TNFR1 signaling converging on FGF14 controls neuronal hyperactivity and sickness behavior in experimental cerebral malaria.

BACKGROUND: Excess tumor necrosis factor (TNF) is implicated in the pathogenesis of hyperinflammatory experimental cerebral malaria (eCM), including gliosis, increased levels of fibrin(ogen) in the brain, behavioral changes, and mortality. However, the role of TNF in eCM within the brain parenchyma, particularly directly on neurons, remains underdefined. Here, we investigate electrophysiological consequences of eCM on neuronal excitability and cell signaling mechanisms that contribute to observed phenotypes. METHODS: The split-luciferase complementation assay (LCA) was used to investigate cell signaling mechanisms downstream of tumor necrosis factor receptor 1 (TNFR1) that could contribute to changes in neuronal excitability in eCM. Whole-cell patch-clamp electrophysiology was performed in brain slices from eCM mice to elucidate consequences of infection on CA1 pyramidal neuron excitability and cell signaling mechanisms that contribute to observed phenotypes. Involvement of identified signaling molecules in mediating behavioral changes and sickness behavior observed in eCM were investigated in vivo using genetic silencing. RESULTS: Exploring signaling mechanisms that underlie TNF-induced effects on neuronal excitability, we found that the complex assembly of fibroblast growth factor 14 (FGF14) and the voltage-gated Na+ (Nav) channel 1.6 (Nav1.6) is increased upon tumor necrosis factor receptor 1 (TNFR1) stimulation via Janus Kinase 2 (JAK2). On account of the dependency of hyperinflammatory experimental cerebral malaria (eCM) on TNF, we performed patch-clamp studies in slices from eCM mice and showed that Plasmodium chabaudi infection augments Nav1.6 channel conductance of CA1 pyramidal neurons through the TNFR1-JAK2-FGF14-Nav1.6 signaling network, which leads to hyperexcitability. Hyperexcitability of CA1 pyramidal neurons caused by infection was mitigated via an anti-TNF antibody and genetic silencing of FGF14 in CA1. Furthermore, knockdown of FGF14 in CA1 reduced sickness behavior caused by infection. CONCLUSIONS: FGF14 may represent a therapeutic target for mitigating consequences of TNF-mediated neuroinflammation.

Fentanyl self-administration impacts brain immune responses in male Sprague-Dawley rats.

Opioid use disorder (OUD) affects over two million in the United States and is an increasing public health crisis. The abuse of fentanyl and the emergence of potent fentanyl derivatives increases the risk for the user to succumb to overdose, but also to develop OUD. While intense attention is currently focused on understanding the complexity of behaviors and neural functions that contribute to OUD, much remains to be discovered concerning the interactions of opioid intake with the immune response in the central nervous system (CNS). In the present studies, we tested the hypothesis that short-term abstinence from fentanyl self-administration associates with altered expression of innate immune markers. Male Sprague-Dawley rats were trained to self-administer fentanyl (0.0032 mg/kg/infusion) to stability followed by 24 h of abstinence. Several innate immune markers, as well as opioid receptors (ORs) and intracellular pattern recognition receptors (PRRs), were interrogated within nodes of the neurocircuitry involved in OUD processes, including the prefrontal cortex (PFC), nucleus accumbens (NAc), caudate putamen (CPu), hippocampus (HIP) and midbrain (MB). In the present study, few immune targets were impacted in the PFC and MB during short-term abstinence from fentanyl (relative to saline) self-administration. However, increased expression of cytokines [e.g., interleukin (IL)1ß, IL5], chemokines [e.g., C-C motif chemokine 20 (MIP3α)], tumor necrosis factor α (TNFα) and interferon (IFN) proteins (e.g., IFNß and IFNγ)] was seen in the NAc, while decreased expression of cytokines (e.g., several ILs), chemokines [e.g., granulocyte-macrophage colony-stimulating factor (GMCSF), monocyte chemoattractant protein (MCP) MCP1, MIP3α], the chemokine ligand 5 (RANTES) and interferons (e.g., IFNß and IFNγ) in the HIP. Positive correlations were observed between cumulative fentanyl intake and expression of IL1ß and IL6 in the NAc, and significant negative correlations with fentanyl intake and IFN ß, IL2, IL5, IL12p70 and IL17 in the HIP. Few changes in OR expression was observed during early abstinence from fentanyl self-administration. Excitingly, the expression of the PRR, stimulator of interferon genes (STING) negatively correlated with cumulative fentanyl intake and significantly correlated to specific cytokines, chemokines and interferon proteins in the HIP. Although the CPu appears relatively invulnerable to changes in innate immune markers, the highest correlations between cumulative fentanyl intake with MAVS and/or STING was measured in the CPu. Our findings provide the first evidence of CNS innate immune responses and implicate STING as novel mechanistic targets of immunomodulation during short-term abstinence from fentanyl self-administration.

The 5-HT2A Receptor (5-HT2AR) Regulates Impulsive Action and Cocaine Cue Reactivity in Male Sprague-Dawley Rats.

Impulsivity and the attentional orienting response to cocaine-associated cues (cue reactivity) promote relapse in cocaine-use disorder (CUD). A time-dependent escalation of cue reactivity (incubation) occurs during extended, forced abstinence from cocaine self-administration in rats. The investigational serotonin (5-HT) 5-HT2A receptor (5-HT2AR) antagonist/inverse agonist M100907 suppresses impulsive action, or the inability to withhold premature responses, and cocaine-seeking behaviors. The present preclinical study was designed to establish the potential for repurposing the Food and Drug Administration-approved selective 5-HT2AR antagonist/inverse agonist pimavanserin as a therapeutic agent to forestall relapse vulnerability in CUD. In male Sprague-Dawley rats, pimavanserin suppressed impulsive action (premature responses) measured in the 1-choice serial reaction time (1-CSRT) task, similarly to M100907. We also used the 1-CSRT task to establish baseline levels of impulsive action before cocaine self-administration and evaluation of cue reactivity (lever presses reinforced by the discrete cue complex previously paired with cocaine delivery). We observed an incubation of cocaine cue reactivity between day 1 and day 30 of forced abstinence from cocaine self-administration. Baseline levels of impulsive action predicted incubated levels of cocaine cue reactivity in late abstinence. We also found that baseline impulsive action predicted the effectiveness of pimavanserin to suppress incubated cue reactivity in late abstinence from cocaine self-administration at doses that were ineffective in early abstinence. These data suggest that integration of clinical measures of impulsive action may inform refined, personalized pharmacotherapeutic intervention for the treatment of relapse vulnerability in CUD.

Anterior insula activity regulates the associated behaviors of high fat food binge intake and cue reactivity in male rats.

Binge eating episodes are characterized by uncontrollable, excessive intake of food and are associated with binge eating disorder and some subtypes of obesity. One factor thought to contribute to binge episodes is a high level of reactivity to food-associated cues (i.e., cue reactivity). The insula is a neural node poised to regulate both binge eating and cue reactivity because of its prominent role in interpretation of internal and external cues. This work established a positive association between high fat food (HFF) binge intake and cue reactivity in male rats. Furthermore, we demonstrated that activation of the anterior insula suppressed both HFF binge intake and cue reactivity, without altering homeostatic intake of food. We further show that attenuation of HFF binge intake and cue reactivity is not due to decreased food-reward efficacy or deficits in motivation. Together, these data establish a key role for the anterior insula in the control of binge eating related-behaviors and support novel avenues for the treatment of binge eating.

PPARγ agonism attenuates cocaine cue reactivity.

Cocaine use disorder is a chronic relapsing condition characterized by compulsive drug seeking and taking even after prolonged abstinence periods. Subsequent exposure to drug-associated cues can promote intense craving and lead to relapse in abstinent humans and rodent models. The responsiveness to these cocaine-related cues, or 'cue reactivity', can trigger relapse and cocaine-seeking behaviors; cue reactivity is measurable in cocaine-dependent humans as well as rodent models. Cue reactivity is thought to be predictive of cocaine craving and relapse. Here we report that PPARγ agonism during abstinence from cocaine self-administration reduced previously active lever pressing in Sprague Dawley rats during cue-reactivity tests, while administration of the PPARγ antagonist, GW9662, reversed this effect. PPARγ agonism also normalized nuclear ERK activity in the medial prefrontal cortex and hippocampus which was reversed with GW9662. Our results support the utility of PPARγ agonism as a relapse prevention strategy to maintain abstinence in the presence of cocaine-associated cues.

Behavioural and neurological symptoms accompanied by cellular neuroinflammation in IL-10-deficient mice infected with Plasmodium chabaudi.

BACKGROUND: Cerebral malaria is one of the most severe complications of Plasmodium falciparum infection and occurs mostly in young African children. This syndrome results from a combination of high levels of parasitaemia and inflammation. Although parasite sequestration in the brain is a feature of the human syndrome, sequestering strains do not uniformly cause severe malaria, suggesting interplay with other factors. Host genetic factors such as mutations in the promoters of the cytokines IL-10 and TNF are also clearly linked to severe disease. Plasmodium chabaudi, a rodent malaria parasite, leads to mild illness in wildtype animals. However, IL-10(-/-) mice respond to parasite with increased levels of pro-inflammatory cytokines IFN-γ and TNF, leading to lethal disease in the absence of sequestration in the brain. These mice also exhibit cerebral symptoms including gross cerebral oedema and haemorrhage, allowing study of these critical features of disease without the influence of sequestration. METHODS: The neurological consequences of P. chabaudi infection were investigated by performing a general behavioural screen (SHIRPA). The immune cell populations found in the brain during infection were also analysed using flow cytometry and confocal microscopy. RESULTS: IL-10(-/-) mice suffer significant declines in behavioural and physical capacities during infection compared to wildtype. In addition, grip strength and pain sensitivity were affected, suggestive of neurological involvement. Several immune cell populations were identified in the perfused brain on day 7 post-infection, suggesting that they are tightly adherent to the vascular endothelium, or potentially located within the brain parenchyma. There was an increase in both inflammatory monocyte and resident macrophage (CD11b(hi), CD45(+), MHCII(+), Ly6C(+/-)) numbers in IL-10(-/-) compared to wildtype animals. In addition, the activation state of all monocytes and microglia (CD11b(int), CD45(-), MHC-II(+)) were increased. T cells making IFN-γ were also identified in the brain, but were localized within the vasculature, and not the parenchyma. CONCLUSIONS: These studies demonstrate exacerbated neuroinflammation concurrent with development of behavioural symptoms in P. chabaudi infection of IL-10(-/-) animals.

Peptide inhibitors disrupt the serotonin 5-HT2C receptor interaction with phosphatase and tensin homolog to allosterically modulate cellular signaling and behavior.

Serotonin (5-hydroxytryptamine; 5-HT) signaling through the 5-HT(2C) receptor (5-HT(2C)R) is essential in normal physiology, whereas aberrant 5-HT(2C)R function is thought to contribute to the pathogenesis of multiple neural disorders. The 5-HT(2C)R interacts with specific protein partners, but the impact of such interactions on 5-HT(2C)R function is poorly understood. Here, we report convergent cellular and behavioral data that the interaction between the 5-HT(2C)R and protein phosphatase and tensin homolog (PTEN) serves as a regulatory mechanism to control 5-HT(2C)R-mediated biology but not that of the closely homologous 5-HT(2A)R. A peptide derived from the third intracellular loop of the human 5-HT(2C)R [3L4F (third loop, fourth fragment)] disrupted the association, allosterically augmented 5-HT(2C)R-mediated signaling in live cells, and acted as a positive allosteric modulator in rats in vivo. We identified the critical residues within an 8 aa fragment of the 3L4F peptide that maintained efficacy (within the picomolar range) in live cells similar to that of the 3L4F peptide. Last, molecular modeling identified key structural features and potential interaction sites of the active 3L4F peptides against PTEN. These compelling data demonstrate the specificity and importance of this protein assembly in cellular events and behaviors mediated by 5-HT(2C)R signaling and provide a chemical guidepost to the future development of drug-like peptide or small-molecule inhibitors as neuroprobes to study 5-HT(2C)R allostery and therapeutics for 5-HT(2C)R-mediated disorders.

Liquid Biopsy-Based Biomarkers of Inflammatory Nociception Identified in Male Rats.

Physicians are challenged in treating pain patients due to the lack of quantifiable, objective methods of measuring pain in the clinic; pain sensation is multifaceted and subjective to each individual. There is a critical need for point-of-care quantification of accessible biomarkers to provide objective analyses beyond the subjective pain scales currently employed in clinical care settings. In the present study, we employed an animal model to test the hypothesis that circulating regulators of the inflammatory response directly associate with an objective behavioral response to inflammatory pain. Upon induction of localized paw inflammation, we measured the systemic protein expression of cytokines, and activity levels of matrix metalloproteinases (MMPs) that are known to participate in the inflammatory response at the site of injury and investigated their relationship to the behavioral response across a 24 h period. Intraplantar injection with 1% λ-carrageenan induced a significant increase in paw thickness across this timespan with maximal effects observed at the 8 h timepoint when locomotor activity was also impaired. Expression of the chemokines C-X-C motif chemokine ligand 1 (CXCL1) and C-C motif chemokine ligand 2 (CCL2) positively correlated with paw inflammation and negatively correlated with locomotor activity at 8 h. The ratio of MMP9 to MMP2 activity negatively correlated with paw inflammation at the 8 h timepoint. We postulate that the CXCL1 and CCL2 as well as the ratio of MMP9 to MMP2 activity may serve as predictive biomarkers for the timecourse of inflammation-associated locomotor impairment. These data define opportunities for the future development of a point-of-care device to objectively quantify biomarkers for inflammatory pain states.

Serotonin 5-HT2C receptor protein expression is enriched in synaptosomal and post-synaptic compartments of rat cortex.

The action of serotonin (5-HT) at the 5-HT(2C) receptor (5-HT(2C)R) in cerebral cortex is emerging as a candidate modulator of neural processes that mediate core phenotypic facets of several psychiatric and neurological disorders. However, our understanding of the neurobiology of the cortical 5-HT(2C)R protein complex is currently limited. The goal of the present study was to explore the subcellular localization of the 5-HT(2C)R in synaptosomes and the post-synaptic density, an electron-dense thickening specialized for post-synaptic signaling and neuronal plasticity. Utilizing multiples tissues (brain, peripheral tissues), protein fractions (synaptosomal, post-synaptic density), and controls (peptide neutralization, 5-HT(2C)R stably-expressing cells), we established the selectivity of two commercially available 5-HT(2C)R antibodies and employed the antibodies in western blot and immunoprecipitation studies of prefrontal cortex (PFC) and motor cortex, two regions implicated in cognitive, emotional and motor dysfunction. For the first time, we demonstrated the expression of the 5-HT(2C)R in post-synaptic density-enriched fractions from both PFC and motor cortex. Co-immunoprecipitation studies revealed the presence of post-synaptic density-95 within the 5-HT(2C)R protein complex expressed in PFC and motor cortex. Taken together, these data support the hypothesis that the 5-HT(2C)R is localized within the post-synaptic thickening of synapses and is therefore positioned to directly modulate synaptic plasticity in cortical neurons.

Discovery of Potent and Brain-Penetrant GPR52 Agonist that Suppresses Psychostimulant Behavior.

The G protein-coupled receptor 52 (GPR52) is an orphan receptor that is selectively expressed in the striatum and regulates various brain functions through activation of cAMP-dependent pathways. GPR52 has been identified as a promising therapeutic target for central nervous system disorders including schizophrenia and substance use disorders. Here, a series of novel GPR52 agonists were designed, synthesized, and evaluated based on compound 4. Several potent and efficacious GPR52 agonists (12c, 23a, 23d, 23e, 23f, and 23h) were identified with nanomolar range potency based on a systematic structure-activity relationship exploration. Further studies of 12c indicate enhanced efficacy, excellent target selectivity, and pharmacokinetic properties including good brain permeability. In vivo proof-of-concept investigations revealed that 12c displayed antipsychotic-like activity by significantly inhibiting amphetamine-induced hyperlocomotor behavior in mice. Collectively, our findings have resulted in an efficacious, brain-penetrant GPR52 agonist as a valuable pharmacological tool for investigating the physiological and therapeutic potential of GPR52 activation.

Suppression of cocaine relapse-like behaviors upon pimavanserin and lorcaserin co-administration.

Cocaine use disorder (CUD) is a major public health challenge for which there are no pharmacotherapeutics approved by the United States Food and Drug Administration (FDA). The propensity to relapse in CUD involves several vulnerability factors including sensitivity to cues associated with cocaine-taking. Serotonin (5-hydroxytryptamine, 5-HT) neurotransmission, particularly through the 5-HT2A receptor (5-HT2AR) and 5-HT2C receptor (5-HT2CR), is mechanistically linked to cocaine-seeking in preclinical models. In the present experiments, we employed self-administration assays in male rats to investigate whether acute and/or repeated administration of the FDA-approved selective 5-HT2AR antagonist/inverse agonist pimavanserin, selective 5-HT2CR agonist lorcaserin or their combination would alter cocaine intake and/or cocaine-seeking behavior. We found that acute administration of lorcaserin, but not pimavanserin, attenuated cocaine intake while pimavanserin plus lorcaserin did not impact cocaine self-administration. In contrast, 10-days of repeated administration of pimavanserin, lorcaserin, or pimavanserin plus lorcaserin during forced abstinence from cocaine self-administration, blunted cocaine-seeking, similar to the acute administration of each ligand. Taken together, these data reveal the efficacy of repeated treatment with pimavanserin plus lorcaserin to attenuate factors important to relapse-like behaviors in rodent models of CUD. This article is part of the special issue entitled 'Serotonin Research: Crossing Scales and Boundaries'.

Endogenous Serotonin 5-HT2A and 5-HT2C Receptors Associate in the Medial Prefrontal Cortex.

The 5-HT2A receptor (5-HT2AR) and 5-HT2CR are localized to the same neurons within the medial prefrontal cortex (mPFC), which regulates executive function, decision-making, and reward-guided learning and memory processes. The 5-HT2AR and 5-HT2CR coimmunoprecipitate in the mPFC of male Sprague-Dawley rats, while in vitro studies demonstrate the presence of a physical interaction between the 5-HT2AR and 5-HT2CR. The purpose of this study was to identify mPFC subregions in which the 5-HT2AR and 5-HT2CR physically interact ex vivo in the male Sprague-Dawley rat. We established the expression patterns of 5-HT2AR and 5-HT2CR in layers I-VI of the anterior cingulate cortex (ACC), prelimbic (PL), and infralimbic (IL) subregions using double-label fluorescence immunohistochemistry in male rats. We then employed the proximity ligation assay (PLA) to test the hypothesis that the 5-HT2AR and 5-HT2CR form a close, physical association within these mPFC subregions. Our results demonstrate subregion- and layer-specific expression of the 5-HT2AR and 5-HT2CR proteins using immunofluorescence and single recognition PLA, and a spatially close (within 40 nm) interaction between the 5-HT2AR and 5-HT2CR that occurs along a dorsal-ventral gradient in the rat mPFC.

Convergent neural connectivity in motor impulsivity and high-fat food binge-like eating in male Sprague-Dawley rats.

Food intake is essential for survival, but maladaptive patterns of intake, possibly encoded by a preexisting vulnerability coupled with the influence of environmental variables, can modify the reward value of food. Impulsivity, a predisposition toward rapid unplanned reactions to stimuli, is one of the multifaceted determinants underlying the etiology of dysregulated eating and its evolving pathogenesis. The medial prefrontal cortex (mPFC) is a major neural director of reward-driven behavior and impulsivity. Compromised signaling between the mPFC and nucleus accumbens shell (NAcSh) is thought to underlie the cognitive inability to withhold prepotent responses (motor impulsivity) and binge intake of high-fat food (HFF) seen in binge eating disorder. To explore the relationship between motor impulsivity and binge-like eating in rodents, we identified high (HI) and low impulsive (LI) rats in the 1-choice serial reaction time task and employed a rat model of binge-like eating behavior. HFF binge rats consumed significantly greater calories relative to control rats maintained on continual access to standard food or HFF. HI rats repeatedly exhibited significantly higher bingeing on HFF vs. LI rats. Next, we employed dual viral vector chemogenetic technology which allows for the targeted and isolated modulation of ventral mPFC (vmPFC) neurons that project to the NAcSh. Chemogenetic activation of the vmPFC to NAcSh pathway significantly suppressed motor impulsivity and binge-like intake for high-fat food. Thus, inherent motor impulsivity and binge-like eating are linked and the vmPFC to NAcSh pathway serves as a 'brake' over both behaviors.

Chronic poly-drug administration damages adult mouse brain neural stem cells.

Cocaine and ethanol are two commonly co-abused substances; however, the neuropathology following chronic dual consumption is poorly understood. Neural stem cells (NSCs) are a subpopulation of cells within the adult brain that are integral to brain maintenance and repair making them an appealing target to reverse neurodegeneration associated with abused substances. Yet, knowledge about NSC response to chronic poly-drug administration of ethanol and cocaine is minimal. Here, we developed a novel chronic poly-drug administration paradigm of ethanol and cocaine using a transgenic mouse model to trace endogenous NSC survival and differentiation in three brain regions from both male and female mice. We report significant and distinct patterns of NSC survival and differentiation among brain regions, as well as between sexes. Additionally, poly-drug administration had synergistic effects on NSC survival. Altered cognitive and hedonic behaviors were also observed, however the extent of these behavioral changes was not proportional to the NSC changes. With this mouse model we can effectively examine cognitive and behavioral changes and correlate them with pathological changes in the brain in response to chronic poly-drug administration, which is of great value in understanding the progression of neurodegeneration in polysubstance use disorders and evaluation potential therapeutics on neuroregeneration.

Estradiol-sertraline synergy in ovariectomized rats.

This study investigated estradiol (E(2)) modulation of the antidepressant effects of a selective serotonin (5-HT) reuptake inhibitor (SSRI; sertraline) and a tricyclic antidepressant (imipramine) as measured by the forced swim test (FST) followed by assessment of gene and protein expression for the 5-HT transporter (SERT) and multiple 5-HT receptors. Female Sprague-Dawley rats were ovariectomized (OVX) and two-thirds of the rats received E(2) implants (OVE). 4 weeks later, implants were withdrawn in half of the OVE rats (OVW) to capture a time point when E(2) levels were rapidly declining. Rats in each hormone group were treated with vehicle, sertraline (10 mg/kg) or imipramine (10 mg/kg), 24, 5 and 1h before the FST. Immediately after the FST, midbrain, hippocampus and prefrontal cortex tissue was removed and frozen for analysis of gene expression via quantitative real-time PCR (midbrain tissue) and protein expression via Western blot (prefrontal cortex and hippocampal tissue). In the FST, sertraline decreased immobility and increased swimming in OVE rats, as well as increased swimming in OVW rats. In contrast, no sertraline effect was observed in OVX rats. Rats treated with imipramine showed increased climbing but no changes in immobility or swimming. No changes in protein expression were detected in any treatment group. However, in vehicle-treated rats, E(2) increased midbrain SERT mRNA expression, with no effect on midbrain mRNA for the 5-HT receptors. In sertraline-treated rats, E(2) decreased 5-HT(2A) receptor mRNA, and E(2)-withdrawal increased 5-HT(1A), 5-HT(2A) and 5-HT(2C) receptor mRNA. In imipramine-treated rats, E(2) (and E(2)-withdrawal) did not affect mRNA expression for any of the target genes. Thus, E(2) synergized behaviorally and neurochemically with an SSRI but not a tricyclic antidepressant.

Serotonin 5-HT2C Receptor Activation Suppresses Binge Intake and the Reinforcing and Motivational Properties of High-Fat Food.

Binge eating disorder (BED) is characterized by dysfunctional hedonic food intake and reward-related processes. Activation of the serotonin (5-HT) 5-HT2C receptor (5-HT2CR) suppresses both food intake and reward-related behaviors and is thus poised to regulate BED. This study assessed the effects of 5-HT2CR activation via the selective 5-HT2CR agonist WAY163909 on binge eating-related behaviors in adult male Sprague-Dawley rats. Low doses of WAY163909 (1.0, 2.0 mg/kg) suppressed high-fat food (HFF) binge intake, but not standard food non-binge intake. WAY163909 (1.0 mg/kg) also attenuated operant responding for self-administered HFF pellets on fixed and progressive ratio schedules of reinforcement, indicating that 5-HT2CR activation suppresses the reinforcing and motivational properties of HFF, respectively. These findings suggest that activation of the 5-HT2CR may be effective at suppressing binge eating in patients with BED via suppression of the reinforcing and motivational properties of HFF. This work supports future studies targeting the 5-HT2CR in the treatment of BED.

A Protocol for Measuring Cue Reactivity in a Rat Model of Cocaine Use Disorder.

Cocaine use disorder (CUD) follows a trajectory of repetitive self-administration during which previously neutral stimuli gain incentive value. Cue reactivity, the sensitivity to cues previously linked with the drug-taking experience, plays a prominent role in human craving during abstinence. Cue reactivity can be assessed as the attentional orientation toward drug-associated cues that is measurable as appetitive approach behavior in both preclinical and human studies. Herein describes an assessment of cue reactivity in rats trained to self-administer cocaine. Cocaine self-administration is paired with the presentation of discrete cues that act as conditioned reinforcers (i.e., house light, stimulus light, infusion pump sounds). Following a period of abstinence, lever presses in the cocaine self-administration context accompanied by the discrete cues previously paired with cocaine infusion are measured as cue reactivity. This model is useful to explore neurobiological mechanisms underlying cue reactivity processes as well as to assess pharmacotherapies to suppress cue reactivity and therefore, modify relapse vulnerability. Advantages of the model include its translational relevance, and its face and predictive validities. The primary limitation of the model is that the cue reactivity task can only be performed infrequently and must only be used in short duration (e.g., 1 hour), otherwise rats will begin to extinguish the pairing of the discrete cues with the cocaine stimulus. The model is extendable to any positively reinforcing stimulus paired with discrete cues; though particularly applicable to drugs of abuse, this model may hold future applications in fields such as obesity, where palatable food rewards can act as positively reinforcing stimuli.

Selective ablation of GABA neurons in the ventral tegmental area increases spontaneous locomotor activity.

Gamma-aminobutyric acid (GABA) neurons in the ventral tegmental area (VTA) provide innervation to cortical and subcortical regions of the brain. To solidify the importance of these VTA GABA neurons in behavioral function, we employed the neurotoxin dermorphin-saporin (DS) to selectively lesion VTA GABA neurons prior to assessing spontaneous motor activity. Rats were bilaterally microinfused with DS (1.0 or 2.0 pmol/200 nl/side) or blank-saporin control (BS, 200 nl/side) into the VTA. Seven days later, DS-treated rats exhibited significantly elevated motility in comparison with BS-treated rats; this elevated motility normalized by Day 14 following pretreatment with 1.0 pmol of DS but was sustained on Day 14 after pretreatment with 2.0 pmol of DS. A selective loss of VTA GABA neurons on Day 14 was demonstrated through reduced expression of mRNA for glutamic acid decarboxylase-67 and micro-opioid receptor, but not tyrosine hydroxylase (a dopamine neuron marker), in the VTA. Thus, a dose- and time-related selective loss of VTA GABA neurons was accomplished using this novel neurotoxin. This loss of GABA VTA neurons was associated with hypermotility, further supporting their important regulatory role in the generation of behavior.

Lorcaserin Suppresses Oxycodone Self-Administration and Relapse Vulnerability in Rats.

Opioid use disorder (OUD) is a major public health problem. High relapse rates and poor treatment retention continue to pose major challenges in OUD treatment. Of the abused opioids, oxycodone is well described to maintain self-administration and evoke the durable conditioned responses ("cue reactivity") that result from pairing of opioid-related stimuli (e.g., paraphernalia) with repeated abuse. Serotonin (5-HT) neurotransmission, particularly through the 5-HT2C receptor (5-HT2CR), regulates psychostimulant reward and cue reactivity, and in the present experiments, we investigated the hypothesis that the selective 5-HT2CR agonist lorcaserin, which is approved by the United States Food and Drug Administration (FDA) for the treatment of obesity, will suppress oxycodone self-administration and oxycodone-associated cue reactivity in rats. We found that lorcaserin inhibited oxycodone intake, an effect blocked by the selective 5-HT2CR antagonist SB242084. Lorcaserin also decreased responding for the discrete cue complex ("cue reactivity") previously associated with delivery of oxycodone (i.e., stimulus lights, infusion pump sounds) in both abstinence and extinction-reinstatement models. The selected dose range of lorcaserin (0.25-1 mg/kg) does not overtly alter spontaneous behaviors nor operant responding on inactive levers in the present study. Taken together, the ability of lorcaserin to reduce the oxycodone self-administration and decrease cue reactivity associated with relapse highlights the therapeutic potential for lorcaserin in the treatment of OUD.

Blockade of the 5-HT transporter contributes to the behavioural, neuronal and molecular effects of cocaine.

BACKGROUND AND PURPOSE: The psychostimulant cocaine induces complex molecular, cellular and behavioural responses as a consequence of inhibiting presynaptic dopamine, noradrenaline and 5-HT transporters. To elucidate 5-HT transporter (SERT)-specific contributions to cocaine action, we evaluated cocaine effects in the SERT Met172 knock-in mouse, which expresses a SERT coding substitution that eliminates high-affinity cocaine recognition. EXPERIMENTAL APPROACH: We measured the effects of SERT Met172 on cocaine antagonism of 5-HT re-uptake using ex vivo synaptosome preparations and in vivo microdialysis. We assessed SERT dependence of cocaine actions behaviourally through acute and chronic locomotor activation, sensitization, conditioned place preference (CPP) and oral cocaine consumption. We used c-Fos, quantitative RT-PCR and RNA sequencing methods for insights into cellular and molecular networks supporting SERT-dependent cocaine actions. KEY RESULTS: SERT Met172 mice demonstrated functional insensitivity for cocaine at SERT. Although they displayed wild-type levels of acute cocaine-induced hyperactivity or chronic sensitization, the pattern of acute motor activation was different, with a bias toward thigmotaxis. CPP was increased, and a time-dependent elevation in oral cocaine consumption was observed. SERT Met172 mice displayed relatively higher levels of neuronal activation in the hippocampus, piriform cortex and prelimbic cortex (PrL), accompanied by region-dependent changes in immediate early gene expression. Distinct SERT-dependent gene expression networks triggered by acute and chronic cocaine administration were identified, including PrL Akt and nucleus accumbens ERK1/2 signalling. CONCLUSION AND IMPLICATIONS: Our studies reveal distinct SERT contributions to cocaine action, reinforcing the possibility of targeting specific aspects of cocaine addiction by modulation of 5-HT signalling.