Pharmacology Biochemistry and Behavior

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 (10-1000{micro}g/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 300{micro}g/mL). Female mice had decreased locomotion following drinking sessions at 100, 300, and 1000{micro}g/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 {micro}g/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.

Current treatments for opioid use disorder (OUD) have major barriers to access. As such, researching new potential therapies for OUD is important to public health. Previous research has implicated glucagon-like peptide-1 (GLP-1) receptor agonists in decreasing the use of addictive substances by animals. In this study, female Wistar rats (N=32) were surgically implanted with jugular catheters and trained to self-administer fentanyl at a fixed-ratio 1 (FR1) schedule of reinforcement for 21 sessions under short- (ShA; 1 hour) or long-access (LgA; 8 hours) conditions. Next, the animals received injections of semaglutide (0.1 mg/kg, s.c.) or saline (0.9% NaCl, s.c.) prior to another FR1 session. The animals underwent a progressive ratio (PR) schedule of reinforcement while receiving saline (i.v.) or fentanyl (0.625-10 {micro}g/kg/inf, i.v.) and semaglutide (0.1 mg/kg, s.c.) or saline (s.c.). Next, the animals underwent a semaglutide (0-0.1 mg/kg, s.c.) dose response procedure at FR1 and a single dose of fentanyl (2.5 {micro}g/kg/inf, i.v.). Following drug discontinuation, spontaneous locomotor activity and withdrawal-like symptoms were measured. Semaglutide dose-dependently decreased fentanyl rewards under ShA and LgA conditions (p<0.05). Under a PR, semaglutide significantly decreased breakpoint (p<0.05), suggesting semaglutide decreases motivation to self-administer fentanyl. Semaglutide-treated ShA animals displayed significantly less withdrawal-like behavior (p<0.05) but not LgA animals. Overall, these findings suggest semaglutide may modulate motivation to seek opioid reward and could be useful in the development of pharmacotherapies to address OUD.

Activity of the mesolimbic dopamine system has long been implicated in encoding primary rewards and contributing to the addictive properties of drugs of abuse. Dopamine neurons in the ventral tegmental area (VTADA) of the midbrain typically show patterns of spontaneous burst activity that align with the onset of salient events or rewarding stimuli, resulting in phasic dopamine release in the nucleus accumbens (NAc). Fiber photometry is increasingly being used as an accessible technique to quantify neural activity with high temporal resolution at sensors offering signal specificity in stable recordings over extended periods of time. It has been well established by multiple techniques that opioids increase mesolimbic dopamine activity, likely through disinhibition of VTADA neurons. Here we used fiber photometry to compare sub-second transient events from VTADA neurons with GCaMP6f and dopamine release in the lateral shell of the NAc with dLight1.3b and GRABDA2h in response to morphine treatment. In weekly sessions, one dose of morphine was administered in escalating order (2.5, 5,7.5, and 10 mg/kg, intraperitoneal). Consistent with prior literature, both GCaMP6f in VTADA neurons and dLight1.3b in NAc showed patterns of increased signal following morphine treatment. In contrast, morphine suppressed transient activity at GRABDA2h sensors. Further analyses of whole signal streams from each sensor showed a generalized increase, but reduction in variability of the GRABDA2h signal, consistent with the interpretation of sensor saturation. Such results emphasize the importance of the inclusion of appropriate controls to contextualize the interpretation of biosensor responses, particularly in response to pharmacological treatment. HIGHLIGHTSO_LIMorphine elicited increased signaling in VTADA GCaMP6f and NAc dLight1.3b, consistent with prior literature C_LIO_LIMorphine suppressed NAc GRABDA2h signaling of transient events, suggesting saturation of GRABDA2h sensor C_LIO_LISensor validation with pharmacological challenges is critical for interpretation of data C_LI

Acute COVID-19 outcomes are exacerbated by substance use, however, the impact of substance use on Long-COVID is unknown. Here, we investigated the impact of chronic cocaine administration on spike-induced Long-COVID-like outcomes in the rat. Rats received intermittent chronic cocaine administration and a single intravenous injection of the SARS-CoV-2 spike protein. Two months following spike administration, Long-COVID-like outcomes were assessed. Exposure to spike protein in the presence of cocaine produced a persistent reduction in weight gain as compared with controls or spike protein alone. Further, cocaine-treated rats exposed to spike had lower withdrawal thresholds compared to control animals as well as their own baseline, suggesting increased pain sensitivity. Spike and/or cocaine increased the ratio of interleukin-6 (IL-6) to interleukin-10 (IL-10) levels in the hippocampus, indicating a shift towards a proinflammatory state. Paw withdrawal thresholds were positively correlated with IL-10 levels in the hippocampus and prefrontal cortex. Regarding olfaction, rats exposed to spike spent less time sniffing an odor attractant. Cocaine produced an anxiolytic-like phenotype during the elevated plus maze test. Further analysis of behaviors on the maze revealed that the latency to enter the open arms was shorter in rats exposed to spike or cocaine, suggesting a possible impulsive-like phenotype in these animals. These findings demonstrate the negative impact of cocaine on Long-COVID-like outcomes suggesting a need for increased clinical observations of people with co-occurring Long-COVID and cocaine use disorder. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=59 SRC="FIGDIR/small/729575v1_ufig1.gif" ALT="Figure 1"> View larger version (10K): org.highwire.dtl.DTLVardef@12fdc19org.highwire.dtl.DTLVardef@11b1b0dorg.highwire.dtl.DTLVardef@8d1e21org.highwire.dtl.DTLVardef@b53d20_HPS_FORMAT_FIGEXP M_FIG C_FIG

Background and PurposeIts been reported that illicit drug supplies increasingly contain the 2-adrenergic agonist, xylazine, alongside fentanyl, yet the pharmacological basis for the greater lethality of this combination remains unclear. Prior research has shown that -opioid (Oprm1) receptors, on which fentanyl acts, and 2-adrenergic (Adra2a) receptors, on which xylazine acts, are both expressed within brainstem circuits that govern autonomic control, especially the parabrachial (PB) and Kolliker-Fuse (KF) nuclei that regulate respiration. Thus, we propose that co-activation of these inhibitory receptors and their respective pathways could potentiate or additively suppress respiratory and thermoregulatory function. Experimental ApproachFreely behaving C57BL/6J mice received intraperitoneal injections of either saline, fentanyl, xylazine, or fentanyl-xylazine (F+X) solutions. Continuous recordings of respiration using whole-body plethysmography, sleep/wake state using EEG/EMG and body temperature using both infrared thermography, and telemetry were collected for several hours following injection. RNAscope was used to identify Oprm1 and Adra2a expression within PB and KF nuclei. ResultsFentanyl alone produced dose-dependent respiratory depression that was not associated with body temperature changes, whereas the dose we used of xylazine alone had no effect on either respiration or body temperature. In contrast, F+X induced a markedly prolonged (>5 h) reduction in respiratory rate and profound hypothermia lasting 7-8 h, exceeding the effects of either drug alone. Mortality increased to 58.8% following F+X exposure. RNAscope revealed that both Oprm1 and Adra2a receptors are expressed in PB/KF FoxP2-positive neurons, identifying a plausible substrate for convergent inhibitory signaling. ImplicationsThis manuscript provides the first direct experimental evidence that fentanyl and xylazine may interact through convergent -opioid and 2-adrenergic receptor signaling to produce additive and sustained suppression of respiratory and thermoregulatory function. These findings address a critical mechanistic gap in understanding the disproportionate lethality of fentanyl-xylazine mixtures, an emerging public-health crisis. The work further identifies the PB/KF FoxP2 population as a plausible site of dual-receptor convergence and highlights a previously unrecognized pharmacodynamic interaction with immediate implications for overdose reversal strategies. Given the novelty, mechanistic insight, and translational urgency of these results, rapid dissemination will help accelerate scientific and clinical responses to this evolving threat. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=75 SRC="FIGDIR/small/719036v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@5b54aeorg.highwire.dtl.DTLVardef@148b7dorg.highwire.dtl.DTLVardef@d1ebccorg.highwire.dtl.DTLVardef@1cfa9c8_HPS_FORMAT_FIGEXP M_FIG Possible convergent -opioid (Oprm1) and 2-adrenergic (Adra2a) signaling within parabrachial FoxP2-expressing neurons likely produces additive suppression of respiratory and thermoregulatory drive during fentanyl-xylazine co-exposure. Fentanyl and xylazine engage parallel inhibitory GPCR pathways in Parabrachial/ Kolliker Fuse nucleus (PB/KF) neurons that project to the pre-Botzinger complex (preBotC) to depress respiratory rhythm and to the dorsomedial hypothalamus (DMH) to blunt thermogenic output. Co-activation of these pathways results in sustained bradypnea, profound hypothermia, and reduced survival, providing a possible mechanistic basis for the increased lethality of fentanyl-xylazine mixtures. C_FIG

BackgroundEvidence suggests steeper accelerating opioid-related overdose, and non-medical use rates, in middle aged men in recent years compared with younger cohorts. Little is known about whether this is driven by age-related differences in the effects of opioids compared with socio-cultural factors driving non-medical consumption. Rodent models can be useful for dissociating biological from psychosocial factors, however, only minimal evidence exists on the effects of opioids in middle-age rats. ObjectiveTo determine if the anti-nociceptive and rewarding effects of opioids differ between adult and middle-age rats. MethodsFemale and male Wistar rats were obtained in early adulthood and examined across 4 to 11 months of age for nociceptive responses to heroin (0-1.56 mg/kg, s.c.) using a warm-water tail withdrawal assay. Subgroups (N=8 per group) were initiated on intravenous self-administration (IVSA) of heroin at either 5 months or 12 months of age. ResultsAnti-nociceptive effects of heroin did not differ across age. Female rats that initiated IVSA in early adulthood or middle-age obtained significantly more infusions of heroin than male rats of the same age during acquisition, and in dose-substitution under a FR1 schedule. Male, but not female, rats that initiated IVSA in middle age self-administered less heroin then rats that initiated in early adulthood; this was observed in acquisition and in dose-substitution. DiscussionThis study shows that opioid reward is diminished in middle aged male rats. It also found that middle age rats can be used effectively to model opioid-related outcomes, including drug seeking using the IVSA procedure.

Opioid withdrawal is associated with heightened pain sensitivity, including allodynia. Although opioid-induced allodynia is well-documented in humans and animal models, the relationship between the severity of opioid withdrawal-induced allodynia and individual addiction-like behaviors remains poorly understood. To address this gap, Heterogeneous Stock rats underwent long access (12 hours/day) intravenous oxycodone self-administration, followed by measurement of mechanical sensitivity at six timepoints across three weeks of abstinence. Rats were stratified by an Addiction Index derived from individual differences in the escalation of oxycodone intake, motivation to consume oxycodone, tolerance to oxycodones analgesic effects, and acute withdrawal-induced mechanical pain sensitivity. Here, we show that oxycodone withdrawal induces significant and prolonged allodynia for up to three weeks, with High Addiction Index rats exhibiting greater intensity and longer duration of pain sensitivity than Low Addiction Index rats. Results remained consistent even when excluding allodynia from the Addiction Index, highlighting the robustness of the association between addiction-like severity and protracted allodynia. Linear regression associations revealed that self-administration behaviors, particularly oxycodone intake escalation and motivation to seek oxycodone, predicted subsequent withdrawal-induced allodynia severity. These findings demonstrate that greater addiction-like severity is associated with more intense and prolonged withdrawal-induced pain, supporting mechanical allodynia as a marker of addiction severity. These results motivate future work to define the mechanisms linking addiction severity to protracted opioid withdrawal-induced pain, with the goal of informing targeted clinical interventions for individuals most susceptible to severe abstinence-related allodynia.

RationaleCannabis withdrawal contributes to relapse in individuals with cannabis use disorder, yet preclinical studies have largely focused on withdrawal induced by injected cannabinoids rather than inhaled cannabis, which remains the most common route in humans. The behavioural effects of chronic exposure to vapourized cannabis flower and resulting withdrawal after cessation of exposure remain poorly characterized. ObjectivesTo determine the behavioural effects of chronic vapourized high-THC cannabis flower exposure on cannabinoid tetrad, somatic withdrawal and behavioural transition networks in rats following both chronic vapour exposure and administration of the cannabinoid receptor 1 (CB1) receptor antagonist SR141716A (rimonabant). MethodsTwo studies were conducted using adult male Sprague Dawley rats. The first study (N = 16) exposed rats to either air or vapourized high-THC cannabis flower three times a day for seven days using a Volcano vapourizer, followed by intraperitoneal administration of the CB1 antagonist SR141716A (3 mg/kg). The second study (N = 24) included two air controls and two cannabis groups, with one of each receiving either saline or SR141716A. Behavioural assessments included triad measurements to confirm the cannabis effect, along with withdrawal assessment via a sucrose preference test and somatic signs 30 minutes following rimonabant administration. ResultsRepeated cannabis vapour exposure produced reduced locomotor activity, hypothermia, and increased tail-flick latency. Rimonabant administration precipitated withdrawal characterized by increased total withdrawal scores and somatic signs, including blinking, body shakes/tremors, and grooming-related behaviours. Behavioural network analyses revealed substantial reorganization of behavioural transition structure during both chronic cannabis exposure and withdrawal. Chronic cannabis exposure was associated with reduced network modularity, a condensed behavioural repertoire, and altered behavioural centrality measures. At the same time, precipitated withdrawal further increased the influence of exploratory behaviours, particularly sniffing, and reduced the network prominence of locomotor-associated behaviours, such as walking, beyond that detected using conventional behavioural measures alone. ConclusionChronic exposure to vapourized cannabis flower followed by CB1 receptor antagonism produces reliable withdrawal symptoms in rats. Behavioural network analyses further reveal that cannabis exposure and withdrawal are both associated with widespread reorganization of behavioural dynamics, suggesting that withdrawal alters not only individual behaviours but also the structure of behavioural transitions. These findings establish a translational model of cannabis withdrawal using inhaled cannabis flower vapour and identify behavioural network analysis as a sensitive approach for characterizing withdrawal-related behavioural states.

The addictive properties of opioids are due in part to these drugs ability to alter ventral tegmental area (VTA) activity via activation of mu opioid receptors (MORs) on local and distal inputs. Prior studies have identified numerous opioid-modulated afferents to the VTA, some of which show differing levels of functional modulation by opioids, but the degree to which this parallels differences in receptor expression is not known. Hence, we used retrograde labeling combined with RNAscope to examine oprm1 mRNA expression in VTA-projecting afferents arising from a variety of distal brain regions. Because opioids are thought to be particularly influential on GABAergic afferents to the VTA, we also examined colocalization of oprm1 with GABAergic markers in VTA-projecting neurons. Interestingly, we found that oprm1 mRNA is present in both GABAergic and non-GABAergic VTA-projecting neurons. However, many (though not all) GABAergic afferents expressed higher levels of oprm1 compared to most non-GABAergic afferents (especially those arising from the cortex). These results complement previous anatomical studies that had examined oprm1 expression in these regions but in a non-quantitative way and without regard to their efferent targets. Our findings encourage future work to examine the functional implications of MOR sensitivity within these afferent pathways.

Alcohol use disorder (AUD) is a significant public health concern. In Canada, about 18% of individuals aged 15 or older will meet the clinical criteria for AUD at some point in their lives (CAMH, 2023). Treatment options for AUD are limited, and the high relapse rates highlight the urgent need for innovative methods to study and address AUD. Zebrafish (Danio rerio) is an emerging model for exploring the neurobiological impacts of alcohol. Previous studies have demonstrated that zebrafish respond to the rewarding effects of alcohol, but most research methods rely on passive administration, such as immersion, which does not reflect the typical routes of alcohol intake in humans. We previously showed that zebrafish can learn to self-administer drugs of abuse in small groups and conditioned animals are displaying key features of substance abuse disorders. However, group-based conditioning limits our understanding of individual drug preference and intake profile. In this study, we improved upon our previous design by establishing an individual self-administration protocol to measure voluntary alcohol intake and model alcohol use disorder. In this novel assay, individual adult fish learn to discriminate between two zones to self-administer a 5% ethanol solution. Moreover, animals conditioned in this assay can perform progressive ratio and display signs of withdrawal upon cessation of ethanol intake. These results suggest zebrafish can develop ethanol abuse-like behaviour, providing a powerful platform to study genetic predisposition and screen for therapeutic compounds.

The endogenous peptide dynorphin (Dyn) and its target the kappa opioid receptor (KOR) play a crucial role in regulating factors related to stress and reward. The KOR is expressed in multiple cell types in the nucleus accumbens (NAc), including presynaptic dopamine (DA) terminals, where it inhibits DA release modulates the function of the DA transporter (DAT). The Dyn/KOR system is upregulated by exposure to drugs of abuse including the DAT inhibitor, cocaine, and their activity is integrally involved in negative affective states associated with withdrawal from substance abuse. We aimed to better understand the impact of the Dyn/KOR system on presynaptic DA terminals and potential effects on DAT interactions with cocaine by measuring the impact of the KOR agonist U50,488 on electrically-evoked DA release and subsequent reuptake in NAc slices from C57BL6/J mice. We showed that superfusion of U50,488 inhibited DA release and markedly reduced cocaine-induced inhibition of DA reuptake, indicating tolerance to cocaine effects. We replicated this finding in the NAc of rhesus macaques using the DAT/NET inhibitor nomifensine, demonstrating that these mechanisms are conserved across DAT inhibitors and in non-human primates. KOR activation results in phosphorylation of the Threonine-53 site on the DAT, a process thought to mediate its impact on DAT function. We tested whether this phosphorylation site is required for the KOR-mediated reduction cocaine effects. To tackle this question, we employed a knock-in mouse line with an Alanine-53 on the DAT (DAT-T53A), rendering that residue insensitive to phosphorylation. We show that DAT-T53A mice have enhanced DA release and uptake, and U50,488 has a reduced inhibitory effect on peak DA release. Remarkably, U50,488 no longer modified the effect of cocaine on uptake in these mice, demonstrating the dependence of this effect on phosphorylated Threonine-53 and highlighting a potential mechanism underlying cocaine tolerance.

BACKGROUNDThe proliferation of the potent synthetic opioid fentanyl has exacerbated the ongoing crisis of substance use disorder and associated overdose deaths, yet the neurobiological mechanisms that underlie individual vulnerability to addiction and relapse remain poorly understood, particularly in the context of fentanyl use. The prefrontal cortex (PFC) has been identified as a key brain structure important for cognitive functions impacted in addiction, including inhibitory control of behavior and association of drug experience with specific cues, contexts, or actions. Although the heterogenous neuronal composition of the PFC complicates attribution of addiction-related behavioral regulation to specific cortical cell types and circuits, application of cell-type-specific methods in translationally relevant rodent models have begun to elucidate the key neural substrates of opioid addiction. METHODSWe used an intermittent access fentanyl self-administration (IntA SA) model to characterize individual variation and sex differences in addiction vulnerability in male and female rats. Longitudinal wireless fiber photometry recording was used to track calcium activity patterns in intratelencephalic (IT) neurons of the prelimbic cortex across acquisition of self-administration, escalation of fentanyl intake, extinction training, and cue-induced reinstatement of fentanyl seeking. RESULTSWe found that our fentanyl IntA SA paradigm produces distinct low- and high-risk addiction severity phenotypes and that female rats exhibited a greater propensity for high-risk classification, which was characterized by abundant and consistent fentanyl intake, robust responsiveness to conditioned and discriminative fentanyl-associated cues, and high levels of fentanyl-seeking during periods of drug unavailability, extinction training, and a cue-induced reinstatement test. Fiber photometry recordings revealed dynamic encoding of fentanyl-associated stimuli by prelimbic IT neurons across the IntA SA paradigm with event-related calcium transients observed in association with lever presses, fentanyl infusions, and presentation of conditioned and discriminative cues. CONCLUSIONSOur data indicate that fentanyl IntA SA is a translationally relevant paradigm that enables investigation of phenotypic diversity and the role of sex in fentanyl addiction. Longitudinal cell-type-selective calcium recordings revealed dynamic representation of fentanyl-associated stimuli by IT neurons of the prelimbic cortex consistent with a role for this cortical subpopulation in addiction-related behaviors.

BackgroundNeuropathic pain is a major source of disability and distress with few pharmacological options for treatment. Opioid drugs can be effective, but high doses are needed, leading to unwanted effects. BMS-986122 is a positive allosteric modulator of the mu opioid receptor that potentiates acute opioid antinociception without increasing opioid-induced constipation, reward, or respiratory depression. Therefore, we asked if BMS-986122 could increase the effects of low-dose opioid analgesics in chronic neuropathic pain. MethodsWe employed the spared nerve injury and tibial neuroma models in rats and assessed the tactile hypersensitivity of the hind paw and site of neuroma, respectively. ResultsAdministration of low doses of (R)-methadone, morphine, or buprenorphine slightly reduced the tactile hypersensitivity of the hind paw the in spared nerve injury model. Pretreatment with BMS-986122 significantly enhanced the reversal of hypersensitivity, reaching the effect of high-dose gabapentin, a standard of care in neuropathic pain. Pretreatment with BMS-986122 similarly increased the anti-allodynic effects of low dose (R)-methadone on neuroma pain. A similar effect of (R)-methadone in the absence of BMS-986122 was only observed at a dose where respiratory distress was seen. ConclusionsThese findings show that allosteric modulators of the mu opioid receptor such as BMS-986122 can enhance opioid activity that could translate to a safe and effective treatment for chronic neuropathic pain.

BackgroundSex differences in psychostimulant-related behaviors are often attributed to biological sex; however, individual variability may also strongly influence behavioral outcomes. The new MISSING (Mapping Intrinsic Sex Similarities as an Integral quality of Normalized Groups) model identifies mixed-sex behavioral groups in which differences are driven primarily by individual variability rather than sex. The goal of this study was to validate the MISSING model for psychostimulant/sucrose self-administration. MethodsLong Evans rats self-administered methamphetamine (METH, male n = 25, female n = 32, 0.1 mg/kg/infusion, FR1, 6h per day for 20 days), sucrose (male n = 20, female n = 22, one-20 mg pellet/delivery, all other conditions being equal) and saline (male n = 3, female n = 10, other things being equal). We developed a new Quantitative Structure of Curve Analytical (QSCAn) model (using exponential-plateau and linear fit) for the assessment of individual drug self-administration time curve profiles irrespective of biological sex. We analyzed our data using regression analysis and ANOVA. ResultsQSCAn identified three distinct self-administration profiles (consisting of both sexes), which we named exponential-plateau negative (EP-), exponential-plateau positive (EP+), and undefined (EP0). There were no differences in self-administration profiles when we compared males and females within the same group. Differences between sexes (when observed) were due to mismatched comparisons (males from one group versus females from a different group). ConclusionsOur study reinforces the MISSING model for psychostimulant and sucrose self-administration by indicating that differences between males and females (when observed) may not necessarily be driven by biological sex. Significance StatementCurrent approaches often interpret variability in psychostimulant self-administration between males and females primarily through the lens of biological sex. However, this framework may overlook meaningful behavioral phenotypes shared across sexes. The present quantitative model suggests that individual patterns of behavior may better account for variability than sex alone, particularly in behaviors not strongly driven by sex-hormone-dependent mechanisms such as drug self-administration. By classifying animals according to behavioral profiles rather than biological sex, this approach may foster the identification of clinically and biologically relevant phenotypes underlying psychostimulant reinforcement.

RationaleAdolescence is a period of heightened vulnerability to nicotine reinforcement. While zebrafish are a valuable model for investigating drug reward, standard conditioned place preference (CPP) assays typically test subjects in isolation. In this highly social species, solitary testing may act as an environmental stressor that confounds behavioral readouts. ObjectivesThis study examined how social context during testing (isolated vs. grouped) affects experimental attrition, behavioral stability, and nicotine CPP expression in late juvenile zebrafish. MethodsZebrafish housed in groups of four were tested either individually (isolated) or in their housing groups (grouped) during daily 20-minute sessions. Following baseline preference assessments, subjects underwent six days of conditioning pairing their initially non-preferred compartment with fish water or nicotine (0.5, 1.6, or 5.0 {micro}mol/L). Place preference, locomotion, and thigmotaxis were assessed on a drug-free test day. ResultsIsolated testing reduced distance traveled, decreased swimming speed, and increased time spent near tank walls, indicating heightened anxiety-like behavior. Experimental attrition was significantly higher in isolated (38.9%) than grouped (2.5%) subjects. Grouped subjects developed significant place preference at 1.6 and 5.0 {micro} mol/L nicotine, whereas preference was not detectable in isolated subjects. ConclusionsSolitary testing acts as a stressor that increases experimental attrition and masks place preference. Conversely, testing in the presence of conspecifics stabilizes behavior and facilitates the detection of nicotine reward in late juvenile zebrafish.

BackgroundCocaine produces cardiovascular toxicity through intense sympathetic stimulation and direct myocardial injury, generating presentations ranging from hypertension and tachycardia to coronary vasospasm, arrhythmia, and myocardial depression. {beta}-blockers are foundational therapies in acute coronary syndromes, yet their use after cocaine exposure remains controversial due to concerns about unopposed -adrenergic stimulation. MethodsA systematic review was conducted in accordance with PRISMA guidelines. PubMed and Google Scholar (2000-2026) were searched for observational studies of adults ([&ge;]18 years) presenting to acute care with recent cocaine use that compared outcomes between {beta}-blocker recipients and non-recipients. Eligible studies reported in-hospital mortality, myocardial infarction/troponin rise, clinically significant arrhythmia, or haemodynamic instability. Risk of bias was assessed using the Newcastle-Ottawa Scale, and certainty of evidence using GRADE. ResultsFour retrospective ED cohorts (n = 1,140) met inclusion criteria; 503 patients received at least one {beta}-blocker dose. Across studies, {beta}-blocker use was not associated with increased in-hospital mortality or malignant arrhythmias. Myocardial infarction was heterogeneous and sensitive to definition and timing. Haemodynamic data showed no hypertensive surge and modest systolic blood pressure reductions. Risk of bias was moderate, and certainty of evidence very low to low. ConclusionsIn typical ED presentations of recent cocaine use, {beta}-blocker administration does not appear to increase mortality, myocardial infarction, or malignant arrhythmias, and available haemodynamic data do not support a reproducible unopposed- response. However, mechanistic and preclinical evidence suggests potential harm in severe intoxication or myocardial depression. A selective, phenotype-guided approach is warranted, and prospective mechanistic studies are needed. Key PointsO_LIBeta-blockers did not increase deaths, heart attacks, or dangerous heart rhythms in adults who came to the emergency department after recent cocaine use. C_LIO_LIBlood pressure generally decreased after beta-blocker treatment, and no consistent "unopposed alpha" reaction was seen in typical presentations. C_LIO_LICaution is still needed in severe intoxication or cases with heart muscle weakness, but for most emergency presentations, beta-blockers appear safe when used appropriately. C_LI

It is becoming increasingly clear that chronic exposure to lower levels of ethanol impact learning and behavior. To determine the impact of chronic low-dose ethanol exposure on sensitivity to changes in stimulus value, a conditioned taste aversion procedure was used. Adult male and female mice underwent a sucrose two bottle-choice drinking paradigm. Each day, mice received an injection of either low-dose ethanol (0.5g/kg) or saline two hours after sucrose access for 20 days. This was followed by a lithium chloride (LiCl)-induced conditioned taste aversion (CTA) paradigm in which 0.15M LiCl or vehicle injection was administered immediately after sucrose consumption for three days. On the fourth day, changes in sucrose consumption were analyzed. Chronic exposure to low-dose ethanol did not affect sucrose consumption in either female of male mice during two-bottle choice. In female mice, a history of chronic low-dose ethanol exposure blocked the development of LiCl-induced CTA. A history of chronic low-dose ethanol did not impact LiCl-induced CTA in male mice as both ethanol-naive and -exposed male mice who underwent LiCl pairing reduced sucrose consumption. This suggests that low-dose ethanol alters aversion-related learning in female mice which may have implication for development of aberrant behavior and risk for alcohol use disorder (AUD).

Background and PurposeWhile inflammation has been generally considered to exacerbate symptoms of schizophrenia, some clinical observations suggest that acute inflammation may alleviate positive symptoms. However, animal models often use excessive inflammatory stimuli, and the effects of acute inflammation--comparable to levels observed in patients--remain unknown. Experimental ApproachTo address this, we examined whether acute inflammation induced under relatively mild, clinically relevant conditions suppresses behavioural sensitization in methamphetamine (METH)-sensitized mice, a model of psychostimulant-induced psychosis with relevance to certain aspects of positive symptoms of schizophrenia. We used a repeated METH (1 mg/kg) sensitized model to evaluate the effects of acute inflammation on behavioural sensitization. Acute inflammation was induced via two methods using either lipopolysaccharides (LPS; 1 g/kg) to mimic peripheral immune activation or restraint stress (RS; single 2-h exposure) to model the neuroinflammation induced by psychological stress. LPS doses were adjusted with reference to the magnitude of peripheral cytokine elevation reported in patients, and RS was applied in short single sessions to avoid excessive inflammation. Key ResultsBoth LPS and RS significantly suppressed behavioural sensitization, without inducing other behavioural abnormalities. This suppression was dependent on toll-like receptor-4 activation. LPS-mediated suppression involved cyclooxygenase-2, whereas RS-mediated suppression was linked to the microglia-derived tumour necrosis factor-. LPS did not alter, whereas RS significantly reduced the striatal extracellular dopamine levels. Conclusion and ImplicationsThese findings suggest that acute inflammation suppresses behavioural sensitization through distinct mechanisms depending on the inflammatory trigger, providing a framework for understanding how inflammation may influence psychosis-related processes, with potential relevance to schizophrenia.

Background/ObjectivesPersistent cognitive impairments are prevalent in methamphetamine (meth) use disorder and contribute to maladaptive decision-making and increased relapse vulnerability. There are currently no effective treatments for meth-associative cognitive deficits, and their neurobiological underpinnings remain incompletely understood. This study investigated the effects of chronic meth self-administration on episodic-like recognition memory and evaluated whether pharmacological potentiation of metabotropic glutamate receptor subtype 2 (mGlu2) could rescue these deficits. MethodsAdult male Sprague-Dawley rats underwent 7 days of limited- (1h/day) followed by 14 days of extended-access (6h/day) meth self-administration, followed by 30 days of abstinence. Recognition memory was assessed using the object-in-place (OIP) task. A positive allosteric modulator of mGlu2 receptors, LY-487379 (25 mg/kg, s.c.), was administered prior to the memory test. In parallel, changes in total and surface mGlu2/3 protein levels in the prelimbic and perirhinal cortices were evaluated. ResultsRats with extended access to meth self-administration exhibited escalated drug intake and persistent deficits in OIP memory. Administration of LY-487379 reversed this deficit. Total mGlu2/3 protein levels were unaltered; however, meth exposure was associated with a significant increase in surface mGlu2/3 receptor expression in both cortical regions examined. ConclusionsThese results demonstrate that chronic meth produces persistent cognitive dysfunction that can be rescued by mGlu2 receptor potentiation. The observed increase in surface mGlu2/3 expression may represent a compensatory response to chronic glutamatergic dysregulation, but it appears to be insufficient to restore cognitive function alone, without pharmacological enhancement. The current data encourage further exploration of mGlu2 role in stimulant-associated cognitive dysfunction. HighlightsChronic methamphetamine self-administration produced persistent deficits in episodic-like recognition memory in male rats and dysregulation of mGlu2/3 receptors in the prelimbic and perirhinal cortices. Systemic pharmacological potentiation of mGlu2 receptors rescued meth-associated memory deficits. mGlu2 receptor potentiation may represent a promising therapeutic strategy for treating stimulant-associated cognitive dysfunction. Increased surface mGlu2/3 expression may represent a compensatory adaptation to post-methamphetamine glutamatergic dysfunction, but it is not sufficient to restore cognition alone, without pharmacological enhancement.