Basolateral amygdala noradrenergic activity mediates corticosterone-induced enhancement of auditory fear conditioning

The present experiment examined whether posttraining noradrenergic activity within the basolateral complex of the amygdala (BLA) is required for mediating the facilitating effects of acutely administered glucocorticoids on memory for auditory-cue classical fear conditioning. Male Sprague-Dawley rats received five pairings of a single-frequency auditory stimulus and footshock, followed immediately by bilateral infusions of the beta1-adrenoceptor antagonist atenolol (0.5 microg in 0.2 microl) or saline into the BLA together with a subcutaneous injection of either corticosterone (3.0 mg/kg) or vehicle. Retention was tested 24 h later in a novel test chamber and suppression of ongoing motor behavior served as the measure of conditioned fear. Corticosterone facilitated memory as assessed by suppression of motor activity during the 10-s presentation of the auditory stimulus and intra-BLA administration of atenolol selectively blocked this corticosterone-induced memory enhancement. These findings provide evidence that, as found with other emotionally arousing tasks, the enhancing effects of corticosterone on memory consolidation of auditory-cue fear conditioning require posttraining noradrenergic activity within the BLA.     

Immunotoxic cholinergic lesions in the basal forebrain reverse the effects of entorhinal cortex lesions on conditioned odor aversion in the rat

Conditioned odor aversion (COA) corresponds to the avoidance of an odorized-tasteless solution (conditioned stimulus, CS) previously paired with toxicosis. COA occurs only when the interstimulus interval (ISI) is kept short, suggesting that the memory trace of the odor is subject to rapid decay. Previous experiments have shown that the entorhinal cortex (EC) is involved in the acquisition of COA, since lesion of the EC rendered COA tolerant to long ISI. Because EC lesions induce a septo-hippocampal cholinergic sprouting, the present experiment investigated whether COA tolerance to long ISI may be linked to this sprouting reaction. In a first experiment, male Long-Evans rats subjected to bilateral excitotoxic EC lesions combined to intracerebroventricular infusions of the selective cholinergic immunotoxin 192 IgG-saporin were exposed to odor-toxicosis pairing using a long ISI (120 min). Results showed that EC-lesioned rats displayed COA with the long ISI but not the control groups. In rats with EC combined to 192 IgG-saporin lesions, histological analysis demonstrated no evidence for cholinergic septo-hippocampal sprouting. In a second experiment, animals with 192-IgG saporin lesion showed a marked COA with a short ISI (5 min). These results suggest that the COA with the long ISI found in rats with EC lesions might involve a functional activity related to the EC lesion-induced hippocampal cholinergic sprouting. As the injection of 192 IgG-saporin alone did not affect COA with a short ISI, our data also point to a possible role of hippocampal cholinergic neurons in the modulation of memory processes underlying COA.     

Inactivation of the basolateral amygdala impairs the retrieval of recent and remote taste-potentiated odor aversion memory

Memory reorganization as a time-dependent process can be investigated using various learning tasks such as the taste-potentiated odor aversion (TPOA). In this paradigm rats acquire a strong aversion to an olfactory cue presented simultaneously with a gustatory cue. Together these cues are paired with a delayed visceral illness. The basolateral amygdaloid nucleus (BLA) plays a key role in TPOA acquisition but its involvement in retrieval remains unclear. We investigated the involvement of the BLA in either recent or remote retrieval of TPOA. In each case, the number of licks observed in response to the presentation of either the odor or the taste was used to assess retrieval. Before the retrieval test, rats received a bilateral infusion of lidocaine to inactivate the BLA. We observed that both recent and remote TPOA retrieval tests induced by the odor presentation were disrupted in the lidocaine-injected rats. By contrast, the BLA inactivation had no effect upon the aversion towards the taste cue regardless of the time of retrieval. The present study provides evidence that BLA functioning is necessary for retrieval of aversive odor memory, even with a long post-acquisition delay.     

Rho-associated kinase in the gustatory cortex is involved in conditioned taste aversion memory formation but not in memory retrieval or relearning

Rho-associated kinase (ROCK) is intimately involved in cortical neuronal morphogenesis. The present study explores the roles of ROCK in conditioned taste aversion (CTA) memory formation in gustatory cortex (GC) in adult rat. Microinjection of the ROCK inhibitor Y-27632 into the GC 30 min before CTA training or 10 min after the conditioned stimulus (CS) impaired long-term CTA memory (LTM) formation. ROCK inhibitor had no effect on taste aversion when injected before the first LTM test day and did not alter taste aversion on subsequent test days. Microinjection of ROCK inhibitor into GC 30 min before preexposure to the taste CS had no effect on latent inhibition of CTA learning suggesting that ROCK is involved in CS-US association rather than taste learning per se. Cumulatively, these results show that ROCK is needed for normal CTA memory formation but not retrieval, relearning or incidental taste learning.     

Post-trial stimulation and the acquisition of conditioned suppression in the rat

Three experiments investigated the effect of post-trial stimulation on conditioning. Rats were trained in a conditioned suppression procedure in which only half of the CS presentations were followed immediately by a shock reinforcer. Presenting a second, post-trial, shock 8 s after the end of reinforced CS presentations increased resistance to extinction in Experiment I and facilitated conditioning in Experiment II relative to a condition in which the same post-trial shock was delivered 8 s after non-reinforced trials. Experiment III demonstrated that prior exposure to pairings of the shock reinforcer and the post-trial shock also enhanced subsequent conditioning, and thus ruled out an explanation of the facilitation in terms of the surprising nature of the post-trial stimulation.     

The effect of three procedures for eliminating a conditioned taste aversion in the rat

Although extinction procedures have been the most common techniques used to eliminate conditioned taste aversions, several studies have employed postconditioning exposure to the US instead. To date, a comparison of the effectiveness of these two techniques has not been possible, because there were differences in the conditioning phases of these studies. In the present study, after an aversion to saccharin had been established, rats were administered 1, 5, or 10 extinction trials, 1, 5, or 10 postconditioning exposures to the US, or a period of no treatment. Then, all rats received seven two-bottle extinction tests. Five or ten extinction trials reduced the aversion to saccharin most effectively. Five or ten postconditioning presentations of the US were also effective. However, this effect was not noticeable until the fourth of seven test trials, suggesting an elimination procedure-test trials interaction. Neither the extinction nor the US postexposure procedure completely eliminated the aversion. In addition: (i) a single postconditioning exposure to either the CS or the US was ineffective in attenuating the aversion; and (ii) when no postconditioning treatment was administered, the strength of the aversion was undiminished for 20 days.     

Short-term memory reactivation of a weak CS–US association promotes long-term memory persistence in conditioned odor aversion

In conditioned odor aversion (COA), the association of a tasteless odorized solution (the conditioned stimulus [CS]) with an intraperitoneal injection of LiCl (the unconditioned stimulus [US[), which produces visceral malaise, results in its future avoidance. The strength of this associative memory is mainly dependent on two parameters, that is, the strength of the US and the interstimuli interval (ISI). In rats, COA has been observed only with ISIs of ≤15 min and LiCl (0.15 M) doses of 2.0% of bodyweight, when tested 48 h after acquisition (long-term memory [LTM]). However, we previously reported a robust aversion in rats trained with ISIs up to 60 min when tested 4 h after acquisition (short-term memory [STM]). Since memories get reactivated during retrieval, in the current study we hypothesized that testing for STM would reactivate this COA trace, strengthening its LTM. For this, we compared the LTM of rats trained with long ISIs or low doses of LiCl initially tested for STM with that of rats tested for LTM only. Interestingly, rats conditioned under parameters sufficient to produce STM, but not LTM, showed a reliable LTM when first tested for STM. These observations suggest that under suboptimal training conditions, such as long ISIs or low US intensities, a CS–US association is established but requires reactivation in the short-term in order to persist in the long-term.

The dynamic and malleable nature of memories is a well-studied phenomenon. Traditionally, for memory formation to occur, a set of processes collectively known as consolidation are thought to be needed in order to stabilize memories, making them susceptible to modification during this period (Dudai et al. 2015). More recently a slightly distinct theory, known as memory integration, was proposed according to which memories are rapidly formed during learning without the need for consolidation, but any relevant information around the event can be integrated modifying them (Gisquet-Verrier and Riccio 2019). Common to both theories though, is that memories alternate between an inactive and an active state and modifications can mostly occur during the active state, which lasts for some time after learning, or during its reactivation due to retrieval (Lee et al. 2017; Albo and Gräff 2018; Gisquet-Verrier and Riccio 2019). Thus, memory malleability is explained either because consolidation can be altered or because additional information can be integrated with the initial memory (Bailey et al. 1996; Dudai 2004; Wixted 2004; Alberini et al. 2006; Lee et al. 2008, 2017; McGaugh and Roozendaal 2009; Roesler and Schröder 2011; Dudai et al. 2015; Nader 2015; Crossley et al. 2019; Gisquet-Verrier and Riccio 2019).In conditioned odor aversion (COA), an odorized tasteless solution (conditioned stimulus, CS) whose ingestion is followed by gastrointestinal malaise (unconditioned stimulus, US) is rejected in future encounters (conditioned response, CR). In most COA studies, a robust aversion has been observed only when the interstimulus interval (ISI) is ∼5 min, and no significant aversion can be seen when the ISI is >15 min (Hankins et al. 1973; Palmerino et al. 1980; Ferry et al. 1995, 1996; Ferry and di Scala 1997; Ferry et al. 2006; Chapuis et al. 2007). This observation has been attributed to a short-lasting memory of the odor that becomes unavailable for its association with the US after ISI >15 min. However, in all these instances the CR was measured 48 h after conditioning (LTM test), leaving up the possibility that CS–US association was formed but somehow did not last till the long-term. In keeping with this possibility, we previously reported a significant aversion during a test performed 4 h after conditioning (i.e., STM test) in rats trained with ISIs up to 60 min, three times longer than previously described (Tovar-Díaz et al. 2011). The LTM, however, was not tested so no further insight was provided regarding its persistence due to STM reactivation.Thus, in the current paper we hypothesized that a STM test would reactivate the initial memory, allowing it to further consolidate/integrate the information and to persist in the LTM. To test this possibility, we trained independent groups of rats with reduced US intensities or prolonged ISIs in a standard two-bottle choice COA paradigm and tested them twice at 4 and 48 h after conditioning. Our findings suggest that COA takes place under milder US and longer ISIs than previously thought and reactivating this memory during the STM test promotes its persistence in the LTM test.     

Involvement of the insular cortex in retention of conditioned taste aversion is not time dependent

In fear-associated learning paradigms, hippocampal lesions induce memory deficits of recent but not remote memories, while amygdala lesions produce retention deficits irrespective of the age of the memory. In conditioned taste aversion (CTA), non-hippocampal mediated learning paradigm, the insular vortex (IC) has shown to have a crucial role in consolidation and storage of CTA memory. Due to the functional and anatomical similarities to the hippocampus, a time dependent role of the IC in CTA retention cannot be ruled out. To test whether the IC shows a time dependent role in CTA memory retention, male Wistar rats were CTA trained on saccharin 0.1% (LiCl 0.15M, 2% b/w, 40 min after drinking) and lesioned with ibotenic acid (200-300 nL, 5mg/mL) unilaterally into the IC 1 week or bilaterally 1 or 6 weeks after CTA. CTA memory was completely disrupted in both bilateral lesion groups but unaffected in the unilateral lesioned group. The resulting preference was comparable to that of the bilaterally IC lesioned animals exposed to the taste for the first time, proving that in these animals a complete amnesic state was achieved. Bilaterally IC lesioned rats showed normal discrimination between preferred (sucrose 5%) and non-preferred (quinone) tastes. Our data indicates that the involvement of the IC in CTA is not time dependent and that CTA memories are stored in each hemisphere separately.     

Effects of prior exposure on conditioned taste aversion in the rat: androgen- and estrogen-dependent events

The effects of preexposure and gonadal hormone manipulation on the extinction of a conditioned taste aversion were investigated. In Experiment 1, male rats were given one prior exposure to sucrose at some selected time (Days 4, 2, or 1) before a second exposure (Day 0) to sucrose and an LiCl injection. Controls received no prior exposure to sucrose but experienced only the sucrose + LiCl condition (Day 0). under the single exposure condition (Day 0) castrated animals extinguished the aversion faster than either testosterone-treated castrated rats or sham-operated rats. Sham-operated and castrated rats that had received preexposure treatment (Days 4, 2, or 1) were not distinguishable on a within-groups comparison of behavior but differed from their respective controls by exhibiting faster rates of extinction. However, in the testosterone-treated group only the Day 4 preexposure group exhibited a faster extinction rate. In Experiment 2, estradiol, dihydrotestosterone, and testosterone were studied by using only a Day 1 preexposure condition. The testosterone-treated group maintained the aversion for the longest period of time, followed by dihydrotestosterone-treated, sham, castrated, and estradiol-treated groups. It appears that estradiol augments the castration effect whereas dihydrotestosterone attenuates the effect. In Experiment 3, estradiol was administered alone or in combination with two different doses of dihydrotestosterone, and a Day 1 preexposure condition was used. The findings indicate that the outcome of behavior is dependent on the ratio of estradiol to dihydrotestosterone, with variations in this ratio resulting in fast (estrogen effect) to slow (androgen effect) rates of extinction.     

Stimulus generalization of conditioned taste aversion in rats

Relatively little information is available regarding the intradimensional stimulus generalization of conditioned taste aversion (CTA). Experiment 1 employed a between-groups generalization test to examine the extent to which conditioned flavor aversion to one sucrose solution generalized to other concentrations of sucrose in adult rats. Evidence of a gradient of aversion was obtained. Because generalization gradients in other tasks have been found to flatten over a retention interval, Experiment 2 investigated the effects of delayed testing (2, 7, or 21 days) upon the slope of the generalization gradient. The generalization gradient flattened at the intervals, suggesting that subjects forgot the specific attributes of the conditioning concentration and avoided generalized stimuli as if they were the original CS. Experiment 3 used a long delay between taste and toxicosis to degrade the associative contingency and found no evidence that the generalization gradients found in the first two experiments could be explained in terms of enhanced neophobia due to poisoning. These findings provide further evidence (cf. A. W. Logue, 1979, Psychological Bulletin, 86, 276-296; M. Domjan, 1980, in J. S. Rosenblatt, R. A. Hinde, C. Beer, & M. Busnel (Eds.), Advances in the study of behavior, Vol. 11, New York: Academic Press) that CTA shares a number of similarities with other learning processes. Further, they illustrate that stimulus forgetting can be detected in a paradigm considered relatively immune to retention loss.     

The role of the amygdala in conditioned flavor preference

Long-Evans rats with control or amygdala lesions were tested in a conditioned flavor preference task. Half of the rats in each lesion group received an unsweetened grape-flavored solution on odd-numbered days and a sweetened cherry-flavored solution on even-numbered days. The remaining rats received a sweetened grape-flavored solution on odd-numbered days and an unsweetened cherry-flavored solution on even-numbered days. The appropriate solution was presented once a day for 15 min to each rat in the homecage. After six days of testing, each rat received unsweetened cherry and grape flavored solutions simultaneously for 15 min daily across four days. When the two unsweetened flavored solutions were presented simultaneously control rats showed a significant preference for the flavor that was sweetened during training compared to the unsweetened solution. However, amygdala-lesioned rats did not show a preference. The data suggest that the amygdala may be critically involved in mediating reward-based conditioned flavor preference.     

Resistance to extinction of conditioned odor perceptions: evaluative conditioning is not unique

A tasteless odor will smell sweeter after being sampled by mouth with sucrose and will smell sourer after being sampled with citric acid. This tasty-smell effect was found in experiments that compared odor-taste and color-taste pairings. Using odors and colors with minimal taste (Experiment 1), the authors found that repeated experience of odor-taste mixtures produced conditioned changes in odor qualities that were unaffected by intermixed color-taste trials (Experiment 2). An extinction procedure, consisting of postconditioning presentations of the odor in water, had no detectable effect on the changed perception of an odor (Experiments 3 and 4). In contrast, this procedure altered judgments about the expected taste of colored solutions. Evaluative conditioning (conditioned changes in liking) is claimed to be resistant to extinction. However, these results suggest that resistance to extinction in odors is related to the way they are encoded rather than to their hedonic properties.     

The amygdala is involved in affective priming effect for fearful faces

The object of this study was to investigate whether the amygdala is involved in affective priming effect after stimuli are encoded unconsciously and consciously. During the encoding phase, each masked face (fearful or neutral) was presented to participants six times for 17ms each, using a backward masking paradigm. During the retrieval phase, participants made a fearful/neutral judgment for each face. Half of the faces had the same valence as that seen during encoding (congruent condition) and the other half did not (incongruent condition). Participants were divided into unaware and aware groups based on their subjective and objective awareness assessments. The fMRI results showed that during encoding, the amygdala elicited stronger activation for fearful faces than neutral faces but differed in the hemisphere according to the awareness level. During retrieval, the amygdala showed a significant repetition priming effect, with the congruent faces producing less activation than the incongruent faces, especially for fearful faces. These data suggest that the amygdala is important in unconscious retrieving of memories for emotional faces whether they are encoded consciously or unconsciously.     

Amnesia produced by anisomycin in an appetitive task is not due to conditioned aversion

Two experiments investigated the effects of lithium chloride (LiCl) and anisomycin (ANI) in a water reward Y-maze task. In Experiment 1, male CD-1 mice given weak or strong training were injected post-training with either saline or LiCl (150 mg/kg), which has been reported to produce conditioned aversion in mice. One day after training, both LiCl groups avoided the rewarded arm of the maze and drank less water than saline-injected controls. Two days after training, the strongly trained LiCl mice showed avoidance, while both LiCl groups drank less water. In Experiment 2, weakly trained mice given pre- and post-training ANI (30 mg/kg) were amnesic on the second test day compared to mice that received post-trial saline. However, water consumption was increased on the test day for both groups. LiCl produced a different pattern of results than ANI in this task. On the basis of these results, it is suggested that amnesia produced by ANI is due to impaired memory formation and not to conditioned aversion.     

Enhancement of inhibitory avoidance and conditioned taste aversion memory with insular cortex infusions of 8-Br-cAMP: involvement of the basolateral amygdala

There is considerable evidence that in rats, the insular cortex (IC) and amygdala are involved in the learning and memory of aversively motivated tasks. The present experiments examined the effects of 8-Br-cAMP, an analog of cAMP, and oxotremorine, a muscarinic agonist, infused into the IC after inhibitory avoidance (IA) training and during the acquisition/consolidation of conditioned taste aversion (CTA). Posttraining infusion into the IC of 0.3 microg oxotremorine and 1.25 microg 8-Br-cAMP enhanced IA retention. Infusions of 8-Br-cAMP, but not oxotremorine, into the IC enhanced taste aversion. The experiments also examined whether noradrenergic activity in the basolateral amygdala (BLA) is critical in enabling the enhancement of CTA and IA memory induced by drug infusions administered into the IC. For both CTA and IA, ipsilateral infusions of beta-adrenergic antagonist propranolol administered into the BLA blocked the retention-enhancing effect of 8-Br-cAMP or oxotremorine infused into the IC. These results indicate that the IC is involved in the consolidation of memory for both IA and CTA, and this effect requires intact noradrenergic activity into the BLA. These findings provide additional evidence that the BLA interacts with other brain regions, including sensory cortex, in modulating memory consolidation.