Addiction Neuroscience

RationalePrenatal alcohol exposure (PAE) can result in Fetal Alcohol Spectrum Disorder (FASD), which consists of a group of diagnosable medical conditions that can include an increased risk for anxiety disorders and/or alcohol misuse, and sensory issues, such as increased mechanical sensitivity. ObjectiveThis study investigated how a single moderate PAE on gestational day 12 (G12) alters anxiety-like behavior, ethanol (EtOH) intake, and mechanical sensitivity across the lifespan of Sprague Dawley rats. MethodsPregnant dams were exposed to vaporized EtOH or room air (control) for 6 hours (BECs [~]108 mg/dL). Testing in male and female offspring began at three different ages: juveniles ([~]postnatal day (P) 25), adolescents ([~]P45) and adults ([~]P80). ResultsThe greatest PAE effects were observed in adolescent animals, with alterations in anxiety-like behaviors demonstrated in the light-dark box and elevated plus maze. Additionally, adolescent female animals consumed more sweetened EtOH compared to males. However, PAE adolescent animals consuming less sweetened EtOH compared to their counterparts, which was also observed in adult PAE females. Interestingly, this effect is reversed in juvenile and adolescent males when tested with unsweetened EtOH, with juvenile females consuming more EtOH also. Finally, PAE and air animals exhibited increased mechanical sensitivity following post-natal EtOH consumption across all ages. ConclusionThese data demonstrate that there are age- and sex-specific effects of PAE on anxiety-like behaviors, EtOH intake, and mechanical sensitivity that are more distinct in adolescent animals.

BackgroundDisturbance of circadian rhythms is a hallmark of substance use disorders, with depressant drugs often causing soporific effects such as reduced sleep latency. The suprachiasmatic nucleus (SCN) of the hypothalamus is the central circadian pacemaker in mammals, regulating daily rhythms in physiology and behavior. However, the cellular mechanisms through which depressants alter SCN function remain poorly defined. MethodsWe used whole-cell patch clamp electrophysiology in acute brain slices to examine how alcohol and opioids modulate excitatory glutamatergic transmission onto SCN neurons. Ethanol effects were examined both acutely and following chronic exposure paradigms. Optogenetic stimulation was used to activate either RHT input or -opioid receptor-expressing (MOR) terminals, and MOR agonists were used to assess opioid-mediated effects on synaptic transmission. ResultsWe show that acute application of ethanol paradoxically enhances SCN firing rates. In contrast, chronic alcohol exposure reduces glutamatergic drive. We also found that activating MOR+ terminals produced bidirectional modulation of SCN firing and that MOR+ inputs formed functional glutamatergic synapses onto SCN neurons. Notably, this transmission could be suppressed by the MOR agonists DAMGO and fentanyl. ConclusionsTogether, these findings reveal that both alcohol and opioids modulate glutamatergic input to the SCN. This work establishes the SCN as a novel target of depressant substances and highlights glutamatergic transmission as a key point of vulnerability in circadian dysregulation associated with substance use.

BackgroundLoss of control over drinking is a hallmark feature of alcohol use disorder (AUD) that is modeled preclinically through escalation of ethanol consumption and aversion-resistant drinking. Prior work with other reinforcers suggests that within-session unpredictable, intermittent access (uIntA) promotes loss of control over intake. However, the effect of uIntA on voluntary ethanol consumption is unknown. MethodsMale and female Long-Evans rats (n=9-10/group) underwent seven weeks of daily voluntary ethanol (20% v/v) drinking sessions under either a continuous access (ContA) or uIntA schedule. Following four weeks of baseline, rats were rendered dependent using a two-week chronic intermittent ethanol vapor exposure procedure. Daily testing was maintained through one week into withdrawal from vapor exposure. On the final day of testing, ethanol was adulterated with quinine (30 mg/L) to assess aversion-resistant drinking. ResultsRats drinking under ContA and uIntA exhibited similar levels of average daily ethanol consumption at baseline. However, uIntA elicited a more robust dependence-induced escalation of ethanol consumption compared to ContA, with uIntA sustaining escalation through early protracted withdrawal. Additionally, while rats with ContA to ethanol remained sensitive to quinine even after chronic ethanol vapor exposure, uIntA promoted aversion-resistant drinking in ethanol dependent rats. ConclusionsThese results demonstrate that, compared to ContA, uIntA maintains ethanol drinking and exacerbates AUD-related symptomatology while also providing researchers with the ability to capture additional measures of motivation and drinking patterns without increasing experimental burden. This work positions uIntA as a powerful tool to assess psychological and neurobiological factors underlying loss of control over drinking.

BackgroundAlcohol use disorder is a chronic relapsing condition characterized by excessive drinking and withdrawal symptoms. Alcohol dependence disrupts function across multiple brain regions, and recent evidence implicates the cortical amygdala (CoA) as a critical node in alcohol-related circuits. However, how CoA activity influences alcohol intake and brain-wide network function during withdrawal remains unclear. MethodsAlcohol dependence was induced using chronic intermittent ethanol vapor (CIE). In one cohort, electrophysiological activity of CoA neurons was assessed during withdrawal. In a second cohort, mice underwent CIE paired with two-bottle choice drinking, and inhibitory DREADDs (hM4Di) were used to suppress CoA activity during drinking and withdrawal while behavioral outcomes were measured. Brains were then collected for Fos immunolabeling and iDISCO+ based whole-brain activity mapping to determine how CoA inhibition during withdrawal altered network organization. ResultsRepeated CIE increased alcohol sensitivity in CoA neurons during withdrawal. Chemogenetic inhibition of the CoA reduced alcohol intake in dependent mice without affecting withdrawal-related behaviors. Whole-brain Fos mapping showed that CoA inhibition reduced activity within the CoA while enhancing functional connectivity across multiple brain regions, particularly in the isocortex, thalamus, and anterior hypothalamic nucleus. During withdrawal without CoA inhibition, thalamic regions exhibited negative connectivity, consistent with disrupted network function; CoA inhibition reversed this pattern, producing strongly positive thalamic and medial prefrontal cortex connectivity. ConclusionsThese findings demonstrate that alcohol dependence alters CoA sensitivity, alcohol dependence-induced drinking and brain-wide network organization during withdrawal. The CoA appears to selectively regulate withdrawal-associated alcohol drinking, and its inhibition may reduce intake by restoring thalamic and cortical connectivity. HighlightsO_LIThis study identifies the cortical amygdala as a previously underexplored brain region involved in alcohol-related behaviors. C_LIO_LIBy integrating chemogenetic inhibition with brain-wide network analysis, the study reveals candidate circuit connections through which the CoA may regulate alcohol dependence-related brain activity. C_LIO_LIThis study establishes the CoA as a potential driver of excessive alcohol drinking and alcohol-related network dysfunction. C_LI

There is a dearth of information on how different cocktails sweetened with different sugars impact brain activity. Glucose enters the brain faster and in greater concentration than fructose and directly affects neuronal activity of melanin-concentrating hormone (MCH) neurons. MCH signaling promotes both glucose drinking and alcohol intake by integrating central and sensory inputs, but it is currently unknown how MCH neuronal activity relates to sweetened cocktail drinking. This study sought to investigate the relationship between MCH activity and sugar-sweetened alcoholic cocktail drinking. We also sought to compare MCH neuronal responses to the sugar solutions without alcohol as well as their response to sensory stimuli. In female and male rats, we used fiber photometry to monitor MCH neurons in response to sensory stimuli and during drinking of 10% glucose, 10% fructose, and glucose or fructose cocktails with 1.25% or 10% alcohol. We found that MCH activity rises in response to a variety of sensory stimuli and peaks before the start of drinking for all cocktails, before returning to baseline near the start of drinking. The cocktail type impacted the dynamics of MCH activity, where increased alcohol concentration resulted in earlier MCH activity for fructose but not glucose cocktails. Finally, we found that peak MCH activity during drinking is correlated with approach behavior for all sugar and cocktail types. These findings suggest that glucose and alcohol may interact to directly influence MCH activity. Further, MCH neurons may regulate cocktail drinking in response to sugar type and alcohol concentration. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=118 SRC="FIGDIR/small/719280v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@54685org.highwire.dtl.DTLVardef@59003eorg.highwire.dtl.DTLVardef@11f0358org.highwire.dtl.DTLVardef@114b524_HPS_FORMAT_FIGEXP M_FIG C_FIG New and noteworthyFiber photometry was used to monitor lateral hypothalamic melanin-concentrating hormone (MCH) neurons in male and female rats during sensory stimuli and drinking of glucose, fructose, or glucose- or fructose-sweetened alcoholic cocktails. Subsecond-scale changes in MCH activity occurred after stimuli. Peak MCH activity during drinking was correlated with approach behavior. Alcohol concentration only impacted MCH activity with fructose cocktails. We discuss the implications of MCH dynamics towards brain function, associative learning, and alcohol use disorder.

Background: Alcohol use disorder is associated with dysregulated glutamatergic signaling within mesocorticolimbic circuits that govern reinforcement and excessive ethanol intake. Group II metabotropic glutamate receptors (mGlu2/3) act primarily as presynaptic autoreceptors that regulate glutamate release. However, how voluntary alcohol intake alters mGlu2/3 expression within reward circuitry remains unclear. Methods and Results: We examined the effects of operant alcohol self-administration on mGlu2/3 protein expression and assessed the functional impact of group II receptor modulation on binge-like ethanol intake. Male C57BL/6J mice self-administered sweetened ethanol or sucrose under behaviorally matched conditions for 35 days. Immediately after the final session, tissue punches from the nucleus accumbens (NAc), amygdala, and prefrontal cortex were collected for Western blot analysis. Operant ethanol self-administration selectively reduced mGlu2/3 protein expression in the NAc, with no changes detected in the amygdala or prefrontal cortex. Both monomeric and dimeric mGlu2/3 protein levels were reduced, and a composite index revealed coordinated downregulation of receptor expression. In separate cohorts, systemic administration of the mGlu2/3 agonist LY379268 dose-dependently reduced binge-like ethanol intake in a limited-access home-cage drinking model, whereas positive allosteric modulation of mGlu2 receptors with LY487379 was ineffective. Conclusions: These results show that low-dose operant ethanol self-administration produces an ethanol- and region-specific reduction of mGlu2/3 protein expression in the NAc and that pharmacological activation of group II receptors, potentially involving mGlu3-specific receptors, is sufficient to suppress binge-like ethanol consumption. These data identify presynaptic mGlu2/3 dysregulation as a mechanism contributing to ethanol-related behaviors and support group II metabotropic glutamate receptors as therapeutic targets for alcohol use disorder.

Neuroimmune signaling is increased in postmortem brain tissue from individuals with alcohol use disorder (AUD), and growing evidence suggests that it contributes to persistent alcohol-related neuroadaptations. Interferon regulatory factor 7 (IRF7), a transcription factor downstream of endosomal Toll-like receptor signaling, is induced in alcohol-relevant brain regions and may contribute to escalated drinking. Here, we tested whether chronic intermittent ethanol (CIE) vapor exposure engages IRF7 signaling during subsequent alcohol self-administration and whether this is associated with altered molecular E/I balance in the aIC and altered functional E/I balance in aICnucleus accumbens projection neurons. Female Wistar rats (n=30) were trained to self-administer alcohol (15% v/v; FR2 vs inactive lever) during 30-minute sessions. After establishing baseline drinking, rats underwent 1-3 cycles of CIE, which increased alcohol self-administration at the 72 h post vapor test. This increase positively correlated with IRF7 levels in the anterior insular cortex (aIC) and nucleus accumbens, while molecular, and immunofluorescence showed that CIE shifted aIC excitatory/inhibitory (E/I) balance toward reduced excitation. Electrophysiological recordings further showed reduced functional E/I balance in aIC neurons projecting to the nucleus accumbens. Knockdown of IRF7 in the aIC attenuated CIE induced escalation of alcohol self-administration, supporting a role for insular IRF7 signaling in alcohol related neuroadaptations that promote escalated drinking.

RationaleThe progression of psychostimulant abuse is associated with a shift from recreational to habitual use (R2H-shift). Because this R2H-shift can be modeled using behavioral economics, we developed a novel Behavioral Economic model for the Analysis of Self-administration Time-curve (BEAST) to obtain R2H-shift variable(s). The relationship(s) between R2H-shift variables and drug intake (under normal and/or punishment conditions) is/are unknown. Our goal was to determine if the R2H-shift variable and intake variables obtained during the initial self-administration training phase were related to 1) drug intake at that time, and subsequent drug intake under 2) normal, 3) punishment, 4) post-punishment, and 5) price-constrained conditions. MethodLong Evans rats self-administered methamphetamine (METH, males n = 16, females n = 14), sucrose (males n = 22, females n = 22) and/or saline (males n = 3, females n = 10) under FR1 for 6 h per day for 20 days to obtain 1) followed by the assessment of subsequent drug intake under different conditions (2-5 above). We obtained all variables referenced above. We determined the relationships between all variables (multivariate analysis). ResultsThere were no sex differences detected in the METH and sucrose studies. For METH and sucrose, prior drug intake levels could predict drug intake under normal/punishment but not under price-constrained conditions. The R2H-shift variable could predict drug intake under a consumption-price curve but could not predict intake under normal/punishment conditions. ConclusionsWhile related to economic demand, the recreational-to-habitual shift rate was unrelated to drug intake levels (under normal and punishment conditions).

Alternative polyadenylation (APA) is a common posttranscriptional mechanism to regulate gene expression. APA generates mRNAs with varying lengths of 3' UTRs or transcripts that encode distinct protein carboxy-terminal ends. APA is especially important in neurons, where different mRNA variants are often asymmetrically localized to dendrites and axons, and can be locally translated into proteins. Local protein synthesis is crucial for axon guidance, synaptic plasticity, and learning and memory, key processes associated with the development of alcohol use disorder (AUD). We investigated the role of APA in AUD using a mouse model of alcohol dependence characterized by increased voluntary drinking after chronic intermittent ethanol (CIE) exposure. We examined APA during protracted withdrawal from alcohol in three brain regions of male and female mice. Our analyses revealed hundreds of genes undergoing APA in males, but substantially fewer in females, suggesting sex-specific effects of CIE on APA. Notably, male and female mice displayed distinct APA signatures. APA genes were different from differentially expressed genes (DEGs), suggesting that these molecular processes are regulated independently. We also determined that the expression of APA genes was associated with neurons, while DEGs were associated with non-neuronal cells. Many of the APA genes were involved in synaptic integrity, neuroplasticity, and neuronal maintenance, which was consistent with their enrichment in neurons. Our study suggests that APA is a crucial sex- and cell type-specific mechanism in AUD with the potential to influence localized neuronal protein expression during protracted withdrawal and to modify alcohol consumption behavior. HIGHLIGHTSO_LIChronic ethanol exposure in mice results in profound changes of APA genes in brain. C_LIO_LICommonly regulated cleavage and polyadenylation sites and genes were identified in male but not in female mice. C_LIO_LIThere was a minimal overlap between APA and differentially expressed genes (DEGs). C_LIO_LIAPA genes were primarily associated with neurons, whereas DEGs were associated with non-neuronal cells. C_LI

Craving - the powerful urge to seek and consume alcohol in response to alcohol-associated cues does not diminish after drinking cessation but rather is magnified throughout abstinence. This phenomenon, termed "incubation of craving", contributes to the relapsing nature of alcohol use disorder. Despite its occurrence in human populations and being well-studied in rodent models of psychostimulant drug relapse, the underlying neural mechanisms and potential treatments remain largely unexplored for alcohol-related incubation of craving. Our research seeks to meet this gap, and this particular study investigated the neural correlates of the incubation of craving for alcohol-associated cues and assessed whether exercise could prevent increased relapse propensity in rats. Male Long Evans rats were trained to lever press for an alcohol reward delivered with simultaneous presentation of a discrete cue. This response was then extinguished and reinstated by presenting the discrete cue alone when rats pressed the lever. Cue-induced reinstatement occurred either on day 1 following extinction (No Abstinence) or on day 29 (Abstinence). A third group was tested on day 29 and had 4-hour daily voluntary running wheel access throughout this abstinence period (Exercise). All rats were perfused 90 minutes following test, and relative activation across the brain was estimated by quantifying c-Fos protein immunoreactivity. The brain-wide coordination of neural activity was also mapped. We found a robust incubation of craving effect for alcohol-associated cues, which was mitigated by exercise. Immunohistochemistry revealed that the Abstinence group demonstrated higher c-Fos immunoreactivity compared to the No Abstinence group in multiple reinstatement-related brain regions. This effect was reversed in the Exercise group. Brain-wide neural mapping demonstrated that the Abstinence group had decreased modularity (groups of coordinating brain regions) compared to the no-abstinence group. Although network connectivity profile in the exercise group was different from no abstinence, we found that overall neural activation returned to a similar modularity profile of clustered regions as this condition, indicating that exercise does not attenuate the incubation of craving effect by reversing all the neural effects of abstinence. Rather, exercise may be acting upon select brain regions or pathways to exert relapse protective effects by restoring widespread interconnectivity. This is the first study to investigate neural activation in incubated alcohol-seeking, and provides supporting evidence for promoting voluntary exercise as an adjunctive treatment for relapse prevention in alcohol-use disorder.

Opioid use disorder (OUD) is a chronic, relapsing disease driven by the reinforcing properties of opioids and perpetuated by avoidance of the negative affective states associated with the absence of the drug. Most available OUD treatments directly engage the {micro}-opioid receptor and may induce side effects that can compromise their therapeutic efficacy, thus underscoring the need for novel therapeutic alternatives. Calcitonin gene-related peptide (CGRP) is produced by a small population of neurons in the parabrachial nucleus (PBN) that has been shown to modulate itch, pain, as well as appetitive behaviors. Using a cell-specific nuclear labeling approach coupled with RNA-sequencing, we generated a baseline transcriptome of CGRPPBN neurons and confirmed expression of multiple genes associated with behavioral responses to appetitive stimuli, as well as enrichment of the {micro}-opioid receptor, suggesting that CGRPPBN neuron function may be sensitive to the presence of opioids. Indeed, cFos immunostaining showed that CGRPPBN neuron activity increases during early morphine abstinence and reduces gradually over 48 hours. Given the inhibitory effects of opioids on CGRPPBN neuron activity, we next tested whether these neurons could regulate opioid reinforcement. Using a mouse model of morphine intravenous self-administration, we found that chemogenetic inhibition of CGRPPBN neurons significantly reduced the number of morphine rewards earned in both single-dose and dose-response tests but did not affect context-induced morphine seeking after 21 days of abstinence. These results suggest that CGRPPBN neurons are sensitive to opioid administration and can regulate appetitive behaviors such as morphine-taking. Considering that CGRP signaling is regulated by opioid administration, molecular targets that regulate CGRP neurotransmission without direct -opioid receptor engagement may therefore serve as novel therapeutic avenues for the treatment of OUD. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=195 SRC="FIGDIR/small/712659v1_ufig1.gif" ALT="Figure 1"> View larger version (56K): org.highwire.dtl.DTLVardef@1fb9c9borg.highwire.dtl.DTLVardef@1e6ba79org.highwire.dtl.DTLVardef@dc60f5org.highwire.dtl.DTLVardef@61adaf_HPS_FORMAT_FIGEXP M_FIG C_FIG

BackgroundMaladaptive drinking is often sustained by automatic approach tendencies toward alcohol cues that override conscious self-control. While cognitive and behavioral modification techniques show some promise, their effects remain limited, highlighting the need for alternative neuromodulatory strategies. The current study examined the feasibility of a single session of 10 Hz repetitive transcranial magnetic stimulation (rTMS) to the right dorsolateral prefrontal cortex (dLPFC) as a targeted approach to reduce automatic alcohol approach tendencies. MethodForty-five healthy alcohol-using participants completed an alcohol approach- avoidance task (A-AAT) with concurrent electroencephalographic recording before and after active or sham stimulation. Primary analyses focused on participants with baseline alcohol approach tendencies (n = 35). ResultsAt baseline, individuals with approach tendencies exhibited attenuated N2 and P3b amplitudes to alcohol relative to non-alcohol cues, indicating reduced cognitive control and attentional mechanisms irrespective of group. Following stimulation, active rTMS selectively facilitated alcohol avoidance responses and enhanced prefrontal N2 amplitudes, suggesting strengthened top-down control and protection against repetition-induced automaticity, which was evident in the sham group. ConclusionThese findings suggest that high-frequency rTMS over the right dLPFC can modulate automatic alcohol-related action tendencies by strengthening neural control mechanisms, supporting its further evaluation as a neuromodulatory adjunct for maladaptive drinking. Baseline motivational profiles may additionally influence rTMS response and warrant consideration when tailoring such approaches.

The misuse of opioid medications is a significant health issue in the United States. Very few studies have investigated the effect of opioids on perineuronal nets (PNNs), scaffold-like structures that surround neurons and are involved in the regulation of plasticity-dependent mechanisms such as development, learning and memory, and acquisition of addiction-like phenotypes. Regulation of PNNs in the orbitofrontal cortex (OFC) during periods of drug intoxication or withdrawal is widely unknown. In this study, male Wistar rats were injected with fentanyl (0.125 mg/kg, s.c.) or 0.9% saline twice daily for 7 days and once on day 8 (7continuous days following by 3 days of abstinence) or twice daily for 15 days (5 continuous days followed by 2 days of abstinence for more than 3 weeks) and twice on day 16. Antinociception was evaluated using the tail immersion test immediately before and 30 minutes after injections. Whole-brain coronal slices were collected, and immunohistochemistry was used to identify Wisteria Floribunda Agglutinin (WFA)-positive PNNs and parvalbumin (PV)-expressing cells. Results confirmed that repeated fentanyl injections induced tolerance to the antinociceptive effects, which normalized following acute abstinence periods. WFA intensity decreased following 8 days of injections. Analyses confirmed significant correlations between PV+ density and tail withdrawal latency following 8 days of fentanyl injections. These data confirm that repeated fentanyl injections modulate both WFA+ and PV+ expression in the rodent brain and antinociceptive tolerance in a duration-dependent manner. Overall, these data suggest that perineuronal nets may mediate opioid-induced behavioral effects, such as antinociceptive tolerance, following repeated administration and abstinence in rats.

BackgroundAlcohol use disorder (AUD) is linked to increased neuroinflammation. Alcohol (ethanol) may activate toll-like receptors, which leads to the release of inflammatory molecules that could influence AUD-related behaviors, such as increased alcohol intake. Activation of toll-like receptor 3 (TLR3) by Polyinosinic:polycytidylic acid (Poly(I:C) or PIC) is associated with escalation of alcohol consumption in male, but not female F1 hybrid mice from reciprocal crosses between FVB/NJ (FVB) and C57BL/6J (B6) strains. Little is known about the underlying mechanisms of these sex-specific behavioral effects. In this study, we investigated the effects of TLR3 activation by PIC on temporal profiles of several pro- and anti-inflammatory molecules in the blood and brain of FVB/B6 F1 hybrid male and female mice at multiple time points. We hypothesized that TLR3 - dependent immune profiles would differ between males and females, which may, at least in part, explain the observed differences in drinking behavior. MethodsMale and female FVB/B6 F1 hybrid alcohol-naive mice were injected intraperitoneally with PIC (10 mg/kg) or saline. Blood and perfused brain tissues from the prefrontal cortex (PFC) and striatum were collected at 6-, 24-, and 48-hours post-injection. The expressions of Ccl2, Ccl5, Tnf, Il-6, Il-1{beta}, Ifng, Ifnb1, and Mmp9 genes were analyzed using qPCR. Protein levels of a subset of these molecules and IL-17r/a, IL-4, and IL-10 were measured in striatal samples from the same animals using ELISA. ResultsActivation of TLR3 by PIC triggered time-dependent, sex- and tissue-specific responses in immune genes and their proteins. PIC induced a time-dependent increase in expression of majority of the genes peaking at the 6 hr time point. Temporal immune profiles for pro-inflammatory chemokines, Ccl2 and Ccl5 differed between males and females in the PFC and striatum, suggesting possible sex-specific effects of these molecules on behavior. Protein levels of CCL2, CCL5, and IL-6 increased in the striatum of both sexes and correlated strongly with gene expression, with females showing somewhat higher protein fold changes. MMP-9, a key regulator of blood-brain barrier (BBB) permeability and synaptic plasticity, showed an increase in protein levels, but not mRNA levels in striatum. This pattern suggests altered blood-brain barrier (BBB) permeability, although this would require further investigation. ConclusionOur results revealed distinct TLR3-dependent immune gene and protein expression profiles in blood and brain between males and females and suggested different roles for these molecules in regulating alcohol consumption. We identified CCL2, CCL5 and MMP-9 as target molecules for investigating sex-specific behavior in the immune modulation of alcohol consumption.

"Dry Hitting" is a unique phenomenon of e-cigarette use that has been shown to produce toxic chemical degradants and byproducts. Although it is widely understood that nicotine exposure during adolescence impacts neurobiological and behavioral function, little is known about how dry hitting may impact users. We hypothesized that subjects repeatedly exposed to nicotine dry hit vapor would exhibit distinct behavioral responses compared with saturated nicotine vapor and would differentially alter the expression of perineuronal nets (PNNs) in the rodent brain. Using a customized system of e-cigarette vapor inhalation, adolescent male Wistar rats (PND 31-40) received vaporized nicotine (30 or 60 mg/mL; [~]2.5-3 mL/cage), nicotine with dry hits (60 mg/mL; 1.75-2 mL/cage), or propylene glycol (PG) vehicle for 30 minutes over 7 daily sessions. Locomotor activity, antinociception, and elevated plus maze testing were used to assess behavioral response to drug intoxication and tolerance. Immunohistochemistry was used to identify Wisteria Floribunda Agglutinin (WFA)-positive PNN structures in the amygdala and insular cortex. Rats exposed to dry hits exhibited behavioral responses (locomotor sensitization, antinociception) similar to those of rats exposed to saturated nicotine vapor, but spent more time in the open arms of the elevated plus maze. Immunohistochemical analyses confirmed significantly greater WFA intensity in the central nucleus of the amygdala, but not the basolateral amygdala or insular cortex, of rats exposed to dry hits. Overall, these data confirm the impact of dry hit vapor on behavioral responses and perineuronal net expression in rats during adolescence.

Astrocytes are the most abundant glial cells in the brain and an integrative component of the neural network. Studies have shown that ethanol altered expression of an astrocyte marker, i.e., glial fibrillary acidic protein (GFAP), in two key corticolimbic regions, the medial prefrontal cortex (mPFC) and nucleus accumbens (NAc). These regions comprise anatomically and functionally different subregions, i.e., the prelimbic (PL) and infralimbic (IL) cortex of the mPFC, the shell and core subregions of the NAc. However, ethanol effects on GFAP expression within these subregions remain largely unknown. In addition, effects of pharmacological manipulation of astrocytes on alcohol drinking have been understudied. Western blot was conducted to determine GFAP expression in subregions of the mPFC and NAc after chronic ethanol drinking. Fluorocitrate, an astrocyte-specific metabolic inhibitor, was administered to inhibit astrocytes and was tested on ethanol drinking. Ethanol drinking enhanced GFAP protein expression in the PL cortex and NAc core, but not in the IL cortex or NAc shell. Intra-ventricular administration of fluorocitrate reduced ethanol intake and preference, but increased water consumption during choice ethanol drinking. In addition, fluorocitrate did not affect total fluid consumption or basal locomotor activity. These results indicate that chronic ethanol drinking induced GFAP elevation in a subregion-specific manner within the mPFC and NAc, and that metabolic inhibition of astrocytes selectively attenuated ethanol drinking without non-specific effects on water drinking or general activity. Together, these results suggest that astrocytes may play an important role in ethanol drinking. HighlightsO_LIEthanol drinking enhanced GFAP levels in the PL cortex and NAc core. C_LIO_LIFluorocitrate inhibition of astrocytes reduced intermittent ethanol drinking. C_LIO_LIFluorocitrate did not alter total fluid consumption or basal locomotor activity. C_LI

BackgroundPersistent automatic approach tendencies toward alcohol cues that resist goal-directed control are a key feature of harmful alcohol use, yet the causal neural mechanisms underlying this imbalance remain poorly understood. Converging evidence implicates the frontoparietal network (FPN) in actively regulating alcohol approach-avoidance behavior, but whether its constituent nodes make dissociable causal contributions has not been established. MethodsIn a within-subject, active-sham counterbalanced design, inhibitory continuous theta burst stimulation (cTBS) was applied to right dorsolateral prefrontal cortex (rDLPFC) and right posterior parietal cortex (rPPC) in separate groups of non-clinical alcohol users (rDLPFC: n = 29; rPPC: n = 28), followed by an Alcohol Approach-Avoidance Task. ResultsActive rDLPFC cTBS selectively slowed down alcohol push responses, whereas rPPC suppression produced a bidirectional action-specific shift in response to alcohol cues, where pull responses accelerated, and push slowed simultaneously. Suppression of either node shifted automatic tendencies toward greater alcohol approach through mechanistically distinct routes. ConclusionThese dissociable profiles indicate that rDLPFC is causally necessary for effortful top-down avoidance control, while rPPC supports the priority-based selection of alcohol cue-driven actions. These findings provide the first node-specific causal evidence for functional specialization within the FPN in the context of automatic tendencies towards alcohol. Alcohol avoidance emerges as an active, prefrontal-dependent process, whereas priority-based regulation emerges as a parietal-dependent process, together indicating rDLPFC and rPPC as mechanistically independent targets for intervention in maladaptive alcohol approach behavior.

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

Endocannabinoid (eCB) signaling is a key regulator of reward-related dopaminergic signaling, particularly in response to drugs of abuse, such as cocaine. To date, our understanding of this mechanism has primarily been limited to male subjects. Prior work establishes that female cocaine users have more adverse outcomes, and female rats show greater sensitivity to cannabinoid type 1 receptor (CB1R) regulation of cocaine self-administration. Therefore, we hypothesize that female rats exhibit enhanced eCB regulation of cocaine-evoked dopamine (DA). We used in vivo fiber photometry recording of the dopamine biosensor, dLight 1.3b, in the nucleus accumbens medial shell (NAcms) in response to cocaine in male and female rats. Rats were pretreated with cannabinoid-targeting drugs to investigate the effects of CB1R inactivation or augmentation of the eCB 2-AG on cocaine-evoked DA. Our results revealed that CB1R inactivation attenuates cocaine-evoked DA in male and female rats, but females showed enhanced sensitivity for CB1R regulation of cocaine-evoked DA. Cocaine-evoked DA was enhanced by augmenting 2-AG levels, and females again showed increased sensitivity to this manipulation. Finally, females show greater cocaine-evoked DA when in a non-estrous cycle compared to estrous, reinforcing that estrous cycle is a determinant of cocaine-evoked DA. These data indicate that females show enhanced eCB regulation of cocaine-evoked DA signaling, underscoring the importance of sex as a biological variable in our understanding of endocannabinoid regulation of drug reward. HighlightsO_LICB1R inactivation attenuates cocaine-evoked DA in NAcms, preferentially in females C_LIO_LI2-AG augmentation via MAGL inhibition enhances cocaine-evoked DA, with female bias C_LIO_LIEstrous phase modulates the dopamine response to a high dose of cocaine in females C_LIO_LIMale and female rats show similar baseline DA and locomotor responses to cocaine C_LI

Prenatal alcohol exposure (PAE) causes fetal alcohol spectrum disorders (FASDs), which are neurodevelopmental conditions characterized by behavioral dysregulation, learning deficits, and cognitive inflexibilities. Alcohol exposure is harmful at all stages of human gestation, including the third trimester. This developmental window--characterized by rapid brain growth, myelination, and neural circuit formation--may be particularly vulnerable, yet the long-lasting behavioral and sensory consequences of exposure during this period remain poorly understood. In this study, neonatal mouse pups were exposed to ethanol (EtOH) or air vapor from postnatal day (P) 4 to P8, which is equivalent to a third-trimester alcohol exposure (TTAE) in humans. Blood ethanol concentrations measured at P8 reached approximately 250 mg/dL, consistent with binge-level exposure. Air- and EtOH-exposed mice were then assessed as adults at 5-6 months of age for locomotor activity, anxiety-related risky behaviors, recognition memory, and increased susceptibility to peripheral neuropathy, as indicated by sensitization to light touch following minor chronic constriction injury (mCCI) of the sciatic nerve. We found that TTAE was sufficient to produce long-lasting behavioral outcomes in a sex-dependent manner. Notably, EtOH-exposed males exhibited increased spontaneous locomotor activity and risky behavior, whereas EtOH-exposed females showed minimal or decreased changes compared to their respective controls. However, both EtOH-exposed male and female mice exhibited marked increases in light-touch sensitization, referred to as mechanical allodynia, following mCCI, a response absent in air-exposed controls. Together, these findings reveal that TTAE is highly detrimental to behavioral regulation and creates a vulnerability to developing neuropathic pain in adulthood.