Effects of coupled perirhinal cortex and medial septal area, fimbria-fornix, entorhinal cortex tetrodotoxin inactivations on passive avoidance consolidation in the rat.

In order to ascertain the rat perirhinal cortex (PC) function during early consolidation of a passive avoidance response (PAR), and to ascertain whether there are some functional interactions with the medial septal area (MSA), the fimbria-fornix complex (FF) and the entorhinal cortex (EC), PC-MSA, PC-FF, and PC-EC coupled inactivations were performed immediately after the PAR acquisition session. Anesthetized male adult Wistar rats aged 60 days were treated with stereotaxical bilateral injections of TTX (5 ng in 0.5 microl saline) in the appropriate sites. Retrieval testing was performed 48 h later. It was shown that all three coupled inactivations were followed by significant PAR disruption. It may be concluded that PC is somehow active even during the first mnemonic phase following the acquisition session, thus better defining PC mnemonic involvement chronology. These results may be taken as indicating that during initial consolidation the engram is concurrently processed in more than one septal and parahippocampal site, each of which by itself is not absolutely necessary for the final engram formation.

Effects of combined medial septal area, fimbria-fornix and entorhinal cortex tetrodotoxin inactivations on passive avoidance response consolidation in the rat.

On the basis of previous experimental evidence, it has been concluded that the entorhinal cortex (EC), the fimbria-fornix (FF) complex and medial septal area (MSA) do not take part in the consolidation phase of passive avoidance response (PAR) memorization. On the other hand, a mnemonic role during consolidation of at least two of these structures has been argued, based on several considerations. In order to ascertain whether the EC and FF are still involved in PAR memorization during consolidation, the coupled fully reversible functional tetrodotoxin (TTX) inactivation of MSA, FF and EC was performed in rats having undergone a PAR training. In Experiment 1 MSA, FF and EC were inactivated pair-wise (FF and EC always bilaterally). Permanently cannulated animals were injected stereotaxically with TTX (5 ng in 0.5 microliter saline) or saline (0.5 microliter) immediately following PAR acquisition. It was shown that combined FF-EC inactivation induced PAR retention impairment, whereas FF-MSA and EC-MSA inactivation was not followed by amnesic effects. Having obtained a positive result, in Experiment 2 the combined FF-EC inactivation was performed at different post-acquisition delays (0.25 h, 1.5 h, 6 h), so as to assess the duration of their involvement in PAR consolidation. It was shown that only the coupled inactivation performed at the shortest post-acquisition delay was followed by amnesic effects. Thus EC and FF play a definite role during early consolidation. The results are discussed in relation to EC, FF, MSA, and hippocampal involvement in PAR memorization, as reported in previous studies, and to their connectivity.

Neural topography and chronology of memory consolidation: a review of functional inactivation findings.

Findings on the role of subcortical and cortical structures in mnemonic processes, obtained by means of the reversible functional inactivation technique, are reviewed. The main advantage of this method (subcortical or cortical administration of local anesthetics or tetrodotoxin) is that it provides information not only on "where" but also "when" and for "how long" these processes take place, thus adding to the topographical dimension the chronological one. The review covers several types of memory (e.g., passive avoidance and spatial memory) studies examining the neural substrates of memory consolidation on the basis of the functional inactivation of the nucleus of the solitary tract, parabrachial nuclei, substantia nigra, hippocampus (dorsal and ventral), nucleus basalis magnocellularis, amygdala, medial septal area, striatum, olfactory bulb, and neocortex. The data are discussed in relation to earlier research and with respect to the anatomical and functional connectivity of the examined centers.

Temporal characterization of subcortical nuclei in mnemonic processes: results of tetrodotoxin reversible inactivation studies in the rat.

By means of permanent lesion techniques it has been possible to ascertain whether a given subcortical neural structure is involved in memory processing. These results, however, are useful only to build a topography of memory, i.e. to provide information only on the "where" such processes take place. Memory being, per se, a temporal process, organized in at least three putative phases (acquisition, consolidation, retrieval) it is of paramount importance to know not only the "where", but also the "when", and, possibly, the "how long" of a given site involvement. The fully reversible inactivation technique has been employed to assess the chronological involvement of subcortical sites. By means of the stereotaxic administration of tetrodotoxin (TTX) it has been possible to inactivate known volumes of nervous tissue for given periods of time. In this way, it has been possible to measure the amnesic effects (disruption of the performance of a passive avoidance response, PAR) after inactivation of discrete neural sites. The data so far obtained by these means are presented and discussed. The comparison of results is justified by the constancy of the experimental subjects (young adult male rats of the same age), the surgical interventions, and the conditioning paradigm (passive avoidance responding in the light-dark box). The parabrachial nuclei, substantia nigra, ventral hippocampus, dorsal hippocampus, nucleus basalis magnocellularis, amygdala, globus pallidus, nucleus caudate-putamen (anterior, median, posterior), medial septal area and nucleus accumbens have been investigated. From these studies, data have been acquired on all three phases of memorization. The most detailed findings concern consolidation. In particular, it was shown that the functional integrity duration necessary to avoid amnesic damages varies greatly from site to site, from at least 15 min to no less than 48 hours. The results confirm and amplify previous experimental work, by defining the chronology of mnemonic involvement of many neural sites. The results are discussed in terms of comparison between sites and connectivity between the investigated sites and other sites or neuronal systems.

Entorhinal cortex and fimbria-fornix role in rat's passive avoidance response memorization.

The stereotaxic administration of tetrodotoxin (TTX) was employed to induce the fully reversible inactivation of the fimbria-fornix complex (FF) and of the entorhinal cortex (EC), in order to ascertain the role of these structures in the memorization of a passive avoidance response (PAR). On permanently cannulated rats TTX (5 ng in 0.5 microliter saline) or saline (0.5 microliter) was injected uni- or bilaterally, respectively, in the FF and in the EC, 60 min before PAR acquisition, immediately after PAR acquisition and 60 min before PAR retrieval, always performed 48 h after the acquisition trial. It was shown that EC unilateral or bilateral pre-acquisition inactivation was followed by amnesia, while TTX inactivation in post-acquisition and pre-retrieval had no effects. Identical results were obtained by TTX administration in FF. The experimental evidence indicates that both EC and FF play a role during acquisition of PAR engram. The results are discussed in comparison with previous ones concerning dorsal and ventral hippocampus TTX inactivation effects on rat's PAR, and in relation to hippocampal and medial septal area connectivity.

Analysis of mnemonic processing by means of totally reversible neural inactivations.

The irreversible lesions technique precludes the analysis of the possibly critical role played by discrete brain sites in the several distinct stages of mnemonic processing (acquisition, consolidation, retrieval) during which these may be specifically but transiently active. On the contrary, the reversible functional inactivation techniques, by means of stereotaxic local microinjection of active compounds, make it possible to suppress the neuronal function of a discrete volume of nervous tissue, for a pre-determined time, with the assurance of complete functional recovery within a known duration. This technique makes it possible to block the neural activity of a chosen neural site at a given stage of memory processing without any interference with the function of the same structure either during earlier or later stages of the same process. Thus, the reversible ablation results may provide information not only on the qualitative topographical but also on the quantitative temporal dimension of learning and memory. The technique employed to cause totally reversible neural inactivation is detailed. The employment of several agents to obtain functional inactivation is discussed. Of these, perhaps the safest and most manageable is tetrodotoxin when a fairly long functional inactivation (e.g., 1 h) is desired. The effects of a reversible inactivation can be quite easily and accurately assessed by observing the severity of the amnesic disruption, if any, of a conditioned response. In order to do this as well as possible, it is advantageous to employ a very simple behavioral paradigm. The passive avoidance response in the light-dark box apparatus fulfills this requirement. Moreover, this paradigm, being one-trial, provides the necessary condition of a single well-defined temporal beginning. The present protocol has been successfully employed in learning and memory research, to assess when the functional integrity of a given neural structure is necessary in order that a conditioned response may be acquired, consolidated or retrieved. The employment of this protocol in relation to the intrinsic functional characteristics of a given subcortical neural site is discussed.

Role of ventral hippocampus in acquisition, consolidation and retrieval of rat's passive avoidance response memory trace.

By means of local administration of tetrodotoxin (TTX) a fully reversible functional inactivation of rat's ventral hippocampus (VH) was obtained in order to characterize the role of this structure in the memorization of a conditioned passive avoidance response (PAR). In Experiment 1, on permanently cannulated animals, TTX (10 ng in 1.0 microl saline) or saline (1.0 microl) was injected uni- or bilaterally in the VH, respectively, 1 h before PAR acquisition, immediately after PAR acquisition, and 1 h before PAR retrieval, always performed 48 h after the acquisition trial. It was shown that both pre-acquisition and pre-retrieval VH uni- or bilateral blockades were followed by significant PAR retention impairment, while in post-acquisition only the bilateral blockade determined PAR retention impairment. In Experiment 2, on three different groups of rats, TTX (10 ng in 1 microl saline) was bilaterally administered, under general ketamine anesthesia (100 mg/kg b.w.), into the VH at different post-acquisition delays (0.25, 1.5, 6 h). Retrieval testing, 48 h after treatment, showed that post-acquisition bilateral VH blockade caused PAR impairment only when performed 0.25 h after acquisition. The results clearly indicate a role of VH during acquisition, consolidation and retrieval of PAR engram. The experimental evidence is discussed in comparison to previous results concerning TTX dorsal hippocampus blockade effects on rat's PAR and in relation to hippocampal connectivity with the medial septal area and the amygdala.

Post-training nucleus basalis magnocellularis functional tetrodotoxin blockade effects on passive avoidance consolidation in the rat.

The tetrodotoxin (TTX) functional ablation technique was employed in order to evaluate the temporal coordinates of the rat's nucleus basalis magnocellularis (NBM) involvement in memory trace processing. Under ketamine general anesthesia, TTX (10 ng in 1 microliter saline) was stereotaxically administered to rats, either in one or both NBMs. TTX was injected to different groups of rats, respectively 15 min, 6, 24, 48, 96 h after passive avoidance acquisition testing. The rats underwent retrieval testing 48 h later, i.e. after full recovery from TTX effects. Results show that: (1) monolateral TTX blockade significantly impairs PAR conditioned responding if induced up to 6 h but not 24 h after acquisition testing; (2) bilateral TTX blockade dramatically impairs passive avoidance responding up to a 48-h delay but not 96 h after acquisition testing. The results indicate a very profound involvement of NBM in passive avoidance response consolidation. The experimental evidence is discussed together with previous functional ablation findings concerning amygdala, parabrachial nuclei and neocortex.

Passive avoidance response distribution by post-training substantia nigra functional tetrodotoxin inactivation in the rat.

The tetrodotoxin (TTX) functional ablation technique was employed to assess the temporal coordinates of rat's substantia nigra (SN) in memory processing. TTX (10 ng in 1 microliter saline) was stereotaxically administered to rats under general ketamine anesthesia, either bilaterally or unilaterally. TTX was injected in different groups of rats respectively 0.25, 6, 24, and 48 hours after passive avoidance acquisition testing. Rats always underwent retrieval testing 48 hours later, after full recovery from TTX effects. The results show that: i) unilateral TTX blockade significantly impairs PAR only up to 0.25 h and not 6 h after acquisition testing, and ii) bilateral TTX blockade dramatically disrupts passive avoidance responding up to 24 but not 48 hours after acquisition testing. The results indicate a much more important SN role in memory processing than was previously assessed. The experimental evidence is discussed both in relation to previous TTX functional ablation findings (amygdala, parabrachial nuclei, nucleus basalis magnocellularis) and in relation to SN anatomical and functional connections with other subcortical structures.

Physical and optical shelter characteristics influence rat's preferences in a multiple Y-maze.

Rat's preference for covered or uncovered sections of a multiple Y-maze, measured as time spent under cover, was investigated. Surface area of covered and uncovered sections was the same. There were no light-intensity differences between uncovered and covered sections. Coverings were of two types: transparent or sanded plexiglas, affording respectively only physical or physical and optical protection. Both types of covering were placed either over discontinuous sections of the maze or continuously over one entire half of it. Male adult Wistar rats were employed. Rats exhibited maximal preference for the continuous sanded covering. They also exhibited a very similar significant preference for the continuous transparent covering and the discontinuous sanded one. Equal permanence time was measured in uncovered sections and under discontinuous transparent coverings. The results show that rats can recognize and choose shelter even when there is no light diminution under it. In fact they can very well discriminate between the several types of shelter, as shown by their significant longer permanence under the most protective and most continuous one. Finally, results are taken as basis for discussing whether the accepted "dark preference" of rats may be due solely to photophobia or also to the fact that normally darkness indicates a shelter.

Forced extinction as a means to evaluate consolidation gradient of a passive avoidance response in the rat.

Passive avoidance response (PAR) consolidation gradient, and US (footshock) intensity/engram strength relationship were investigated by means of specific forced extinction procedure (30 min detention in the shock box without receiving punishment) in Wistar rats trained in the light-dark box apparatus. Different groups of rats (punished either with 0.8 or 1.2 mA footshock intensity) underwent detention at different postacquisition time delays: immediately or 1, 2, 4 days after acquisition training. By means of this purely behavioral paradigm, designed to investigate a specific PAR memory trace, previous results obtained by using diverse and sometimes unspecific memory-disrupting agents were fully confirmed: PAR strength and consolidation gradient are positively related to US intensity. The influence of differential generalization effects on extinction is discussed. An unexpected finding was that from engrams that are experimentally shown to be of unequal resistance to disruption, equal conditioned responses are obtained.

Minaprine cancels scopolamine effects on the rat's acquisition of passive avoidance responses in two multitrial paradigms.

The antiamnesic activity of minaprine has been studied in male Wistar rats. Two multitrial paradigms were employed: the light-dark box test (aversive stimulus: 0.6-mA foot-shocks) and the tail-handling test (aversive stimulus: manual tail-handling). In both paradigms, intraperitoneal scopolamine administration 30 min before testing significantly impaired the acquisition of the passive avoidance conditioned response. There were no significant differences in either paradigm between control rats and those to whom scopolamine and minaprine were simultaneously administered. These results show that minaprine fully protects the acquisition process of conditioned responses against scopolamine impairment not only in one-trial tests but also in multitrial paradigms. The effects of minaprine in reversing memory deficits are discussed in relation to its stimulating activity on central cholinergic systems.

Effects of nucleus basolateralis amygdalae neurotoxic lesions on some spontaneous activities in the rat.

Drinking, feeding, locomotion and exploratory activity of male Wistar rats were assessed after bilateral stereotaxic administration of ibotenic acid in the nucleus basolateralis amygdalae. Feeding, drinking and locomotion were measured in an activity cage, while exploratory activity was determined in a multiple Y-maze. In the 24-hour cycle, lesioned animals exhibited unvaried feeding, decreased drinking and increased locomotion. Exploration was also increased. The results show that this nucleus is not involved in quantitative feeding control, while it does exert a significant facilitatory influence on drinking. It also exerts an inhibitory influence on exploration and on locomotion.

The behavior of the homozygous and heterozygous sub-types of rats which are genetically-selected for diabetes insipidus: a comparison with Long Evans and Wistar stocks.

Several aspects of spontaneous and conditioned behavior (food and water intake, locomotion and emotionality, passive and active avoidance acquisition and retention) of standard (albino and pigmented) rats, and rats heterozygous (HEDI) and homozygous (HODI) for diabetes insipidus, are reviewed. As would be expected, HODI rats have been repeatedly found to consume far more fluid than either HEDI or control rats. Pigmented rats appear to be more active than albinos. HODI rats exhibit less marked emotional responses than do control rats, among which the pigmented ones exhibit the highest emotionality. Light aversion is more evident in albino than in pigmented rats. No differences are found among HEDI, HODI and normal Long Evans rats. It is quite difficult to provide a clear-cut statement concerning inter-strain differences in passive avoidance behavior, possibly because of the variety of techniques employed. In any case, HODI rats do not perform worse than normal controls do. In one-way active avoidance paradigms, pigmented rats perform better than albinos, and the performance of HODI rats does not differ from that of controls. In two-way avoidance paradigms, albinos appear to outperform pigmented rats. Once again, there are no obvious differences between HODI and control animals. In addition to indicating that HODI rats may actually be less emotional than the other groups of rats reviewed here, the studies described once again fail to confirm the previously alleged functions of vasopressin in memory consolidation.

Effects of nucleus basolateralis amygdalae neurotoxic lesions on aversive conditioning in the rat.

After bilateral stereotaxic administration of ibotenic acid on the n. basolateralis amygdalae, male adult rats were tested in the light-dark box apparatus to measure the time-course of the acquisition and retention of passive and active avoidance responses. The results show that after the lesions both passive avoidance and active avoidance acquisition were impaired. Passive avoidance responses were retained quite well, while active avoidance responses disappeared quickly. Conditioned freezing was almost completely absent. Thus it appears that the n. basolateralis plays a facilitatory role in all the conditioned responses which were investigated.

The rat's right-left preference during free exploration of a multiple Y-maze.

The right-left preference of Wistar, Sprague-Dawley, and Long Evans rats of both sexes was investigated in a multiple Y-maze in which the animals had to perform three consecutive right-left choices in order to go from the common starting point to one of the eight goal arms. The rats were free to explore the maze without being subjected to punishments or rewards. Care was taken to minimize inhibitory influences on exploration (low illumination, no handling to return the rats to home cage). The statistical analysis of the total right-left choices performed by the rats showed that three groups (Wistar females, Sprague-Dawley females, and Long Evans males) exhibited a preference for the right. This preference was not related either to sex or strain, but was possibly a species characteristic.

Aversive conditioning of homozygous and heterozygous D.I. Brattleboro rats in the light-dark box.

Active and passive avoidance, and conditioned freezing acquisition and retention were studied in HODI and HEDI Brattleboro rats. All animals were from the same source and of the same age and sex. The light-dark box test was employed. 0.6 and 2.0 mA footshocks were administered for the same number (7) of daily trials. Extinction time-course was followed for seven consecutive daily trials. Passive avoidance: the conditioned response was acquired and retained equally well by all Ss and for both shock intensities. Active avoidance: for 0.6 mA shocks HODI Ss acquired and retained the response significantly better than HEDI Ss; for 2.0 mA shocks the response was acquired equally by both groups of Ss, and retained significantly better by HODI Ss. Freezing: in general, HODI Ss exhibited less freezing then HEDI Ss. The diverse conditioned behavior of HODI and HEDI Ss in this paradigm, which allows the contemporaneous investigation of several aversive responses, does not support the hypothesis that vasopressin deficiency impairs learning and memory in the rat.

Spontaneous and conditioned behavior of Wistar and Long Evans rats.

Spontaneous behavior (locomotion, feeding, drinking, and exploration in a two box apparatus) as well as conditioned behavior (passive and active avoidance responding, and freezing in the light-dark box apparatus) were studied in naive male Wistar and Long Evans rats. Concerning spontaneous behavior, Long Evans rats were more active during both light and dark periods, and showed better exploratory performance than Wistar rats. Concerning conditioned behavior, Long Evans rats acquired and retained better active and passive avoidance responses, and exhibited longer initial freezing than Wistar rats in the range of 0.6-1.4 mA footshocks. The results better define the important behavioral differences existing between the two strains, Long Evans rats showing consistently a higher level of alertness and a better conditioned performance.

Effects of detention and illumination on rats' exploratory behavior in a two-box apparatus.

The effects of detention and lighting intensity on the exploratory behavior of male Wistar rats have been investigated by means of several types of two-box apparatus. Experiments consisted of six consecutive daily trials. Step-through latency values were taken up to 180 sec. Even a very short detention (10 sec) within the starting box exerted a powerful inhibitory influence on the exploratory behavior of the subjects. Uniformly well-lighted surroundings (both boxes) exerted an equally powerful inhibitory influence on the same behavior. This inhibition was only slightly decreased by the availability of visual cues. Only in the apparatus in which at least one box was dark was the exploratory behavior well maintained. Both detention and bright lighting inhibited selectively the exploratory, locomotor behavior of these animals and did not inhibit other motor activities. In fact, groomings and attempts (abortive passages from one box into the other) were repeatedly performed during the increased step-through latency. These activities are discussed as indicators of a conflict between drives.