Effects of xylazine on naloxone-precipitated fentanyl withdrawal in male and female rats.

PURPOSE: The combination of fentanyl and xylazine (i.e., "tranq-dope") was recently declared an emerging national health threat in the United States. Given the recency of this development, very little is known regarding the behavioral pharmacology of fentanyl-xylazine combinations. The purpose of this study was to characterize the somatic and affective withdrawal symptoms of this drug combination. METHODS: Male and female Long Evans rats were given twice daily (08:00 and 20:00) subcutaneous injections of fentanyl, xylazine, or combined fentanyl-xylazine for five days. On the sixth (testing) day, rats were given a final injection at 08:00. Four hours later, rats were injected intraperitoneally with either saline or a naloxone challenge before behavioral observation. Somatic withdrawal was examined using the Gellert-Holtzman scale and anxiety-like behavior was examined using the elevated plus maze. RESULTS: Naloxone administration did not induce somatic or affective symptoms in rats treated with fentanyl alone, a low dose of xylazine alone, or a high dose of xylazine alone. Naloxone induced somatic but not affective withdrawal symptoms in rats treated with both fentanyl and xylazine. CONCLUSION: Chronic co-exposure to fentanyl and xylazine produces an opioid-like somatic withdrawal syndrome at doses that are not apparent with either drug alone. These results corroborate clinical reports that xylazine worsens fentanyl withdrawal and suggest that novel interventions may be required to treat withdrawal from fentanyl-xylazine combinations in humans.

The positive reinforcing effects of cocaine and opposite-sex social contact: roles of biological sex and estrus.

RATIONALE: Preclinical studies report that drug use and social contact mutually influence the reinforcing effects of one another. Most of these studies have used same-sex dyads exclusively, and the role of factors related to biological sex and hormonal fluctuations are not well understood. OBJECTIVES: The purpose of this study was to examine the reinforcing effects of cocaine and social contact with an opposite-sex partner in male and female rats, and how these effects are modulated by ovarian hormones. METHODS: Male and female rats were trained in a nonexclusive choice procedure in which cocaine and social contact with an opposite-sex partner were simultaneously available on concurrent progressive ratio schedules of reinforcement. To examine the effects of ovarian hormones related to estrous cycling, Experiment 1 used naturally cycling, gonadally intact females, whereas Experiment 2 used ovariectomized females, and estrus was artificially induced with exogenous hormones. RESULTS: In both experiments, cocaine and social contact functioned as robust reinforcers, and there were no significant effects of biological sex or estrus status of the females. The positive reinforcing effects of both cocaine and social contact increased as a function of cocaine dose, indicating that contingent cocaine administration increases the reinforcing effects of social contact. CONCLUSIONS: These data suggest that cocaine use among opposite-sex partners may enhance factors that contribute to social bonding.

"Tranq-dope" overdose and mortality: lethality induced by fentanyl and xylazine.

Introduction: The recreational use of fentanyl in combination with xylazine (i.e., "tranq-dope") represents a rapidly emerging public health threat characterized by significant toxicity and mortality. This study quantified the interactions between these drugs on lethality and examined the effectiveness of potential rescue medications to prevent a lethal overdose. Methods: Male and female mice were administered acute doses of fentanyl, xylazine, or their combination via intraperitoneal injection, and lethality was determined 0.5, 1.0, 1.5, 2.0, and 24 h after administration. Both fentanyl and xylazine produced dose-dependent increases in lethality when administered alone. Results: A nonlethal dose of fentanyl (56 mg/kg) produced an approximately 5-fold decrease in the estimated LD50 for xylazine (i.e., the dose estimated to produce lethality in 50% of the population). Notably, a nonlethal dose of xylazine (100 mg/kg) produced an approximately 100-fold decrease in the estimated LD50 for fentanyl. Both drug combinations produced a synergistic interaction as determined via isobolographic analysis. The opioid receptor antagonist, naloxone (3 mg/kg), but not the alpha-2 adrenergic receptor antagonist, yohimbine (3 mg/kg), significantly decreased the lethality of a fentanyl-xylazine combination. Lethality was rapid, with death occurring within 10 min after a high dose combination and generally within 30 min at lower dose combinations. Males were more sensitive to the lethal effects of fentanyl-xylazine combinations under some conditions suggesting biologically relevant sex differences in sensitivity to fentanyl-xylazine lethality. Discussion: These data provide the first quantification of the lethal effects of "tranq-dope" and suggest that rapid administration of naloxone may be effective at preventing death following overdose.

"Tranq-Dope" Overdose and Mortality: Lethality Induced by Fentanyl and Xylazine.

The recreational use of fentanyl in combination with xylazine (i.e., "tranq-dope") represents a rapidly emerging public health threat characterized by significant toxicity and mortality. This study quantified the interactions between these drugs on lethality and examined the effectiveness of potential rescue medications to prevent a lethal overdose. Male and female mice were administered acute doses of fentanyl, xylazine, or their combination via intraperitoneal injection, and lethality was determined 30, 60, 90, 120, and 1440 min (24 hr) after administration. Both fentanyl and xylazine produced dose-dependent increases in lethality when administered alone. A nonlethal dose of fentanyl (56 mg/kg) produced an approximately 5-fold decrease in the estimated LD50 for xylazine (i.e., the dose estimated to produce lethality in 50% of the population). Notably, a nonlethal dose of xylazine (100 mg/kg) produced an approximately 100-fold decrease in the estimated LD50 for fentanyl. The opioid receptor antagonist, naloxone (3 mg/kg), but not the alpha-2 adrenergic receptor antagonist, yohimbine (3 mg/kg), significantly decreased the lethality of a fentanyl-xylazine combination. Lethality was rapid, with death occurring within 10 min after a high dose combination and generally within 30 min at lower dose combinations. Males were more sensitive to the lethal effects of fentanyl-xylazine combinations under some conditions, suggesting biologically relevant sex differences in sensitivity to fentanyl-xylazine lethality. These data provide the first quantification of the lethal effects of "tranq-dope" and suggest that rapid administration of naloxone may be effective at preventing death following overdose.

Chemogenetic inhibition of a monosynaptic projection from the basolateral amygdala to the ventral hippocampus selectively reduces appetitive, but not consummatory, alcohol drinking-related behaviours.

Alcohol use disorder (AUD) and anxiety/stressor disorders frequently co-occur and this dual diagnosis represents a major health and economic problem worldwide. The basolateral amygdala (BLA) is a key brain region that is known to contribute to the aetiology of both disorders. Although many studies have implicated BLA hyperexcitability in the pathogenesis of AUD and comorbid conditions, relatively little is known about the specific efferent projections from this brain region that contribute to these disorders. Recent optogenetic studies have shown that the BLA sends a strong monosynaptic excitatory projection to the ventral hippocampus (vHC) and that this circuit modulates anxiety- and fear-related behaviours. However, it is not known if this pathway influences alcohol drinking-related behaviours. Here, we employed a rodent operant self-administration regimen that procedurally separates appetitive (e.g. seeking) and consummatory (e.g., drinking) behaviours, chemogenetics and brain region-specific microinjections, to determine if BLA-vHC circuitry influences alcohol and sucrose drinking-related measures. We first confirmed prior optogenetic findings that silencing this circuit reduced anxiety-like behaviours on the elevated plus maze. We then demonstrated that inhibiting the BLA-vHC pathway significantly reduced appetitive drinking-related behaviours for both alcohol and sucrose while having no effect on consummatory measures. Taken together, these findings provide the first indication that the BLA-vHC circuit may regulate appetitive reward seeking directed at alcohol and natural rewards and add to a growing body of evidence suggesting that dysregulation of this pathway may contribute to the pathophysiology of AUD and anxiety/stressor-related disorders.

The maladaptive alcohol self-administration task: An adapted novel model of alcohol seeking with negative consequences.

The progression of recreational drinking to alcohol use disorder is characterized by loss of control over seeking, which involves continued use of alcohol despite negative consequences. The present study proposes a novel maladaptive alcohol self-administration task in which animals are trained to withhold alcohol drinking in the presence of an auditory cue signaling consequence (conflict phase) but to drink freely when there is no consequence (neutral phase). These phases are performed within trial; successful performance involves waiting for the conflict phase to end and drinking during the neutral phase. We discuss the background and implementation of the task, its relation to existing models, and its relevance to the field of translational alcohol research. Importantly, we also present evidence of its efficacy. Both male and female Long-Evans rats are capable of performing the maladaptive alcohol self-administration task for both sweetened and unsweetened alcohol solutions. Finally, we show that acute injection of a pharmacological stressor (yohimbine) significantly disrupted performance of the task in both sexes and reinforcers. We suggest the maladaptive alcohol self-administration task may prove particularly useful in models of alcohol use disorder or vulnerability to this disorder where its application may reveal maladaptive neural circuit adaptations responsible for motivational perturbations associated with loss of control over alcohol seeking.

Stimulation of mu opioid, but not GABAergic, receptors of the lateral habenula alters free feeding in rats.

Overconsumption, or eating beyond the point of homeostasis, is a key feature in the development of obesity. Although people are consuming beyond the point of homeostasis, they are not consuming constantly or indefinitely. Thus, there is likely a mechanism that acts to terminate periods of food intake at some point beyond satiation and prior to aversion, or the negative effects of extreme excess (nausea, bloating, etc.). The purpose of the present study was to assess the lateral habenula as a candidate region for such a mechanism, due to its connectivity to midbrain reward circuitry, sensitivity to metabolic signaling, and pronounced role in drug-related motivated behaviors. Two groups of male Sprague-Dawley rats were surgically implanted with bilateral guide cannula targeting the LHb. Rats were then habituated to feeding chambers, wherein locomotion and food intake were monitored throughout a two-hour session. One experimental group was tested in the presence of rat chow; the second group was instead given access to a sweetened fat diet. Each subject separately received a 0.2 µL vehicle (0.9% saline solution) and baclofen-muscimol (50 ng/0.2 µL of each drug dissolved in 0.9% saline) injection. Additionally, on a third injection day, each rat received an injection of mu-opioid agonist DAMGO (0.1 µg/0.2 µL) prior to placement in the chamber. LHb inactivation did not result in significant alterations in feeding behavior, but produced a consistent increase in locomotor activity in both experimental groups. Mu-opioid receptor stimulation increased feeding on standard chow, but decreased intake of the sweetened-fat diet. Although LHb inactivation did not increase feeding as predicted, the novel finding that mu opioid receptor stimulation decreased feeding on a highly palatable diet, but increased intake of rat chow, highlights a differential role for the LHb in regulating hedonic consummatory behavior.

Chronic intermittent ethanol promotes ventral subiculum hyperexcitability via increases in extrinsic basolateral amygdala input and local network activity.

The hippocampus, particularly its ventral domain, can promote negative affective states (i.e. stress and anxiety) that play an integral role in the development and persistence of alcohol use disorder (AUD). The ventral hippocampus (vHC) receives strong excitatory input from the basolateral amygdala (BLA) and the BLA-vHC projection bidirectionally modulates anxiety-like behaviors. However, no studies have examined the effects of chronic alcohol on the BLA-vHC circuit. In the present study, we used ex vivo electrophysiology in conjunction with optogenetic approaches to examine the effects of chronic intermittent ethanol exposure (CIE), a well-established rodent model of AUD, on the BLA-vHC projection and putative intrinsic vHC synaptic plasticity. We discovered prominent BLA innervation in the subicular region of the vHC (vSub). CIE led to an overall increase in the excitatory/inhibitory balance, an increase in AMPA/NMDA ratios but no change in paired-pulse ratios, consistent with a postsynaptic increase in excitability in the BLA-vSub circuit. CIE treatment also led to an increase in intrinsic network excitability in the vSub. Overall, our findings suggest a hyperexcitable state in BLA-vSub specific inputs as well as intrinsic inputs to vSub pyramidal neurons which may contribute to the negative affective behaviors associated with CIE.

The neural, behavioral, and epidemiological underpinnings of comorbid alcohol use disorder and post-traumatic stress disorder.

Alcohol use disorder (AUD) and (PTSD) frequently co-occur and individuals suffering from this dual diagnosis often exhibit increased symptom severity and poorer treatment outcomes than those with only one of these diseases. Although there have been significant advances in our understanding of the neurobiological mechanisms underlying each of these disorders, the neural underpinnings of the comorbid condition remain poorly understood. This chapter summarizes recent epidemiological findings on comorbid AUD and PTSD, with a focus on vulnerable populations, the temporal relationship between these disorders, and the clinical consequences associated with the dual diagnosis. We then review animal models of the comorbid condition and emerging human and non-human animal research that is beginning to identify maladaptive neural changes common to both disorders, primarily involving functional changes in brain reward and stress networks. We end by proposing a neural framework, based on the emerging field of affective valence encoding, that may better explain the epidemiological and neural findings on AUD and PTSD.

Shifting motivational states: The effects of nucleus accumbens dopamine and opioid receptor activation on a modified effort-based choice task.

The nucleus accumbens (NAc) is critical for regulating the appetitive and consummatory phases of motivated behavior. These experiments examined the effects of dopamine and opioid receptor manipulations within the NAc during an effort-based choice task that allowed for simultaneous assessment of both phases of motivation. Male Sprague-Dawley rats received bilateral guide cannulas targeting the NAc core and were tested in 1-hr sessions with free access to rat chow and the choice to work for sugar pellets on a progressive ratio 2 (PR2) reinforcement schedule. Individual groups of rats were tested following stimulation or blockade of NAc D1-like or D2-like receptors, stimulation of µ-, δ-, or κ-opioid receptors, or antagonism of opioid receptors. Behavior was examined under ad libitum conditions and following 23-h food restriction. NAc blockade of the D1-like receptors or stimulation of the D2 receptor reduced break point for earning sugar pellets; D2 receptor stimulation also modestly lowered chow intake. NAc µ-opioid receptor stimulation increased intake of the freely-available chow while simultaneously reducing break point for the sugar pellets. In non-restricted conditions, δ-opioid receptor stimulation increased both food intake and breakpoint. There were no effects of stimulating NAc D1 or κ receptors, nor did blocking D2 or opioid receptors affect task behavior. These data support prior literature linking dopamine to appetitive motivational processes, and suggest that µ- and δ-opioid receptors affect food-directed motivation differentially. Specifically, µ-opioid receptors shifted behavior towards consumption, and δ-opioid receptor enhanced both sugar-seeking and consumption of the pabulum chow when animals were not food restricted.