Pharmacology Biochemistry and Behavior

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.

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

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.

RationaleAppetitive Pavlovian cues can drive maladaptive reward seeking via stimulus-reward memories. Disrupting memory reconsolidation offers a potential strategy to reduce their influence, but evidence for {beta}-adrenergic blockade with propranolol is inconsistent across behavioural paradigms, particularly relative to NMDA receptor antagonism. ObjectivesWe tested whether propranolol disrupts reconsolidation of appetitive sucrose memories in a discriminative goal-tracking paradigm, and compared its effects with those of the most commonly used NMDA receptor antagonist, MK-801. MethodsAdult Lister hooded rats underwent discriminative Pavlovian conditioning. Thirty minutes before a brief memory reminder (non-reinforced or reinforced), rats received systemic drug treatment or saline control. In study 1, MK-801 (0.1 mg/kg) was administered to male rats. In study 2, propranolol (10 mg/kg) was administered to equal numbers of male and female rats. Goal-tracking was tested drug-free at 1 and 8 days. ResultsIn study 1, MK-801 impaired subsequent discriminated responding at test. These effects were observed not only when reminder was non-reinforced as in previous successful demonstrations, but also with reinforced reminder. In study 2, Propranolol also impaired subsequent goal-tracking, regardless of reminder type, and the effects were consistent across sexes. ConclusionsPropranolol can disrupt reconsolidation of appetitive goal-tracking memories to a similar extent as MK-801 under conditions that promote memory destabilisation. These findings demonstrate that {beta}-adrenergic blockade can impair appetitive memory reconsolidation in a goal-tracking paradigm, challenging prior null findings and revitalising the potential for propranolol-based interventions in maladaptive reward-seeking behaviours.

Background: Compulsive alcohol use despite negative outcomes is a defining characteristic of alcohol use disorder. Rats exposed to long-term intermittent alcohol access (IAA) demonstrate sustained motivation for ethanol despite presence of the bitter additive quinine, offering a useful preclinical model of compulsive alcohol use. However, little is known about the role of habenular circuitry in the development of this phenotype. Here, we employed chemogenetic techniques targeting basal forebrain (BF) input to the lateral habenula (LHb) to probe the involvement of this neural circuitry in aversion-resistant alcohol consumption. Methods: Following long-term IAA or control conditions, male and female Long-Evans rats underwent surgery for the expression of designer receptors in BF-to-LHb projections. We then excited this pathway in rats with IAA history, or inhibited this pathway in rats with more limited ethanol history, before testing consumption of unadulterated and quinine-adulterated ethanol as well as unadulterated and quinine-adulterated sucrose. Results: Long-term IAA elevated ethanol drinking in all rats and aversion-resistant ethanol preference in males. Chemogenetic activation of BF-to-LHb neurons in rats with IAA history produced different effects in males and females: excitation enhanced ethanol intake in females, but reduced ethanol preference in males, regardless of quinine adulteration. Activation also led to a relative insensitivity to quinine-adulteration of sucrose when compared to controls, particularly in females. Chemogenetic inhibition in rats with limited prior ethanol exposure did not alter either ethanol or sucrose consumption with or without quinine. Conclusions: Our results suggest a differential role for BF-to-LHb circuitry in ethanol drinking based on sex, and a potential role for this circuitry in the sensitivity to quinine in the context of natural reward consumption.

Psilocybin shows therapeutic promise for several psychiatric disorders, but the acute perceptual and cognitive alterations produced by conventional doses (10-25 mg) require in-clinic supervision, which limits scalability. Whether the therapeutically relevant pharmacology of psilocybin can be separated from its hallucinogenic activity remains unresolved. To address this gap, we conducted a Phase 1, randomized, double-blind, placebo-controlled, single ascending dose study to characterize the safety, pharmacokinetics and pharmacodynamics of low doses of psilocybin. Fifty-six healthy adults received a single oral dose of psilocybin (0.5, 1.0, 1.5, 2.5, 3.5 or 4.0 mg) or matching placebo across seven sequential cohorts, with each dose escalation reviewed by a Drug Safety Review Committee. All participants completed the study with no serious adverse events or discontinuations. Treatment-emergent adverse events were comparable to placebo and most prominently arose as somnolence. Plasma psilocin appeared rapidly with a median time to maximum concentration < 1 h with dose-proportional exposure and a short terminal half-life. Subjective drug effects were dose-related and became distinguishable from placebo at doses at or below 2.5 mg. Peak subjective ratings increased with dose, while any signs of hallucinations or altered-states scores remained low and not different than placebo. Psychophysiological engagement was confirmed by a clear dose-dependent pupillary dilation while cognitive performance (attention, vigilance, working memory, impulse control) showed no dose-dependent decrement and state anxiety did not increase at any dose. These findings indicate that the perceptible pharmacology of psilocybin can be dissociated from significant perceptual alterations and cognitive impairment at low doses. They further support controlled investigations in outpatient Phase 2 studies evaluating the safety and feasibility of repeated, self-administered low-dose psilocybin. ClinicalTrials.gov #NCT07710027

Serotonin (5-HT) 2A receptors (5-HT2AR) modulate corticolimbic circuits regulating fear extinction. Although activation of these receptors has been shown to facilitate fear extinction, the behavioral consequences of 5-HT2AR antagonism during extinction is not well defined. Here, we examined the systemic effects of two 5-HT2A receptor antagonists, the mixed 5-HT2A/2C antagonist MDL 11,939 (Glemanserin) and the selective 5-HT2A antagonist MDL 100,907 (Volinanserin) on fear extinction in adult C57BL/6J mice. Prior to drug administration, mice assigned to future treatment groups acquired comparable conditioned freezing responses during delay fear conditioning. Twenty-four hours later, acute administration of MDL 11,939 (1.0 mg/kg) or MDL 100,907 (0.01 mg/kg) increased freezing to the first conditioned stimulus (CS) presentation on Extinction Day 1, indicating enhanced expression of conditioned fear. However, acquisition of fear extinction differed between the respective cohorts of mice treated with the two 5-HT2AR antagonists. Repeated administration of MDL 11,939 significantly impaired extinction, as evidenced by increased freezing across extinction trials and an increased number of trials required to reach extinction criterion. In contrast, MDL 100,907 has reported affinity for did not significantly alter extinction under either acute or repeated dosing conditions. Because MDL 11,939 has reported affinity for 5-HT2C receptors, we tested potential contributions of 5-HT2C receptor antagonism in a separate cohort of mice using two doses of the selective 5-HT2C antagonist, SB 242084. Neither dose affected conditioned fear expression, extinction learning, or trials required to reach extinction criterion. Together, these findings demonstrate ligand-specific and dose-dependent effects of 5-HT2AR antagonism on fear extinction and suggest that distinct intracellular receptor signaling pathways may differentially regulate extinction-related behavior.

20% of Americans are at risk for environmental circadian rhythm disruptions (CRD) due to shift work, leading to substantial negative health outcomes. However, females are especially affected with greater vulnerability for substance use (SU) and adverse outcomes associated with pregnancy, including for offspring at birth and later in life. In mice, prenatal CRD (pCRD) recapitulates these risks, but it is unknown whether pCRD affects SU in mature offspring. To investigate this, C57BL/6J dams were disrupted by reversing the light/dark cycle during gestation. Following pCRD, reward- (cocaine conditioned place preference, intravenous self-administration) and mood-related behaviors (open field, elevated plus maze, light/dark box, forced swim) were measured in adult offspring. Adult female offspring of dams exposed to CRD developed an anhedonic-like phenotype with decreased food self-administration, cocaine intake and reinforcing properties of cocaine. Opposingly, pCRD male offspring showed a SU-like phenotype with increased cocaine preference, higher order food self-administration and cocaine reinforcement. Interestingly, these divergent behavioral outcomes were not specific to reward. While female pCRD mice showed increased anxiety-like behavior, pCRD males showed decreased anxiety/increased risk-taking behavior, as well as decreased immobility in the forced swim test. Rhythms in corticosterone were also sex-specifically affected by pCRD. These results suggest that pCRD may predispose individuals to distinct psychiatric disorders based on sex with mood disorders developing in females and SU disorders developing in males. By better understanding how disrupted rhythms during pregnancy affect behavior in adulthood, we can develop novel therapeutic approaches for SU and mood disorders in adults.

Synthetic opioids like fentanyl are a leading cause of overdose mortality. Although the hallmark of fentanyl overdose is ventilatory depression, fentanyl also induces tonic activation of skeletal musculature, including the diaphragm, which may advance progression of overdose towards death. While tonicity may further restrict diaphragmatic contractility, phase-specific dysregulation may also reflect a larger state of discoordination in respiratory control. Using urethane-anesthetized mice exposed to fentanyl, we test the hypothesis that fentanyl-induced diaphragm tonicity results from a loss of coordinated motor activity. Fentanyl produced two distinct phases: an initial phase of maximal ventilatory depression with preserved phasic activity, and a later phase characterized by unstable ventilation that partially rebounds, tonic diaphragmatic activation with loss of inspiratory phase dominance in EMG activity, and diminished bilateral diaphragmatic coordination. Carotid body denervation eliminated tonic activity and expiratory-phase EMG elevation, but it did not prevent hemi-diaphragm discoordination or ventilatory instability. Rhythmic brainstem slice recordings showed that bilateral preBotzinger complex burst-amplitude coupling was disrupted by u-opioid receptor (MOR) agonism. Furthermore, disordered diaphragm activity was reversed by administration of the MOR antagonist, Naloxone. Our findings reframe fentanyl overdose as a temporally evolving syndrome that involves distinct mechanisms to disrupt respiratory motor coordination.

Alcohol demand is commonly assessed using hypothetical alcohol purchase tasks (APTs), from which individual demand curves are constructed and yield multiple indices of reinforcing value. Procedurally, APTs can confer participant burden, and existing brief alternatives cannot produce demand curves or derived indices. Thus, we evaluated a novel, adjusting APT that efficiently and idiographically assesses alcohol demand while preserving the benefits of a full task. Adults reporting past-six-month alcohol use (n=897) completed either the adjusting or full APT, the former utilizing a binary-search-style algorithm to administer six prices from the full APT's price set based on level of alcohol demand. The adjusting APT reduced item burden by 49% and produced well-fitting individual demand curves. Average demand intensity and elasticity estimates did not differ significantly by modality, whereas Omax and breakpoint estimates were significantly higher on the adjusting APT, though only by $3 each. All demand indices from both APTs were positively associated with alcohol use and problems, with similar magnitude by modality. Results provide support for the adjusting APT as a brief measure of alcohol demand that retains demand-curve-based indices of reinforcing value.

Neonatal hypoxia-ischemia (HI) injury is a major risk factor for lifelong cognitive impairments. Given its systemic impact, the neural mechanisms of impairments associated with HI injury remain unclear. In this study, we used a mouse model of neonatal HI injury to study its impact on goal-directed behavior and neural activity in adulthood using a head-fixed visual discrimination task. While neonatal HI injury did not impair discriminability or learning, it was associated with increased motor output in form of licking, faster reaction times and liberal decision bias, indicating an impulsive-like phenotype. These behavioral changes were accompanied by suppressed neuronal activity in the primary visual cortex (V1) and elevated cue-driven fluctuations in trial-to-trial firing variability in the prefrontal cortex (PFC), the latter of which was predictive of decision bias in HI mice. Our findings identify the long term impact of neonatal HI injury on goal-directed behavior, describe in detail the task-related patterns of neural activity in HI mice, and implicate abnormal neural variability in the PFC as a driver of impulsive-like behavior in adults that suffered neonatal HI injury.

PVT is involved in stress responses, fear, anxiety, arousal, aversion and reward. Anterior and posterior PVT (aPVT and pPVT) have different neuronal connections, molecular contents, and functional roles. PVT expressed a high level of KOR. Herein we mapped the projection targets of aPVT KOR(+) neurons and explored the behavioral significance of aPVT KOR. Using KOR-iCre mice and Cre-dependent anterograde tracer, we found that KOR(+) glutamatergic neurons in aPVT primarily projected to NAc, CeA, BNST, PFC, and reticular nucleus of the thalamus (RT). 3-D images showed the pathway emanating from aPVT to the ventral RT, through NAc, and out to the other regions, indicating widespread axonal collateralization. We conditionally knock-downed KOR (KOR cKD) in aPVT by injection of AAV-eGFP-Cre or AAV-eGFP (control) into aPVT of Oprk1lox/lox mice. [3H]U69,593 receptor autoradiography revealed substantial KOR cKD in aPVT. In both male and female mice, the KOR cKD in aPVT significantly reduced anxiety-like behaviors in the elevated plus-maze test, cue-induced freezing after fear conditioning and naloxone-precipitated morphine withdrawal-associated jumps. KOR cKD attenuated U50,488H-induced conditioned place aversion in males only, while having no effect on forced swim immobility or the U50,488H-induced visceral analgesic and antipruritic effects in either sex. Thus, our results reveal for the first time that KOR-mediated inhibition of aPVT neurons may mediate morphine withdrawal, anxiety, and cue-induced fear in both sexes but contribute to KOR agonist-induced aversion only in males. Notably, our findings reveal a previously unrecognized role for aPVT in regulating morphine withdrawal, acting in a manner distinct from pPVT.

Early-life adversity durably alters neural development through complex mother-offspring interactions whose underlying mechanisms remain poorly understood. We investigated how neonatal maternal separation (NMS) affects the large repertoire of maternal behaviors and subsequently influences spinal nociceptive circuit development and pain responses in rat offspring. Rat dams underwent NMS from postnatal day 2 (P2) to P12, 3h/day, and maternal behaviors were assessed before and after the separation period. These behaviors were compared to those of control (non-separated) dams. Offspring spinal cord and dorsal root ganglia were analyzed at P14 and P24 for several neurotrophic, glutamatergic, and GABAergic gene expression patterns. Offspring nociceptive sensitivity was also assessed at P24. NMS induced increased maternal behaviors (including longer arched-back nursing, higher nest occupancy, and better pup retrieval efficiency), alongside reduced self-care behaviors. These behavioral adaptations were correlated with spinal gene reprogramming in offspring, characterized by a biphasic developmental pattern. At P14, we observed elevated neurotrophic signaling alongside increased GABAergic and glutamatergic markers. By P24, neurotrophic factors decreased while compensatory changes emerged, yet persistent excitatory-inhibitory imbalances remained evident. Parallel to these results, NMS rats also showed mechanical and thermal hot hypersensitivity at P24. These findings reveal that despite apparent maternal behavioral compensation following NMS, offspring exhibit neurotrophic-driven developmental dysregulation resulting in persistent spinal circuit alterations. The disconnect between maternal behavioral normalization and sustained molecular changes suggests that early separation stress triggers enduring neurobiological cascades independent of ongoing maternal care quantity, with long-term consequences for sensory processing and pain sensitivity.

Treatment-resistant depression and anxiety remain major challenges in psychiatry, particularly in female patients, who are disproportionately affected yet remain underrepresented in preclinical ketamine research. The present study investigated short- and long-term anxiolytic effects of acute subanesthetic ketamine administration in female Wistar-Kyoto (WKY) rats, a validated genetic model of treatment-resistant affective dysfunction. Subjects received a single intraperitoneal injection of saline vehicle or racemic ketamine (5, 10, or 15 mg/kg), followed by acoustic startle response (ASR) testing 24 hours and 7 days later. Oxidative stress was assessed using 8-oxo-2'-deoxyguanosine (8-oxo-dG) immunofluorescence in the basolateral amygdala (BLA), prefrontal cortex (PFC), and hippocampus, alongside analysis of parvalbumin-positive (PV+) interneurons. Ketamine treatment produced dose- and time-dependent behavioral effects with 10 mg/kg eliciting the strongest delayed anxiolytic-like response at 7 days, while 15 mg/kg showed more immediate behavioral effects at 24 hours. While ketamine did not alter PV+ cell count, it significantly increased oxidative stress markers globally in the BLA and prelimbic region of the PFC and specifically in the PV+ interneurons in the BLA. The findings suggest that ketamine's therapeutic effects in female WKY rats may involve region-specific modulation of stress circuitry and oxidative signaling rather than gross interneuron loss. Overall, the study provides evidence for sex-dependent and temporally dynamic effects of ketamine in a translational model of treatment-resistant anxiety and depression.

BackgroundSerotonergic psychedelics are advancing as psychiatric treatments, yet acute responses vary between individuals and the contribution of sex, a fundamental biological variable, remains largely unexamined. MethodsWe pooled two double-blind, placebo-controlled studies in healthy volunteers (N = 72; 31 male, 41 female) comparing psilocybin 15 mg, 2C-B 20 mg, and LSD 50 {micro}g. Linear mixed models tested sex differences in acute subjective effects (visual analogue scales), retrospective altered-states ratings (5D- and 11-ASC), empathy (Multifaceted Empathy Test), and peak plasma blood concentrations (Cmax, AUC), with treatment, sex, their interaction, as fixed factors, and age as a covariate. ResultsFemale participants reported numerically higher subjective ratings than male participants on most measures. After adjustment for age, sex differences remained significant for feeling under the drugs influence, reduced vigilance, and impaired control and cognition, with medium-to-large effects. These effects were largely consistent across the three drugs. No sex differences emerged on any empathy measure or in peak drug concentrations. ConclusionsFemale participants may experience more intense acute subjective effects and greater perceived impairment under psychedelics, independent of age and not explained by drug exposure. These preliminary findings, implicating pharmacodynamic rather than pharmacokinetic mechanisms, have implications for dosing, informed consent, and safety monitoring, and underscore the need to treat sex as a biological variable in adequately powered psychedelic trials.

Psychoactive and psychotoxic drugs are particularly harmful, if their use coincides with critical developmental windows of brain maturation. Methamphetamine is one such stimulant with developmental exposure increasing seizure susceptibility and long-term neuronal maladaptation in children. Nevertheless, the extent at which infant and adult vulnerability to methamphetamine could differ in time-course and severity remains incompletely understood. Here, we developed a method to monitor methamphetamine-induced hyperactivity in infant mice at high temporal resolution, differentiate it from a biphasic response in adults, and link it to activity changes in cortical areas executing goal-directed (escape) behaviors in infant subjects when using Fos expression as a molecular surrogate. Subsequently, we hypothesized that methamphetamine could alter the expression and cellular distribution of inhibitory neuropeptides, which, when co-released with fast neurotransmitters, could protect circuit plasticity by counteracting methamphetamine-induced hyperexcitability. Methamphetamine differentially altered somatostatin, cholecystokinin, and galanin expression in corticolimbic areas. These data suggest that methamphetamine can evoke age-specific neurocircuit modifications, at least in mice.

Bisphenol S (BPS), a widely used substitute for bisphenol A, is increasingly detected in aquatic environments; however, its neurodevelopmental effects remain insufficiently understood. This study investigated whether developmental exposure to an environmentally relevant concentration of BPS disrupts social behaviour and underlying neurobiological pathways in zebrafish (Danio rerio). At 21 days post-fertilization, BPS-exposed larvae exhibited a significant reduction in social preference, indicating impaired conspecific interactions. Neurochemical analysis revealed a marked increase in serotonin (5-HT) levels, whereas lipid peroxidation (MDA) remained unchanged, suggesting the absence of overt oxidative damage. Gene expression profiling demonstrated a dysregulated antioxidant response, suppression of apoptotic signaling, and pronounced upregulation of serotonergic receptors and transporters. To resolve system-level mechanisms, protein-protein interaction (PPI) network analysis identified BDNF and CREB1 as dominant regulatory hubs, with the serotonergic synapse pathway as the most significantly enriched term. Molecular docking further demonstrated direct binding of BPS to multiple serotonergic targets, including HTR1A and TPH2, supporting receptor-level interference. Expanded network and pathway analyses revealed coordinated enrichment of monoamine GPCR, oxidative stress, and inflammatory pathways. These findings demonstrate that BPS induces serotonergic dysregulation and network-level reprogramming rather than significant oxidative damage, leading to behavioural impairment. This study provides a multi-scale mechanistic framework linking molecular perturbations to neurobehavioural outcomes, identifying serotonergic signaling and BDNF-CREB1 pathways as central targets of BPS neurotoxicity.

It is well established that activating the mGluR2 metabotropic glutamate receptor (mGluR2), which is the main presynaptic autoreceptor for glutamate in the brain, attenuates the behavioral and electrophysiological actions of LSD and other psychedelics. However, the mechanisms responsible for these actions are controversial. The two competing mechanistic hypotheses have been proposed to explain this phenomenon are: (1) direct actions mediated by mGluR2/5-HT2A heterodimers, and (2) inhibition of 5-HT2A-mediated excitation of pyramidal neurons via presynaptic inhibition of glutamate release by mGluR2 receptors. Consistent with prior reports, we show mGluR2 agonist pretreatment attenuates the head twitch response induced by the psychedelic drug 1-(2,5-Dimethoxy-4-iodophenyl)-2-aminopropane (DOI) in these mice. We next employed multiple orthogonal in vivo and in vitro approaches to explore the potential for direct physical interactions between mGluR2 and 5-HT2A receptors. We next engineered mice to express mGluR2-mCherry-CT and 5-HT2A-eGFP-CT tagged receptors and found no evidence for receptor colocalization or oligomerization under basal or 5-HT2A agonist-exposed conditions in vitro or in vivo. Radioligand binding and kinetic analyses revealed no evidence for mGluR2-mediated modulation of 5-HT2A ligand binding in vitro or in vivo. Collectively, our findings support models in which mGluR2 signaling modulates the activity of Gq-coupled 5-HT2A receptors in layer V pyramidal neurons, rather than models positing the requirement of mGluR2/5-HT2A multimers.

The excitability of afferents involved in nociceptive signaling reflects the interaction of several co-expressed membrane receptors. Current studies have shown that Toll-like receptor-4 (TLR-4) signaling can exacerbate excitation evoked by transient receptor potential vanilloid type 1 (TRPV1) activity, and this interaction plays a key role in driving and sustaining facilitated pain states. The mechanism by which this potentiated TRPV1 activity secondary to TLR-4 agonism occurs in sensory neurons remains unknown, although intracellular kinase activity is a strong candidate. To address this hypothesized linkage, neuronal cell cultures prepared from dorsal root ganglia (DRG) of male wildtype (WT) and Tlr4-/- mice were used to evaluate calcium transients of neurons after capsaicin administration in culture, pre-treated for 30 minutes with the TLR-4 agonist, lipopolysaccharide (LPS). TRPV1 protein expression at the neuron surface in cultured DRG cells with or without LPS treatment was quantified by flow cytometry assay. The roles of protein kinase A (PKA) and C were assessed using selective inhibitors (KT5720 for PKA and Chelerythrine chloride for PKC) applied to WT-DRG neurons or administered in vivo by intraplantar or intrathecal injection, prior to LPS and capsaicin administration. Behavioral effects of in vivo TRPV1 activation were assessed through paw flinch responses evoked by intraplantar capsaicin injection and by hind paw tactile thresholds measured by von Frey filaments. LPS incubation in cultured DRG neurons enhances the intensity of calcium influx following TRPV1 activation in WT but not Tlr4-/ cells. The augmented calcium influx evoked by capsaicin was prevented by the inhibition of PKA but not PKC. Similarly, mice treated with LPS in the hind paw displayed greater nociceptive responding after capsaicin and increased tactile allodynia. The facilitated component was prevented by the local pre-treatment with the PKA inhibitor. Correspondingly, lumbar spinal blockade of PKA resulted in temporary reversal of hyperalgesia induced by intrathecal LPS injection in mice. Together, these results demonstrate the relevance of TLR-4 in modulating the excitability of nociceptor signaling by regulating TRPV1, thereby influencing pain transmission through PKA signaling.

Abstract Objectives To evaluate and compare the neuro-behavioural safety profiles of piroxicam and nitroglycerine by investigating their differential effects on cognitive function, spatial and recognition memory, and hippocampal neurochemistry in a di-oestrous female Wistar rat model. Methods Female Wistar rats at di-oestrous were randomly assigned to receive distilled water, piroxicam, or nitroglycerine orally for four consecutive days. Following treatment, spatial and recognition memory were evaluated using standard behavioural paradigms. Hippocampal tissues were analysed for acetylcholinesterase and glutamate activity, oxidative stress markers, and neuroinflammatory indices. Results Piroxicam improved recognition memory and was associated with increased glutamatergic activity and a compensatory rise in superoxide dismutase. However, it also elicited elevated nitric oxide signaling, lipid peroxidation, and localized neuroinflammatory markers in the hippocampus. In contrast, nitroglycerine impaired non-spatial memory during di-oestrous. Although both treatments preserved working memory, they produced distinct effects on object recognition, memory discrimination, oxidative stress parameters, and neuroinflammatory mediators. Conclusions Piroxicam and nitroglycerine exert differential effects on cognition and hippocampal neurochemistry during di-oestrous. Piroxicam improved recognition memory and produced distinct hippocampal neurochemical alterations, whereas nitroglycerine impaired recognition memory. These findings highlight the influence of menstrual pain therapeutics on cognitive function and hippocampal physiology under hormonally sensitive conditions. Keywords: cognitive function; cognitive impairment; cyclooxygenase inhibitors; neuroinflammation; neurochemistry