Ketone Supplementation Dampens Subjective and Objective Responses to Alcohol: Evidence From a Preclinical Rat Study and a Randomized, Cross-Over Trial in Healthy Volunteers.

BACKGROUND: Previous preclinical and human studies have shown that a high-fat ketogenic diet and ketone supplements (KS) are efficacious in reducing alcohol craving, alcohol consumption, and signs of alcohol withdrawal. However, the effects of KS on alcohol sensitivity are unknown. METHODS: In this single-blind, cross-over study, 10 healthy participants (3 females) were administered a single, oral dose of a KS (25 g of ketones from D-ß-hydroxybutyric acid and R-1,3-butanediol) or placebo 30 minutes before an oral alcohol dose (0.25 g/kg for women; 0.31 g/kg for men). Assessments of breath alcohol concentration and blood alcohol levels (BAL) and responses on the Drug Effect Questionnaire were repeatedly obtained over 180 minutes after alcohol consumption. In a parallel preclinical study, 8 Wistar rats (4 females) received an oral gavage of KS (0.42 g ketones/kg), water, or the sweetener allulose (0.58 g/kg) followed 15 minutes later by an oral alcohol dose (0.8 g/kg). BAL was monitored for 240 minutes after alcohol exposure. RESULTS: In humans, the intake of KS before alcohol significantly blunted breath alcohol concentration and BAL, reduced ratings of alcohol liking and wanting more, and increased disliking for alcohol. In rats, KS reduced BAL more than either allulose or water. CONCLUSION: KS altered physiological and subjective responses to alcohol in both humans and rats, and the effects were likely not mediated by the sweetener allulose present in the KS drink. Therefore, KS could potentially reduce the intoxicating effects of alcohol.

Ketone supplementation dampens subjective and objective responses to alcohol in rats and humans.

Previous preclinical and human studies have shown that high-fat ketogenic diet and ketone supplements (KS) are efficacious in reducing alcohol craving, alcohol consumption, and signs of alcohol withdrawal. However, the effects of KS on alcohol sensitivity are unknown. In this single-blind, cross-over study, 10 healthy participants (3 females) were administered a single, oral dose of a KS (25 g of ketones from D-ß-hydroxybutyric acid and R-1,3-butanediol) or placebo 30 min prior to an oral alcohol dose (0.25 g/kg for women; 0.31 g/kg for men). Assessments of breath alcohol concentration (BrAC) and blood alcohol levels (BAL) and responses on the Drug Effect Questionnaire were repeatedly obtained over 180 min after alcohol consumption. In a parallel preclinical study, 8 Wistar rats (4 females) received an oral gavage of KS (0.42 g ketones/kg), water, or the sweetener allulose (0.58 g/kg) followed 15 min later by an oral alcohol dose (0.8 g/kg). BAL were monitored for 240 min after alcohol exposure. In humans, the intake of KS prior to alcohol significantly blunted BrAC and BAL, reduced ratings of alcohol liking and wanting, and increased disliking for alcohol. In rats, KS reduced BAL more than either allulose or water. In conclusion, KS altered physiological and subjective responses to alcohol in both humans and rats and the effects were likely not mediated by the sweetener allulose present in the KS drink. Therefore, KS could potentially reduce the intoxicating and rewarding effects of alcohol and thus be a novel intervention for treating alcohol use disorder.

Heroin- and Fentanyl-Induced Respiratory Depression in a Rat Plethysmography Model: Potency, Tolerance, and Sex Differences.

The opioid overdose death toll in the United States is an ongoing public health crisis. We characterized the magnitude and duration of respiratory depression, the leading cause of death in opioid overdose cases, induced by heroin or fentanyl and the development of tolerance in male and female rats. We used whole-body plethysmography to first establish dose-response curves by recording breathing for 60 minutes post-intravenous opioid injection. We then tested the development of respiratory tolerance to acute heroin or fentanyl over several weeks and to chronic fentanyl with acute fentanyl or heroin challenge. Heroin and fentanyl each provoked dose-dependent respiratory depression. Heroin caused prolonged (45-60 minute) respiratory depression in female and male rats, characterized by decreased frequency, tidal volume, and minute ventilation and increased inspiratory time and apneic pause. Fentanyl produced similar changes with a shorter duration (10-15 minutes). High-dose heroin or fentanyl produced robust respiratory depression that was slightly more severe in females and, when given intermittently (acute doses 2 to 3 weeks apart), did not lead to tolerance. In contrast, chronic fentanyl delivered with an osmotic minipump resulted in tolerance to acute fentanyl and heroin, characterized by a shorter duration of respiratory depression. This effect persisted during withdrawal in males only. Our model and experimental design will allow for investigation of the neurobiology of opioid-induced respiratory depression and for testing potential therapeutics to reverse respiratory depression or stimulate breathing. SIGNIFICANCE STATEMENT: Fentanyl was more potent and had shorter duration in producing respiratory depression than heroin in both sexes, whereas female rats were more sensitive than males to heroin-induced respiratory depression. Tolerance/cross-tolerance develops in chronic fentanyl administration but is minimized with long interadministration intervals.

Effects of Alcohol Withdrawal on Sleep Macroarchitecture and Microarchitecture in Female and Male Rats.

The prevalence of sleep disruptions is higher among people with alcohol use disorder (AUD), particularly during alcohol withdrawal, compared to non-AUD individuals. Although women generally have a higher risk of developing sleep disorders, few studies have investigated sex differences in sleep disruptions following chronic alcohol exposure. The present study examined sleep macroarchitecture (time spent asleep or awake and sleep onset latency) and microarchitecture (bout rate and duration and sleep spindle characterization) prior to alcohol vapor exposure (baseline), during acute withdrawal, and through protracted abstinence in female and male rats. Females and males showed reduced time in rapid eye movement (REM) sleep during acute withdrawal, which returned to baseline levels during protracted abstinence. REM sleep onset latency was decreased during protracted abstinence in females only. Furthermore, there was a sex difference observed in overall REM sleep bout rate. Although there were no changes in non-REM sleep time, or to non-REM sleep bout rate or duration, there was an increase in non-REM sleep intra-spindle frequency during acute withdrawal in both females and males. Finally, there was increased wakefulness time and bout duration during acute withdrawal in both females and males. The results demonstrate both macroarchitectural and microarchitectural changes in sleep following chronic alcohol exposure, particularly during acute withdrawal, suggesting the need for therapeutic interventions for sleep disturbances during withdrawal in individuals with AUD. Furthermore, sex differences were observed in REM sleep, highlighting the importance of including both sexes in future alcohol-related sleep studies.

Corticosteroid sensitization drives opioid addiction.

The global crisis of opioid overdose fatalities has led to an urgent search to discover the neurobiological mechanisms of opioid use disorder (OUD). A driving force for OUD is the dysphoric and emotionally painful state (hyperkatifeia) that is produced during acute and protracted opioid withdrawal. Here, we explored a mechanistic role for extrahypothalamic stress systems in driving opioid addiction. We found that glucocorticoid receptor (GR) antagonism with mifepristone reduced opioid addiction-like behaviors in rats and zebrafish of both sexes and decreased the firing of corticotropin-releasing factor neurons in the rat amygdala (i.e., a marker of brain stress system activation). In support of the hypothesized role of glucocorticoid transcriptional regulation of extrahypothalamic GRs in addiction-like behavior, an intra-amygdala infusion of an antisense oligonucleotide that blocked GR transcriptional activity reduced addiction-like behaviors. Finally, we identified transcriptional adaptations of GR signaling in the amygdala of humans with OUD. Thus, GRs, their coregulators, and downstream systems may represent viable therapeutic targets to treat the "stress side" of OUD.

Ketogenic diet reduces alcohol withdrawal symptoms in humans and alcohol intake in rodents.

Individuals with alcohol use disorder (AUD) show elevated brain metabolism of acetate at the expense of glucose. We hypothesized that a shift in energy substrates during withdrawal may contribute to withdrawal severity and neurotoxicity in AUD and that a ketogenic diet (KD) may mitigate these effects. We found that inpatients with AUD randomized to receive KD (n = 19) required fewer benzodiazepines during the first week of detoxification, in comparison to those receiving a standard American (SA) diet (n = 14). Over a 3-week treatment, KD compared to SA showed lower "wanting" and increased dorsal anterior cingulate cortex (dACC) reactivity to alcohol cues and altered dACC bioenergetics (i.e., elevated ketones and glutamate and lower neuroinflammatory markers). In a rat model of alcohol dependence, a history of KD reduced alcohol consumption. We provide clinical and preclinical evidence for beneficial effects of KD on managing alcohol withdrawal and on reducing alcohol drinking.

Converging Structural and Functional Evidence for a Rat Salience Network.

BACKGROUND: The salience network (SN) is dysregulated in many neuropsychiatric disorders, including substance use disorder. Though the SN was initially described in humans, identification of a rodent SN would provide the ability to mechanistically interrogate this network in preclinical models of neuropsychiatric disorders. METHODS: We used modularity analysis on resting-state functional magnetic resonance imaging data of rats (n = 32) to parcellate rat insula into functional subdivisions and to identify a potential rat SN based on functional connectivity patterns from the insular subdivisions. We then used mouse tract tracing data from the Allen Brain Atlas to confirm the network's underlying structural connectivity. We next compared functional connectivity profiles of the SN across rats, marmosets (n = 10), and humans (n = 30). Finally, we assessed the rat SN's response to conditioned cues in rats (n = 21) with a history of heroin self-administration. RESULTS: We identified a putative rat SN, which consists of primarily the ventral anterior insula and anterior cingulate cortex, based on functional connectivity patterns from the ventral anterior insular division. Functional connectivity architecture of the rat SN is supported by the mouse neuronal tracer data. Moreover, the anatomical profile of the identified rat SN is similar to that of nonhuman primates and humans. Finally, we demonstrated that the rat SN responds to conditioned cues and increases functional connectivity to the default mode network during conditioned heroin withdrawal. CONCLUSIONS: The neurobiological identification of a rat SN, together with a demonstration of its functional relevance, provides a novel platform with which to interrogate its functional significance in normative and neuropsychiatric disease models.

PCSK9 inhibition as a novel therapeutic target for alcoholic liver disease.

Alcoholic liver disease (ALD) causes significant morbidity and mortality, and pharmacological treatment options are limited. In this study, we evaluated the PCSK9 inhibitor alirocumab, a monoclonal antibody that robustly reduces low-density lipoprotein cholesterol (LDL-C), for the treatment of ALD using a rat model of chronic alcohol exposure. Alirocumab (50 mg/kg) or vehicle was administered weekly for 6 weeks to rats receiving a 12% alcohol liquid diet or an isocaloric control diet. At the end of the alcohol exposure protocol, serum and liver samples were obtained for molecular characterization and histopathological analysis. PCSK9 inhibition with alirocumab attenuated alcohol-induced hepatic triglyceride accumulation through regulation of lipid metabolism (mRNA expression of modulators of fatty acid synthesis (FAS) and catabolism (PPARα and CPT1)), hepatocellular injury (ALT), hepatic inflammation (mRNA expression of pro-inflammatory cytokines/chemokines (TNFa, IL-1ß, IL-22, IL-33, IL-17α, IL-2, MIP-2, and MCP-1), and neutrophil infiltration (myeloperoxidase staining)). Alirocumab treatment also attenuated alcohol-induced PCSK9 mRNA elevation and upregulated LDL-receptor (LDL-R) via modulation of the transcription factors (SREBP-1, SREBP-2, and E2F1) in liver. We demonstrated that chronic anti-PCSK9 treatment using the monoclonal antibody alirocumab attenuated alcohol-induced steatohepatitis in the rat model. Given the large unmet clinical need for effective and novel treatments for ALD, anti-PCSK9 treatment with the monoclonal antibody that spares liver metabolism is a viable new therapeutic possibility. Future studies are needed to elucidate the exact role of PCSK9 in ALD and alcohol use disorder (AUD) and to evaluate efficacy and safety of anti-PCSK9 treatment in clinical populations with ALD/AUD.

Oxytocin blocks enhanced motivation for alcohol in alcohol dependence and blocks alcohol effects on GABAergic transmission in the central amygdala.

Oxytocin administration has been reported to decrease consumption, withdrawal, and drug-seeking associated with several drugs of abuse and thus represents a promising pharmacological approach to treat drug addiction. We used an established rat model of alcohol dependence to investigate oxytocin's effects on dependence-induced alcohol drinking, enhanced motivation for alcohol, and altered GABAergic transmission in the central nucleus of the amygdala (CeA). Intraperitoneal oxytocin administration blocked escalated alcohol drinking and the enhanced motivation for alcohol in alcohol-dependent but not nondependent rats. Intranasal oxytocin delivery fully replicated these effects. Intraperitoneal administration had minor but significant effects of reducing locomotion and intake of non-alcoholic palatable solutions, whereas intranasal oxytocin administration did not. In dependent rats, intracerebroventricular administration of oxytocin or the oxytocin receptor agonist PF-06655075, which does not cross the blood-brain barrier (i.e., it would not diffuse to the periphery), but not systemic administration of PF-06655075 (i.e., it would not reach the brain), decreased alcohol drinking. Administration of a peripherally restricted oxytocin receptor antagonist did not reverse the effect of intranasal oxytocin on alcohol drinking. Ex vivo electrophysiological recordings from CeA neurons indicated that oxytocin decreases evoked GABA transmission in nondependent but not in dependent rats, whereas oxytocin decreased the amplitude of spontaneous GABAergic responses in both groups. Oxytocin blocked the facilitatory effects of acute alcohol on GABA release in the CeA of dependent but not nondependent rats. Together, these results provide converging evidence that oxytocin specifically and selectively blocks the enhanced motivation for alcohol drinking that develops in alcohol dependence likely via a central mechanism that may result from altered oxytocin effects on CeA GABA transmission in alcohol dependence. Neuroadaptations in endogenous oxytocin signaling may provide a mechanism to further our understanding of alcohol use disorder.

Heroin addiction engages negative emotional learning brain circuits in rats.

Opioid use disorder (OUD) is associated with the emergence of persistent negative emotional states during drug abstinence that drive compulsive drug taking and seeking. Functional magnetic resonance imaging (fMRI) in rats identified neurocircuits that were activated by stimuli that were previously paired with heroin withdrawal. The activation of amygdala and hypothalamic circuits was related to the degree of heroin dependence, supporting the significance of conditioned negative affect in sustaining compulsive-like heroin seeking and taking and providing neurobiological insights into the drivers of the current opioid crisis.

Compulsive-Like Sufentanil Vapor Self-Administration in Rats.

Opioid misuse is at historically high levels in the United States, with inhalation (ie, smoking and vaping) being one of the most common routes of consumption. We developed and validated a novel preclinical model of opioid self-administration by inhalation that does not require surgery and reliably produces somatic and motivational signs of dependence. Rats were trained to perform an operant response (nosepoke) to receive 10 s of vaporized sufentanil, a potent opioid, in 2 h daily sessions. Rats readily and concentration-dependently self-administered vaporized sufentanil. Rats exhibited a significant increase in responding for sufentanil when given the preferential µ-opioid receptor inverse agonist naloxone, suggesting the participation of µ-opioid receptors in the reinforcing properties of sufentanil vapor. Serum sufentanil concentrations significantly correlated with the number of sufentanil vapor deliveries. Rats that were given long access (LgA; 12 h/day) but not short access (ShA; 1 h/day) to vaporized sufentanil escalated their drug intake over time and exhibited both naloxone-precipitated somatic signs of opioid withdrawal and spontaneous withdrawal-induced mechanical hypersensitivity. After 6 months of forced drug abstinence, LgA rats returned to pre-escalation baseline levels of responding for sufentanil and mechanical sensitivity. Upon subsequent re-escalation (ie, after the return to extended access to sufentanil vapor), LgA rats again developed naloxone-precipitated somatic signs of withdrawal and spontaneous withdrawal-induced mechanical hypersensitivity. These findings demonstrate that the operant sufentanil vapor self-administration model has both face and construct validity and therefore will be useful for investigating the neurobiological basis of opioid addiction.

Atypical dopamine transporter inhibitors attenuate compulsive-like methamphetamine self-administration in rats.

Methamphetamine (METH) is a highly addictive drug, but no pharmacological treatment is yet available for METH use disorders. Similar to METH, the wake-promoting drug (R)-modafinil (R-MOD) binds to the dopamine transporter (DAT). Unlike METH, R-MOD is not a substrate for transport by DAT and has low abuse potential. We tested the hypothesis that the atypical DAT inhibitor R-MOD and compounds that are derived from modafinil would decrease METH intake by reducing the actions of METH at the DAT. We tested the effects of systemic injections of R-MOD and four novel modafinil-derived ligands with increased DAT affinity (JJC8-016, JJC8-088, JJC8-089, and JJC8-091) on intravenous (i.v.) METH self-administration in rats that were allowed short access (ShA; 1 h) or long access (LgA; 6 h) to the drug. ShA rats exhibited stable METH intake over sessions, whereas LgA rats exhibited an escalation of drug intake. R-MOD decreased METH self-administration in ShA and LgA rats (in the 1st hour only). JJC8-091 and JJC8-016 decreased METH self-administration in both ShA and LgA rats. JJC8-089 decreased METH self-administration in LgA rats only, whereas JJC8-088 had no effect on METH self-administration in either ShA or LgA rats. These findings support the potential of atypical DAT inhibitors for the treatment of METH use disorders and suggest several novel compounds as candidate drugs.

Sex, strain, and estrous cycle influences on alcohol drinking in rats.

Although women appear to be more vulnerable to alcohol-induced pathophysiology than men, the neurobiological basis for sex differences is largely unknown, partially because most studies on alcohol drinking are conducted in male subjects only. The present study examined sex differences in alcohol consumption in two rat strains, Long Evans and Wistar, using multiple behavioral paradigms. The effects of the estrous cycle on alcohol consumption were monitored throughout the study. The results indicated that females drank more alcohol than males when given either continuous or intermittent access to alcohol (vs. water) in their home cages (voluntary drinking). Under operant conditions, no sex or strain differences were found in drinking prior to development of alcohol dependence. However, upon dependence induction by chronic, intermittent alcohol vapor exposure, Wistar rats of both sexes substantially escalated their alcohol intake compared with their nondependent drinking levels, whereas Long Evans rats only exhibited a moderate escalation of drinking. Under these conditions, the estrous cycle had no effect on alcohol drinking in any strain and drinking model. Thus, strain, sex, and drinking conditions interact to modulate nondependent and dependent alcohol drinking. The present results emphasize the importance of including sex and strain as biological variables in exploring individual differences in alcohol drinking and dependence.

A conjugate vaccine using enantiopure hapten imparts superior nicotine-binding capacity.

A leading nicotine conjugate vaccine was only efficacious for one-third of clinical trial participants, likely due in part to its use of racemic nicotine hapten, (±)-3'-AmNic. Immunization of male Wistar rats with (+)-, (-)-, or (±)-3'-AmNicSucTT and subsequent antibody immunoassays suggest that a vaccine using enantiopure (-)-3'-AmNic hapten imparts superior capacity to bind (-)-nicotine. Future nicotine vaccine clinical candidates must incorporate this design consideration (i.e., hapten enantiopurity) in order to maximize efficacy.

Reduced alcohol drinking in adult rats exposed to sucrose during adolescence.

Intake of sweet-alcoholic drinks during adolescence is believed to favor alcohol abuse and dependence in adulthood. This study examined the influence of early exposure to ethanol with or without sucrose on the consumption of sweet or alcoholic solutions in adulthood. Adolescent rats (from post-natal day 30-46) were given continuous free access to tap water and either 5% sucrose, 5% ethanol or mixed 5% sucrose-5% ethanol. The control group was given access to water only. Upon reaching adulthood (post-natal day 60), rats were tested for saccharin (sweet), quinine (bitter) and ethanol consumption using a two-bottle free-choice paradigm. The results indicated that pre-exposure to ethanol did not alter the intake of sweet or ethanol solutions in adulthood. However, rats exposed to sucrose during adolescence showed a decreased consumption of both sweet and ethanol solutions. Because alcohol has a sweet taste component, an additional group of rats, pre-exposed to either 5% sucrose or water during adolescence, was tested for intravenous ethanol self-administration (preventing oral sensory stimulation) and in a new model of simultaneous access to oral saccharin and intravenous ethanol that results in higher total ethanol intake. Relative to controls, sucrose-exposed rats showed reduced operant self-administration of saccharin, yet no differences were found for intravenous ethanol self-administration. Altogether, these findings indicate that sucrose exposure during adolescence persistently affected the perception of sweet taste reward and thereby alcohol's acceptance in adulthood.