Recruitment of neurons in basolateral amygdala after intense training produces a stronger memory trace.

Typical amnestic treatments are ineffective when administered to subjects trained in aversively-motivated tasks using relatively high foot-shock intensities. This effect has been found when treatments that disrupt neuronal activity are administered to different regions of the brain, including the amygdala. However, the molecular mechanisms induced by this intense training are unknown. We made a detailed mapping of c-Fos-expressing neurons in four regions of the amygdala after moderate and intense one-trial inhibitory avoidance training. Rats were sacrificed 90 min after training or after appropriate control procedures, and their brains were prepared for immunohistochemical c-Fos protein detection in the central, lateral, and in the anterior and posterior parts of the basolateral amygdaloid nucleus. We found a high percentage of neurons expressing c-Fos in the anterior part of the basolateral nucleus after moderate training, and this percentage increased further after intense training. Moderate and intense training did not induce changes in c-Fos expression in the other explored amygdaloid regions. These results show that inhibitory avoidance training produces a localized expression of c-Fos in the basolateral anterior nucleus of the amygdala, which is dependent upon the intensity of training, and indicate that synaptic plastic changes in this region may be required for the formation of memory of moderate and intense aversive learning.

Hippocampal mechanisms in impaired spatial learning and memory in male offspring of rats fed a low-protein isocaloric diet in pregnancy and/or lactation.

Maternal nutritional challenges during fetal and neonatal development result in developmental programming of multiple offspring organ systems including brain maturation and function. A maternal low-protein diet during pregnancy and lactation impairs associative learning and motivation. We evaluated effects of a maternal low-protein diet during gestation and/or lactation on male offspring spatial learning and hippocampal neural structure. Control mothers (C) ate 20% casein and restricted mothers (R) 10% casein, providing four groups: CC, RR, CR, and RC (first letter pregnancy, second lactation diet). We evaluated the behavior of young adult male offspring around postnatal day 110. Corticosterone and ACTH were measured. Males were tested for 2 days in the Morris water maze (MWM). Stratum lucidum mossy fiber (MF) area, total and spine type in basal dendrites of stratum oriens in the hippocampal CA3 field were measured. Corticosterone and ACTH were higher in RR vs. CC. In the MWM acquisition test CC offspring required two, RC three, and CR seven sessions to learn the maze. RR did not learn in eight trials. In a retention test 24 h later, RR, CR, and RC spent more time locating the platform and performed fewer target zone entries than CC. RR and RC offspring spent less time in the target zone than CC. MF area, total, and thin spines were lower in RR, CR, and RC than CC. Mushroom spines were lower in RR and RC than CC. Stubby spines were higher in RR, CR, and RC than CC. We conclude that maternal low-protein diet impairs spatial acquisition and memory retention in male offspring, and that alterations in hippocampal presynaptic (MF), postsynaptic (spines) elements and higher glucocorticoid levels are potential mechanisms to explain these learning and memory deficits.

Effect of 17ß-estradiol on zinc content of hippocampal mossy fibers in ovariectomized adult rats.

Sex hormones such as estrogen (17ß-estradiol) may modulate the zinc content of the hippocampus during the female estrous cycle. The mossy fiber system is highly plastic in the adult brain and is influenced by multiple factors including learning, memory, and stress. However, whether 17ß-estradiol is able to modulate the morphological plasticity of the mossy fibers throughout the estrous cycle remains unknown. Ovariectomized (Ovx) female 70- to 90-day-old Sprague-Dawley rats without or with estrogen supplement (OvxE) were compared with control rats in three stages of the estrous cycle: diestrus, proestrus, and estrus. The brain tissue from each of the five groups was processed with Timm's silver sulfide technique using the Image J program to measure the mossy fiber area in the stratum lucidum of CA3. Total zinc in the hippocampus was measured using Graphite Furnace Atomic Absorption Spectrophotometry. Two additional (Ovx and OvxE) groups were examined in spatial learning and memory tasks using the Morris water maze. Similar increases in total zinc content and mossy fiber area were observed. The mossy fiber area decreased by 26 ± 2 % (difference ± SEM percentages) in Ovx and 23 ± 4 % in estrus as compared to the proestrus group and by 18 ± 2 % in Ovx compared to OvxE. Additionally, only the OvxE group learned and remembered the task. These results suggest that estradiol has a significant effect on zinc content in hippocampal CA3 during the proestrus stage of the estrous cycle and is associated with correct performance in learning and memory.

[The consolidation of memory, one century on]. / La consolidacion de la memoria, un siglo despues.

INTRODUCTION: The theory of memory consolidation, based on the work published by Georg Elias Muller and Alfons Pilzecker over a century ago, continues to guide research into the neurobiology of memory, either directly or indirectly. In their classic monographic work, they concluded that fixing memory requires the passage of time (consolidation) and that memory is vulnerable during this period of consolidation, as symptoms of amnesia appear when brain functioning is interfered with before the consolidation process is completed. Most of the experimental data concerning this phenomenon strongly support the theory. DEVELOPMENT: In this article we present a review of experiments that have made it possible to put forward a model that explains the amnesia produced in conventional learning conditions, as well as another model related to the protection of memory when the same instances of learning are submitted to a situation involving intensive training. CONCLUSIONS: Findings from relatively recent studies have shown that treatments that typically produce amnesia when they are administered immediately after a learning experience (during the period in which the memory would be consolidating itself) no longer have any effect when the instances of learning involve a relatively large number of trials or training sessions, or relatively high intensity aversive events. These results are not congruent with the prevailing theories about consolidation.

Spared retention of inhibitory avoidance learning after posttraining amygdala lesions.

Previous findings indicate that the memory-impairing effects of posttraining amygdala lesions are attenuated by increasing the number of training trials given prior to the induction of the lesion. The aim of this experiment was to determine whether the degree of impairment is also influenced by the footshock intensity used during training. Rats were given 1 trial of inhibitory avoidance (IA) training with either no footshock or a footshock at 1 of 3 intensities. Sham or neurotoxic amygdala lesions were induced 1 week later. On a retention test performed 4 days after surgery, the performance of all amygdala-lesioned rats given footshock training, including those given the lowest training footshock, was better than that of amygdala-lesioned rats given no training footshock. These findings of preserved retention of IA learning in rats given posttraining amygdala lesions do not support a general hypothesis that the amygdala is a locus of permanent changes underlying aversively motivated learning.

Dose- and time-dependent scopolamine-induced recovery of an inhibitory avoidance response after its extinction in rats.

The present investigation was aimed at elucidating the dose and time dependency of scopolamine-induced recovery of inhibitory avoidance after its extinction. Two experiments were conducted: in the first, we analyzed the effects of four doses (1, 2, 4, and 8 mg/kg) of the musacrinic receptor antagonist scopolamine, on the expression of this conditioned response once it had been extinguished. Independent groups of rats were trained in a one-trial, step-through inhibitory avoidance task and submitted to daily retention (extinction) tests. After extinction had occurred, animals were injected intraperitoneally 10 min before retention testing, either with saline or scopolamine. Results show that scopolamine produced a dose-dependent recovery of the avoidance response. The second experiment was carried out in the same animals, which were now tested for retention of inhibitory avoidance at 1, 2, 3, 6, and 9 months after completion of the first experiment. All rats received counterbalanced injections of saline or scopolamine 10 min before testing at each time interval. Reliable recovery of the avoidance response was observed at the 1-month interval with a clear dose dependency while, after the second month, only the groups treated with the two higher doses continued responding. The results indicate that recovery of the extinguished response produced by muscarinic blockade follows dose- and time-dependent curves, and can be achieved long after a single training session. These data suggest that the inhibitory avoidance memory trace is retained in the brain after behavioural extinction of this response, thus supporting the view of extinction as new learning that affects the retrieval of the original memory, but does not modify its storage.

Norepinephrine release in the amygdala in response to footshock and opioid peptidergic drugs.

These experiments used in vivo microdialysis and high-performance liquid chromatography to examine, in rats, norepinephrine (NE) release in the amygdala induced by footshock and systemic administration of drugs affecting the opioid peptidergic system. A microdialysis probe was inserted into a previously implanted guide cannula aimed at the amygdala and the rat was placed in a box with a stainless-steel grid floor through which a single footshock was delivered. Samples were collected and analyzed at 15-min intervals. Footshock stimulation increased NE levels and the magnitude of the increase varied with footshock intensity. Relative to baseline levels, intensities of 0.3, 0.7 and 1.2 mA (3 s) induced increases of 41, 64 and 97%, respectively. NE levels returned to baseline within 30 min after footshock stimulation. The opioid peptidergic antagonist naloxone (1 mg/kg, i.p.) administered immediately after footshock (0.55 mA for 1 s) potentiated NE release. In contrast, the opioid peptidergic agonist beta-endorphin (10 microgram/kg, i.p.) administered after the footshock blocked the footshock-induced increase in NE levels. The magnitude of NE release was less when the drugs were administered without prior footshock and when the injections were given 30 min after footshock. The findings are consistent with previous evidence that acute, mildly stressful stimulation induces the release of NE in the amygdala as well as with extensive pharmacological evidence indicating that amygdala NE released by arousing stimulation is involved in regulating memory storage and that the opioid peptidergic system influences memory storage by modulating the release of NE in the amygdala.

Selective M1 muscarinic receptor antagonists disrupt memory consolidation of inhibitory avoidance in rats.

The effect of three different M1 muscarinic antagonists, pirenzepine, biperiden, and trihexyphenidyl on memory consolidation was investigated. Rats were trained in a one-trial step-through inhibitory avoidance task and injected intraperitoneally immediately afterwards, either with pirenzepine, biperiden, or trihexyphenidyl (dose range from 0 to 16 mg/kg). The non-selective antimuscarinic compound scopolamine, was also administered for comparison. One day later, rats were tested for retention. Results show that biperiden, trihexyphenidyl and scopolamine produced a dose-dependent impairment of inhibitory avoidance consolidation, while pirenzepine had no effect. The amnestic state produced by biperiden and trihexyphenidyl was comparable to that observed after the administration of scopolamine. These results indicate that the selective blockade of the central M1 muscarinic receptors interfere with memory consolidation of inhibitory avoidance and suggest that this receptor subtype is critically involved in mnemonic functions.

Protective effect of under-reinforcement of passive avoidance against scopolamine-induced amnesia.

Administration of antimuscarinic drugs induces amnesia of aversively motivated behaviors. However, when relatively high intensities of footshock are used during training (over-reinforcement), animals become protected against such amnesic state. Moreover, the protective effect is established in a none-or-all fashion, i.e., within a series of increasing intensities a minute augmentation of footshock intensity is sufficient to reach the protective threshold. In the present experiment it was found that very low intensities of aversive stimulation (under-reinforcement), sufficient to produce learning, also protected animals from scopolamine-induced amnesia. These results suggest that acetylcholine is critically involved in memory consolidation of passive avoidance, but only within a limited range of training intensities.

Effects of GABA antagonists on inhibitory avoidance.

Experimental data indicate that GABA is involved in memory processes. However there are marked inconsistencies in the reported effects of interference with GABA synaptic activity on memory consolidation of aversively-motivated tasks. Both amnesia and improvement of performance have been reported after treatment with GABA antagonists. These contradictory effects could be explained by procedural differences in training. To test for this possibility rats were trained in passive avoidance using two levels of footshock and injected with a wide range of doses of picrotoxin and bicuculline. Picrotoxin did not modify the conditioned response while bicuculline induced amnesia only with the lower doses at both low and high footshock intensities. It was concluded that GABA is involved in memory consolidation, and that the conflicting results in the literature are indeed due, in part, to procedural differences, and also to the mode of action of these drugs.

Reversal of extinction by scopolamine.

The aim of this experiment was to determine the effects of muscarinic blockade on extinction of passive avoidance conditioning. Rats were trained with a foot shock of 2.5, 3.0, or 6.0 mA and were tested for retention for 8 weeks (once weekly). Five minutes before the seventh test they were injected with 8 mg/kg scopolamine. The groups that had been trained with 2.5 and 3.0 mA showed extinction, which was reversed by the scopolamine; the overreinforced group (6.0 mA) did not show extinction and the scopolamine did not alter the conditioned response. The data support the hypothesis that extinction represents the learning of a new response sustained by a set of cholinergic neurons, different from that which mediated original passive avoidance learning.

Increase of mushroom spine density in CA1 apical dendrites produced by water maze training is prevented by ovariectomy.

Dendritic spine density increases after spatial learning in hippocampal CA1 pyramidal neurons. Gonadal activity also regulates spine density, and abnormally low levels of circulating estrogens are associated with deficits in hippocampus-dependent tasks. To determine if gonadal activity influences behaviorally induced structural changes in CA1, we performed a morphometric analysis on rapid Golgi-stained tissue from ovariectomized (Ovx) and sham-operated (Sham) female rats 7 days after they were given a single water maze (WM) training session (hidden platform procedure) or a swimming session in the tank containing no platform (SC). We evaluated the density of different dendritic spine types (stubby, thin, and mushroom) in three segments (distal, medial, and proximal) of the principal apical dendrite from hippocampal CA1 pyramidal neurons. Performance in the WM task was impaired in Ovx animals compared to Sham controls. Total spine density increased after WM in Sham animals in the proximal and distal CA1 apical dendrite segments but not in the medial. Interestingly, mushroom spine density consistently increased in all CA1 segments after WM. As compared to the Sham group, SC-Ovx rats showed spine pruning in all the segments, but mushroom spine density did not change significantly. In Ovx rats, WM training increased the density of stubby and thin, but not mushroom spines. Thus, ovariectomy alone produces spine pruning, while spatial learning increases spine density in spite of ovariectomy. Finally, the results suggest that mushroom spine production in CA1 after spatial learning requires gonadal activity, whereas this activity is not required for mushroom spine maintenance.

Glucocorticoid enhancement of memory storage involves noradrenergic activation in the basolateral amygdala.

Evidence indicates that the modulatory effects of the adrenergic stress hormone epinephrine as well as several other neuromodulatory systems on memory storage are mediated by activation of beta-adrenergic mechanisms in the amygdala. In view of our recent findings indicating that the amygdala is involved in mediating the effects of glucocorticoids on memory storage, the present study examined whether the glucocorticoid-induced effects on memory storage depend on beta-adrenergic activation within the amygdala. Microinfusions (0.5 microg in 0.2 microl) of either propranolol (a nonspecific beta-adrenergic antagonist), atenolol (a beta1-adrenergic antagonist), or zinterol (a beta2-adrenergic antagonist) administered bilaterally into the basolateral nucleus of the amygdala (BLA) of male Sprague-Dawley rats 10 min before training blocked the enhancing effect of posttraining systemic injections of dexamethasone (0.3 mg/kg) on 48-h memory for inhibitory avoidance training. Infusions of these beta-adrenergic antagonists into the central nucleus of the amygdala did not block the dexamethasone-induced memory enhancement. Furthermore, atenolol (0.5 microg) blocked the memory-enhancing effects of the specific glucocorticoid receptor (GR or type II) agonist RU 28362 infused concurrently into the BLA immediately posttraining. These results strongly suggest that beta-adrenergic activation is an essential step in mediating glucocorticoid effects on memory storage and that the BLA is a locus of interaction for these two systems.

Differential effects of unilateral lidocaine infusion into the globus pallidus on consolidation and performance of inhibitory avoidance.

The striatum is involved in memory consolidation; also involved in this process is one of its two major efferent targets, namely, the substantia nigra. It is not clear, however, if the other target, the globus pallidus, participates in storage and/or performance of learned information. To examine this problem, male Wistar rats were trained in an inhibitory avoidance task and tested for retention 24 h afterward. Independent groups were infused, unilaterally, with 2% lidocaine in the pallidus either 2 min after training or 2 min before testing. No disturbances of memory were detected with posttraining infusion, but a significant deficit in retention was observed as a consequence of pretest infusion. Infusion of isotonic saline into the globus pallidus, or of lidocaine before testing into the parietal cortex, after training into the ventral thalamic nucleus, and both before training and testing into this thalamic nucleus were without effect. Taken together, the data indicate that unilateral inactivation of the GP interferes with retrieval of information derived from inhibitory avoidance training, but not with the early stages of memory consolidation of this task, and other work indicates that the pallidus may be involved in a late phase of this process.

Post-training injection of GABAergic antagonists into the striatum produces retrograde amnesia.

Although it is well established that the striatum contains both local and projection GABA neurons, little is known about their possible role in memory storage. We now report that in the Wistar rat, intrastriatal post-training injection of microgram quantities of bicuculine or picrotoxin, two GABA antagonists with different modes of action, induced retrograde amnesia of inhibitory avoidance, in concordance with the effects of injections of picrotoxin into the substantia nigra. These results suggest that GABA activity of the nigrostriatal system is involved in cognitive functions.

Effects of regional GABAergic blockade of the striatum on memory consolidation.

The prediction was made that blocking the action of gamma-aminobutyric acid (GABA) in different regions of the striatum, through local injection of picrotoxin, would produce differential amnestic effects. Rats were trained in an inhibitory avoidance task, and a retention test was carried out 24 h later. Posttraining injection of picrotoxin (1 microgram/1 microliter) into posteroventral and lateral regions produced strong amnesia; an intermediate degree of impairment was found when delivered at dorsomedial regions; and no retention deficit was found when given to the ventromedial aspect of the anterior striatum. In sum, the retention impairments were consistently higher in posterior and lateral striatal regions than in the anterior and medial regions. The present findings support the concept that striatal GABAergic activity is involved in memory functions and also provide further evidence of neurochemical heterogeneity within the striatum regarding memory consolidation.

High level of footshock during inhibitory avoidance training prevents amnesia induced by intranigral injection of GABA antagonists.

Disruption of synaptic activity of a number of cerebral structures (e.g., neostriatum, amygdala, and thalamus) produces marked deficits in retention of instrumentally conditioned behaviors. When animals are given a relatively high number of training trials or high intensities of footshock during learning, however, such disruption is considerably less effective. Since there is a close anatomical and functional relationship between the neostriatum and the substantia nigra, it was of interest to determine whether enhanced training with a high level of footshock would prevent the reported amnesic state induced by injections of GABA antagonists into the latter structure. Rats were trained in a one-trial inhibitory task, using 0.2 or 0.4 mA, and then injected with microgram quantities of picrotoxin or bicuculline into the substantia nigra and posterior region of the zona incerta; retention was measured 24 h later. Only those groups that had been injected into the nigra and trained with 0.2 mA showed amnesia. These results support the hypotheses that (a) the normal activity of a set of structures is essential for the development of memory consolidation and (b) after an enhanced learning experience these structures may participate in memory consolidation, but are not necessary for the occurrence of this process.

Effects of central muscarinic blockade on passive avoidance: anterograde amnesia, state dependency, or both?

It was recently reported that administration of relatively high intensities of footshock (overreinforcement) during training of passive avoidance protected animals against the amnesic effect of scopolamine, injected 5 min after training. This was interpreted in terms of a lesser involvement of acetylcholine in memory consolidation. An alternative explanation was that overreinforcement accelerated the consolidation process, which could have taken place before the injection of scopolamine. To test for this possibility, male Wistar rats were injected with 4, 8, or 12 mg/kg of scopolamine, 5 min before training with low or high levels of footshock and then tested for retention of the task. Scopolamine induced the expected memory deficit after the low-intensity footshock; after overreinforcement the higher doses of scopolamine induced state dependency, while no deficits were produced with the lower dose. It was concluded that: (a) acetylcholine is indeed involved in memory consolidation of passive avoidance; (b) scopolamine interacts with high footshock levels to produce state dependency; and (c) when relatively low doses of scopolamine are used in conditions of overreinforcement, protection against scopolamine-induced amnesia becomes evident.