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Versión / Versió:Preprint Conflict of Interest (COI) StatementThe authors of the present manuscript declare no conflict of interest. AbstractDue to its capital role in drug seeking, consumption and addictive behaviour there is a growing interest in identifying the neural circuits and molecular mechanisms underlying the formation, maintenance and retrieval of drug related memories. Human studies focused on neuronal systems that store and control drug-conditioned memories have found cerebellar activations during the retrieval of drug-associated cues memory. However, at the preclinical level, almost no attention has been paid to a possible role of the cerebellum in drug-related memories. In the present study, we ought to fill this gap by aiming to investigate the pattern of neuronal activation (as revealed by cFos expression) in different regions of the prefrontal cortex and cerebellum of mice trained to develop conditioned preference for an olfactory stimulus (CS+) paired with cocaine.Our results indicate that CS+ preference was directly associated with cFos expression in cells at the apical region of the granule cell layer of the cerebellar vermis, this relationship being more prominent in some specific lobules. Conversely, cFos+ immunostaining in other cerebellar regions seems unrelated to CS+ preference but to other aspects of the conditioning procedure. At the prefrontal cortex, cFos expression seemed to be related to cocaine administration rather than to its ability to establish conditioned preference. The present results suggest that as it has been observed in some clinical studies, the cerebellum might be an important and largely overlooked part of the neural circuits involved in generating, maintaining and/or retrieving drug memories.

Despite the fact that several data have supported the involvement of the cerebellum in the functional alterations observed after prolonged cocaine use, this brain structure has been traditionally ignored and excluded from the circuitry affected by addictive drugs. In the present study, we investigated the effects of a chronic cocaine treatment on molecular and structural plasticity in the cerebellum, including BDNF, D3 dopamine receptors, ΔFosB, the Glu2 AMPA receptor subunit, structural modifications in Purkinje neurons and, finally, the evaluation of perineuronal nets (PNNs) in the projection neurons of the medial nucleus, the output of the cerebellar vermis. In the current experimental conditions in which repeated cocaine treatment was followed by a 1‐week withdrawal period and a new cocaine challenge, our results showed that cocaine induced a large increase in cerebellar proBDNF levels and its expression in Purkinje neurons, with the mature BDNF expression remaining unchanged. Together with this, cocaine‐treated mice exhibited a substantial enhancement of D3 receptor levels. Both ΔFosB and AMPA receptor Glu2 subunit expressions were enhanced in cocaine‐treated animals. Significant pruning in Purkinje dendrite arborization and reduction in the size and density of Purkinje boutons contacting deep cerebellar projection neurons accompanied cocaine‐dependent increase in proBDNF. Cocaine‐associated effects point to the inhibitory Purkinje function impairment, as was evidenced by lower activity in these cells. Moreover, the probability of any remodelling in Purkinje synapses appears to be decreased due to an upregulation of extracellular matrix components in the PNNs surrounding the medial nuclear neurons.

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Pavlovian conditioning tunes the motivational drive of drug-associated stimuli, fostering the probability of those environmental stimuli to promote and trigger drug seeking and taking.Interestingly, different areas in the cerebellum are involved in the formation and longlasting storage of Pavlovian emotional memory. Very recently, we have shown that conditioned preference for an odour associated with cocaine was directly correlated with cFOS expression in cells at the dorsal region of the granule cell layer of the cerebellar vermis. The main goal of the current investigation was to further extend the description of cFOS-IR patterns in cerebellar circuitry after training mice in a cocaine-odour Pavlovian conditioning procedure, including now the major inputs (the inferior olive and pontine nuclei) and one of the output nuclei (the medial deep nucleus) of the cerebellum. The results showed that the cerebellar hallmark of preference towards an odour cue associated to cocaine is an increase in cFOS expression in the dorsal part of the granule cell layer. cFOS-IR levels expressed in the granule cell layer of mice that did not show cocaine conditioned preference did not differ from the basal levels. Remarkably, mice subjected to a random cocaine-odour pairing procedure (the unpaired group) exhibited higher cFOS-IR in the inferior olive, the pontine nuclei and in the deep medial nucleus. Therefore, our findings suggest that inputs and the output of cerebellar circuitry are enhanced when contingency between the CS+ and cocaine is lacking.

Although speculative, it is possible that these cocaine-dependent cerebellar changes were incubated during withdrawal and manifested by the last drug injection. Importantly, the present findings indicate that cocaine is able to promote plasticity modifications in the cerebellum of sensitised animals similar to those in the basal ganglia.

Nicotine, a major psychoactive component of tobacco smoke, alters GABA modulation of dopamine (DA) neurons in the ventral tegmental area (VTA). Changes in structural neuroplasticity can occur in GABAergic parvalbumin (PRV) positive neurons, which are enveloped by structures of the extracellular matrix called perineuronal nets (PNNs). In the current study, rats were trained to self-administer IV nicotine (0.03 mg/kg/infusion) for 21 days in 1-h daily sessions with an incrementing fixed ratio requirement; a control group received saline infusions. At either 45 min or 72 hr after the last session, immunofluorescence measurements for PNNs, PRV and c-Fos were conducted. In VTA, nicotine self-administration reduced the number of PRV+ cells surrounded by PNNs at 45 min, as well as reducing the intensity of PNNs, suggesting a remodeling of GABA interneurons in this region; the number of PRV+ cells surrounded by PNNs was also reduced at 72 hr. A similar reduction of PNNs occurred in orbitofrontal cortex (OFC), but not in medial prefrontal cortex (PrL or IL), 45 min after the last session; PNNs were not detected in nucleus accumbens (shell or core). The reduction of PNNs in VTA and OFC was unrelated to c-Fos+ cells, as the percent of WFA+ cells co-expressing c-Fos was decreased in OFC, but not in VTA. Thus, nicotine self-administration remodeled PNNs surrounding GABA interneurons in VTA and its indirect connections to OFC, suggesting a new possible molecular target where nicotine-induced neuroplasticity takes place. PNN manipulations may prevent or reverse the different stages of tobacco addiction.