A bevy of clinical and preclinical evidence has established that the prefrontal cortex (PFC) is negatively affected by heavy alcohol consumption during adolescence, yet the specific neuroadaptations to the PFC associated with adolescent alcohol are not well understood. Using a retrograde viral approach and ex vivo whole-cell patch-clamp electrophysiology, we examined the impact of adolescent alcohol consumption on neuronal excitability in specific projections from the prelimbic PFC to the contralateral PFC (PFCcPFC), nucleus accumbens (PFCNAc), or mediodorsal thalamus (PFCMdT). Following adolescent alcohol consumptions, we observed several electrophysiological effects specifically to PFCMdt pyramidal neurons (PNs) including increased intrinsic excitability due to a reduction in the hyperpolarization-activated, nonselective cation current (Ih) and decreased excitatory synaptic neurotransmission. Many of these effects were persistent in male mice as they were observed after 10 days of abstinence in PFCMdT PNs and were not seen when IA EtOH was initiated in adulthood or even in late adolescence. To further characterize the consequences of adolescent alcohol consumption on PFC dependent behaviors, we performed touchscreen-based operant tasks aimed at more directly assessing PFC-dependent cognition. We found that cognitive flexibility and working memory performance were not largely affected by adolescent alcohol, however, additional analyses revealed blunted task shifting and delayed response latencies. The PFCMdT circuit has previously been implicated in response timing and attentional processes and therefore exposure to alcohol during the early adolescent window may disrupt executive function through adaptations to PFCMdT circuitry.

Across one's lifetime, heavy alcohol use during adolescence confers the highest risk for developing an alcohol use disorder (AUD), and therefore understanding its impact on the adolescent brain is essential for advancing effective interventions. Preclinical models are critical for investigations into the long-term neurobiological consequences of adolescent alcohol exposure with current mouse paradigms capturing either modest alcohol levels through voluntary consumption or high alcohol levels through passive exposure methods. The current study investigates the outcome of a mixed model of adolescent alcohol exposure by combining volitional adolescent alcohol intake using 2-bottle choice interleaved with alcohol vapor exposure. Our results show that 2 cycles of alcohol vapor elevate alcohol consumption and compulsive-like drinking behavior when measured in late adolescence, without affecting adult alcohol consumption. Neural activation patterns measured through c-Fos expression following adult alcohol consumption demonstrated that adolescent alcohol vapor exposed mice had increased activity in NAc core and CeA, with sex-dependent effects seen in PFC activation. We conclude that mixed alcohol consumption and vapor paradigms have short-term behavioral consequences and long-term neural effects.

Traumatic brain injury (TBI) and alcohol misuse are major contributors to the global health care burden, both linked to persistent cognitive deficits and mitochondrial dysfunction. This study examined whether post-TBI alcohol exposure exacerbates mitochondrial impairment and contributes to cognitive decline. Adult male Wistar rats were assigned to four groups: Sham, Sham + ethanol (EtOH), TBI, or TBI + EtOH. TBI was induced via lateral fluid percussion 3 days post-craniotomy. Starting 2 days post-injury, EtOH-treated animals received 2.0 g/kg EtOH (i.p.) daily for 5 days. Behavioral testing (Y-maze and open field) was conducted on day 6; brain tissue was collected on day 7 for mitochondrial analysis using the Agilent Seahorse XF Pro Analyzer in the prefrontal cortex (PFC) and retrosplenial cortex (RSC). Rats in the EtOH, TBI, and TBI + EtOH groups showed reduced spontaneous alternation in the Y-maze, suggesting impaired spatial memory. The EtOH group also showed decreased exploratory activity. In the PFC, baseline respiration was unaffected; however, EtOH exposure reduced maximal respiration and glycolytic reserve. In the RSC, TBI and EtOH independently decreased basal respiration, maximal respiration, adenosine triphosphate production, and glycolytic reserve. These findings reveal that alcohol and TBI independently impair mitochondrial bioenergetics in brain regions relevant to cognition and that these deficits parallel behavioral impairments. Disrupted mitochondrial energy metabolism may thus be a key mechanism underlying cognitive dysfunction following TBI and alcohol exposure.

Alcohol use disorder (AUD) is a chronic, relapsing disease affecting ∼10% of individuals in the United States that is notoriously difficult to manage, with ∼60% untreated and ∼40% treated individuals relapsing within 3 years ([Moos et al., 2006][1]; Substance Abuse and Mental Health Services

Many of the behavioral symptoms that define alcohol use disorder (AUD) are thought to be mediated by amplified glutamatergic activity. As a result, previous preclinical studies have investigated glutamate receptor inhibition as a potential pharmacotherapy for AUD, particularly the metabotropic glutamate receptor 5 (mGlu5). In rodents, mGlu5 negative allosteric modulators (NAMs) have been shown to decrease alcohol selfadministration. However, their effect on non-human primates has not previously been explored. To bridge this gap, the effects of mGlu5 NAM pretreatment on sweetened alcohol (8% w/v in diluted KoolAid) selfadministration in female baboons were evaluated. Two different mGlu5 NAMs were tested: 1) 3-2((-Methyl-4thiazolyl) ethynyl) pyridine (MTEP) which was administered at a dose of 2 mg/kg IM; and 2) auglurant (N-(5fluoropyridin-2-yl)-6-methyl-4-(pyrimidin-5-yloxy)picolinamide), a newly developed NAM, which was tested under two different routes (0.001, 0.01, 0.03, 0.1 mg/kg IM and 0.1, 0.3, 1.0 mg/kg PO). MTEP decreased both fixed ratio and progressive ratio responding for sweetened alcohol. Auglurant, administered IM, decreased alcohol self-administration at doses that did not affect self-administration of an alcohol-free sweet liquid reward (0.01 to 0.1 mg/kg). Oral administration of auglurant was not effective in decreasing alcohol self-administration. Our results extend positive findings from rodent studies on mGlu5 regulation of alcohol drinking to female baboons and further strengthen the rationale for targeting mGlu5 in clinical trials for AUD.

The prefrontal cortex (PFC) is involved in executive ("top-down") control of behavior and its function is especially susceptible to the effects of alcohol, leading to behavioral disinhibition that is associated with alterations in decision making, response inhibition, social anxiety and working memory. The circuitry of the PFC involves a complex interplay between pyramidal neurons (PNs) and several subclasses of inhibitory interneurons (INs), including somatostatin (SST)-expressing INs. Using in vivo calcium imaging, we showed that alcohol dose-dependently altered network activity in layers 2/3 of the prelimbic subregion of the mouse PFC. Low doses of alcohol (1 g/kg, intraperitoneal, i.p.) caused moderate activation of SST INs and weak inhibition of PNs. At moderate to high doses, alcohol (2-3 g/kg) strongly inhibited the activity of SST INs in vivo, and this effect may result in disinhibition, as the activity of a subpopulation of PNs was simultaneously enhanced. In contrast, recordings in brain slices using ex vivo electrophysiology revealed no direct effect of alcohol on the excitability of either SST INs or PNs over a range of concentrations (20 and 50 mM) consistent with the blood alcohol levels reached in the in vivo experiments. This dose-dependent effect of alcohol on SST INs in vivo may reveal a neural basis for the disinhibitory effect of alcohol in the PFC mediated by other neurons within or external to the PFC circuitry.

Excessive alcohol consumption is a global health burden and requires a better understanding of its neurobiology. A lower density of brain microtubules is found in alcohol-related human brain disease postmortem and in rodent models of chronic alcohol consumption. Here, we report in vivo imaging studies of microtubules in brain using our recently reported Positron Emission Tomography (PET) tracer, [11C]MPC-6827, in chronic alcohol-consuming adult male C57BL/6 J mice and control mice. In vivo PET imaging studies of [11C]MPC-6827 (3.7  ±  0.8 MBq) were performed in two groups of adult male mice: (1) water-consuming control mice (n  =  4) and (2) mice that consumed 20% alcohol (w/v) for 4 months using the intermittent 2-bottle choice procedure that has been shown to lead to signs of alcohol dependence. Dynamic 63 min PET images were acquired using a microPET Inveon system (Siemens, Germany). PET images were reconstructed using the 3D-OSEM algorithm and analyzed using VivoQuant version 4 (Invicro, MA). Tracer uptake in ROIs that included whole brain, prefrontal cortex (PFC), liver and heart was measured and plotted as %ID/g over time (0–63 min) to generate time-activity curves (TACs). In general, a trend for lower binding of [11C]MPC-6827 in the whole brain and PFC of mice in the chronic alcohol group was found compared with control group. No group difference in radiotracer binding was found in the peripheral organs such as liver and heart. This pilot study indicates a trend of loss of microtubule binding in whole brain and prefrontal cortex of chronic alcohol administered mice brain compared to control mice, but no loss in heart or liver. These results indicate the potential of [11C]MPC-6827 as a PET ligand for further in vivo imaging investigations of AUD in human.

Optogenetic genome engineering tools enable spatiotemporal control of gene expression and provide new insight into biological function. Here, we report the new version of genetically encoded photoactivatable (PA) Cre recombinase, PA-Cre 3.0. To improve PA-Cre technology, we compare light-dimerization tools and optimize for mammalian expression using a CAG promoter, Magnets, and 2A self-cleaving peptide. To prevent background recombination caused by the high sequence similarity in the dimerization domains, we modify the codons for mouse gene targeting and viral production. Overall, these modifications significantly reduce dark leak activity and improve blue-light induction developing our new version, PA-Cre 3.0. As a resource, we have generated and validated AAV-PA-Cre 3.0 as well as two mouse lines that can conditionally express PA-Cre 3.0. Together these new tools will facilitate further biological and biomedical research.

The hyperpolarization-activated cyclic nucleotide-gated channel (HCN), which underlies the hyperpolarization-activated cation current (Ih), has diverse roles in regulating neuronal excitability across cell types and brain regions. Recently, HCN channels have been implicated in preclinical models of substance abuse including alcohol. In the prefrontal cortex of rodents, HCN expression and Ih magnitude are developmentally regulated during adolescence and may be vulnerable to alcohol’s effects. In mice, binge alcohol consumption during the adolescent period results in a sustained reduction in Ih that coincides with increased alcohol consumption in adulthood, yet the direct role HCN channels have on alcohol consumption are unknown. Here, we show that the genetic deletion of Hcn1 causes an increase in alcohol preference on intermittent 2-bottle choice task in homozygous null (HCN1−/−) male mice compared to wild-type littermates without affecting saccharine or quinine preference. The targeted viral deletion of HCN1 in pyramidal neurons of the medial prefrontal cortex resulted in a gradual loss of Hcn1 expression and a reduction in Ih magnitude during adolescence, however, this did not significantly affect alcohol consumption or preference. We conclude that while HCN1 regulates alcohol preference, the genetic deletion of Hcn1 in the medial prefrontal cortex does not appear to be the locus for this effect.

Animal studies indicate that the kappa-opioid receptor/dynorphin system plays an important role in cocaine binges and stress-induced relapse. Our goal was to investigate changes in kappa-opioid receptor (KOR) availability in the human brain using positron emission tomography (PET), before and after a cocaine binge. We also investigated the correlation between KOR and stress-induced cocaine self-administration. PET imaging was performed with the KOR selective agonist [11C]GR103545. Subjects with cocaine-use disorder (CUD) underwent PET scans and performed two types of cocaine self-administration sessions in the laboratory as follows: (1) choice sessions following a cold pressor test, to induce stress, and (2) binge dosing of cocaine. This allowed us investigate the following: (1) the association between KOR binding and a laboratory model of stress-induced relapse and (2) the change in KOR binding following a 3-day cocaine binge, which is thought to represent a change in endogenous dynorphin. A group of matched healthy controls was included to investigate between group differences in KOR availability. A significant association between [11C]GR103545 binding and cocaine self-administration was seen: greater KOR availability was associated with more choices for cocaine. In addition, the 3-day cocaine binge significantly reduced [11C]GR103545 binding by 18% in the striatum and 14% across brain regions. No difference in [11C]GR103545 binding was found between the CUD subjects and matched controls. In the context of previous studies, these findings add to the growing evidence that pharmacotherapies targeting the KOR have the potential to significantly impact treatment development for cocaine-use disorder.