Reactivation enables memory updating, precision-keeping and strengthening: exploring the possible biological roles of reconsolidation.

Although much has been learned regarding the molecular and cellular mechanisms of memory reconsolidation, its actual biological function remains unclear. In this work we investigate the possibility that three different mnemonic processes - updating, precision-keeping and trace strengthening - are mediated by reconsolidation in contextual fear conditioning. Reconsolidation involves the activation of calcium channels for the destabilization during the reactivation. Our results show that when memory is reactivated in a situation that does not match the original information, content is modified, i.e., "updated". However, when the contextual condition matches the original one, memory reactivation contributes either to its strengthening or to the maintenance of its precision content over time. Since the L-type voltage-gated calcium channel antagonist nimodipine blocked these effects, we suggest that reconsolidation is the mechanism supporting these processes.

Early life handling decreases serotonin turnover in the nucleus accumbens and affects feeding behavior of adult rats.

In our previous studies, we reported that neonatally handled rats have an increased ingestion of sweet food but are resistant to the damaging effects of a chronic exposure to a highly palatable diet. Accumbal serotonin (5-HT) is important for feeding behavior and plays a role in the vulnerability to diet-induced obesity. Therefore, our hypotheses were (1) 5-HT turnover in the nucleus accumbens is altered in neonatally handled animals and plays a role in their differential feeding behavior and (2) if this is so, a chronic pharmacological treatment affecting 5-HT reuptake (chronic imipramine) would be able to revert the behavioral findings. Litters were divided into nonhandled and handled (10 min/day, Days 1-10 after birth). In Experiment 1, we demonstrated that a decreased 5-HT metabolism in the nucleus accumbens was observed in adult handled animals. In Experiment 2, the two previous groups were subdivided and assigned to receive imipramine diluted in water or water alone. After 30 days of treatment, we evaluated their weight gain and feeding behavior. Handled rats weighed less than nonhandled rats, and all imipramine-treated rats showed a reduction in weight gain after 60 days of treatment. Imipramine reverted the increased sweet food consumption seen in neonatally handled rats. We conclude that serotonin is involved in the altered feeding behavior of neonatally handled rats, and this protocol is an important tool for studying the mechanisms by which early life events have a long-term impact on feeding preferences.

Early life experience alters behavioral responses to sweet food and accumbal dopamine metabolism.

Neonatal handling in rats persistently alters behavioral parameters and responses to stress. Such animals eat more sweet food in adult life, without alterations in lab chow ingestion. Here, we show that neonatally handled rats display greater incentive salience to a sweet reward in a runway test; however they are less prone to conditioned place preference and show less positive hedonic reactions to sweet food. When injected with methylphenidate (a dopamine mimetic agent), non-handled rats increase their sweet food ingestion in the fasted state, while neonatally handled rats do not respond. We did not observe any differences regarding baseline general ambulatory activity between the groups. A lower dopamine metabolism in the nucleus accumbens was observed in handled animals, without differences in norepinephrine content. We suggest that early handling leads to a particular response to positive reinforcers such as palatable food, in a very peculiar fashion of higher ingestion but lower hedonic impact, as well as higher incentive salience, but diminished dopaminergic metabolism in the nucleus accumbens.

Opposite action of hippocampal CB1 receptors in memory reconsolidation and extinction.

Retrieval of a consolidated memory triggers a number of processes which depend, among other factors, on the duration of the reactivation session: reconsolidation requires a brief reactivation session, and extinction, a prolonged one. The scope of this study is to explore the potential role of the hippocampal endocannabinoid system on reconsolidation and extinction processes. Bilateral infusion of the CB1 cannabinoid receptor antagonist, N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2, 4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251) into the CA1 region of the dorsal hippocampus of Wistar rats after memory reactivation facilitated the reconsolidation of the contextual fear conditioning memory. The inhibition of protein synthesis with DRB in the same brain region blocked memory reconsolidation. Both effects were persistent, lasting up to 7 days after the first retrieval experience. In contrast, the local infusion of anandamide blocked memory reconsolidation, an effect that was antagonized by the combined administration of anandamide with a subthreshold dose of a CB1 antagonist, supporting a CB1-mediated role of the hippocampal endocannabinoid system in the modulation of the memory reconsolidation. Local infusion of AM251 into CA1 blocked memory extinction whereas the administration of anandamide facilitated it; however, when combined with a subthreshold concentration of the CB1 antagonist, anandamide did not affect the extinction process. The clear-cut, opposite effects observed in each situation suggest a possible role of the hippocampal endocannabinoid system as a switching mechanism deciding which processes will take place, either maintaining the original memory (reconsolidation) or promoting a new learning (extinction).

Reliable short-term memory in the trion model: toward a cortical language and grammar.

It has previously been shown that Hebb learning in a single column in the trion model of cortical organization occurs by selection. Motivated by von Neumann's solution for obtaining reliability and by models of circulating cortical activity, we introduce Hebb intercolumnar couplings to achieve dramatic enhancements in reliability in the firing of connected columns. In order for these enhancements to occur, specific temporal phase differences must exist between the same inherent spatial-temporal memory patterns in connected columns. We then generalize the criteria of large enhancements in the global firing of the entire connected columnar network to investigate the case when different inherent memory patterns are in the columns. The spatial rotations as well as the temporal phases now are crucial. Only certain combinations of inherent memory patterns meet these criteria with the symmetry properties playing a major role. The columnar order of these memory patterns not in the same symmetry family can be extremely important. This yields the first higher-level architecture of a cortical language and grammar within the trion model. The implications of this result with regard to an innate human language and grammar are discussed.

Muscarinic toxins: novel pharmacological tools for the muscarinic cholinergic system.

Muscarinic receptors are widely spread throughout the body, and are involved in the regulation of fundamental physiological processes, like the modulation of the heart rate, control of motor systems and modulation of learning and memory. In the central nervous system the cholinergic transmission is mainly mediated by muscarinic receptors; there are five subtypes that are all expressed in the brain of mammals (m1-m5). There are regional differences in their concentrations in the brain and more than one subtype is expressed in the same cell. It has been difficult to study their localization and function in vivo due to the lack of ligands that exclusively act on one subtype of the receptor. We studied the action of the muscarinic toxins MT1, MT2 and MT3, from the venom of the snake Dendroaspis angusticeps, on muscarinic receptors, by using the classical muscarinic radioligand 3H-NMS as reporter of the inhibition of its own binding, to either native or cloned receptors. We have also studied the in vivo effects on memory retention of the injection of the toxins into discrete brain regions. The muscarinic toxins appear to be invaluable tools to study receptor pharmacology, physiology and structure/function relationships. They would enable the design of new, more selective, pharmacological agents.

S100B infusion into the rat hippocampus facilitates memory for the inhibitory avoidance task but not for the open-field habituation.

Adult male Wistar rats were bilaterally implanted with indwelling cannulae in the hippocampus. Forty-eight hours after surgery, animals were habituated to an open-field box during 2 min, being tested 24 h later; next they were trained in a step-down inhibitory avoidance task (3.0 s, 0.4 mA foot-shock), being tested again 24 h later. Immediately after the training session of each task, animals received a 0.5-microl infusion of calcium-phosphate-buffered saline (PBS) and S100B (20, 200, 2000, or 20,000 nM). In the inhibitory avoidance task, animals infused with the two highest concentrations of S100B, 2 and 20 microM, obtained higher scores of retention relative to controls in the test session (p<0.05), and a trend toward an increase was observed in animals infused with 200 nM (p<0. 10). In both sessions of the habituation task, groups were not different regarding crossings, rearings, and time for leaving the first square (p>0.10). These results indicate that, in rats, post-training increased hippocampal levels of S100B right after training facilitate, in a dose-dependent way, long-term memory for an inhibitory avoidance task, but not for an open-field habituation.

What can toxins tell us for drug discovery?

Toxins are of interest in drug design because the toxins provide three-dimensional templates for creating small molecular mimics with interesting pharmacological properties. Toxins are also useful in drug discovery because they can be used as pharmacological tools to uncover potential therapeutic targets. With their high potency and selectivity, toxins are often more useful in functional experiments than standard pharmacological agents. We have used two groups of neurotoxins, the dendrotoxins and the muscarinic toxins (MTs), to explore the involvement of subtypes of potassium ion channels and muscarinic receptors, respectively, in processes involved in cognition and the changes in neuronal properties with aging. From our current work, quantitative autoradiographic studies with radiolabelled dendrotoxins reveal widespread distribution of binding sites throughout rat brain sections, but few differences exist between young adult and aged rats. However, displacement studies with toxin K, which preferentially binds to the Kv1.1 subtype of cloned potassium channel, show the selective loss of such sites in regions of the hippocampus and septohippocampal pathway with aging. MTs have been tested for effects on performance of rats in memory paradigms. MT2, which activates m1 receptors, improves performance of rats in a step-down inhibitory avoidance test, whereas MT3, which blocks m4 receptors, decreases performance when given into the hippocampus. This is the first clear demonstration of a role for m4 muscarinic receptors in cognition.

L-type voltage-dependent calcium channel blocker nifedipine enhances memory retention when infused into the hippocampus.

Wistar rats with cannulae bilaterally implanted in the CA1 region of the dorsal hippocampus were trained in a step-down inhibitory avoidance task. Through these cannulae they received an infusion of 28 or 280 ng per side of the L-type voltage-dependent calcium channel antagonist nifedipine, or of its vehicle (20% dimethyl sulfoxide in saline). The two doses of the drug were studied by administration 0 or 30 min after training; in addition, the higher dose was studied by infusion 10 min before training. A retention test was carried out 24 h after the training session. The highest dose of nifedipine administered 0 min post-training enhanced test session performance of the animals compared to the control group; the effect of the lower dose was not statistically significant. There was no effect of the drug given 30 min post-training or 10 min pretraining. Despite the inability to discriminate direct neural from indirect vascular effects, these results are consistent with previous reports on nootropic actions of the dihydropyridine class of calcium channel blockers. The data are at variance with the amnestic effect of intrahippocampal nifedipine described by Lee and Lin (1991, Life Sciences, 48, 1333-1340), which may be attibuted to the different range of doses studied here. This might resemble the inverted U-shaped dose-response curve observed with another dihydropyridine, nimodipine, by other authors.

Muscarinic toxin selective for m4 receptors impairs memory in the rat.

The selectivity of the muscarinic toxin MT3 from green mamba snake venom was corroborated by inhibition of the binding of [3H]NMS, a classical muscarinic radioligand, to native and cloned muscarinic receptors, showing 214-fold higher affinity for m4 than for m1 subtype, without significant binding to the others. The highest concentrations of MT3 sites (putative m4 receptors) in the rat brain were found in striatum and olfactory tubercle, intermediate concentration in dentate gyrus and CA1, and lower but still conspicuous levels in CA3 and frontal cortex. MT3 caused retrograde amnesia of an inhibitory avoidance task, when injected into the dorsal hippocampus of rats after training, suggesting a positive role of these MT3 sensitive sites, which are probably m4 muscarinic receptors, in memory consolidation of this task.

Sequential role of hippocampus and amygdala, entorhinal cortex and parietal cortex in formation and retrieval of memory for inhibitory avoidance in rats.

The hippocampus and amygdala, the entorhinal cortex and the parietal cortex participate, in that sequence, both in the formation and in the expression of memory for a step-down inhibitory avoidance task in rats. Bilateral infusion of AP5 or muscimol caused retrograde amnesia when given 0 min after training into both hippocampus and amygdala, when given or 180 min after training into the entorhinal cortex, or when given 180 min after training into the parietal cortex. Therefore, memory formation requires the sequential and integrated activity of all these areas mediated by glutamate NMDA receptors in each case. Pre-test administration of CNQX 1 day after training into hippocampus and amygdala, 1 or 31 days after training in entorhinal cortex, or 1, 31 or 60 days after training in the parietal cortex temporarily blocked retention test performance. Therefore, 1 day after training, all these brain structures are necessary for retrieval; 1 month later, the hippocampus and amygdala are no longer necessary for retrieval but the entorhinal and parietal cortex still are; and 60 days after training only the parietal cortex is needed. In all cases the mechanisms of retrieval require intact glutamate AMPA receptors.

Involvement of the hippocampus, amygdala, entorhinal cortex and posterior parietal cortex in memory consolidation.

A total of 182 young adult male Wistar rats were bilaterally implanted with cannulae into the CA1 region of the dorsal hippocampus and into the amygdaloid nucleus, the entorhinal cortex, and the posterior parietal cortex. After recovery, the animals were trained in a step-down inhibitory avoidance task. At various times after training (0, 30, 60 or 90 min) the animals received a 0.5-microliter microinfusion of vehicle (saline) or 0.5 microgram of muscimol dissolved in the vehicle. A retention test was carried out 24 h after training. Retention test performance was hindered by muscimol administered into both the hippocampus and amygdala at 0 but not at 30 min posttraining. The drug was amnestic when given into the entorhinal cortex 30, 60 or 90 min after training, or into the parietal cortex 60 or 90 min after training, but not before. These findings suggest a sequential entry in operation, during the posttraining period, of the hippocampus and amygdala, the entorhinal cortex, and the posterior parietal cortex in memory processing.

Involviment of the hippocampus, amygdala, entorhinal cortex and posterior parietal cortex in memory consolidation

A total of 182 young adult male Wistar rats were bilaterally implanted with cannulae into the CA1 region of the dorsal hippocampus and into the amygdaloid nucleus, the entorhinal cortex, and the posterior parietal cortex. After recovery, the animals were trained in a stepdown inhibitory avoidance task. At various times after training (0, 30, 60 or 90 min) the animals received a 0.5-mul microinfusion of vehicle (saline) or O.5 mug of muscimol dissolved in the vehicle. A retention test was carried out 24 h after training. Retention test performance was hindered by muscimol administered into both the hippocampus and amygdala at 0 but not at 30 min posttraining. The drug was amnestic when given into the entorhinal cortex 30, 60 or 90 min after training, or into the parietal cortex 60 or 90 min after training, but not before. These findings suggest a sequential entry in operation, during the posttraining period, of the hippocampus and amygdala, the entorhinal cortex, and the posterior parietal cortex in memory processing.

Age-related effects of diazepam on retention of inhibitory avoidance and shuttle avoidance tasks in rats.

We investigated the effect of diazepam on memory of 30 days-old and 60-70 days-old female Wistar rats, using two behavioral tasks: step-down inhibitory avoidance (IA) and shuttle avoidance (SA). Diazepam (0.2, 1.0 or 5.0 mg/kg) or its vehicle were given i.p., 60 min prior to the training session. Training-test interval was 24 h. Diazepam impaired the retention of IA in 30 days-old rats at the three doses used, while retention of SA was not impaired by any dose. In the 60-70 days-old animals, diazepam at the dose of 0.2 mg/kg was facilitatory in IA and had no effect on SA, while doses of 1.0 mg/kg and 5.0 mg/kg impaired retention of both tasks. We suggest that these age-dependent effects of diazepam on memory of IA and SA could be related to developmental changes in brain GABAA receptors.

Involvement of mechanisms dependent on NMDA receptors, nitric oxide and protein kinase A in the hippocampus but not in the caudate nucleus in memory.

The effects of the NMDA receptor antagonist AP5, the nitric oxide synthase (NO) inhibitor NO-arg or the protein kinase A (PKA) inhibitor KT5720 on memory were evaluated. Rats bilaterally implanted in the CA1 region of the dorsal hippocampus were trained and tested in a step-down inhibitory avoidance task, and rats unilaterally implanted in the left posteroventral region of the caudate nucleus were trained and tested in a cued water maze task. Previous findings from this and other laboratories had found that lesions or pharmacological treatments of these sites significantly altered memory of these two tasks. Immediately after training, animals received intrahippocampal or intracaudate 0.5 microliter microinfusions of saline, AP5, NO-arg or KT5720. All three drugs impaired retention of inhibitory avoidance, but did not affect retention of the cued water maze. The findings suggest that NMDA receptor-, NO- and PKA-mediated processes in the dorsal hippocampus, but not in the caudate nucleus, are involved in memory.

Different brain areas are involved in memory expression at different times from training.

Rats were trained in a step-down inhibitory avoidance task and tested for retention 1, 31, or 60 days later. Three to 7 days prior to testing, they were bilaterally implanted with cannulae in the CA1 region of the dorsal hippocampus and in the amygdaloid nucleus (H + A), in the entorhinal cortex (EC), and in the posterior parietal cortex (PPC). Ten minutes prior to testing, the animals received, through the cannulae, 0.5-microliter microinfusions of vehicle (20% dimethylsulfoxide in saline) or of 0.5 microgram of CNQX dissolved in the vehicle. A second test session was carried out 90 min after the first. CNQX blocked retention test performance when given into H + A 1 day after training but not later; when given into EC 1 or 31 days after training, but not later; and when given into PPC 1, 31, or 60 days after training. In all cases performance returned to normal levels in the second test session. The data suggest that H and A are involved in memory expression for only a few days after acquisition; that EC is involved in memory expression for up to 31, but less than 60, days after acquisition; and that PPC is involved in memory expression for up to at least 2 months after acquisition.

Sequential involvement of NMDA receptor-dependent processes in hippocampus, amygdala, entorhinal cortex and parietal cortex in memory processing.

Rats bilaterally implanted with cannulae in the CA1 region of the dorsal hippocampus and/or in the amygdaloid nucleus, in the entorhinal cortex, and in the posterior parietal cortex, were trained in a step-down inhibitory avoidance task. At various times after training (immediately, 30, 60 or 90min) they received, through the cannulae, 0.5µl microinfusions of saline or of 5.0µg of AP5 dissolved in saline. A retention test was carried out 24h after training. Retention test performance was hindered by AP5 given into hippocampus, amygdala, or both hippocampus and amygdala immediately but not 30min post-training. The drug was amnestic when given into the entorhinal cortex 30, 60 or 90min after training, or into the parietal cortex 60 or 90min after training, but not at earlier times. The findings suggest a sequential entry in operation, in the post-training period, of NMDA-receptor mediated mechanisms involved in memory processing; first in hippocampus and amygdala, 30min later in entorhinal cortex, and 30min later in posterior parietal cortex.

Memory enhancement by intrahippocampal, intraamygdala, or intraentorhinal infusion of platelet-activating factor measured in an inhibitory avoidance task.

Platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine), which is thought to be a retrograde messenger in long-term potentiation (LTP), enhances glutamate release and LTP through an action on presynaptic nerve endings. The PAF antagonist BN 52021 blocks CA1 LTP in hippocampal slices, and, when infused into rat dorsal hippocampus pre- or posttraining, blocks retention of inhibitory avoidance. Here we report that memory is affected by pre- or posttraining infusion of the PAF analog 1-O-hexadecyl-2-N-methylcarbamoyl-sn-glycerol-3-phosphocholine (mc-PAF) into either rat dorsal hippocampus, amygdala, or entorhinal cortex. Male Wistar rats were implanted bilaterally with cannulae in these brain regions. After recovery from surgery, the animals were trained in step-down inhibitory avoidance or in a spatial habituation task and tested for retention 24 h later. mc-PAF (1.0 microgram per side) enhanced retention test performance of the two tasks when infused into the hippocampus before training without altering training session performance. In addition, mc-PAF enhanced retention test performance of the avoidance task when infused into (i) the hippocampus 0 but not 60 min after training; (ii) the amygdala immediately after training; and (iii) the entorhinal cortex 100 but not 0 or 300 min after training. In confirmation of previous findings, BN 52021 (0.5 microgram per side) was found to be amnestic for the avoidance task when infused into the hippocampus or the amygdala immediately but not 30 or more minutes after training or into the entorhinal cortex 100 but not 0 or 300 min after training. These findings support the hypothesis that memory involves PAF-regulated events, possibly LTP, generated at the time of training in hippocampus and amygdala and 100 min later in the entorhinal cortex.

Effect of antagonists of platelet-activating factor receptors on memory of inhibitory avoidance in rats.

Platelet-activating factor (PAF) is present in the brain. It enhances glutamate release and long-term potentiation (LTP) through an action on synaptic membrane receptors sensitive to the antagonist, BN 52021, and has been proposed as a retrograde messenger in the genesis of LTP. In addition, PAF has other, metabolic actions mediated by microsomal receptors sensitive to the antagonist, BN 50730. We investigated the effect on memory of the pre- or post-training infusion of BN 52021 or BN 50730 into the hippocampus and that of BN 52021 in the amygdala and the entorhinal cortex. Male Wistar rats were implanted bilaterally with cannulae aimed at these brain regions. After recovery from surgery, the animals were trained in step-down inhibitory avoidance using a 0.5-mA foot shock and tested for retention 24 h later. BN 52021 (0.5 microgram/side) was amnestic when given into the hippocampus or the amygdala either before or immediately after training but not 30 or 100 min later. BN 52021 was also amnestic when given into the entorhinal cortex 100 but not 0 or 300 min after training. Intrahippocampally administered BN 50730 had no effect on memory. The findings are compatible with the suggestion from previous findings that memory of this task depends on the generation of LTP at the time of training in hippocampus and amygdala and, 90-180 min later, in the entorhinal cortex.

CNQX infused into entorhinal cortex blocks memory expression, and AMPA reverses the effect.

Rats were trained in a step-down inhibitory avoidance task using a 0.8-mA foot shock and tested for retention 26 days later. Three to five days prior to the retention test they were bilaterally implanted with cannulae aimed at the entorhinal cortex. Ten minutes before testing they received an infusion, into the entorhinal cortex, of vehicle, ciano-nitro-quinoxaline-dione (CNQX; 0.5 micrograms), amino-hydroxy-methyl-isoxalone-propionate (AMPA; 1.0 or 2.5 micrograms), or AMPA (1.0 micrograms) plus CNQX (0.5 micrograms). CNQX blocked memory expression; the effect lasted less than 90 min. AMPA had no effect of its own, but at the lower dose level it counteracted the depressant influence of CNQX. It is not likely that the effect of CNQX could have been due to an influence on performance: In separate sets of experiments the bilateral intraentorhinal infusion of CNQX (0.5 micrograms) 10 min before training did not affect either acquisition or retention of the avoidance task or general activity during 3 min of free exploration in the training box. The results indicate that the integrity of AMPA receptors in the entorhinal cortex is necessary for memory expression.