Xylazine is a pervasive adulterant in clandestine opioid supplies. This trend is troubling, as xylazine carries its own acute side effects and its long-term cognitive and motivational effects are not known. Thus, we developed and validated a model of oral xylazine self-administration that is conducive to polysubstance studies and can easily be implemented in animal research labs. Mice underwent 4-hour drinking sessions where their only source of drinking water was adulterated with xylazine (10-1000μg/mL). Oral bioavailability and brain penetrance were validated using mass spectrometry on brain and plasma samples collected after a drinking session. Male and female mice decreased fluid intake at high concentrations of xylazine, with male mice consuming less than female mice at intermediate concentrations (100 and 300μg/mL). Female mice had decreased locomotion following drinking sessions at 100, 300, and 1000μg/mL. After a 4-hour drinking session, all mice received brain xylazine concentrations that are above the Ki (affinity) and EC50 (potency) of several known binding targets of xylazine. We then performed a three-bottle choice experiment where mice had the option of drinking from water with xylazine, water with fentanyl, or plain water. In three-bottle choice, mice consumed less xylazine-containing water than fentanyl-containing or plain water. Our results indicate that xylazine is orally bioavailable in mice, that mice will readily drink xylazine to pharmacologically and behaviorally relevant doses, and that mice prefer consuming fentanyl and water over xylazine.

The ongoing opioid epidemic underscores the need for scalable and translational preclinical models of voluntary opioid intake and dependence. We therefore sought to establish and validate a voluntary two-bottle choice drinking-in-the-dark (DID) model of oral opioid intake in mice and to determine relationships between experimental parameters and behaviors during and after withdrawal. Male and female C57BL/6J mice were given daily access to two bottles during the dark phase for 24 drinking sessions over 5 weeks. Control mice received two bottles containing water. Experimental mice received one water bottle and one bottle containing oxycodone (0.1-1 mg/mL) or fentanyl (10-100 μg/mL) under varying session durations and concentrations. On the final day, physical dependence was assessed using naloxone-precipitated withdrawal and then a behavioral battery to assess negative affect was performed in the following week. Mice voluntarily consumed both oxycodone and fentanyl without taste adulteration and maintained drug preference across most concentrations. Oxycodone intake produced minimal withdrawal symptoms. In contrast, fentanyl intake resulted in naloxone-precipitated withdrawal that was modulated by session duration and concentration. Four-hour sessions produced stronger withdrawal than two-hour sessions at equivalent concentrations. Escalating high-concentration fentanyl exposure revealed emerging sex differences, with females exhibiting greater intake and withdrawal at higher concentrations. Affective behavioral assays following withdrawal revealed minimal persistent alterations in any cohort. These findings establish key parameters for a scalable voluntary fentanyl model that produces dose- and session-dependent physical dependence in male and female mice. This paradigm provides a cost-effective and straightforward platform for future investigations of opioid use and dependence.

The mu-opioid receptor (MOR) and the nociceptin/orphanin FQ receptor (NOPR) are closely related yet functionally distinct modulators of rewards, motivation, and affect. Within the mesocorticolimbic system, including the prefrontal cortex (PFC), the ventral tegmental area (VTA) and the nucleus accumbens (NAc), these receptors exhibit divergent, cell type–specific expression patterns that drive opposing behavioral outcomes. For example, MOR activation enhances rewards processing and reinforcement by facilitating dopamine transmission, whereas NOPR signaling in the VTA can reduce dopamine cell activity. In addition, MOR and NOPR are positioned within cortical circuits to preferentially reduce GABA and glutamate transmission, respectively. This review synthesizes current knowledge on how MOR and NOPR coordinate motivational and affective states through distinct neuronal populations across the mesocorticolimbic circuit. We also discuss emerging evidence for functional interactions between these systems and the therapeutic implications of pharmacological strategies targeting both receptors, including dual-acting MOR/NOPR ligands that enhance analgesic efficacy with reduced abuse liability. By integrating behavioral, molecular, and circuit-level findings, this synthesis aims to clarify how MOR and NOPR signaling jointly shape rewards and stress pathways, and provide insight into the development of safer and more effective treatments for opioid use disorders.

Binge drinking continues to pose major societal and health challenges. Understanding the molecular mechanisms that drive problematic alcohol use is critical for developing novel therapeutics. Parvalbumin-expressing interneurons (PV-INs), a key subpopulation of interneurons within the prefrontal cortex (PFC) and other brain areas, have been implicated in a variety of alcohol-seeking behaviors. Recent work has identified the metabotropic glutamate 5 (mGlu5) receptor as a critical sex-specific regulator of PV-IN activity. Genetic deletion of mGlu5 from PV-INs alters PFC-dependent cognitive behaviors and sex-specifically decreases volitional ethanol intake, but its role in motivated drinking remains unknown. Here, we use operant self-administration to examine how disruption of mGlu5 in PV-INs alters motivated drinking. Under a fixed ratio schedule, PV-mGlu5-/- mice show impairments in the acquisition of ethanol self-administration. By contrast, PV-mGlu5-/- mice readily acquire sucrose self-administration, suggesting a specific deficit to ethanol reinforcement. With sucrose administration, we found that males produced more active nosepokes and consumed more sucrose than females, an effect attenuated in male PV-mGlu5-/- mice. Using a fading procedure, mice were gradually transitioned to respond for 20% ethanol. Following the fade, male wildtypes maintained elevated responding relative to females and to male PV-mGlu5-/- mice. Progressive ratio and quinine adulteration suggest that these sex- and genotype-dependent effects reflect differences in motivation rather than altered taste sensitivity. Finally, we found that PV-mGlu5-/- mice displayed reduced expression of perineuronal nets and PV protein, with a greater disruption in males than females. Together, these data highlight mGlu5 receptors and PV-INs as critical regulators of motivated drinking.

Critical evaluation and refinement of animal models is essential for neuroscientists to understand complex physiological and pathological processes related to psychiatric diseases. In general, preclinical studies modeling drug dependence and problematic substance use have been limited to the administration of a single substance; however, there is a growing appreciation that this approach has failed to capture the complexities of humans and has stifled translational efforts. Polysubstance use represents the overwhelmingly common patterns of alcohol and drug use in humans. For example, epidemiological studies generally determine that between 70-95% of individuals with alcohol use disorder use tobacco daily, and upwards of 60% of individuals who use cocaine have a comorbid alcohol use disorder. Based on this, it is imperative for preclinical researchers to consider incorporating nicotine, alcohol, and other drugs into preclinical models of drug use. Here, we discuss the complexities of polysubstance use in the real-world and in rodent models, describing core findings from recent studies that illustrate how the neurobiological mechanisms that drive polysubstance use can differ critically from monosubstance use. Despite these compelling data that justify the support for polysubstance use research, these studies face systemic challenges and barriers to funding that have throttled research in this area. We bring these challenges to light and identify new opportunities for improving the rigor and reproducibility of polysubstance use research in animal models.

Open source devices are becoming widely used in behavioral neuroscience. Despite their advantages in cost effectiveness, modularity, and customization, measurements obtained using newly developed devices may not always recapitulate measurements from existing and validated equipment, potentially due to the materials used in manufacture. In this study, we evaluated a commonly used open-source optical lickometer that delivers fluid via a Hydropac® plastic valve in a multi-site intermittent access two-bottle choice (IA2BC) paradigm for alcohol consumption. Mice were tested with both traditional metal sippers and plastic sippers equipped with Hydropac® valves to assess differences in alcohol intake, preference, and total fluid consumption. Our findings revealed that mice displayed reduced intake and preference for alcohol (10-20% v/v) delivered via the Hydropac® containing plastic sippers. Notably, the effect was observed at both testing sites, suggesting a generalizable phenomenon. The decreased intake was also specific to alcohol, as water, quinine, and sucrose consumption were unaffected by sipper type. To investigate the underlying cause of the reduced alcohol consumption, we pre-incubated Hydropac® valves in 20% alcohol and found that the pre-treated alcohol reduced intake even when delivered via metal sippers. This suggests that prolonged interaction between alcohol and the components of the Hydropac® valves alter the fluid, likely by generating unpalatable contaminants. These results highlight a limitation of using plastic sippers in long-term alcohol self-administration studies. While these devices may remain suitable for limited access paradigms their use in extended access protocols may compromise data integrity. Our study underscores the need for rigorous validation of open-source hardware in each research project.

Aberrant signaling within cortical inhibitory microcircuits has been identified as a common signature of neuropsychiatric disorders. Interneuron (IN) activity is precisely regulated by neuromodulatory systems that evoke widespread changes in synaptic transmission and principal cell output. Cortical interneurons express high levels of opioid receptors, positioning opioid signaling as a critical regulator of inhibitory transmission; however, we lack a complete understanding of how classical opioid receptor systems regulate prefrontal cortex (PFC) microcircuitry. Here, we combine whole-cell patch-clamp electrophysiology, optogenetics, and viral tools to provide an extensive characterization of how the Mu opioid receptor (MOR), Delta opioid receptor (DOR), and Kappa opioid receptor (KOR) regulate inhibitory transmission in male and female mice. We show that across these receptor systems, DOR activation is more effective at suppressing spontaneous inhibitory transmission in layer 2/3 of the prelimbic PFC, while MOR causes a greater acute suppression of electrically evoked GABA release, and KOR plays a minor role in inhibitory transmission. Cell type-specific optogenetics revealed that MOR and DOR differentially regulate inhibitory transmission from parvalbumin, somatostatin, cholecystokinin, and vasoactive intestinal peptide-expressing INs. Finally, we demonstrate that DOR regulates inhibitory transmission through simultaneous pre- and postsynaptic modifications to IN physiology, whereas MOR function varies between somato-dendritic or presynaptic signaling depending on cell type.

Several neurochemical systems converge in the prefrontal cortex (PFC) to regulate cognitive and motivated behaviors. A rich network of endogenous opioid peptides and receptors spans multiple PFC cell types and circuits, and this extensive opioid system has emerged as a key substrate underlying reward, motivation, affective behaviors, and adaptations to stress. Here, we review the current evidence for dysregulated cortical opioid signaling in the pathogenesis of psychiatric disorders. We begin by providing an introduction to the basic anatomy and function of the cortical opioid system, followed by a discussion of endogenous and exogenous opioid modulation of PFC function at the behavioral, cellular, and synaptic level. Finally, we highlight the therapeutic potential of endogenous opioid targets in the treatment of psychiatric disorders, synthesizing clinical reports of altered opioid peptide and receptor expression and activity in human patients and summarizing new developments in opioid-based medications.This article is part of the Special Issue on “PFC circuit function in psychiatric disease and relevant models”.

Despite established sex differences in the prevalence and presentation of psychiatric disorders, little is known about the cellular and synaptic mechanisms that guide these differences under basal conditions. The proper function of the prefrontal cortex (PFC) is essential for the top-down regulation of motivated behaviors. The activity of the PFC is tightly controlled by parvalbumin-expressing interneurons (PV-INs), a key subpopulation of fast-spiking GABAergic cells that regulate cortical excitability through direct innervations onto the perisomatic regions of nearby pyramidal cells. Recent rodent studies have identified notable sex differences in PV-IN activity and adaptations to experiences such as binge drinking. Here, we investigated the cellular and molecular mechanisms that underlie sex-specific regulation of PFC PV-IN function. Using whole-cell patch-clamp electrophysiology and selective pharmacology, we report that PV-INs from female mice are more excitable than those from males. Moreover, we find that mGlu1 and mGlu5 metabotropic glutamate receptors regulate cell excitability, excitatory drive, and endocannabinoid signaling at PFC PV-INs in a sex-dependent manner. Genetic deletion of mGlu5 receptors from PV-expressing cells abrogates all sex differences observed in PV-IN membrane and synaptic physiology. Lastly, we report that female, but not male, PV-mGlu5-/- mice exhibit decreased voluntary drinking on an intermittent access schedule, which could be related to changes in ethanol's stimulant properties. Importantly, these studies identify mGlu1 and mGlu5 receptors as candidate signaling molecules involved in sex differences in PV-IN activity and behaviors relevant to alcohol use.

At the core of value-based learning is the nucleus accumbens (NAc). D1- and D2-receptor-containing medium spiny neurons (MSNs) in the NAc core are hypothesized to have opposing valence-based roles in behavior. Using optical imaging and manipulation approaches in mice, we show that neither D1 nor D2 MSNs signal valence. D1 MSN responses were evoked by stimuli regardless of valence or contingency. D2 MSNs were evoked by both cues and outcomes, were dynamically changed with learning, and tracked valence-free prediction error at the population and individual neuron level. Finally, D2 MSN responses to cues were necessary for associative learning. Thus, D1 and D2 MSNs work in tandem, rather than in opposition, by signaling specific properties of stimuli to control learning.

Cellular responses to metabotropic glutamate (mGlu) receptor activation are shaped by mechanisms of receptor-receptor interaction. mGlu receptor subtypes form homodimers, intra- or inter-group heterodimers, and heteromeric complexes with other G protein-coupled receptors (GPCRs). In addition, mGlu receptors may functionally interact with other receptors through the βγ subunits released from G proteins in response to receptor activation or other mechanisms. Here, we discuss the interactions between (i) mGlu1 and GABAB receptors in cerebellar Purkinje cells; (ii) mGlu2 and 5-HT2Aserotonergic receptors in the prefrontal cortex; (iii) mGlu5 and A2A receptors or mGlu5 and D1 dopamine receptors in medium spiny projection neurons of the indirect and direct pathways of the basal ganglia motor circuit; (iv) mGlu5 and A2A receptors in relation to the pathophysiology of Alzheimer's disease; and (v) mGlu7 and A1 adenosine or α- or β1 adrenergic receptors. In addition, we describe in detail a novel form of non-heterodimeric interaction between mGlu3 and mGlu5 receptors, which appears to be critically involved in mechanisms of activity-dependent synaptic plasticity in the prefrontal cortex and hippocampus. Finally, we highlight the potential implication of these interactions in the pathophysiology and treatment of cerebellar disorders, schizophrenia, Alzheimer's disease, Parkinson's disease, l-DOPA-induced dyskinesias, stress-related disorders, and cognitive dysfunctions. This article is part of the Special Issue on "The receptor-receptor interaction as a new target for therapy".

The prefrontal cortex (PFC) regulates drinking behaviors and affective changes following chronic alcohol use. PFC activity is dynamically modulated by local inhibitory interneurons (INs), which can be divided into non-overlapping groups with distinct functional roles. Within deeper layers of neocortex, INs that express either parvalbumin or somatostatin directly inhibit pyramidal cells. By contrast, the plurality of all remaining INs express vasoactive intestinal peptide (VIP), reside within superficial layers, and preferentially target other types of INs. While recent studies have described adaptations to PFC parvalbumin-INs and somatostatin-INs in alcohol use models, whether ethanol or drinking affect the physiology of PFC VIP-INs has not been reported. To address this gap, we used genetically engineered female and male mice to target VIP-INs in layers 1-3 of prelimbic PFC for whole-cell patch-clamp electrophysiology. We found that ethanol (20 mM, ∼0.09 BEC) application to PFC brain slices enhances VIP-IN excitability. We next examined effects following chronic drinking by providing mice with 4 weeks of intermittent access (IA) ethanol two-bottle choice in the home cage. In these studies, VIP-INs from female and male IA ethanol mice displayed reduced excitability relative to cells from water-only controls. Finally, we assessed whether these effects continue into abstinence. After 7-11 days without ethanol, the hypo-excitability of VIP-INs from male IA ethanol mice persisted, whereas cells from female IA ethanol mice were not different from their controls. Together, these findings illustrate that acute ethanol enhances VIP-IN excitability and suggest these cells undergo pronounced homeostatic changes following long-term drinking.

Metabotropic glutamate (mGlu) receptors are promising targets for the treatment of affective disorders and alcohol use disorder (AUD). Nonspecific ligands for Group II (mGlu2 and mGlu3) mGlu receptors have demonstrated consistent therapeutic potential for affective disorders in preclinical models. Disentangling the specific roles of mGlu2 versus mGlu3 receptors in these effects has persisted as a major challenge, in part due to pharmacological limitations. However, the recent development of highly specific allosteric modulators for both mGlu2 and mGlu3 receptors have enabled straightforward and rigorous investigations into the specific function of each receptor. Here, we review recent experiments using these compounds that have demonstrated both similar and distinct receptor functions in behavioral, molecular, and electrophysiological measures associated with basal function and preclinical models of affective disorders. Studies using these selective drugs have demonstrated that mGlu2 is the predominant receptor subclass involved in presynaptic neurotransmitter release in prefrontal cortex. By contrast, the activation of postsynaptic mGlu3 receptors induces a cascade of cellular changes that results in AMPA receptor internalization, producing long-term depression and diminishing excitatory drive. Acute stress decreases the mGlu3 receptor function and dynamically alters transcript expression for both mGlu2 (Grm2) and mGlu3 (Grm3) receptors in brain areas involved in reward and stress. Accordingly, both mGlu2 and mGlu3 negative allosteric modulators show acute antidepressant-like effects and potential prophylactic effects against acute and traumatic stressors. The wide array of effects displayed by these new allosteric modulators of mGlu2 and mGlu3 receptors suggest that these drugs may act through improving endophenotypes of symptoms observed across several neuropsychiatric disorders. Therefore, recently developed allosteric modulators selective for mGlu2 or mGlu3 receptors show promise as potential therapeutics for affective disorders and AUD.

While many new programs bridge the arts and sciences, a data-based examination of artscience program design can lead to more efficient programming. The Vanderbilt Institute for Infection, Immunology, and Inflammation Artist-in-Residence program is a virtual program that brings together undergraduate student "artists" and faculty-level "scientists" to generate science-art content. We have recruited over 80 artists and 50 scientists to collaborate in creating visual science communication content. Using self-reported data from both groups, we performed qualitative and quantitative analyses to define sources for negative and positive experiences for artists and scientists. We also identify areas for improvement and key features for in producing a positive experience. We found that artists participants had more positive responses about "learning something new" from the program than scientists. We also found that for both artists and scientists the length of the program and the virtual nature were identified as key features that could be improved. However, the most surprising aspect of our analysis suggests that for both "way of thinking" and "science communication to the public or general audience," were seen as significant beneficial gains for scientists compared to artists. We conclude this analysis with suggestions to enhance the benefits and outcomes of an art-science program and ways to minimize the difficulties, such as communication and collaboration, faced by participants and program designers.