Adolescent sleep loss and alcohol: enduring effects on brain and behavior in msP rats

Chronic sleep restriction (CSR) has become an epidemic among adolescents and has been proposed as a contributing factor to the development of alcohol use disorders (AUD) and related neuropsychiatric comorbidities later in life. However, the biological underpinnings of this link remain unclear. Here, we map the enduring effects of adolescent CSR on concurrent and adult alcohol intake, behaviour, glutamatergic synapses, and microglial cells in various brain regions of Marchigian Sardinian alcohol-preferring (msP) rats. Adolescent msP rats were divided into Sleep+Water, CSR+Water, SLEEP+Alcohol and CSR+Alcohol. The CSR groups were subjected to 22hrs of automatic sleep restrictions for 4 consecutive days, whereas the Sleep groups had the same manipulation for 6 hrs during the dark phase. On the fifth day, SLEEP+Alcohol and CSR+Alcohol were exposed to a two-bottle choice between 10% alcohol and water to measure voluntary alcohol drinking. Sleep+Water and CSR+Water had access to water only. This cycle was repeated 6 times throughout adolescence, then rats were allowed to recover for 5 weeks. Behavioural tests were conducted in late adolescence and adulthood. Brain sections were processed for immunofluorescence to assess the density of vglut1+ puncta and microglia in the medial orbitofrontal cortex (mORB), prelimbic cortex (PL) and nucleus Accumbens (NAc). Two-way ANOVA showed that CSR triggered a faster escalation of alcohol consumption (F[1, 36] = 15.59, p=0.0004) relative to Sleep in adolescence and adulthood. Adolescent CSR led to shorter latency to consume food in the arena of the novelty-suppressed feeding test relative to sleep controls (F[1, 55] = 2.085, p=0.0010), both in adolescents and adult rats. Furthermore, a trend for longer time spent in the light chamber of the light-dark box test (p=0.09) was observed in CSR+Alcohol relative to SLEEP+Alcohol. No signs of anhedonia or despair were associated with CSR. Linear mixed model revealed a significant effect of alcohol (p=0.0077), CSR (p=0.035), and their interaction (p=0.03561) on the density of vglut1+ puncta in the mORB. Sidak’s test revealed CSR+Alcohol was associated with higher density relative to other groups (CSR+Alcohol vs SLEEP+Alcohol p=0.019, vs CSR+water p=0.021, vs Sleep+water p=0.04). Albeit not significant, we observed a similar trend in the deep layers of the PL. In the NAc, alcohol drinking had the opposite effect and significantly reduced vglut1+ puncta density in both CSR+Alcohol and SLEEP+Alcohol groups relative to the water-only groups (alcohol p=0.0041). The density of microglial cells in PL deep layers was higher in the CSR+Alcohol group relative to SLEEP+Alcohol (p=0.029), whereas in the NAc the density was lower in the alcohol groups relative to water (p=0.0045). In summary, our findings support the longitudinal link between adolescent insufficient sleep and higher adult alcohol consumption, coupled with elevated risk-taking behaviours in anxiety- inducing contexts and altered density of glutamatergic inputs and microglial cells in the mORB, PL, and NAc of msP rats.