Benedetto Sacchetti

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.

Role of Secondary Sensory Cortices in Emotional Memory Storage and Retrieval in Rats

The Storage of Emotions The neural mechanisms involved in emotional learning are well understood. However, how and where emotional memories are stored is still largely unclear. Sacco and Sacchetti (p. 649) now show that Pavlovian fear memories are stored in a modality-specific way in the secondary, but not primary, sensory cortices. The site of storage depended on whether the conditioned stimulus was visual, auditory, or olfactory. Only “old,” not new, memories were stored in this way, and lesions of secondary cortices, while disrupting the old memories, did not prevent the acquisition of new memories. An emotional memory gradually becomes widely distributed throughout the cortex. Visual, acoustic, and olfactory stimuli associated with a highly charged emotional situation take on the affective qualities of that situation. Where the emotional meaning of a given sensory experience is stored is a matter of debate. We found that excitotoxic lesions of auditory, visual, or olfactory secondary sensory cortices impaired remote, but not recent, fear memories in rats. Amnesia was modality-specific and not due to an interference with sensory or emotional processes. In these sites, memory persistence was dependent on ongoing protein kinase Mζ activity and was associated with an increased activity of layers II–IV, thus suggesting a synaptic strengthening of corticocortical connections. Lesions of the same areas left intact the memory of sensory stimuli not associated with any emotional charge. We propose that secondary sensory cortices support memory storage and retrieval of sensory stimuli that have acquired a behavioral salience with the experience.

Learning-related feedforward inhibitory connectivity growth required for memory precision

In the adult brain, new synapses are formed and pre-existing ones are lost, but the function of this structural plasticity has remained unclear. Learning of new skills is correlated with formation of new synapses. These may directly encode new memories, but they may also have more general roles in memory encoding and retrieval processes. Here we investigated how mossy fibre terminal complexes at the entry of hippocampal and cerebellar circuits rearrange upon learning in mice, and what is the functional role of the rearrangements. We show that one-trial and incremental learning lead to robust, circuit-specific, long-lasting and reversible increases in the numbers of filopodial synapses onto fast-spiking interneurons that trigger feedforward inhibition. The increase in feedforward inhibition connectivity involved a majority of the presynaptic terminals, restricted the numbers of c-Fos-expressing postsynaptic neurons at memory retrieval, and correlated temporally with the quality of the memory. We then show that for contextual fear conditioning and Morris water maze learning, increased feedforward inhibition connectivity by hippocampal mossy fibres has a critical role for the precision of the memory and the learned behaviour. In the absence of mossy fibre long-term potentiation in Rab3a−/− mice, c-Fos ensemble reorganization and feedforward inhibition growth were both absent in CA3 upon learning, and the memory was imprecise. By contrast, in the absence of adducin 2 (Add2; also known as β-adducin) c-Fos reorganization was normal, but feedforward inhibition growth was abolished. In parallel, c-Fos ensembles in CA3 were greatly enlarged, and the memory was imprecise. Feedforward inhibition growth and memory precision were both rescued by re-expression of Add2 specifically in hippocampal mossy fibres. These results establish a causal relationship between learning-related increases in the numbers of defined synapses and the precision of learning and memory in the adult. The results further relate plasticity and feedforward inhibition growth at hippocampal mossy fibres to the precision of hippocampus-dependent memories.

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.

Long-term synaptic changes induced in the cerebellar cortex by fear conditioning

To better understand learning mechanisms, one needs to study synaptic plasticity induced by behavioral training. Recently, it has been demonstrated that the cerebellum is involved in the consolidation of fear memory. Nevertheless, how the cerebellum contributes to emotional behavior is far from known. In cerebellar slices at 10 min and 24 hr following fear conditioning, we found a long-lasting potentiation of the synapse between parallel fibers and Purkinje cells in vermal lobules V-VI, but not in the climbing fiber synapses. The mechanism is postsynaptic, due to an increased AMPA response. In addition, in hotfoot mice with a primary deficiency of the parallel fiber to Purkinje cell synapse, cued (but not contextual) fear conditioning is affected. We propose that this synapse plays an important role in the learned fear and that its long-term potentiation may represent a contribution to the neural substrate of fear memory.

Cerebellum and emotional behavior.

Fear conditioning involves learning that a previously neutral stimulus (CS) predicts an aversive unconditioned stimulus (US). Lesions of the cerebellar vermis may affect fear memory without altering baseline motor/autonomic responses to the frightening stimuli. Reversible inactivation of the vermis during the consolidation period impairs retention of fear memory. In patients with medial cerebellar lesions conditioned bradycardia is impaired. In humans, cerebellar areas around the vermis are activated during mental recall of emotional personal episodes, if a loved partner receives a pain stimulus, and during learning of a CS-US association. Moreover, patients with cerebellar stroke may fail to show overt emotional changes. In such patients, however, the activity of several areas, including ventromedial prefrontal cortex, anterior cingulate, pulvinar and insular cortex, is significantly increased relative to healthy subjects when exposed to frightening stimuli. Therefore, other structures may serve to maintain fear response after cerebellar damage. These data indicate that the vermis is involved in the formation of fear memory traces. We suggest that the vermis is not only involved in regulating the autonomic/motor responses, but that it also participates in forming new CS-US associations thus eliciting appropriate responses to new stimuli or situations. In other words, the cerebellum may translate an emotional state elaborated elsewhere into autonomic and motor responses.

The Cerebellum: Synaptic Changes and Fear Conditioning

In addition to coordinating movement, the cerebellum participates in motor learning, emotional behavior, and fear memory. Fear learning is reflected in a change of autonomic and somatic responses, such as heart rate and freezing, elicited by a neutral stimulus that has been previously paired with a painful one. Manipulation of the vermis affects these responses, and its reversible inactivation during the consolidation period impairs fear memory. The neural correlate of cerebellar involvement in fear consolidation is provided by a behaviorally induced long-term increase of synaptic efficacy between parallel fibers and a Purkinje cell. Similar synaptic changes after fear conditioning are well documented in the amygdala and hippocampus, providing a link between emotional experiences and changes in neural activity. In addition, in hotfoot mice, with a primary deficiency of parallel fiber to Purkinje cell synapse, short- and long-term fear memories are affected. All these data support the idea that the cerebellum participates in fear learning. The functional interconnection of the vermis with hypothalamus, amygdala, and hippocampus suggests a more complex role of the cerebellum as part of an integrated network regulating emotional behavior.

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.

Involvement of the intracellular ion channel CLIC1 in microglia-mediated beta-amyloid-induced neurotoxicity

It is widely believed that the inflammatory events mediated by microglial activation contribute to several neurodegenerative processes. Alzheimers disease, for example, is characterized by an accumulation of β-amyloid protein (Aβ) in neuritic plaques that are infiltrated by reactive microglia and astrocytes. Although Aβ and its fragment 25-35 exert a direct toxic effect on neurons, they also activate microglia. Microglial activation is accompanied by morphological changes, cell proliferation, and release of various cytokines and growth factors. A number of scientific reports suggest that the increased proliferation of microglial cells is dependent on ionic membrane currents and in particular on chloride conductances. An unusual chloride ion channel known to be associated with macrophage activation is the chloride intracellular channel-1 (CLIC1). Here we show that Aβ stimulation of neonatal rat microglia specifically leads to the increase in CLIC1 protein and to the functional expression of CLIC1 chloride conductance, both barely detectable on the plasma membrane of quiescent cells. CLIC1 protein expression in microglia increases after 24 hr of incubation with Aβ, simultaneously with the production of reactive nitrogen intermediates and of tumor necrosis factor-α (TNF-α). We demonstrate that reducing CLIC1 chloride conductance by a specific blocker [IAA-94 (R(+)-[(6,7-dichloro-2-cyclopentyl-2,3-dihydro-2-methyl-1-oxo-1H-inden-5yl)-oxy] acetic acid)] prevents neuronal apoptosis in neurons cocultured with Aβ-treated microglia. Furthermore, we show that small interfering RNAs used to knock down CLIC1 expression prevent TNF-α release induced by Aβ stimulation. These results provide a direct link between Aβ-induced microglial activation and CLIC1 functional expression.