Cholinergic Interneurons in the Accumbal Shell Region Regulate Binge Alcohol Self-Administration in Mice: An In Vivo Calcium Imaging Study.

Recently, we and others have shown that manipulating the activity of cholinergic interneurons (CIN) present in the NAc can modulate binge alcohol consumption. The present study is designed to examine the relationship between binge alcohol consumption and the activity of the CIN in real time by using an in vivo microendoscopic technique. We hypothesized that mice exposed to Drinking in the Dark (DID)-a recognized mouse model for binge drinking-would exhibit increased activity in the accumbal shell region (NAcSh). To test this hypothesis, male mice expressing Cre-recombinase in the cholinergic neurons were exposed to binge alcohol consumption (alcohol group), employing the DID method, and utilized in vivo calcium imaging to observe CIN activity in real time during alcohol consumption. The control (sucrose) group was exposed to 10% (w/v) sucrose. As compared to sucrose, mice in the alcohol group displayed a significant increase in the frequency and amplitude of discharge activity, which was measured using calcium transients in the CIN present in the NAcSh. In summary, our findings suggest that the activity of CIN in the NAcSh plays a crucial role in alcohol self-administration. These results emphasize the potential significance of targeting CIN activity as a therapeutic approach for addressing AUD.

Cholinergic interneurons in the shell region of the nucleus accumbens regulate binge alcohol consumption: A chemogenetic and genetic lesion study.

BACKGROUND: Binge drinking, characterized by heavy episodic alcohol consumption, poses significant health hazards and increases the likelihood of developing an alcohol use disorder (AUD). Given the growing prevalence of this behavior and its negative consequences, there is a need to explore novel therapeutic targets. Accumulating evidence suggests that cholinergic interneurons (CIN) within the shell region of the nucleus accumbens (NAcSh) play a critical role in reward and addiction. However, their specific involvement in binge alcohol administration remains unclear. We hypothesized that CIN in the NAcSh regulates binge alcohol consumption. METHODS: To test this hypothesis, we used male ChAT-cre mice expressing Cre-recombinase in cholinergic neurons. We performed chemogenetic manipulation using Designer Receptor Exclusively Activated by Designer Drugs (DREADD) to examine the activity, and genetic ablation of CIN in the NAcSh to examine the amount of alcohol consumed in mice exposed to binge alcohol consumption using the 4-Days Drinking-in-Dark (DID) paradigm. The impact of CIN manipulations in the NAcSh on sucrose self-administration was used to control for taste and caloric effects. Additionally, in a separate group of mice, c-Fos immunofluorescence was employed to verify chemogenetic activation or inhibition. Histological and immunohistochemical techniques were used to verify microinfusion sites, DREADD expression in CINs, and genetic ablation. RESULTS: We found that, while chemogenetic activation of CIN in the NAcSh caused a significant increase in alcohol consumption, chemogenetic inhibition or genetic ablation of CIN significantly reduced the amount of alcohol consumed without affecting sucrose self-administration. The chemogenetic inhibition caused a significant reduction, whereas activation caused a significant increase, in the number of c-Fos-labeled CIN in the NAcSh. CONCLUSIONS: Our findings highlight the crucial involvement of CIN in the NAcSh in modulating binge alcohol consumption, suggesting that targeting these neurons could serve to modify alcohol-related behaviors.

Ischemic Stroke Disrupts Sleep Homeostasis in Middle-Aged Mice.

Sleep disturbances, including insomnia and excessive daytime sleepiness, are highly prevalent in patients with ischemic stroke (IS), which severely impacts recovery and rehabilitation efforts. However, how IS induces sleep disturbances is unclear. Three experiments were performed on middle-aged C57BL/6J mice, instrumented with sleep recording electrodes and/or subjected to 1 h of middle cerebral artery (MCAO; Stroke group) or sham (Sham group) occlusion to induce IS. After 48 h of reperfusion (a) experiment 1 verified sensorimotor deficit (using Garcia scale) and infarction (using TTC staining) in this mouse model; (b) experiment 2 examined the effects of IS on the quality (sleep latency and NREM delta power) and quantity (duration) of sleep; and (c) experiment 3 determined the effects of IS on sleep homeostasis using sleep deprivation (SD) and recovery sleep (RS) paradigm. Stroke mice display (a) a significant correlation between sensorimotor deficit and cerebral infarction; (b) insomnia-like symptoms (increased sleep latency, reduced NREM duration and delta power) during the light (inactive) period and daytime sleepiness-like symptoms during the dark (active) period mimicking sleep in IS patients; and (c) impairments in the markers of sleep pressure (during SD) and sleep dissipation (during RS). Our results suggest that IS disrupts sleep homeostasis to cause sleep disturbances.

Sleep, sleep homeostasis and arousal disturbances in alcoholism.

The effects of alcohol on human sleep were first described almost 70 years ago. Since then, accumulating evidences suggest that alcohol intake at bed time immediately induces sleep [reduces the time to fall asleep (sleep onset latency), and consolidates and enhances the quality (delta power) and the quantity of sleep]. Such potent sleep promoting activity makes alcohol as one of the most commonly used "over the counter" sleep aid. However, the somnogenic effects, after alcohol intake, slowly wane off and often followed by sleep disruptions during the rest of the night. Repeated use of alcohol leads to the development of rapid tolerance resulting into an alcohol abuse. Moreover, chronic and excessive alcohol intake leads to the development of alcohol use disorder (AUD). Alcoholics, both during drinking periods and during abstinences, suffer from a multitude of sleep disruptions manifested by profound insomnia, excessive daytime sleepiness, and altered sleep architecture. Furthermore, subjective and objective indicators of sleep disturbances are predictors of relapse. Finally, within the USA, it is estimated that societal costs of alcohol-related sleep disorders exceed $18 billion. Thus, although alcohol associated sleep problems have significant economic and clinical consequences, very little is known about how and where alcohol acts to affect sleep. In this review, a conceptual framework and clinical research focused on understanding the relationship between alcohol and sleep is first described. In the next section, our new and exciting preclinical studies, to understand the cellular and molecular mechanism of how acute and chronic alcohol affects sleep, are described. In the end, based on observations from our recent findings and related literature, opportunities for the development of innovative strategies to prevent and treat AUD are proposed.

Activation of dopamine D2 receptors in the medial shell region of the nucleus accumbens increases Per1 expression to enhance alcohol consumption.

Circadian genes, including Per1, in the medial shell region of nucleus accumbens (mNAcSh), regulate binge alcohol consumption. However, the upstream mechanism regulating circadian genes-induced alcohol consumption is not known. Since activation of dopamine D2 receptors (D2R) increases Per1 gene expression, we hypothesised that local infusion of quinpirole, a D2R agonist, by increasing Per1 gene expression in the mNAcSh, will increase binge alcohol consumption in mice. We performed two experiments on male C57BL/6J mice, instrumented with bilateral guide cannulas above the mNAcSh, and exposed to a 4-day drinking-in-dark (DID) paradigm. The first experiment determined the effects of bilateral infusion of quinpirole (100 ng/300 nl/site) or DMSO (Vehicle group) in the mNAcSh on Per1 gene expression and alcohol consumption. The second experiment determined the effect of antisense-induced downregulation of Per1 in the mNAcSh on the quinpirole-induced increase in alcohol consumption. Control experiments were performed by exposing the animals to sucrose (10% w/v). After the experiment, animals were euthanised, brains removed and processed for localisation of injection sites and analysis of Per1 gene expression in the mNAcSh. As compared with the DMSO, local bilateral infusion of quinpirole significantly increased the expression of Per1 in the mNAcSh along with an increase in the amount of alcohol consumed in mice exposed to DID paradigm. In addition, local antisense-induced downregulation of Per1 significantly attenuated the effects of intro-accumbal infusion of quinpirole on alcohol consumption. Our results suggest that Per1 in the mNAcSh mediates D2R activation-induced increase in alcohol consumption.

Antisense-induced downregulation of major circadian genes modulates the expression of histone deacetylase-2 (HDAC-2) and CREB-binding protein (CBP) in the medial shell region of nucleus accumbens of mice exposed to chronic excessive alcohol consumption.

Circadian genes in the medial accumbal shell (mNAcSh) region regulate binge alcohol consumption. Here, we investigated if antisense-induced knockdown of major circadian genes (Per1, Per2, and NPAS2) in the mNAcSh of mice exposed to intermittent access two-bottle choice (IA2BC) paradigm modulates the expression of histone deacetylase-2 (HDAC-2) and CREB-binding protein (CBP), key epigenetic modifiers associated with withdrawal-associated behaviors such as anxiety. Adult male C57BL/6J mice (N = 28), surgically implanted with bilateral guide cannulas above the mNAcSh, were chronically (4 weeks) exposed to alcohol (20% v/v) or saccharin (0.03%) via IA2BC paradigm. In the fourth week, a mixture of antisense (AS-ODNs; N = 14/group) or nonsense (NS-ODNs; N = 14/group) oligodeoxynucleotides against circadian genes were bilaterally infused into the mNAcSh. Subsequently, alcohol/saccharin consumption and preference were measured followed by euthanization of animals and verification of microinjection sites by visual inspection and the expression of HDAC-2 and CBP by using RT-PCR along with the verification of antisense-induced downregulation of circadian genes in the mNAcSh. As compared with NS-ODNs, AS-ODNs infusion significantly attenuated the alcohol-induced increase in HDAC-2 and reduction in CBP expression in the mNAcSh along with a significant reduction in alcohol consumption and preference. No significant effect was observed on either saccharin consumption or preference. Our results suggest that circadian genes in the mNAcSh may have a causal to play in mediating epigenetic changes observed after chronic alcohol consumption.

Intrapancreatic Accessory Spleen Diagnosed As Neuroendocrine Tumor: The Dangers of False Positives and Their Implications in Subsequent Management.

This case serves as a reminder to consider ectopic splenic tissue in the differential diagnosis of pancreatic masses. The literature shows a lack of awareness and overtreatment of this condition due to clinical and radiologic concern for malignancy, namely neuroendocrine tumors (NETs) identified on positron emission tomography (PET)-CT NETSPOT. Given the vast difference in management and prognosis of ectopic splenic anomalies and malignant neoplasms involving the pancreas, accurate diagnosis is imperative to avoid unnecessary invasive procedures such as Whipple or distal pancreatectomy and splenectomy, which are associated with increased morbidity and mortality.

Antisense-induced knockdown of cAMP response element-binding protein downregulates Per1 gene expression in the shell region of nucleus accumbens resulting in reduced alcohol consumption in mice.

INTRODUCTION: We recently showed that circadian genes expressed in the shell region of nucleus accumbens (NAcSh) play a key role in alcohol consumption, though, the molecular mechanism of those effects is unclear. Because CREB-binding protein (CBP) promotes Per1 gene expression, we hypothesized that alcohol consumption would increase CBP expression in the NAcSh and antisense-induced knockdown of CBP would reduce Per1 expression and result in a reduction in alcohol consumption. METHODS: To test our hypothesis, we performed two experiments. The Drinking-in-the-dark (DID) paradigm was used to evaluate alcohol consumption in male C57BL/6J mice. In Experiment 1 we examined the effects of alcohol consumption on CBP gene expression in the NAcSh. Control animals were exposed to, sucrose [10% (w/v) taste and calorie] and water (consummatory behavior). In Experiment 2 examined the effects of CBP gene silencing on the expression of the Per1 gene in the NAcSh and alcohol consumption in mice exposed to alcohol using the DID paradigm. CBP gene silencing was achieved by local infusion of two doses of either CBP antisense oligodeoxynucleotides (AS-ODNs; Antisense group) or nonsense ODNs (NS-ODNs; Nonsense group) bilaterally microinjected into the NAcSh within 24 h before alcohol consumption on Day 4 of the DID paradigm. The microinfusion sites were verified by cresyl violet staining. RESULTS: Compared to sucrose, alcohol consumption, under the DID paradigm, significantly increased the expression of CBP in the NAcSh. Compared to Controls, bilateral infusion of CBP AS-ODNs significantly reduced the expression of Per1 in the NAcSh and alcohol consumption without affecting the amount of sucrose consumed. CONCLUSIONS: Our results suggest that CBP is an upstream regulator of Per1 expression in the NAcSh and may act via Per1 to modulate alcohol consumption.

Antisense-Induced Downregulation of Clock Genes in the Shell Region of the Nucleus Accumbens Reduces Binge Drinking in Mice.

INTRODUCTIONS: Binge drinking is a deadly pattern of alcohol consumption. Evidence suggests that genetic variation in clock genes is strongly associated with alcohol misuse; however, the neuroanatomical basis for such a relationship is unknown. The shell region of the nucleus accumbens (NAcSh) is well known to play a role in binge drinking. Hence, we examined whether clock genes in the NAcSh regulate binge drinking. METHODS: To address this question, 2 experiments were performed on male C57BL/6J mice. In the first experiment, mice exposed to alcohol or sucrose under the 4-day drinking-in-the-dark (DID) paradigm were euthanized at 2 different time points on day 4 [7 hours after light (pre-binge drinking) or dark (post-binge drinking) onset]. The brains were processed for RT-PCR to examine the expression of circadian clock genes (Clock, Per1, and Per2) in the NAcSh and suprachiasmatic nucleus (SCN). In the second experiment, mice were exposed to alcohol, sucrose, or water as described above. On day 4, 1 hour prior to the onset of alcohol exposure, mice were bilaterally infused with either a mixture of circadian clock gene antisense oligodeoxynucleotides (AS-ODNs; antisense group) or nonsense/random ODNs (R-ODNs; control group) through surgically implanted cannulas above the NAcSh. Alcohol/sucrose/water consumption was measured for 4 hours. Blood alcohol concentration was measured to confirm binge drinking. Microinfusion sites were histologically verified using cresyl violet staining. RESULTS: As compared to sucrose, mice euthanized post-binge drinking (not pre-binge drinking) on day 4 displayed a greater expression of circadian genes in the NAcSh but not in the SCN. Knockdown of clock genes in the NAcSh caused a significantly lower volume of alcohol to be consumed on day 4 than in the control treatment. No differences were found in sucrose or water consumption. CONCLUSIONS: Our results suggest that clock genes in the NAcSh play a crucial role in binge drinking.

Unilateral versus bilateral hilar stents for the treatment of cholangiocarcinoma: a multicenter international study.

BACKGROUND: Endoscopic placement of hilar stents is an accepted palliative therapy for patients with advanced, unresectable cholangiocarcinoma. However, whether unilateral versus bilateral stent placement provides optimal relief continues to be a subject of debate. The aim of this study was to compare the technical and clinical outcomes in patients with inoperable cholangiocarcinoma who received unilateral or bilateral self-expanding metal stents (SEMS). METHODS: We conducted a multicenter, international retrospective study of 187 patients with cholangiocarcinoma who received unilateral or bilateral SEMS. Outcomes included, but were not limited to, technical success, clinical success, adverse events, stent occlusion, and survival time. Results were further stratified based on the Bismuth classification. RESULTS: Fifty patients received unilateral stents and 137 patients received bilateral stents. All patients achieved technical success. The clinical success rates were 86% for unilateral stents and 82.5% for bilateral stents (P>0.99). Clinical success was not statistically different for either group when stratified by the Bismuth classification (P=0.62 and P=0.72 respectively). There were significantly more adverse events in the bilateral stents group (11.7% vs. 0%, P=0.007). There was no greater risk of stent occlusion when bilateral stents were used (unadjusted P=0.71, adjusted P=0.81). There was a greater risk of death for patients who received bilateral SEMS (hazard ratio 1.78, 95% confidence interval 1.09-2.89; P=0.02). CONCLUSIONS: Unilateral and bilateral drainage had similar technical and clinical success rates. However, bilateral stents had a higher risk of death and more adverse events. Therefore, unilateral SEMS placement is sufficient for relief of biliary obstruction secondary to cholangiocarcinoma.

Use of fully covered self-expanding metal stents for benign biliary etiologies: a large multi-center experience.

BACKGROUND: Fully-covered self-expandable metal stents (FCSEMS) have been used in benign biliary diseases although reported data is limited. These devices are most commonly used to treat biliary leaks, strictures, or both. The aim of this study was to evaluate effectiveness of FCSEMS in treating benign biliary disease and recognize the associated complications. METHODS: We performed a multicenter longitudinal retrospective cohort study of patients with benign biliary disease needing FCSEMS between 2011 and 2016. Descriptive statistics were performed using SPSS version 24 (SPSS Inc, Chicago, IL, USA) and continuous variables were presented as mean±standard deviation. RESULTS: 75, 57% M/43% F, with a mean age of 58.5±14.9 years, were included. 64 (85%) had benign strictures, 7 patients had leaks, and 4 patients had both a leak and a stricture. Chronic pancreatitis was the most common cause of BBS (47%) and cholecystectomy was the most common cause of leaks. FCSEMS placement was technically successful in all patients. Four patients died of unrelated causes. A recurrent stricture was observed in 24 (32%) of the patients. Recurrent strictures were most commonly seen in patients with chronic pancreatitis 12/35 (34%). Stent migration occurred in 8/75 patients (10.7%). Seven patients (9.3%) had adverse events, acute pancreatitis (N.=4) was most common. CONCLUSIONS: FCSEMS are safe and effective for treating biliary strictures and leaks. We report decreased rates of stent migration compared to previous studies. Prospective studies are needed to compare plastic stents with FCSEMS, determine optimal stent in-dwell times and cost effectiveness of FCSEMS.