Safe taste memory consolidation is disrupted by a protein synthesis inhibitor in the nucleus accumbens shell.

Consolidation is the process by which a new memory is stabilized over time, and is dependent on de novo protein synthesis. A useful model for studying memory formation is gustatory memory, a type of memory in which a novel taste may become either safe by not being followed by negative consequences (attenuation of neophobia, AN), or aversive by being followed by post-digestive malaise (conditioned taste aversion, CTA). Here we evaluated the effects of the administration of a protein synthesis inhibitor in the nucleus accumbens (NAc) shell for either safe or aversive taste memory trace consolidation. To test the effects on CTA and AN of protein synthesis inhibition, anisomycin (100microg/microl) was bilaterally infused into the NAc shell of Wistar rats' brains. We found that post-trial protein synthesis blockade impaired the long-term safe taste memory. However, protein synthesis inhibition failed to disrupt the long-term memory of CTA. In addition, we infused anisomycin in the NAc shell after the pre-exposure to saccharin in a latent inhibition of aversive taste. We found that the protein synthesis inhibition impaired the consolidation of safe taste memory, allowing the aversive taste memory to form and consolidate. Our results suggest that protein synthesis is required in the NAc shell for consolidation of safe but not aversive taste memories, supporting the notion that consolidation of taste memory is processed in several brain regions in parallel, and implying that inhibitory interactions between both taste memory traces do occur.

Spatial long-term memory is related to mossy fiber synaptogenesis.

Structural synaptic changes have been suggested to underlie long-term memory formation. In this work, we investigate if hippocampal mossy fiber synaptogenesis induced by water maze overtraining can be related with long-term spatial memory performance. Rats were trained in a Morris water maze for one to five identical daily sessions and tested for memory retrieval 1 week and 1 month after training. After the last test session, the rat brains were obtained and processed for Timm's staining to analyze mossy fiber projection. The behavioral results showed that with more training, animals showed a better performance in the memory tests, and this performance positively correlates with Timm's staining in the stratum oriens. Furthermore, with the use of the NMDA antagonist MK801 before, but not after acquisition, water maze spatial memory was impaired. Increased Timm's staining in the stratum oriens was observed in the animals treated with MK801 after acquisition but not in those treated before. Finally, we observed that mossy fiber synaptogenesis occurs mainly in the septal region of the dorsal hippocampus, supporting the idea that this anterior region is important for spatial memory. Altogether, these results suggest that mossy fiber synaptogenesis can be related with spatial long-term memory formation.

Redundant basal forebrain modulation in taste aversion memory formation.

Mnemonic deficits resulting from excitotoxic lesion of the basal forebrain have been classically attributed to the resulting depletion of cortical acetylcholine activity. It has been demonstrated that in spite of the strong cholinergic depletion after injections into the basal forebrain of the immunotoxin 192IgG-saporin, no detectable deficit can be found in the acquisition of several learning tasks, including conditioned taste aversion. Conversely, NMDA-induced lesions of the basal forebrain strongly impair taste aversion learning. In this study we show that 192IgG-saporin produces an efficient and selective cholinergic deafferentation of the rat neocortex but not the amygdala. Furthermore, a stronger relationship between severity of memory impairment after NMDA lesions and basoamygdaloid cholinergic deafferentation was found. Therefore, in a second experiment, we show that combining NMDA-induced lesions into the basolateral amygdala with 192IgG-saporin injections into the basal forebrain results in a strong disruption of taste aversion learning, whereas none of these treatments were by themselves capable of producing any detectable impairment in this learning task. The double lesion effect was only paralleled by simple NMDA lesions into the basal forebrain, suggesting that the learning deficits associated to excitotoxic lesions of the basal forebrain are the result of the simultaneous destruction of the corticopetal and basoamygdaloid interaction. A model is proposed, according to which the modulation of learning processes exerted by the basal forebrain can be redundantly performed by both the basocortical and basoamygdaloid pathway.

Blockade of N-methyl-D-aspartate receptors in the insular cortex disrupts taste aversion and spatial memory formation.

The present experiments examined the effects of direct intracortical microinjections of the N-methyl-D-aspartate receptor antagonist 2-amino-5-phosphonovaleric acid directly into the insular cortex of rats, before or immediately after training of conditioned taste aversion and the water maze spatial learning task. In the first series of experiments animals received bilateral injections of 2-amino-5-phosphonovaleric acid prior to taste aversion conditioning or spatial training. A strong disruptive effect was found in the acquisition of training tasks. To determine the possible involvement of N-methyl-D-aspartate receptors in the early post-training processes taking place in the cortex during both learning paradigms, in a second series of experiments, animals received bilateral 2-amino-5-phosphonovaleric acid microinjections 30, 60 or 120 min after the acquisition trial, and 15 min before the retention test. For spatial learning successive treatments were independently done either starting at the onset of the asymptotic phase of the learning curve, 0, 30 or 120 min after finishing the training session, as well as 15 min before the retention test trial. The conditioned taste aversion task remained sensitive to N-methyl-D-aspartate blockade during a period of at least 2 h after the first presentation of the gustatory stimulus, while in the case of the spatial learning task, a gradually decreasing effect was observed from the onset of the asymptotic phase onwards. Taken together, these results provide direct evidence for N-methyl-D-aspartate receptor involvement in cortical regulation of memory formation. Furthermore, our results suggest that in the same cortical region, a different time-course for the activation of N-methyl-D-aspartate-dependent mechanisms occurs during the early formation of cortically mediated memories, depending on the particular behavioural task.

Taste-potentiated noise-illness associations.

In five experiments we attempted to establish aversions to a noise presented each time thirsty rats licked a water source. Using an apparatus and a procedure similar to those of previous studies which reported noise-illness associations, weak taste-potentiated noise-illness associations were found (Experiment 1). However, when the apparatus and procedure were varied, noise-illness associations were not observed (Experiments 3, 4, and 5). The noise used in all of these failures was readily associated with shock (Experiment 2), which indicates that this noise was salient to the rats and could be associated with an aversive event. These failures to find noise-illness associations occurred despite the fact that the conditions were chosen to maximize the likelihood of observing these associations. Taste-illness associations always developed normally. The results are consistent with the idea that conditioned aversions to all exteroceptive stimuli are not equally well potentiated by taste.

The conditioned stimulus-unconditioned stimulus-feedback feeding sequence: reply to Ellins, von Kluge, and Cramer (1990).

Taste-potentiated noise aversions have been demonstrated in rats (Ellins, Cramer, & Whitmore, 1985; Ellins & von Kluge, 1987; Holder, Bermudez-Rattoni, & Garcia, 1988). However, these aversions are apparently less readily established than taste-potentiated odor aversions suggesting that all exteroceptive stimuli are not equally well potentiated by taste (Holder et al., 1988). Despite the claims of Ellins, von Kluge, and Cramer (1990), we replicated their findings and see no inconsistency between our findings and theirs. Both sets of results are explained by a common theory of conditioning (Garcia, 1989; Garcia & Holder, 1985).

Long-term memory retrieval deficits of learned taste aversions are ameliorated by cortical fetal brain implants.

In this study, the effects that fetal brain implants have on the ability to retrieve the memory for a previously acquired conditioned taste aversion (CTA) in insular cortex (IC) lesioned rats were tested. Several groups of rats were trained for a CTA, were lesioned in the IC 4 days later, were implanted with different fetal cortical tissues, were treated or untreated with nerve growth factor (NGF), and then were tested for recall either 15 or 45 days later. Rats were then retrained and tested with a different taste and in the inhibitory avoidance (IA) task. All implanted animals recovered the retrieval of CTAs learned before IC lesions; however, only the homotopic IC implants at 45 days or NGF supplemented at 15 days induced recovery of the ability to learn CTA. The latter effect was also true for IA learning. The results suggest that the brain mechanisms for recovery of memory functions are different from those of learning abilities.

Odor and taste aversions conditioned in anesthetized rats.

Conditioned flavor aversions (CFA) are acquired by anesthetized rats but effects of various anesthetics on acquisition of aversions for separate odor and taste components are unknown. In Experiment 1, rats drank tomato juice and then were tranquilized with "Innovar-Vet" or "Rompun" before receiving injections of lithium chloride. Neither drug interfered with acquisition of aversions. Innovar-Vet alone produced no aversions; Rompun alone produced mild aversions but did not enhance aversions when combined with lithium. In Experiments 2 and 3, rats received a compound odor/taste cue as they drank and then were anesthetized with pentobarbital before lithium injections. Anesthesia alone produced negligible aversions but facilitated taste-lithium aversions. During odor tests, odor aversions were weaker than taste aversions. These data extend previous work and suggest that CFA does not result from ordinary classical conditioning. A tripartite notation that unites CFA and classical conditioning is discussed.

Morphometric study of fetal brain transplants in the insular cortex and NGF effects on neuronal and glial development.

Homotopic grafts supplemented with nerve growth factor (NGF) speed the recovery from learning deficits observed following electrolytic lesions of the insular cortex in rats. NGF also reduces the time in which the activity of choline acetyltransferase (ChAT) is first detected inside the graft by histochemical techniques. It is not known whether this behavioral and biochemical recovery correlates with an advanced maturation of the cellular elements within the graft, presumably induced by NGF. To investigate the degree of maturation of neurons, glial cells and blood vessels in NGF-supplemented grafts, adult rats were lesioned electrolytically in the insular cortex, and homotopic embryonic grafts (E16) with or without NGF supplementation were transplanted into the lesion. Fifteen days post grafting, the rats were perfused and the brains stained using silver impregnation techniques. Our results showed that neuronal maturation, as evaluated through several morphometric parameters, was advanced in NGF-supplemented grafts when compared with other experimental groups. Furthermore, grafts supplemented with NGF also showed significant increases in the number of neurons, oligodendrocytes, astrocytes and blood vessels. These observations indicated that the addition of NGF to insular cortex grafts promoted the maturation of neuronal and glial elements within the graft. They also support the possibility that the advanced morphological maturation of insular cortex grafts supplemented with NGF underlies the accelerated functional and biochemical recovery of animals with lesions of the insular cortex.

Differential participation of the NBM in the acquisition and retrieval of conditioned taste aversion and Morris water maze.

Deficits in both learning and memory after lesions of the cholinergic basal forebrain, in particular the nucleus basalis magnocellularis (NBM), have been widely reported. However, the participation of the cholinergic system in either acquisition or retrieval of memory process is still unclear. In this study, we tested the possibility that excitotoxic lesions of the NBM affect either acquisition or retrieval of two tasks. In the first experiment, animals were trained for two conditioned taste aversion tasks using different flavors, saccharine and saline. The acquisition of the first task was before NBM lesions (to test retrieval) and the acquisition of the second task was after the lesions (to test acquisition). Accordingly, in the first part of the second experiment, animals were trained in the Morris water maze (MWM), lesioned and finally tested. In the final part of this experiment, another set of animals was lesioned, then trained in the MWM and finally tested. All animals were able to retrieve conditioned taste aversion (CTA) and MWM when learned before NBM lesions; however, lesions disrupted the acquisition of CTA and MWM. The results suggest that the NBM and cholinergic system may play an important role in acquisition but not during retrieval of aversive memories.

Differential effects of bicuculline and muscimol microinjections into the nucleus basalis magnocellularis in taste and place aversive memory formation.

The role of the nucleus basalis magnocellularis (NBM) in learning and memory has been demonstrated in different learning paradigms such as conditioned taste aversion (CTA) and inhibitory avoidance (IA). This participation has been related to the cholinergic system, but recent studies have reported the potential role of other neurotransmitters such as GABA. The effects of acute intracerebral administration of the GABAergic antagonist bicuculline (0.05 microg) and the GABAergic agonist muscimol (0.05 microg) into the NBM of male Wistar rats were assessed in CTA and IA learning. In both learning tasks, the drug administration was performed before the acquisition. Taste aversion learning was not affected by the infusion of any of the drugs administered. IA acquisition was not affected by the administration of bicuculline or muscimol, requiring similar number of trials to reach the learning criterion. However, when the rats were tested 24 h later, those injected with bicuculline or muscimol showed an impairment of the IA learning. The present results support a role of the GABAergic system in the consolidation process of IA learning.

Long-term potentiation in the insular cortex enhances conditioned taste aversion retention.

Long-lasting changes in synaptic strength, such as long-term potentiation (LTP), are thought to underlie memory formation. Recent studies on the insular cortex (IC), a region of the temporal cortex implicated in the acquisition and retention of conditioned taste aversion (CTA), have demonstrated that tetanic stimulation of the basolateral nucleus of the amygdala (Bla) induce LTP in the IC of adult rats in vivo, as well as, that blockade of N-methyl-D-aspartate (NMDA) receptors disrupts CTA and IC-LTP induction in vivo. Here, we present experimental data showing that induction of LTP in the Bla-IC projection previous to CTA training enhances the retention of this task. These findings are of particular interest since they provide support for the view that the neural mechanisms underlying neocortical LTP may contribute to memory related functions performed by the IC.

Insular cortex and amygdala lesions differentially affect acquisition on inhibitory avoidance and conditioned taste aversion.

These experiments examined the effects of NMDA-induced lesions of the amygdala and insular (gustatory) cortex (IC) on inhibitory avoidance learning and conditioned taste aversion (CTA) in rats. IC lesions, but not amygdala lesions, disrupted CTA. In contrast, lesions of either brain region disrupted inhibitory avoidance learning. These findings support the view that the IC is strongly involved in the acquisition of external as well as visceral aversively motivated behavior. Despite extensive functional interconnections, these 2 brain regions appear to have different roles in mediating different forms of aversively based learning.

In vivo long-term potentiation in the insular cortex: NMDA receptor dependence.

It has been demonstrated that the insular cortex (IC) plays an important role in the acquisition and storage of different aversive motivated learning tasks like conditioned taste aversion, spatial maze and inhibitory avoidance. It is of particular interest to investigate whether activity-dependent modification of synaptic efficacy, a presumptive mechanism for learning and memory, is present in this cortical region. Here, we address this issue by examining the induction of synaptic plasticity, long-term potentiation (LTP) in in vivo preparations. The results showed that high frequency stimulation of the basolateral amygdaloid nucleus (Bla) induced LTP in the IC. The LTP induced by tetanus was blocked by application of the N-methyl-D-aspartate (NMDA) receptor antagonists CPP and MK-801, indicating that NMDA receptors were responsible for its induction. These results suggest that in vivo tetanus induced LTP of the Bla-IC projection is a possible mechanism for the memory-related functions performed by the IC.

Hypothalamic but not cortical grafts induce recovery of sexual behavior and connectivity in medial preoptic area-lesioned rats.

We have previously shown that hypothalamic fetal brain grafts induced recovery of sexual behavior in medial preoptic area (MPOA)-lesioned male rats. In the present series of experiments, male rats with completely abolished sexual behavior by MPOA lesions received either hypothalamic or frontal cortical fetal grafts. The animals that received hypothalamic grafts showed a gradual recovery of sexual behavior. In contrast, those animals who received cortical grafts did not recover sexual behavior during the 15 weeks after the graft. In addition, to evaluate the connectivity of the grafted tissue with the host brain, a retrograde tracer, fluorogold, was injected in the dorsal tegmental area. Fluorogold-labeled cells were found in the hypothalamic, but not in the cortical grafts. These results suggest that specificity of the grafted tissue and connectivity between brain grafts and host tissue are necessary for the recovery of male sexual behavior in MPOA-lesioned rats.

Recovery of taste aversion learning induced by fetal neocortex grafts: correlation with in vivo extracellular acetylcholine.

Rats showing disrupted taste aversion due to insular cortex lesions, received either homotopic or heterotopic (occipital) cortical fetal brain grafts. Behavioral results showed that the recovery of the ability to acquire conditioned taste aversions induced by fetal grafts depended on post-graft time (45 but not at 15 days) and tissue specificity (homotopic but not heterotopic). In vivo analysis of acetylcholine (ACh) release revealed that only the group receiving homotopic grafts and tested 45 days post graft had a release of ACh after KCl stimulation similar to that in the control group. Furthermore, homotopic grafts and lesioned groups showed significantly weaker specific receptor binding of [3H]L-glutamate compared with controls. These results suggest that ACh is specifically involved in the process of behavioral recovery induced by homotopic cortical transplants.

Cortical cholinergic activity is related to the novelty of the stimulus.

A number of studies have related cholinergic activity to the mediation of learning and memory. However, the acetylcholine (ACh) participation has been recently implicated in the early stages of memory formation but not during retrieval. The aim of the present study is to evaluate ACh release in the insular cortex (IC) during presentation of different taste stimuli and during their re-exposition by means of the free-moving microdialysis technique. We evaluated the changes in ACh release when a novel taste, saccharin or quinine was presented to the rat and after several presentations of saccharin. Unilateral microdialysis was performed in the IC 1 h before and 1 h after the presentation of: (1) a familiar stimulus (water), (2) a novel taste (quinine), (3) another novel taste (saccharin), (4) a second presentation, (5) a third presentation, and (6) a fourth presentation of saccharin. The volume consumed by the animals was registered as a behavioral parameter. The ACh levels from the microdialysis fractions were analyzed by an HPLC-ED system. Biochemical results showed a significant increment in the cortical ACh release induced by a novel stimulus compared with the release observed during the presentation of a familiar stimulus. The ACh release observed after several presentations of the stimuli decreased to the same levels as those produced by the familiar taste, indicating an inverse relationship between familiarity and cortical ACh release. These results suggest that the cholinergic system plays an important role in the identification and characterization of different kinds of stimuli.

Release of acetylcholine, gamma-aminobutyrate, dopamine and glutamate, and activity of some related enzymes, in rat gustatory neocortex.

The gustatory neocortex (GN), final relay along the gustatory pathway, is a region of the brain involved in the neural integration of feeding behavior. Since information on the neurotransmitters in this nucleus is scarce, the aim of the present work was to establish whether acetylcholine (ACh), gamma-aminobutyric acid (GABA), dopamine and glutamate may act as transmitters within this structure. It was found that GN slices are able to release labeled GABA, ACh and glutamate but not dopamine. Additionally, it was possible to detect significant glutamic acid decarboxylase, choline acetyltransferase and acetylcholinesterase activities in GN homogenates. The activity of the two enzymes involved in acetylcholine metabolism was higher than that observed in other cortical regions. These findings suggest that GABA, ACh and glutamate probably are neurotransmitters in the GN, whereas dopamine is not.

Fetal brain grafts induce recovery of learning deficits and connectivity in rats with gustatory neocortex lesion.

Three groups of rats showing disrupted taste aversion due to gustatory neocortex lesions, were studied. One group received a transplant of homotopic cortical tissue, another of heterotopic tectal tissue, obtained from 17-day-old fetuses. The third group remained without transplant as a lesioned control group. Comparisons of the taste aversion scores before and after graft, revealed that cortical grafted animals significantly improved the taste aversion, whereas those which received tectal grafts, and the cortical-lesioned controls did not. Moreover, results with horseradish peroxidase (HRP) histochemistry revealed that the homotopic, but not the heterotopic, brain transplants were able to re-establish connections with amygdala and with the ventromedial nucleus of the thalamus areas who normally kept connectivity with the gustatory neocortex. These results support the hypothesis that fetal brain transplants can reestablish cognitive functions, as well as connectivity with its host tissue.

Fetal brain transplants induce recuperation of taste aversion learning.

Rats showing disrupted taste aversion due to gustatory neocortex or amygdala lesions were transplanted into the lesioned area with homologous brain tissue obtained from 17-day-old fetuses. Comparisons of taste aversions scores before and after the graft, revealed that the grafted animals significantly recuperated taste aversions, whereas cortical lesioned animals without grafts did not. Surprisingly, however, amygdala-lesioned animals without graft presented spontaneous recovery. These results not only support the hypothesis that fetal brain transplants can restore cognitive functions, but also that there are some fundamental functional differences between the gustatory neocortex and the amygdala in the regulation of the processes involved in the acquisition and retention of taste aversion.