Conference Agenda

Background: Prenatal alcohol exposure (PAE) affects neuronal networks and brain development causing a range of physical, cognitive and behavioural disorders in newborns that persist into adulthood, a pathological condition referred as Fetal Alcohol Spectrum Disorder (FASD). Unfortunately, there is no cure for FASD and the molecular mechanisms underlying this pathology are still unknown. Here, we explored how EtOH may impact the homeostasis of the glutamatergic synapse from a molecular, structural and functional point of view in hippocampus and prefrontal cortex (PFC), leading to behavioural alterations.

Methods: We used in vitro and in vivo models of PAE. For in vitro study, rat organotypic hippocampal slices, after 2 days in culture (immature), were exposed to EtOH (150 mM) for 7 days and 24h of abstinence. For in vivo study, C57Bl/6 mice were exposed to 10% EtOH during the first trimester of pregnancy and the offspring were analyzed for protein expression in postsynaptic compartment, electrical properties from CA1 neurons and behavioral test to assess memory and sociability.

Results: We observed that in vitro EtOH induces downregulation of AMPA and NMDA receptor expression and alteration of AMPA neurotransmission. Similar results were obtained in adolescent mice exposed to EtOH during the first trimester of gestation. In fact, these animals show a significant reduction of hippocampal AMPAergic response, associated with an altered expression of excitatory synaptic proteins, both in hippocampus and PFC. Our molecular results may be associated to the behavioural alterations observed in PAE offspring, that display reduced memory and sociability. Furthermore, these mice exhibited a significant reduction in growth curve and nose size, consistent with what was observed in infants pre-exposed to EtOH during pregnancy.

Conclusions: Our study suggest that PAE leads to dysfunctional formation of glutamatergic synapses that could be responsible for neurobehavioural impairments, contributing to the understanding of the pathogenesis of FASD.

Binge alcohol drinking is common among adolescents, posing a major concern for the maturation of brain and behaviour, including reward/aversion processing and stress response. Calcitonin gene-related peptide (CGRP) signalling represents the convergence point of multisensory aversive afferents, affective state and stress response, thus we explored CGRP levels in discrete brain regions in two rat models of adolescent alcohol binge drinking. In addition, the effects of the phytocannabinoid cannabidiol, which modulates affective states and stress response, were evaluated.

Rats underwent either non-contingent- or two-bottle choice- intermittent alcohol paradigm during adolescence; reward (i.e. social interaction; sucrose preference tests) and aversion (resident-intruder paradigm; tail-flick test) responses were assessed. Hypothalamic/extrahypothalamic corticotropin-releasing hormone and corticosterone were measured by immunofluorescence and ELISA. CGRP in limbic brain regions and brainstem was measured by radioimmunoassay. Cannabidiol was administered during adolescent binge-like alcohol withdrawal (60 mg/kg) or over the two-bottle choice drinking paradigm (40 mg/kg).

Our results show that repeated binge-like drinking during adolescence decreased CGRP levels in prefrontal cortex and nucleus accumbens, and increased CGRP in brainstem, whereas increased levels were observed at late withdrawal. In addition, dynamic alterations of the behavioural response to rewarding- and aversive stimuli, including compromised social stress coping, were observed. In parallel, a progressive increase in hypothalamic and extrahypothalamic corticotropin-releasing hormone levels and glucocorticoid output was highlighted during withdrawal, together with a blunted glucocorticoid output in response to stress. Cannabidiol during withdrawal ameliorated behavioural, neuroendocrine and CGRP-related abnormalities; when administered during the two-bottle choice paradigm, cannabidiol curbed binge-like alcohol intake and prevented behavioural, neuroendocrine and CGRP consequences.

Overall, repeated alcohol intoxication and withdrawal during adolescence altered CGRP signalling; bruised behavioural responses to affective stimuli, including behavioural and neuroendocrine responses to social stress. On the other hand, cannabidiol displayed promising potential for limiting adolescent vulnerability to binge alcohol drinking and preventing long-lasting detrimental consequences.

Compulsive alcohol seeking and taking, defined by resistance to adverse consequences, represents a major challenge in treatment of alcohol use disorders (AUD). To search for neural substrates of individual differences in compulsive alcohol use, we studied footshock-punished alcohol self-administration in outbred rats.

Using a large cohort of male Wistar rats, we found that a subset of animals (~35%) continued to self-administer alcohol despite the presence of a contingent electric shock (0.2 mA). This behaviour was encoded by activity of a neuronal ensemble within the central amygdala (CeA). Chemogenetic inhibition of activated CeA neurons selectively decreased this behavior. Using double-labeling immunohistochemistry we found that CeA neurons promoting punishment-resistant were all protein kinase Cd (PKCδ)-expressing neurons. We established a causal role of these neurons in sustaining compulsive alcohol self-administration, by showing that this behaviour was attenuated following an shRNA knock-down of PKCδ in CeA.

Next, combining RNAscope in situ hybridization and fluorescent immunohistochemistry, we found that expression of PKCd colocalizes with that of the GABA_B receptor in the CeA. To test for the functional role of GABA_B receptors in PKCδ-expressing neurons in compulsive alcohol self-administration, we administered the GABA_B agonist, baclofen, by systemic (1, 3 mg/kg) and central amygdala injections (70 ng/0.3 ul), in shock-resistant and -sensitive rats.

Baclofen reduced CeA activity measured as Fos expression, and decreased activity of PKCδ-expressing neurons by reducing their firing frequency. This was also associated with a selective suppression of punishment resistant self-administration. Systemic and CeA baclofen injections had no effect on saccharin self-administration or locomotor activity.

Our findings demonstrate that PKCδ downregulation or inhibition of neuronal activity through activation of GABA_B receptors in the CeA are sufficient to reduce compulsive alcohol self-administration. Because PKCδ is also expressed in the human CeA, this mechanism is potentially accessible for medications in a vulnerable population that develops AUD.

Alcohol use disorder (AUD) is the most demanding area of unmet medical needs in psychiatry. Improving the predictive validity of AUD animal models is crucial to bring forward novel and effective medications. Based on the DSM-IV/5 diagnostic criteria, we used a multidimensional operant model to characterize an alcohol addiction prone phenotype in outbred Wistar rats combined with electrophysiological assessments. In line with clinical studies rats as humans showed individual variability in the propensity to develop AUD that includes the motivation to seek for alcohol (crit1), increased effort to obtain the substance (crit2) and continued alcohol drinking despite negative consequences (crit 3). Only a subset of rats met all the aforementioned criteria for AUD (3crit, AUD-prone phenotype), while a larger fraction was considered AUD-resilient (0 crit).

Compulsive alcohol seeking was encoded by specific neuronal changes within the medial prefrontal cortex (mPFC) where presynaptic activity was altered in the alcohol addiction-prone phenotype compared to the resilient phenotype. The latter did not differ in neuronal properties compared to water drinking controls. Moreover, we observed a trend toward as increase in neuronal excitability specifically in the AUD-prone phenotype, indicating potential changes in the postsynaptic activity of glutamatergic cells within the mPFC.

Ex vivo pharmacological manipulation of the mPFC, accumbens core and basolateral amygdala further indicated adaptations in mGluR2/3 signalling within the mPFC, specifically in the AUD prone phenotype. Our findings suggest that glutamatergic changes within the mPFC contribute to the vulnerability to develop AUD. The mGluR2/3 receptor system could thus represent an ideal pharmacological intervention for a target-phenotypic-based treatment in AUD.