Carlo Ambrogi Lorenzini

Role of dorsal hippocampus in acquisition, consolidation and retrieval of rat's passive avoidance response: a tetrodotoxin functional inactivation study

By means of local administration of tetrodotoxin (TTX) fully reversible functional inactivation of rats dorsal hippocampus (DH) was obtained in order to define 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 microliter saline) or saline (1.0 microliter) was injected uni- or bilaterally in the DH, 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 DH 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 microliter) saline) was bilaterally administered, under general ketamine anesthesia (100 mg/kg), into the DH at different post-acquisition delays (0.25, 1.5, 6 h). Retrieval testing, 48 h after treatment, showed that post-acquisition bilateral DH blockade caused PAR impairment only when performed 0.25 or 1.5 h after acquisition. The results indicate a well defined mnemonic role of DH during the acquisition, consolidation and retrieval of PAR engram. The experimental evidence is discussed in relation to other reports and to DH connectivity with the medial septal area and the amygdala.

Cerebellar role in fear-conditioning consolidation

Some cerebellar structures are known to be involved in the memorization of several conditioned responses. The role of the interpositus nucleus (IN) and the vermis (VE) in fear-conditioning consolidation was investigated by means of a combined behavioral and neurophysiological technique. The IN and VE were subjected to fully reversible tetrodotoxin (TTX) inactivation during consolidation in adult male Wistar rats that underwent acoustic conditioned stimulus (CS) and context fear training. TTX was injected in different groups of rats at increasing intervals after the acquisition session. Memory was assessed as conditioned freezing duration measured during retention testing, always performed 72 and 96 h after the stereotaxic TTX administration. This schedule ensures that there is no interference with normal cerebellar function during either the acquisition or the retrieval phase so that any amnesic effect may be due only to consolidation disruption. Our results show that IN functional integrity is necessary for acoustic CS fear response memory formation up to the 96-h after-acquisition delay. VE functional integrity was shown to be necessary for memory formation of both context (up to the 96-h after-acquisition delay) and acoustic CS (up to the 192-h after-acquisition delay) fear responses. The present findings help to elucidate the role of the cerebellum in memory consolidation and better define the neural circuits involved in fear memories.

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 rats 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 rats PAR and in relation to hippocampal connectivity with the medial septal area and the amygdala.

Long-lasting hippocampal potentiation and contextual memory consolidation

In order to ascertain whether there are hippocampal electrophysiological modifications specifically related to memory, exploratory activity and emotional stress, extracellular electrical activity was recorded in hippocampal slices prepared from the brains of male adult rats. Several groups of animals were employed: (i) rats which had freely explored the experimental apparatus (8 min exposure); (ii) rats which had been subjected, in the same apparatus, to a fear conditioning paradigm training entailing the administration of aversive electrical footshocks (8 min exposure); (iii) rats to which the same number of aversive shocks had been administered in the same apparatus, but temporally compressed so as to make difficult the association between painful stimuli and the apparatus (30 s exposure); (iv) naïve rats never placed in the apparatus. Half of the rats from each treatment group were used for retrieval testing and the other half for hippocampal excitability testing. The conditioned freezing response was exhibited for no less than 4 weeks. Hippocampal excitability was measured by means of input–output curves (IOC) and paired‐pulse facilitation curves (PPF). Retrieval testing or brain slices preparation were performed at increasing delays after the training sessions: immediately afterwards or after 1, 7 or 28 days. Only the rats subjected to the fear conditioning training exhibited freezing when placed again in the apparatus (retrieval testing). It was found that IOCs, with respect to naïve rats, increased in the conditioned animals up to the 7‐day delay. In free exploration animals the IOCs increased only immediately after the training session. In all other rats no modification of the curves was observed. IOC increases do not appear to imply presynaptic transmitter release modifications, because they were not accompanied by PPF modifications. In conclusion, a clear‐cut correlation was found between the increase in excitability of the Schaffer collateral–CA1 dendrite synapses and freezing response consolidation.

Time‐dependent inhibition of hippocampal LTP in vitro following contextual fear conditioning in the rat

The effects of contextual fear‐learning on hippocampal synaptic excitability were investigated by means of high frequency tetanic stimulation (HFS) in rat brain slices (hippocampal CA1 region), prepared at different intervals (immediately, 24 h or 7 days) after a one‐trial contextual fear conditioning paradigm session. In the latter, rats that had previously received aversive electrical footshocks in the experimental apparatus exhibited freezing (the conditioned response) when placed again in the same apparatus (retrieval test). It was shown that contextual fear‐learning affects the hippocampal synaptic response. In fact, the HFS produced a decrease in the amplitude of short‐term (STP) and long‐term potentiation (LTP) when compared to control ‘naïve’ subject values. This decrease in STP amplitude could be observed only in slices prepared immediately after the training session. A decrease in the amplitude of long‐term but not short‐term potentiation was also observed at 24 h. At 7 days, no decreases in amplitude were observed. These modifications may be thought of as specifically associated with the learning process as they were not recorded in brain preparations from ‘shock‐only’ rats (i.e. those that received the same number of aversive stimuli of equal intensity as the conditioned group but with the shocks compressed temporally so that the shocked subjects could not associate nociceptive stimulation and surroundings – no conditioned freezing during retention testing). In ‘exploration’ preparations (brain slices from rats having only freely explored the experimental apparatus without receiving any adverse stimulation) a decrease in LTP amplitude was recorded only immediately after the training session, and STP was never modified. The synaptic response modifications do not appear to be due to presynaptic events, as they are not associated with paired‐pulse facilitation curve (PPF) modifications. The present results show that contextual fear conditioning and exploration of a novel environment (i) reduce the ability to induce synaptic plasticity; (ii) differentially influence STP and LTP and that (iii) the persistence of synaptic modifications depends on an animals prior experience.

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.

Differential contribution of some cortical sites to the formation of memory traces supporting fear conditioning

The aim of the present work was to assess the role of some cortical sites of the rat (the prefrontal, PFC, frontal, FC, parietal, PAC, and perirhinal, PC, cortices) in the acquisition of classical fear conditioning (CS and context conditioned freezing). Using the reversible ablation technique the sites were inactivated with lidocaine (LIDO), administered before the one-trial training session. The freezing response, taken as memorization index, was measured in conditions of full functional recovery after the short reversible LIDO inactivation either 3 h (“short-term” memory) and/or 72 h (“long-term” memory) later, so as to follow the temporal dimension of mnemonic elaboration. The results of the inactivations performed during the training session show that PFC, FC, PAC and PC play contemporaneous but distinct roles in the memorization of aversive responses to CS and context. PC inactivation weakened the retention of both mnemonic traces at the 3-h delay. At the same delay FC and PAC inactivation weakened only freezing to acoustic CS while PFC inactivation improved the retention of both traces. Inactivation of all four sites was followed by significant amnesia for both traces at the 72-h after-acquisition delay. The present findings show that PFC, FC, PAC and PC reversible inactivation during the acquisition training session diversely interferes with the memorization of conditioned freezing to acoustic CS and context. Moreover, the different results obtained at the two different retention intervals support the hypothesis that “short-term” and “long-term” memories are not necessarily linked, the earlier one not always influencing the subsequent one.

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.

2-Deoxy-D-galactose effects on passive avoidance memorization in the rat.

2-Deoxy-D-galactose (do-gal) hinders glycoprotein fucosylation. This compound was intracerebroventricularly administered to male adult Wistar rats in order to assess whether it could exert amnesic effects on a passive avoidance response (PAR) to be learned in the light-dark box apparatus. Three experiments were performed. In the first, do-gal was administered immediately after the acquisition trials at three dosages (2, 4, and 8 mumol). It was found that only the 4-mumol dosage was followed by PAR disruption. In the second, do-gal was administered at the adequate dosage (4 mumol) either 30 min before the acquisition trial or 30 min before retrieval testing. It was found that only the preretrieval administration was followed by PAR impairment. In the third, do-gal (4 mumol) was administered in postacquisition, at increasing postacquisition delays (0.25, 1.5, 4, and 6 h). It was found that there was PAR disruption only after do-gal administration at the shortest (0.25 h) delay. The results confirm that in the rat, glycoprotein fucosylation is involved in some of the phases of memory trace processing, and they are discussed in relation to other findings in the rat and the chick.

Amnesic Effects of Preacquisition, Postacquisition, or Preretrieval Tetrodotoxin Administration into the Medial Septal Area on Rat's Passive Avoidance Memorization

By means of local administration of tetrodotoxin (TTX) fully reversible functional inactivation of the medial septal area (MSA) of the rat was obtained in order to define the role of this structure in the retention of a conditioned passive avoidance response (PAR). In permanently cannulated animals, TTX (5 ng in 0.5 microl saline) or saline (0.5 microl) was injected in the MSA. TTX or saline was administered to six different groups of rats, respectively 1 h before PAR acquisition, immediately after PAR acquisition, and 1 h before PAR retrieval, performed 48 h after the acquisition trial. It was shown that MSA preacquisition and preretrieval TTX injections were followed by significant PAR retention impairment, while postacquisition TTX administration had no effect on PAR retention. The results indicate a well-defined mnemonic role of MSA during the acquisition and retrieval periods but not during the consolidation of PAR engram. The experimental evidence is discussed in relation to other reports and to MSA connectivity with other subcortical structures.