Although alcoholism and depression are highly comorbid, treatment options that take this into account are lacking, and mouse models of alcohol (ethanol (EtOH)) intake-induced depressive-like behavior have not been well established. Recent studies utilizing contingent EtOH administration through prolonged two-bottle choice access have demonstrated depression-like behavior following EtOH abstinence in singly housed female C57BL/6J mice. In the present study, we found that depression-like behavior in the forced swim test (FST) is revealed only after a protracted (2 weeks), but not acute (24 h), abstinence period. No effect on anxiety-like behavior in the EPM was observed. Further, we found that, once established, the affective disturbance is long-lasting, as we observed significantly enhanced latencies to approach food even 35 days after ethanol withdrawal in the novelty-suppressed feeding test (NSFT). We were able to reverse affective disturbances measured in the NSFT following EtOH abstinence utilizing the N-methyl D-aspartate receptor (NMDAR) antagonist and antidepressant ketamine but not memantine, another NMDAR antagonist. Pretreatment with the monoacylglycerol (MAG) lipase inhibitor JZL-184 also reduced affective disturbances in the NSFT in ethanol withdrawn mice, and this effect was prevented by co-administration of the CB1 inverse agonist rimonabant. Endocannabinoid levels were decreased within the BLA during abstinence compared with during drinking. Finally, we demonstrate that the depressive behaviors observed do not require a sucrose fade and that this drinking paradigm may favor the development of habit-like EtOH consumption. These data could set the stage for developing novel treatment approaches for alcohol-withdrawal-induced mood and anxiety disorders.

Negative affect is a core symptom domain associated with an array of neurological and psychiatric disorders and is only partially targeted by current therapies, highlighting the need for better, more targeted treatment options. This study focuses on negative affective symptoms associated with prolonged alcohol abstinence, one of the leading causes of relapse. Using a mouse model of chronic alcohol consumption followed by forced abstinence (CDFA), prolonged alcohol abstinence increased c-fos expression and spontaneous glutamatergic neurotransmission in the dorsal bed nucleus of the stria terminalis (dBNST), a region heavily implicated in negative affect in both humans and rodents. Further, pharmacologically enhancing endogenous cannabinoids (eCB) with JZL184 prevents abstinence-induced increases in dBNST neuronal activity, underscoring the therapeutic potential of drugs targeting the brain's eCB system. Next, we used a channelrhodopsin-assisted mapping strategy to identify excitatory inputs to the dBNST that could contribute to CDFA-induced negative affect. We identified the insular cortex (insula), a region involved in regulating interoception, as a dense, functional, eCB-sensitive input to the dBNST. Using a chemogenetic strategy to locally mimic eCB signaling, we demonstrate that the insula strongly influences the CDFA behavioral phenotype and dBNST neuronal activity. Lastly, we used an anterograde strategy for transynaptic targeting of Cre expression in combination with a G q -DREADD to selectively recruit dBNST neurons receiving insula projections. Chemogenetic recruitment of these neurons mimicked behavioral and c-fos responses observed in CDFA. Collectively, this study supports a role for the insula-BNST neural circuit in negative affective disturbances and highlights the therapeutic potential of the eCB system for treating negative affective disorders.Neuropsychopharmacology (2019) 44:526-537; https://doi.

i Nucleos(t)ide reverse transcriptase inhibitors (NRTIs) form the backbone of most anti-HIV therapies. We have shown that 4=-ethynyl-2-fluoro-2=-deoxyadenosine (EFdA) is a highly effective NRTI; however, the reasons for the potent antiviral activity of EFdA are not well understood. Here, we use a combination of structural, computational, and biochemical approaches to examine how substitutions in the sugar or adenine rings affect the incorporation of dA-based NRTIs like EFdA into DNA by HIV RT and their susceptibility to deamination by adenosine deaminase (ADA). Nuclear magnetic resonance (NMR) spectroscopy studies of 4=-substituted NRTIs show that ethynyl or cyano groups stabilize the sugar ring in the C-2=-exo/C-3=-endo (north) conformation. Steady-state kinetic analysis of the incorporation of 4=-substituted NRTIs by RT reveals a correlation between the north conformation of the NRTI sugar ring and efficiency of incorporation into the nascent DNA strand. Structural analysis and the kinetics of deamination by ADA demonstrate that 4=-ethynyl and cyano substitutions decrease the susceptibility of adenosinebased compounds to ADA through steric interactions at the active site. However, the major determinant for decreased susceptibility to ADA is the 2-halo substitution, which alters the pK a of N1 on the adenine base. These results provide insight into how NRTI structural attributes affect their antiviral activities through their interactions with the RT and ADA active sites. There are 10 nucleos(t)ide reverse transcriptase inhibitors (NRTIs) that are currently approved for the treatment of human immunodeficiency virus type 1 (HIV-1) infections (1-5). Several more nucleoside analog drugs are approved or being studied for the treatment of viruses, such as herpes simplex virus (HSV), hepatitis C virus (HCV), and hepatitis B virus (HBV), or as anticancer agents (6-10). NRTIs are among the most effective anti-HIV drugs. All approved anti-HIV NRTIs lack a 3=-hydroxyl moiety and, thus, act as chain terminators following their incorporation by the viral reverse transcriptase (RT) into the nascent DNA chain. However, the absence of a 3=-OH, while essential for the inhibition of DNA synthesis, also imparts detrimental properties to these inhibitors, including reduced intracellular phosphorylation to the active triphosphate form and reduced RT binding affinity (11). Prolonged exposure to NRTI-based treatments causes mitochondrial toxicity (12-14) and leads to the development of NRTI resistance mutations (15-18), giving rise to complications in the treatment of HIV-infected patients.Ideally, an NRTI should have a strong binding affinity for the RT target, a high barrier for the development of resistance, and low toxicity. We have reported that a series of 4=-substituted nucleosides in which the 3=-OH is retained has exceptional inhibitory activity against HIV-1 RT (19). Among these compounds, 4=-ethynyl-2-fluoro-2=-deoxyadenosine (EFdA) is a highly active RT inhibitor that prevents translocation of the nucleic acid from the n...

Using hydroxyl-radical footprinting assays, we demonstrated that the distance between the polymerase and RNase H domains in the MoMLV and XMRV RTs is longer than that in the HIV-1 RT by ϳ3.4 Å. We identified one naphthyridinone and one hydroxyisoquinolinedione as potent inhibitors of HIV-1 and XMRV RT RNases H with 50% inhibitory concentrations ranging from ϳ0.8 to 0.02 M. Two acylhydrazones effective against HIV-1 RT RNase H were less potent against the XMRV enzyme. We also solved the crystal structure of an XMRV RNase H fragment at high resolution (1.5 Å) and determined the molecular details of the XMRV RNase H active site, thus providing a framework that would be useful for the design of antivirals that target RNase H.

Corticotropin releasing factor (CRF) is a neuropeptide that plays a key role in behavioral and physiological responses to stress. A large body of animal literature implicates CRF acting at type 1 CRF receptors (CRFR1) in consumption by alcohol dependent subjects, stress-induced reinstatement of alcohol seeking, and possibly binge alcohol consumption. These studies have encouraged recent pilot studies of CRFR1 antagonists in humans with alcohol use disorder (AUD). It was a great disappointment to many in the field that these studies failed to show an effect of these compounds on stress-induced alcohol craving. Here, we examine these studies to explore potential limitations, and discuss preclinical and human literature to ask whether CRFR1 is still a valid drug target to pursue for the treatment of alcohol use disorder.

Stress contributes to numerous psychiatric disorders. Corticotropin releasing factor (CRF) signaling and CRF neurons in the bed nucleus of the stria terminalis (BNST) drive negative affective behaviors, thus agents that decrease activity of these cells may be of therapeutic interest. Here, we show that acute restraint stress increases cFos expression in CRF neurons in the mouse dorsal BNST, consistent with a role for these neurons in stress-related behaviors. We find that activation of ␣ 2A-adrenergic receptors (ARs) by the agonist guanfacine reduced cFos expression in these neurons both in stressed and unstressed conditions. Further, we find that ␣and ␤-ARs differentially regulate excitatory drive onto these neurons. Pharmacological and channelrhodopsin-assisted mapping experiments suggest that ␣ 2A-ARs specifically reduce excitatory drive from parabrachial nucleus (PBN) afferents onto CRF neurons. Given that the ␣ 2A-AR is a G i-linked GPCR, we assessed the impact of activating the G i-coupled DREADD hM4Di in the PBN on restraint stress regulation of BNST CRF neurons. CNO activation of PBN hM4Di reduced stress-induced Fos in BNST Crh neurons. Further, using Prkcd as an additional marker of BNST neuronal identity, we uncovered a female-specific upregulation of the coexpression of Prkcd/Crh in BNST neurons following stress, which was prevented by ovariectomy. These findings show that stress activates BNST CRF neurons, and that ␣ 2A-AR activation suppresses the in vivo activity of these cells, at least in part by suppressing excitatory drive from PBN inputs onto CRF neurons.

Human fMRI studies show that insulin influences brain activity in regions that mediate reward and motivation, including the nucleus accumbens (NAc). Insulin receptors are expressed by NAc medium spiny neurons (MSNs), and studies of cultured cortical and hippocampal neurons suggest that insulin influences excitatory transmission via pre-synaptic and post-synaptic mechanisms. However, nothing is known about how insulin influences excitatory transmission in the NAc. Furthermore, insulin dysregulation accompanying obesity is linked to cognitive decline, depression, anxiety, and aberrant motivation that rely on NAc excitatory transmission. Using whole-cell patch clamp and biochemical approaches we determined how insulin affects NAc glutamatergic transmission in non-obese and obese male rats and the underlying mechanisms. We find that there are concentration-dependent, bi-directional effects of insulin on excitatory transmission, with insulin receptor activation increasing and IGF receptor activation decreasing NAc excitatory transmission. Increases in 2 2 excitatory transmission were mediated by activation of post-synaptic insulin receptors located on MSNs.However, this effect was due to an increase in presynaptic glutamate release. This suggested feedback from MSNs to presynaptic terminals. In additional experiments, we found that insulin-induced increases in presynaptic glutamate release are mediated by opioid receptor-dependent disinhibition. Furthermore, obesity resulted in a loss of insulin receptor-mediated increases in excitatory transmission and a reduction in NAc insulin receptor surface expression, while preserving reductions in transmission mediated by IGF receptors.These results provide the first insights into how insulin influences excitatory transmission in the adult brain, and evidence for a previously unidentified form of opioid receptor-dependent disinhibition of NAc glutamatergic transmission.Significance Statement: Data here provide the first insights into how insulin influences excitatory transmission in the adult brain, and identify previously unknown interactions between insulin receptor activation, opioids, and glutamatergic transmission. These data contribute to our fundamental understanding of insulin's influence on brain motivational systems and have implications for the use of insulin as a cognitive enhancer and for targeting of insulin receptors and IGF receptors to alter motivation. Introduction:Recent studies in humans suggest that insulin may enhance cognition and decision-making processes that

CP-AMPARs in the nucleus accumbens (NAc) mediate cue-triggered motivation for food and cocaine. In addition, increases in NAc CP-AMPAR expression and function can be induced by cocaine or sugary, fatty junk-foods. However, the precise nature of these alterations and the degree to which they rely on the same underlying mechanisms is not well understood. This has important implications for understanding adaptive vs. maladaptive plasticity that drives food-and drug-seeking behaviors. Furthermore, effects of junk-foods on glutamatergic plasticity in females are unknown. Here, we use a combination of protein biochemistry and whole-cell patch clamping to determine effects of diet manipulation on glutamatergic plasticity within the NAc of males and females. We found that junk-food consumption increases silent synapses and subsequently increases CP-AMPAR levels in males in the NAc of male rats. In addition, a brief period of junk-food deprivation is needed for the synaptic insertion of CP-AMPARs and the maturation of silent synapses in males. In contrast, junk-food did not induce AMPAR plasticity in females but may instead alter NMDAR-mediated transmission. Thus, these studies reveal sex differences in the effects of junk-food on NAc synaptic plasticity. In addition, they provide novel insights into how essential food rewards alter NAc function.