on a biconditional contextual discrimination with these same stimuli (AX+, AY−; BY+, BX−). The next stage involved conditioning trials to two new stimuli (W and Z), one in each context, while the animals were actively discriminating contexts A and B by continuing to perform the original biconditional discrimination (AX+, AY−, AW+; BY+, BX−, BZ+). Finally, they were trained on a second biconditional discrimination involving these new stimuli (AX+, AY−, AW+, AZ−; BY+, BX−, BZ+, BW−). The incidental use of context cues was examined by looking at the rate of conditioned responding to cues X, Y, W, and Z in their original training contexts or a different context; HPC/DG lesioned rats differed from controls in being unaffected by a change of context. The contingent use of context cues was examined by looking at performance of each of the two biconditional tasks; HPC/DG lesioned rats reached levels of conditional performance indistinguishable from those of controls. These findings point to two distinct ways in which contextual information is processed in the brain, revealing a dissociation between incidental and contingent processing of contextual cues after HPC/DG lesions. Hippocampus 1998;8:147–159. © 1998 Wiley-Liss, Inc.     

Temporary inactivation of the nucleus accumbens disrupts acquisition and expression of fear-potentiated startle in rats

Recent research suggests that in addition to its prominent role in appetitive learning, the nucleus accumbens (NAC) may also be involved in fear conditioning. In the present study, we investigated whether temporary inactivation of the NAC, by injection of tetrodotoxin (TTX), affects acquisition and expression of conditioned fear, as measured by fear-potentiated startle (FPS). TTX injection into the NAC totally blocked acquisition and markedly decreased expression of conditioned fear to a discrete visual conditioned stimulus (CS). Interestingly, temporary inactivation of the NAC did not affect shock sensitization of startle, indicating that both the perception of the shock and short-term contextual conditioning was not affected by intra-accumbal TTX injection. Taken together, these results show that the NAC is crucial for acquisition and expression of long-term conditioned fear, as measured by fear-potentiated startle, to discrete CSs, but not short-term conditioned fear to a context.     

The hippocampus integrates context and shock into a configural memory in contextual fear conditioning

Contextual fear conditioning involves forming a representation for the context and associating it with a shock, which were attributed by the prevailing view to functions of the hippocampus and amygdala, respectively. Yet our recent evidence suggested that both processes require integrity of the dorsal hippocampus (DH). In view of the DH involvement in uniting multiple stimuli into a configuration, this study examined whether the DH would integrate context and shock into a shocked‐context representation. Male Wistar rats were trained on a two‐phase training paradigm of contextual fear conditioning. They explored a novel context on the first day to acquire a contextual representation, and received a shock in that context on the second day to form the context–shock memory. Tests of conditioned freezing given on the following days revealed two properties of configural memory—direct and mediated pattern completion: First, the contextual fear memory was retrieved in a novel context by a cue embedded in the configural set—a shock that did not elicit significant freezing on its own. Second, freezing was also elicited in a novel context by a transportation chamber that was not directly paired with the shock but could activate the fear memory inferentially. The effects were specific to the cue and not due to context generalization. Infusion of lidocaine into the DH, but not the amygdala, immediately after context–shock training impaired conditioned freezing elicited through either type of pattern completion. Our data suggest that the DH in contextual fear conditioning associates context and shock in parallel with the amygdala by incorporating the shock into an otherwise neutral context representation and turning it into a shocked‐context representation. © 2016 Wiley Periodicals, Inc.     

The effects of ibotenic hippocampal lesions on discriminative fear conditioning to context in mice: impairment or facilitation depending on the associative value of a phasic explicit cue

To what extent the hippocampus is required for contextual conditioning remains a matter of debate. The present experiments examined the effects of ibotenate hippocampal lesions on discriminative fear conditioning to context in mice using measures of freezing in two conditioning paradigms. In both paradigms animals received foot shock as the unconditional stimulus (US) when placed in the (conditioning) context and no foot-shock when placed in the other (neutral) context. In both contexts, animals were presented with a tone as the conditioned stimulus (CS). In the conditioning context there was either no interval (delay condition) or a 30-s interval (trace condition) between tone CS end and shock US onset. These two paradigms were used because theory predicts that in the trace condition animals would learn more about contextual cues as predictors, or not, of shock US occurrence than in the delay condition. In agreement with this, we observed that sham-operated mice learned the context discrimination faster in the trace than in the delay condition. Lesions of the hippocampus significantly retarded, but did not prevent, the acquisition of the context discrimination in the trace condition. In contrast, lesions produced an opposite (facilitatory) effect in the delay condition, which was mainly observed during tone CS presentation. The data suggest that mice used two distinct competing strategies in solving this discrimination task: (i) a strategy relying on the processing of background contextual stimuli allowing direct establishment of context-US associations of different strengths, and (ii) a conditional cue (tone)-based strategy. Hence, hippocampal lesions may impair the use of the former strategy while exacerbating (unmasking) the use of the latter.     

Paradoxical facilitatory effect of fornix lesions on acquisition of contextual fear conditioning in mice

The present study examined the effect of fornix lesions on freezing behavior elicited by contextual and phasic conditioned stimuli. Male mice of the C57Bl/6 strain received electrolytic lesions of the fornix. Ten days following the lesion, they were submitted to acquisition of one-trial classical fear conditioning involving the pairing of an auditory conditioned stimulus (CS) with a footshock unconditioned stimuli (US). Analysis of conditioned fear responses showed that fornix lesions enhanced the freezing response elicited by exposure to the conditioning chamber 24 h after a single tone-shock pairing. In contrast, the two groups did not differ on their fear responses during the auditory cue test. Analysis of the time-course of freezing behavior during re-exposure to the conditioning chamber suggests, however, that the observed fornix lesion-induced facilitation of freezing to the conditioning chamber is more likely due to a facilitation of the processing of a simple (unimodal) rather than polymodal (contextual) CS-US association.     

Mnemonic functions of dorsal striatum and hippocampus in aversive conditioning

These experiments examined the mnemonic functions of hippocampus and dorsal striatum in Pavlovian aversive conditioning. Rats were trained in a single session by presenting three paired tones and footshocks. Immediately after training, they were given microinjections of D-amphetamine or vehicle into either dorsal hippocampus or dorsal striatum. Twenty-four hours later conditioned freezing (measured as cessation of movement by infrared motion detectors) to the experimental context and to the tone were measured separately. Compared to vehicle injections, amphetamine injections into dorsal hippocampus significantly increased conditioned freezing to the context but not to the tone. Injections into dorsal striatum increased conditioned freezing to both the context and the tone. These results converge with findings from lesion experiments suggesting that hippocampus is involved in aversive conditioning with contextual CSs only, and that dorsal striatum is involved in aversive conditioning with both contextual and discrete cue CSs. The functions of the these two structures in relation to that of the amygdala in the mediation of conditioned freezing are discussed.     

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.     

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.     

Basolateral amygdala lesions in the rat produce an abnormally persistent latent inhibition with weak preexposure but not with context shift

Latent inhibition (LI) refers to retarded conditioning to a stimulus as a consequence of its nonreinforced preexposure. We have recently reported that basolateral amygdala (BLA) lesions lead to an abnormally persistent LI under conditions that normally disrupt LI, namely, extended conditioning. This study tested whether BLA lesions would induce abnormally persistent LI under two additional conditions disrupting LI in controls, namely, context shift and weak preexposure. LI was measured in an active avoidance procedure. In the first experiment, rats received 100 nonreinforced preexposures and were conditioned either in the same or in a different context from that of the preexposure stage. In the second experiment, rats received 50 nonreinforced preexposures and were conditioned in the same context as that of preexposure. Sham-operated rats showed LI in the same but not in the different context condition or with low number of preexposures. BLA lesions produced abnormally persistent LI with low number of preexposures but not with context shift. It is suggested that the BLA is involved in LI modulation based on the impact of preexposure and conditioning but not on contextual information.