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Serotonin plays a role in reinforcement learning; however, it is not known which serotonin receptors mediate these effects. Serotonin 6 (5-HT(6)) receptors are abundant in the striatum, a brain area that is involved in reinforcement learning. We previously found that 5-HT(6) receptors in the dorsomedial striatum (DMS) affect reinforcement learning or consolidation over several days. We use viral-mediated gene transfer to discern the role that 5-HT(6) receptors play in mediating post-synaptic responses in anterior versus posterior DMS. Male Long-Evans rats were used to study learning acquisition during a single session of 100 trials on a fixed interval of 20 seconds. In a discrete action-outcome learning task, rats had 10 seconds to press a lever to induce lever retraction and sucrose pellet delivery. In another group of rats, the task had a lever that was continuously extended but only active every 20 seconds, allowing for repetitive, mostly non-reinforced, lever pressing. Results demonstrate that increased expression of 5-HT(6) receptors in the posterior DMS interferes with earning sucrose pellets in only the former task. We take this to indicate that 5-HT(6) receptor signaling in the posterior DMS interferes with acquisition of discrete action-outcome responding. [ABSTRACT FROM AUTHOR]

Experiments to study the biology of addiction have historically focused on the mechanisms through which drugs of abuse drive changes in the functioning of neurons and neural circuits. Glia have often been ignored in these studies, however, and this has left many questions in the field unanswered, particularly, surrounding how glia contribute to changes in synaptic plasticity, regulation of neuroinflammation, and functioning of neural ensembles given massive changes in signaling across the CNS. Omics methods (transcriptomics, translatomics, epigenomics, proteomics, metabolomics, and others) have expanded researchers' abilities to generate hypotheses and carry out mechanistic studies of glial cells during acquisition of drug taking, intoxication, withdrawal, and relapse to drug seeking. Here, we present a survey of how omics technological advances are revising our understanding of astrocytes, microglia, oligodendrocytes, and ependymal cells in addiction biology., (© 2024 Wiley Periodicals LLC.)

Opioid use disorder has become an epidemic in the United States, fuelled by the widespread availability of fentanyl, which produces rapid and intense euphoria followed by severe withdrawal and emotional distress. We developed a new preclinical model of fentanyl seeking in outbred male and female rats using volitional oral self-administration (SA) that can be readily applied in labs without intravascular access. Using a traditional two-lever operant procedure, rats learned to take oral fentanyl vigorously, escalated intake across sessions, and readily reinstated responding to conditioned cues after extinction. Oral SA also revealed individual and sex differences that are essential to studying substance use risk propensity. During a behavioural economics task, rats displayed inelastic demand curves and maintained stable intake across a wide range of fentanyl concentrations. Oral SA was also neatly patterned, with distinct 'loading' and 'maintenance' phases of responding within each session. Using our software DeepSqueak, we analysed ultrasonic vocalizations (USVs), which are innate expressions of current emotional state in rats. Rats produced 50 kHz USVs during loading then shifted quickly to 22 kHz calls despite ongoing maintenance of oral fentanyl taking, reflecting a transition to negative reinforcement. Using fibre photometry, we found that the lateral habenula differentially processed drug cues and drug consumption depending on affective state, with potentiated modulation by drug cues and consumption during the negative affective maintenance phase. Together, these results indicate a rapid progression from positive to negative reinforcement occurs even within an active drug taking session, revealing a within-session opponent process., (© 2023 The Authors. Addiction Biology published by John Wiley & Sons Ltd on behalf of Society for the Study of Addiction.)

The lateral habenula (LHb) is a small, bilateral, epithalamic nucleus which processes aversive information. While primarily glutamatergic, LHb neurons express genes coding for many neuropeptides, such as Adcyap1 the gene encoding pituitary adenylate cyclase-activating polypeptide (PACAP), which itself has been associated with anxiety and stress disorders. Using Cre-dependent viral vectors, we targeted and characterized these neurons based on their anatomical projections and found that they projected to both the raphe and rostromedial tegmentum but only weakly to ventral tegmental area. Using RiboTag to capture ribosomal-associated mRNA from these neurons and reanalysis of existing single cell RNA sequencing data, we did not identify a unique molecular phenotype that characterized these PACAP-expressing neurons in LHb. In order to understand the function of these neurons, we conditionally expressed hM 3 Dq DREADD selectively in LHb PACAP-expressing neurons and chemogenetically excited these neurons during behavioral testing in the open field test, contextual fear conditioning, sucrose preference, novelty suppressed feeding, and conditioned place preference. We found that Gq activation of these neurons produce behaviors opposite to what is expected from the LHb as a whole-they decreased anxiety-like and fear behavior and produced a conditioned place preference. In conclusion, PACAP-expressing neurons in LHb represents a molecularly diverse population of cells that oppose the actions of the remainder of LHb neurons by being rewarding or diminishing the negative consequences of aversive events., (© 2022 The Authors. Genes, Brain and Behavior published by International Behavioural and Neural Genetics Society and John Wiley & Sons Ltd.)

Background: Microglia have recently been implicated in opioid dependence and withdrawal. Mu Opioid (MOR) receptors are expressed in microglia, and microglia form intimate connections with nearby neurons. Accordingly, opioids have both direct (MOR mediated) and indirect (neuron-interaction mediated) effects on microglia function., Methods: To investigate this directly, we used RNA sequencing of ribosome-associated RNAs from striatal microglia (RiboTag-Seq) after the induction of morphine tolerance and followed by naloxone precipitated withdrawal (n=16). We validated the RNA-Seq data by combining fluorescent in-situ hybridization with immunohistochemistry for microglia (n=18). Finally, we expressed and activated the Gi/o-coupled hM 4 Di DREADD receptor in CX3CR1-expressing cells during morphine withdrawal (n=18)., Results: We detected large, inverse changes in RNA translation following opioid tolerance and withdrawal. WGCNA analysis revealed an intriguing network of cAMP-associated genes that are known to be involved in microglial motility, morphology, and interactions with neurons that were downregulated with morphine tolerance and upregulated rapidly by withdrawal. Three-dimensional histological reconstruction of microglia allowed for volumetric, visual colocalization of mRNA within individual microglia that validated our bioinformatics results. Direct activation of Gi/o-coupled DREADD receptors in CX3CR1-expressing cells exacerbated signs of opioid withdrawal rather than mimicking the effects of morphine., Conclusions: These results indicate that Gi-signaling and cAMP-associated gene networks are inversely engaged during opioid tolerance and early withdrawal, perhaps revealing a role of microglia in mitigating the consequences of opioids., Competing Interests: Competing Interests Kevin R. Coffey has no conflicts of interest to disclose. Atom J. Lesiak has no conflicts of interest to disclose. Russell G. Marx has no conflicts of interest to disclose. Emily K. Vo has no conflicts of interest to disclose. Gwenn A. Garden has no conflicts of interest to disclose. John F. Neumaier has no conflicts of interest to disclose.

Stress-induced release of dynorphins (Dyn) activates kappa opioid receptors (KOR) in serotonergic neurons to produce dysphoria and potentiate drug reward; however, the circuit mechanisms responsible for this effect are not known. In male mice, we found that conditional deletion of KOR from Slc6a4 (SERT)-expressing neurons blocked stress-induced potentiation of cocaine conditioned place preference (CPP). Within the dorsal raphe nucleus (DRN), two overlapping populations of KOR-expressing neurons: Slc17a8 (VGluT3) and SERT, were distinguished functionally and anatomically. Optogenetic inhibition of these SERT + neurons potentiated subsequent cocaine CPP, whereas optical inhibition of the VGluT3 + neurons blocked subsequent cocaine CPP. SERT + /VGluT3 - expressing neurons were concentrated in the lateral aspect of the DRN. SERT projections from the DRN were observed in the medial nucleus accumbens (mNAc), but VGluT3 projections were not. Optical inhibition of SERT + neurons produced place aversion, whereas optical stimulation of SERT + terminals in the mNAc attenuated stress-induced increases in forced swim immobility and subsequent cocaine CPP. KOR neurons projecting to mNAc were confined to the lateral aspect of the DRN, and the principal source of dynorphinergic (Pdyn) afferents in the mNAc was from local neurons. Excision of Pdyn from the mNAc blocked stress-potentiation of cocaine CPP. Prior studies suggested that stress-induced dynorphin release within the mNAc activates KOR to potentiate cocaine preference by a reduction in 5-HT tone. Consistent with this hypothesis, a transient pharmacological blockade of mNAc 5-HT 1B receptors potentiated subsequent cocaine CPP. 5-HT 1B is known to be expressed on 5-HT terminals in NAc, and 5-HT 1B transcript was also detected in Pdyn + , Adora2a + and ChAT + (markers for direct pathway, indirect pathway, and cholinergic interneurons, respectively). Following stress exposure, 5-HT 1B transcript was selectively elevated in Pdyn + cells of the mNAc. These findings suggest that Dyn/KOR regulates serotonin activation of 5HT 1B receptors within the mNAc and dynamically controls stress response, affect, and drug reward., (© 2021. The Author(s), under exclusive licence to American College of Neuropsychopharmacology.)

Serotonin is a key mediator of stress, anxiety, and depression, and novel therapeutic targets within serotonin neurons are needed to combat these disorders. To determine how stress alters the translational profile of serotonin neurons, we sequenced ribosome-associated RNA from these neurons after repeated stress in male and female mice. We identified numerous sex- and stress-regulated genes. In particular, Fkbp5 mRNA, which codes for the glucocorticoid receptor co-chaperone protein FKBP51, was consistently upregulated in male and female mice following stress. Pretreatment with a selective FKBP51 inhibitor into the dorsal raphe prior to repeated forced swim stress decreased resulting stress-induced anhedonia. Our results support previous findings linking FKBP51 to stress-related disorders and provide the first evidence suggesting that FKBP51 function may be an important regulatory node integrating circulating stress hormones and serotonergic regulation of stress responses., (© 2020. The Author(s), under exclusive licence to Springer Nature Limited.)

A major problem in the treatment of cocaine addiction is high rates of relapse. Relapse is often provoked by acute reexposure to cocaine-associated cues or to cocaine itself. The lateral habenula (LHb), an epithalamic nucleus, regulates midbrain dopaminergic systems that are known to be involved in cocaine taking and seeking behaviors. However, the role of this nucleus in cocaine self-administration and reinstatement of cocaine seeking has not been entirely parsed out. We used an operant self-administration and reinstatement procedure to explore the effect of Designer Receptors Exclusively Activated by Designer Drug (DREADD)-induced transient inhibition of LHb neurons on cocaine taking and seeking. Firstly, rats were injected with adeno-associated viral vectors expressing hM 4 D i (a G i/o -coupled DREADD) into the LHb, trained to self-administer cocaine (0.75 mg/kg/infusion), and the effect of clozapine-N-oxide (an inert ligand that activates DREADDs) was assessed on cocaine self-administration. Secondly, rats were injected with hM 4 D i into the LHb, trained to self-administer cocaine; the operant response was extinguished, and cue- and cocaine priming-induced reinstatement was assessed. Thirdly, we tested the generality of the effect of inhibiting LHb neurons by assessing the effect of this manipulation on food-taking and seeking. hM 4 D i -induced inhibition of LHb neurons increased cocaine but not food self-administration. In contrast, this manipulation decreased reinstatement of cocaine, but not food-seeking. Taken together, our data suggest that hM 4 D i - induced LHb inhibition specifically mediates taking and seeking behaviors reinforced by cocaine but not by natural reinforcers. Further, our data indicate a dissociation in the role of LHb neurons on cocaine self-administration versus reinstatement of cocaine seeking., (© 2020 Society for the Study of Addiction.)

5-HT receptors expressed throughout the human body are targets for established therapeutics and various drugs in development. Their diversity of structure and function reflects the important role 5-HT receptors play in physiologic and pathophysiological processes. The present review offers a framework for the official receptor nomenclature and a detailed understanding of each of the 14 5-HT receptor subtypes, their roles in the systems of the body, and, where appropriate, the (potential) utility of therapeutics targeting these receptors. SIGNIFICANCE STATEMENT: This review provides a comprehensive account of the classification and function of 5-hydroxytryptamine receptors, including how they are targeted for therapeutic benefit., (Copyright © 2020 by The American Society for Pharmacology and Experimental Therapeutics.)

The lateral habenula (LHb) integrates critical information regarding aversive stimuli that shapes decision making and behavioral responses. The three major LHb outputs innervate dorsal raphe nucleus (DRN), ventral tegmental area (VTA), and the rostromedial tegmental nucleus (RMTg). LHb neurons that project to these targets are segregated and nonoverlapping, and this led us to consider whether they have distinct molecular phenotypes and adaptations to stress exposure. In order to capture a time-locked profile of gene expression after repeated forced swim stress, we used intersectional expression of RiboTag in rat LHb neurons and next-gen RNA sequencing to interrogate the RNAs actively undergoing translation from each of these pathways. The "translatome" in the neurons comprising these pathways was similar at baseline, but diverged after stress, especially in the neurons projecting to the RMTg. Using weighted gene co-expression network analysis, we found one module, which had an overrepresentation of genes associated with phosphoinositide 3 kinase (PI3K) signaling, comprising genes downregulated after stress in the RMTg-projecting LHb neurons. Reduced PI3K signaling in RMTg-projecting LHb neurons may be a compensatory adaptation that alters the functional balance of LHb outputs to GABAergic vs. monoaminergic neurons following repeated stress exposure., (© 2020 The Authors.)

Consumption of high fat, high sugar (western) diets is a major contributor to the current high levels of obesity. Here, we used a multidisciplinary approach to gain insight into the molecular mechanisms underlying susceptibility to diet-induced obesity (DIO). Using positron emission tomography (PET), we identified the dorsal striatum as the brain area most altered in DIO-susceptible rats and molecular studies within this region highlighted regulator of G-protein signaling 4 (Rgs4) within laser-capture micro-dissected striatonigral (SN) and striatopallidal (SP) medium spiny neurons (MSNs) as playing a key role. Rgs4 is a GTPase accelerating enzyme implicated in plasticity mechanisms of SP MSNs, which are known to regulate feeding and disturbances of which are associated with obesity. Compared to DIO-resistant rats, DIO-susceptible rats exhibited increased striatal Rgs4 with mRNA expression levels enriched in SP MSNs. siRNA-mediated knockdown of striatal Rgs4 in DIO-susceptible rats decreased food intake to levels comparable to DIO-resistant animals. Finally, we demonstrated that the human Rgs4 gene locus is associated with increased body weight and obesity susceptibility phenotypes, and that overweight humans exhibit increased striatal Rgs4 protein. Our findings highlight a novel role for involvement of Rgs4 in SP MSNs in feeding and DIO-susceptibility.

The lateral habenula (LHb) processes information about aversive experiences that contributes to the symptoms of stress disorders. Previously, we found that chemogenetic inhibition of rat LHb neurons reduced immobility in the forced swim test, but the downstream target of these neurons was not known. Using an intersectional viral vector strategy, we selectively transduced three different output pathways from the LHb by injecting AAV8-DIO-hM 4 Di into the LHb and CAV2-CRE (a retrograde viral vector) into one of the three target areas as follows: dorsal raphe nucleus (DRN), ventral tegmental area (VTA), or rostromedial tegmentum (RMTg). Using the forced swim test, we found that chemogenetic inhibition of DRN-projecting LHb neurons reduced passive coping (immobility), whereas inhibition of the other pathways did not. Chemogenetic activation of DRN-projecting neurons using hM3Dq in another cohort did not further exacerbate immobility. We next examined the impact of inhibiting DRN-projecting LHb neurons on reward sensitivity, perseverative behavior, and anxiety-like behavior using saccharin preference testing, reward-omission testing, and open-field testing, respectively. There was no effect of inhibiting any of these pathways on reward sensitivity, locomotion, or anxiety-like behavior, but inhibiting DRN-projecting LHb neurons reduced perseverative licking during reward-omission testing, whereas activating these neurons increased perseverative licking. These results support the idea that inhibiting LHb projections to the DRN provides animals with resilience during highly stressful or frustrating conditions but not under low-stress circumstances, and that inhibiting these neurons may promote persistence in active coping strategies.

Cues in the environment can elicit complex emotional states, and thereby maladaptive behavior, as a function of their ascribed value. Here we capture individual variation in the propensity to attribute motivational value to reward-cues using the sign-tracker/goal-tracker animal model. Goal-trackers attribute predictive value to reward-cues, and sign-trackers attribute both predictive and incentive value. Using chemogenetics and microdialysis, we show that, in sign-trackers, stimulation of the neuronal pathway from the prelimbic cortex (PrL) to the paraventricular nucleus of the thalamus (PVT) decreases the incentive value of a reward-cue. In contrast, in goal-trackers, inhibition of the PrL-PVT pathway increases both the incentive value and dopamine levels in the nucleus accumbens shell. The PrL-PVT pathway, therefore, exerts top-down control over the dopamine-dependent process of incentive salience attribution. These results highlight PrL-PVT pathway as a potential target for treating psychopathologies associated with the attribution of excessive incentive value to reward-cues, including addiction., Competing Interests: PC, IC, YK, AP, BK, SL, JN, SF, LS, MS, SF No competing interests declared, (© 2019, Campus et al.)

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.

5-HT 1B receptors modulate synaptic serotonin (5-HT) levels and play a significant role in the regulation of emotional behaviors. These receptors are Gα i/o -coupled and inhibit adenylyl cyclase but have also been reported to activate MAP kinases; however, the details of signaling cascades downstream of 5-HT 1B receptor activation remain unclear, particularly in neuronal cells. We generated a stable 5-HT 1B receptor-expressing Neuro2A (N2A-1B) neuronal cell line and demonstrate that activation of these receptors by the selective 5-HT 1B agonist CP-94253 results in activation of ERK1/2 but not of other closely related MAP kinases. Phosphoproteomics revealed four novel phosphorylation sites on the third intracellular loop of the 5-HT 1B receptor, and mutations of serine-256 and serine-291 to alanine led to reduced levels of ERK1/2 phosphorylation following receptor activation. Inhibition of Gα i/o signaling with pertussis toxin, as well as MEK1/2 inhibition with U0126, also reduced 5-HT 1B -mediated ERK1/2 phosphorylation. Finally, we found that knockout of either β-arrestin 1 or β-arrestin 2 prevented 5-HT 1B -mediated phosphorylation of ERK1/2. Taken together, these results show that 5-HT 1B receptor activation selectively induces ERK1/2 activation through both the Gα i subunit and β-arrestin proteins. This work elucidates the signal transduction pathway of 5-HT 1B receptors, as well as key phosphorylation sites within the receptor that modulate ERK1/2 activation, and further characterizes the intracellular mechanisms that underlie 5-HT 1B receptor function.

Rodents engage in social communication through a rich repertoire of ultrasonic vocalizations (USVs). Recording and analysis of USVs has broad utility during diverse behavioral tests and can be performed noninvasively in almost any rodent behavioral model to provide rich insights into the emotional state and motor function of the test animal. Despite strong evidence that USVs serve an array of communicative functions, technical and financial limitations have been barriers for most laboratories to adopt vocalization analysis. Recently, deep learning has revolutionized the field of machine hearing and vision, by allowing computers to perform human-like activities including seeing, listening, and speaking. Such systems are constructed from biomimetic, "deep", artificial neural networks. Here, we present DeepSqueak, a USV detection and analysis software suite that can perform human quality USV detection and classification automatically, rapidly, and reliably using cutting-edge regional convolutional neural network architecture (Faster-RCNN). DeepSqueak was engineered to allow non-experts easy entry into USV detection and analysis yet is flexible and adaptable with a graphical user interface and offers access to numerous input and analysis features. Compared to other modern programs and manual analysis, DeepSqueak was able to reduce false positives, increase detection recall, dramatically reduce analysis time, optimize automatic syllable classification, and perform automatic syntax analysis on arbitrarily large numbers of syllables, all while maintaining manual selection review and supervised classification. DeepSqueak allows USV recording and analysis to be added easily to existing rodent behavioral procedures, hopefully revealing a wide range of innate responses to provide another dimension of insights into behavior when combined with conventional outcome measures.

Parkinson's disease (PD) is primarily associated with the degeneration of midbrain dopamine neurons, but it is now appreciated that pathological processes like Lewy-body inclusions and cell loss affect several other brain regions, including the central lateral (CL) and centromedian/parafascicular (CM/PF) thalamic regions. These thalamic glutamatergic neurons provide a non-cortical excitatory input to the dorsal striatum, a major projection field of dopamine neurons. To determine how thalamostriatal signaling may contribute to cognitive and motor abnormalities found in PD, we used a viral vector approach to generate mice with loss of thalamostriatal glutamate signaling specifically restricted to the dorsal striatum (CAV2 Cre - Slc17a6 lox/lox mice). We measured motor function and behaviors corresponding to cognitive domains (visuospatial function, attention, executive function, and working memory) affected in PD. CAV2 Cre - Slc17a6 lox/lox mice were impaired in motor coordination tasks such as the rotarod and beam-walk tests compared with controls (CAV2 Cre - Slc17a6 +/+ mice). They did not demonstrate much cognitive impairment in the Morris water maze or a water U-maze, but had slower processing reaction times in those tests and in a two-way active avoidance task. These mice could model an aspect of bradyphrenia, the slowness of thought that is often seen in patients with PD and other neurological disorders., Competing Interests: The authors declare no competing interests.

5-HT 6 (serotonin) receptors are promising targets for a variety of neuropsychiatric disorders and have been linked to several cellular signaling cascades. Endogenous 5-HT 6 receptors are restricted to the primary neuronal cilium, a small sensory organelle stemming from the cell body that receives numerous extrasynaptic signals. Inhibition of 5-HT 6 receptors decreases cilia length in primary neuronal cultures, but the signaling mechanisms involved are still unclear. Intense overexpression of exogenous 5-HT 6 receptors increases the probability for receptors to localize outside the primary cilium and have been associated with changes in cilia morphology and dendritic outgrowth. In the present study, we explore the role of 5-HT 6 R rescue on neuronal morphology in primary neuronal cultures from 5-HT 6 R-KO mice, at the same time maintaining a more physiologic level of expression, wherein the receptor localizes to cilia in 80%-90% of neurons (similar to endogenous 5-HT 6 R localization). We found that rescue of 5-HT 6 R expression is sufficient to increase cilia length and dendritic outgrowth, but primarily in neurons in which the receptor is located exclusively in the primary cilia. Additionally, we found that expression of 5-HT 6 R mutants deficient in agonist-stimulated cAMP or without the predicted Fyn kinase binding domain maintained constitutive activity for stimulating cAMP and still increased the length of cilia, and that the proposed Fyn kinase domain was required for stimulating dendritic outgrowth. These findings highlight the complexity of 5-HT 6 R function and localization, particularly with the use of exogenous overexpression, and provide greater understanding and potential mechanisms for 5-HT 6 R drug therapies., (Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.)

Drug addiction is a chronic disease that is shaped by alterations in neuronal function within the cortical-basal ganglia-thalamic circuit. However, our understanding of how this circuit regulates drug-seeking remains incomplete, and relapse rates remain high. The midline thalamic nuclei are an integral component of the cortical-basal ganglia-thalamic circuit and are poised to mediate addiction behaviors, including relapse. It is surprising that little research has examined the contribution of midline thalamic nuclei and their efferent projections in relapse. To address this, we expressed inhibitory, G i/o -coupled DREADDs (Designer Receptors Exclusively Activated by Designer Drugs) in a subset of the midline thalamic nuclei or in midline thalamic nuclei neurons projecting to either the nucleus accumbens or the amygdala. We examined the effect of transiently decreasing activity of these neuronal populations on cue-induced and cocaine-primed reinstatement of cocaine-seeking. Reducing activity of midline thalamic nuclei neurons attenuated both cue-induced and cocaine-primed reinstatement, but had no effect on cue-induced reinstatement of sucrose-seeking or locomotor activity. Interestingly, attenuating activity of efferent projections from the anterior portion of midline thalamic nuclei to the nucleus accumbens blocked cocaine-primed reinstatement but enhanced cue-induced reinstatement. Decreasing activity of efferent projections from either the posterior midline thalamic nuclei to the nucleus accumbens or the midline thalamic nuclei to amygdala had no effect. These results reveal a novel contribution of subsets of midline thalamic nuclei neurons in drug-seeking behaviors and suggest that modulation of midline thalamic nuclei activity may be a promising therapeutic target for preventing relapse., (© 2017 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Several phosphorylation signaling pathways have been implicated in the pathogenesis of epilepsy arising from both genetic causes and acquired insults to the brain. Identification of dysfunctional signaling pathways in epilepsy may provide novel targets for antiepileptic therapies. We previously described a deficit in phosphorylation signaling mediated by p38 mitogen-activated protein kinase (p38 MAPK) that occurs in an animal model of temporal lobe epilepsy, and that produces neuronal hyperexcitability measured in vitro. We asked whether in vivo pharmacological manipulation of p38 MAPK activity would influence seizure frequency in chronically epileptic animals. Administration of a p38 MAPK inhibitor, SB203580, markedly worsened spontaneous seizure frequency, consistent with prior in vitro results. However, anisomycin, a non-specific p38 MAPK activator, significantly increased seizure frequency. We hypothesized that this unexpected result was due to activation of a related MAPK, c-Jun N-terminal kinase (JNK). Administration of JNK inhibitor SP600125 significantly decreased seizure frequency in a dose-dependent manner without causing overt behavioral abnormalities. Biochemical analysis showed increased JNK expression and activity in untreated epileptic animals. These results show for the first time that JNK is hyperactivated in an animal model of epilepsy, and that phosphorylation signaling mediated by JNK may represent a novel antiepileptic target., (Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.)

The 5-HT 6 receptor has been implicated in a variety of cognitive processes including habitual behaviors, learning, and memory. It is found almost exclusively in the brain, is expressed abundantly in striatum, and localizes to neuronal primary cilia. Primary cilia are antenna-like, sensory organelles found on most neurons that receive both chemical and mechanical signals from other cells and the surrounding environment; however, the effect of 5-HT 6 receptor function on cellular morphology has not been examined. We confirmed that 5-HT 6 receptors were localized to primary cilia in wild-type (WT) but not 5-HT 6 knockout (5-HT 6 KO) in both native mouse brain tissue and primary cultured striatal neurons then used primary neurons cultured from WT or 5-HT 6 KO mice to study the function of these receptors. Selective 5-HT 6 antagonists reduced cilia length in neurons cultured from wild-type mice in a concentration and time-dependent manner without altering dendrites, but had no effect on cilia length in 5-HT 6 KO cultured neurons. Varying the expression levels of heterologously expressed 5-HT 6 receptors affected the fidelity of ciliary localization in both WT and 5-HT 6 KO neurons; overexpression lead to increasing amounts of 5-HT 6 localization outside of the cilia but did not alter cilia morphology. Introducing discrete mutations into the third cytoplasmic loop of the 5-HT 6 receptor greatly reduced, but did not entirely eliminate, trafficking of the 5-HT 6 receptor to primary cilia. These data suggest that blocking 5-HT 6 receptor activity reduces the length of primary cilia and that mechanisms that regulate trafficking of 5-HT 6 receptors to cilia are more complex than previously thought., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The nucleus accumbens (NAc) in the ventral striatum integrates many neurochemical inputs including dopamine and serotonin projections from midbrain nuclei to modulate drug reward. Although D1 and D2 dopamine receptors are differentially expressed in the direct and indirect pathway medium spiny neurons (dMSNs and iMSNs, respectively), 5-HT6 receptors are expressed in both pathways, more strongly than anywhere else in the brain, and are an intriguing target for neuropsychiatric disorders. In the present study, we used viral vectors utilizing dynorphin or enkephalin promoters to drive expression of 5-HT6 receptors or green fluorescent protein (GFP) selectively in the dMSNs or iMSNs of the NAc shell. Rats were then trained to self-administer cocaine. Increased 5-HT6 receptor expression in dMSNs did not change any parameter of cocaine self-administration measured. However, increasing 5-HT6 receptors in iMSNs reduced the amount of cocaine self-administered under fixed-ratio schedules, especially at low doses, increased the time to the first response and the length of the inter-infusion interval, but did not alter motivation as measured by progressive ratio 'break point' analysis. Modeling of cocaine pharmacokinetics in NAc showed that increased 5-HT6 receptors in iMSNs reduced the rat's preferred tissue cocaine concentration at each dose. Finally, increased 5-HT6 receptors in iMSNs facilitated conditioned place preference for a low dose of cocaine. We conclude that 5-HT6 receptors in iMSNs of NAcSh increase the sensitivity to the reinforcing properties of cocaine, particularly at low doses, suggesting that these receptors may be a therapeutic target for the treatment of cocaine addiction.

The medial prefrontal cortex (mPFC) and nucleus accumbens (NAc) are both integral components of the corticobasal ganglia-thalamic circuitry that regulates addiction-related behaviors. However, the role of afferent inputs from mPFC to NAc in these behaviors is unclear. To address this, we used a Cre-recombinase-dependent viral vector approach to express G(i/o)-coupled DREADDs (designer receptors exclusively activated by designer drugs) selectively in mPFC neurons projecting to the NAc and examined the consequences of attenuating activity of these neurons on the induction of amphetamine sensitization and on drug taking and drug seeking during cocaine self-administration. Surprisingly, decreasing mPFC afferent activity to the NAc only transiently reduced locomotor sensitization and had no effect on drug taking during cocaine self-administration. However, inhibiting corticostriatal afferent activity during sensitization subsequently enhanced conditioned responding. In addition, this manipulation during drug self-administration resulted in slower rates of extinction and increased responding during drug prime-induced reinstatement-an effect that was normalized by inhibiting these corticostriatal afferents immediately before the drug prime. These results suggest that dampening cortical control over the NAc during drug exposure may lead to long-term changes in the ability of drugs and associated stimuli to drive behavior that has important implications for guiding treatments to prevent relapse.

Medium spiny neurons (MSNs) constitute 95% of neurons in the dorsal striatum subdivided into direct (striatonigral) and indirect (striatopallidal) pathways. Whereas D1 and D2 receptors and several neuropeptides, including dynorphin and enkephalin, are differentially expressed in these neurons, 5-hydroxytryptamine 6 receptors (5-HT6) are expressed in both pathways. Previous results demonstrate that concurrent 5-HT6 receptor overexpression in MSNs of both pathways in the dorsomedial striatum (DMS) interferes with instrumental learning and that 5-HT6 overexpression in the dorsolateral striatum (DLS) relieves rats from inflexible habitual behaviors. We hypothesized that 5-HT6 receptor-mediated co-activation of both pathways interferes with the differential activation/inhibition of direct/indirect pathways by dopamine. To test this idea, we cloned novel viral vectors to selectively overexpress 5-HT6 receptors in direct or indirect pathway MSNs to deconstruct their role in modulating instrumental learning and habitual responding. We found that increasing 5-HT6 receptor expression in either direct or indirect pathway MSNs of the posterior DMS selectively enhanced or impaired initial acquisition of a discrete instrumental learning task respectively, though all rats were ultimately able to learn the task. In a separate set of experiments, 5-HT6 receptor overexpression in indirect pathway MSNs of the DLS facilitated behavioral flexibility in rats overtrained on a repetitive pressing task using a variable interval schedule of reinforcement, during an omission contingency training session and subsequent probe testing. Together these findings further the notion that 5-HT6 signaling causes balanced activation of opposing MSN pathways by serotonin in sub-regions of the dorsal striatum allowing for more reflective modalities of behavior., (Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.)

Located in the nerve terminals of serotonergic neurons, 5-HT1B autoreceptors are poised to modulate synaptic 5-HT levels with precise temporal and spatial control, and play an important role in various emotional behaviors. This study characterized two novel, complementary viral vector strategies to investigate the contribution of 5-HT1B autoreceptors to fear expression, displayed as freezing, during contextual fear conditioning. Increased expression of 5-HT1B autoreceptors throughout the brain significantly decreased fear expression in both wild-type (WT) and 5-HT1B knockout (1BKO) mice when receptor levels were increased with a cell-type-specific herpes simplex virus (HSV) vector injected into the dorsal raphe nucleus (DRN). Additional studies used an intersectional viral vector strategy, in which an adeno-associated virus containing a double-floxed inverted sequence for the 5-HT1B receptor (AAV-DIO-1B) was combined with the retrogradely transported canine adenovirus-2 expressing Cre (CAV-Cre) in order to increase 5-HT1B autoreceptor expression only in neurons projecting from the DRN to the amygdala. Surprisingly, selective expression of 5-HT1B autoreceptors in just this circuit led to an increase in fear expression in WT, but not 1BKO, mice. These results suggest that activation of 5-HT1B autoreceptors throughout the brain may have an overall effect of attenuating fear expression, but activation of subsets of 5-HT1B autoreceptors in particular brain regions, reflecting distinct projections of serotonergic neurons from the DRN, may have disparate contributions to the ultimate response., (Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.)

Primary neuronal cultures are a useful tool for measuring pharmacological- and transgene-regulated gene expression; however, accurate measurements can be confounded by heterogeneous cell types and inconsistent transfection efficiency. Here we describe our adaptation of a ribosomal capture strategy that was designed to be used in transgenic mice expressing tagged ribosomal subunits (RiboTag) in specific cell types, thereby allowing measurement of translating RNAs from desired cell types within complex tissues. Using this strategy we were able to isolate and analyze neuron-specific RNA despite the presence of glia by co-transfecting experimental plasmids with plasmids that selectively express RiboTag in neurons. RiboTag immunoprecipitation was capable of recovering high integrity RNA from small numbers of transfected cells that can then be interrogated by a variety of methods (e.g., RT-qPCR, PCR array, RNA-Seq) and compared with basal RNA expression of the entire culture. Additionally, we demonstrate how co-transfection of RiboTag with small hairpin RNA (shRNA) constructs can validate and accurately assess the degree of gene expression knockdown, and how RiboTag can be used to measure receptor-mediated gene regulation with transiently expressed designer receptors exclusively activated by designer drugs (DREADDs). RiboTag co-transfection represents a convenient and powerful tool to isolate RNA from a specific subset of cultured cells with a variety of applications for experiments in vitro.

Drug addiction is characterized by compulsive drug-seeking and drug-taking, and a high propensity for relapse. Although the brain regions involved in regulating addiction processes have long been identified, the ways in which individual cell types govern addiction behaviors remain elusive. New technologies for modulating the activity of defined cell types have recently emerged that are allowing us to address these important questions. Here, we review how one such technology, DREADDs (Designer Receptors Exclusively Activated by Designer Drugs), can be used to refine our knowledge of addiction circuitry. These engineered receptors modulate cellular activity by acting on G protein coupled signaling cascades and in this review we pay particular attention to how this slower-onset modulation preferentially regulates behaviors that develop over time.

The ability to map the functional connectivity of discrete cell types in the intact mammalian brain during behavior is crucial for advancing our understanding of brain function in normal and disease states. We combined designer receptor exclusively activated by designer drug (DREADD) technology and behavioral imaging with μPET and [18F]fluorodeoxyglucose (FDG) to generate whole-brain metabolic maps of cell-specific functional circuits during the awake, freely moving state. We have termed this approach DREADD-assisted metabolic mapping (DREAMM) and documented its ability in rats to map whole-brain functional anatomy. We applied this strategy to evaluating changes in the brain associated with inhibition of prodynorphin-expressing (Pdyn-expressing) and of proenkephalin-expressing (Penk-expressing) medium spiny neurons (MSNs) of the nucleus accumbens shell (NAcSh), which have been implicated in neuropsychiatric disorders. DREAMM revealed discrete behavioral manifestations and concurrent engagement of distinct corticolimbic networks associated with dysregulation of Pdyn and Penk in MSNs of the NAcSh. Furthermore, distinct neuronal networks were recruited in awake versus anesthetized conditions. These data demonstrate that DREAMM is a highly sensitive, molecular, high-resolution quantitative imaging approach.

Negative affect is critical for conferring vulnerability to opiate addiction as reflected by the high comorbidity of opiate abuse with major depressive disorder (MDD). Rodent models implicate amygdala prodynorphin (Pdyn) as a mediator of negative affect; however, evidence of PDYN involvement in human negative affect is limited. Here, we found reduced PDYN mRNA expression in the postmortem human amygdala nucleus of the periamygdaloid cortex (PAC) in both heroin abusers and MDD subjects. Similar to humans, rats that chronically self-administered heroin had reduced Pdyn mRNA expression in the PAC at a time point associated with a negative affective state. Using the in vivo functional imaging technology DREAMM (DREADD-assisted metabolic mapping, where DREADD indicates designer receptors exclusively activated by designer drugs), we found that selective inhibition of Pdyn-expressing neurons in the rat PAC increased metabolic activity in the extended amygdala, which is a key substrate of the extrahypothalamic brain stress system. In parallel, PAC-specific Pdyn inhibition provoked negative affect-related physiological and behavioral changes. Altogether, our translational study supports a functional role for impaired Pdyn in the PAC in opiate abuse through activation of the stress and negative affect neurocircuitry implicated in addiction vulnerability.

5-HT1B receptors are densely expressed on terminals of medium spiny neurons projecting from the nucleus accumbens shell (NAccSh) to the ventral tegmental area, where 5-HT1B receptors modulate GABA release directly, and firing of dopaminergic neurons indirectly. While interactions between NAccSh 5-HT1B receptors and stress have been reported in early stages of psychostimulant-induced neuroadaptations, specifically psychomotor sensitization, the effect of this interaction on later stages of drug seeking is currently unknown. Here, we examined the effect of herpes simplex virus (HSV)-mediated overexpression of NAccSh 5-HT1B receptors on reinstatement of cocaine seeking induced by exposure to stress or a cocaine prime. Rats were trained to self-administer cocaine (0.75 mg/kg/infusion) and the operant response was extinguished. Rats were then injected with viral vector expressing 5-HT1B and green fluorescent protein (GFP) or GFP alone into the NAccSh. The effect of 5-HT1B receptor overexpression was assessed on reinstatement induced by intermittent footshock (0.5 mA for 15 min) or a cocaine prime (10mg/kg, ip). Results indicate that NAccSh 5-HT1B receptor overexpression had no effect on footshock reinstatement while significantly decreasing cocaine priming-induced reinstatement. We also found that NAccSh overexpression of 5-HT1B receptors had no effect on saccharin intake following social defeat stress. These results suggest that the efficacy of pharmacological agents targeting 5-HT1B receptors for the treatment of cocaine relapse will depend largely on the nature of the reinstating stimulus. Taken together with previous results, it appears that NAccSh 5-HT1B receptors influence stress responses in early, but not in the later stages of psychostimulant-induced neuroadaptations., (© 2013.)

The dorsal striatum has been implicated in reward-based decision making, but the role played by specific striatal circuits in these processes is essentially unknown. Using cell phenotype-specific viral vectors to express engineered G-protein-coupled DREADD (designer receptors exclusively activated by designer drugs) receptors, we enhanced Gi/o- or Gs-protein-mediated signaling selectively in direct-pathway (striatonigral) neurons of the dorsomedial striatum in Long-Evans rats during discrete periods of training of a high versus low reward-discrimination task. Surprisingly, these perturbations had no impact on reward preference, task performance, or improvement of performance during training. However, we found that transiently increasing Gi/o signaling during training significantly impaired the retention of task strategies used to maximize reward obtainment during subsequent preference testing, whereas increasing Gs signaling produced the opposite effect and significantly enhanced the encoding of a high-reward preference in this decision-making task. Thus, the fact that the endurance of this improved performance was significantly altered over time-long after these neurons were manipulated-indicates that it is under bidirectional control of canonical G-protein-mediated signaling in striatonigral neurons during training. These data demonstrate that cAMP-dependent signaling in direct-pathway neurons play a well-defined role in reward-related behavior; that is, they modulate the plasticity required for the retention of task-specific information that is used to improve performance on future renditions of the task.

The lateral habenula (LHb) is part of the habenular complex in the dorsal diencephalon. The LHb is an important regulator of several neurotransmitter systems in the midbrain; disturbances in this regulation may contribute to mood disorders, abnormalities in cognition, drive, and addiction. Owing to the critical role this nucleus plays in modulating activity of midbrain nuclei, there has been a rapid increase in studies targeting the LHb in the recent years. In this review, we describe studies using traditional approaches to elucidate the function of this brain region, such as lesion, electrical and chemical stimulation, electrophysiology and in vivo microdialysis. We have selected a variety of illustrative studies to discuss each of these methods. Next, we describe studies using methods that are based upon recent advances in molecular biology techniques including recent results from our laboratory using the Designer Receptor Exclusively Activated by Designer Drug (DREADD) technology. Using a Gi/o-coupled DREADD, we found that inhibition of the LHb reduces depression-like behavior in the forced swim test in a manner that suggests enhanced serotonergic activity. The emerging picture reveals that the LHb is likely to be a critical node in the network of subcortical nuclei that regulate aversive learning, motivation, stress responses, etc. We describe how recently developed methods have advanced the study of the LHb and are leading research of this brain region in promising new directions. This article is part of a Special Issue entitled Optogenetics (7th BRES)., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Activation of the dynorphin/κ-opioid receptor (KOR) system by repeated stress exposure or agonist treatment produces place aversion, social avoidance, and reinstatement of extinguished cocaine place preference behaviors by stimulation of p38α MAPK, which subsequently causes the translocation of the serotonin transporter (SERT, SLC6A4) to the synaptic terminals of serotonergic neurons. In the present study we extend those findings by showing that stress-induced potentiation of cocaine conditioned place preference occurred by a similar mechanism. In addition, SERT knock-out mice did not show KOR-mediated aversion, and selective reexpression of SERT by lentiviral injection into the dorsal raphe restored the prodepressive effects of KOR activation. Kinetic analysis of several neurotransporters demonstrated that repeated swim stress exposure selectively increased the V(max) but not K(m) of SERT without affecting dopamine transport or the high-capacity, low-affinity transporters. Although the serotonergic neurons in the dorsal raphe project throughout the forebrain, a significant stress-induced increase in cell-surface SERT expression was only evident in the ventral striatum, and not in the dorsal striatum, hippocampus, prefrontal cortex, amygdala, or dorsal raphe. Stereotaxic microinjections of the long-lasting KOR antagonist norbinaltorphimine demonstrated that local KOR activation in the nucleus accumbens, but not dorsal raphe, mediated this stress-induced increase in ventral striatal surface SERT expression. Together, these results support the hypothesis that stress-induced activation of the dynorphin/KOR system produces a transient increase in serotonin transport locally in the ventral striatum that may underlie some of the adverse consequences of stress exposure, including the potentiation of the rewarding effects of cocaine.

Serotonin-1B (5-HT(1B) ) autoreceptors are located in serotonin (5-HT) terminals, along with serotonin transporters (SERT), and play a critical role in autoregulation of serotonergic neurotransmission and are implicated in disorders of serotonergic function, particularly emotional regulation. SERT modulates serotonergic neurotransmission by high-affinity reuptake of 5-HT. Alterations in SERT activity are associated with increased risk for depression and anxiety. Several neurotransmitter receptors are known to regulate SERT K(m) and V(max) , and previous work suggests that 5-HT(1B) autoreceptors may regulate 5-HT reuptake, in addition to modulating 5-HT release and synthesis. We used rotating disk electrode voltammetry to investigate 5-HT(1B) autoreceptor regulation of SERT-mediated 5-HT uptake into synaptosomes. The selective 5-HT(1B) antagonist SB224289 decreased SERT activity in synaptosomes prepared from wild-type but not 5-HT(1B) knockout mice, whereas SERT uptake was enhanced after pretreatment with the selective 5-HT(1B) agonist CP94253. Furthermore, SERT activity varies as a function of 5-HT(1B) receptor expression-specifically, genetic deletion of 5-HT(1B) decreased SERT function, while viral-mediated overexpression of 5-HT(1B) autoreceptors in rat raphe neurons increased SERT activity in rat hippocampal synaptosomes. Considered collectively, these results provide evidence that 5-HT(1B) autoreceptors regulate SERT activity. Because SERT clearance rate varies as a function of 5-HT(1B) autoreceptor expression levels and is modulated by both activation and inhibition of 5-HT(1B) autoreceptors, this dynamic interaction may be an important mechanism of serotonin autoregulation with therapeutic implications., (2012 Wiley Periodicals, Inc)

Background: The role of serotonin-1B receptors (5-HT(1B)Rs) in modulating cocaine abuse-related behaviors has been controversial due to discrepancies between pharmacological and gene knockout approaches and opposite influences on cocaine self-administration versus cocaine-seeking behavior. We hypothesized that modulation of these behaviors via 5-HT(1B)Rs in the mesolimbic pathway may vary depending on the stage of the addiction cycle., Methods: To test this hypothesis, we examined the effects of increasing 5-HT(1B)R production by microinfusing a viral vector expressing either green fluorescent protein and 5-HT(1B)R or green fluorescent protein alone into the medial nucleus accumbens shell of rats either during maintenance of cocaine self-administration (i.e., active drug use) or during protracted withdrawal., Results: 5-HT(1B)R receptor gene transfer during maintenance shifted the dose-response curve for cocaine self-administration upward and to the left and increased breakpoints and cocaine intake on a progressive ratio schedule, consistent with enhanced reinforcing effects of cocaine. In contrast, following 21 days of forced abstinence, 5-HT(1B)R gene transfer attenuated breakpoints and cocaine intake on a progressive ratio schedule of reinforcement, as well as cue- and cocaine-primed reinstatement of cocaine-seeking behavior., Conclusions: This unique pattern of effects suggests that mesolimbic 5-HT(1B)Rs differentially modulate cocaine abuse-related behaviors, with a facilitative influence during periods of active drug use, in striking contrast to an inhibitory influence during protracted withdrawal. These findings suggest that targeting 5-HT(1B)Rs may lead to a novel treatment for cocaine dependence and that the therapeutic efficacy of these treatments may vary depending on the stage of the addiction cycle., (Copyright © 2012 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.)

Chronic stress contributes to vulnerability for depression and drug addiction. The function of the serotonergic system has been found to be modified by chronic stress and these changes may play an important role in stress-related relapses to drug craving. The 5-HT(1B) receptor is expressed in nucleus accumbens (NAc) projection neurons and modulates drug reward mechanisms and there is evidence suggesting that stress alters the regulation and function of these receptors. To examine the role of these receptors in integrating the effects of stress on reward mechanisms, we examined whether chronic or acute social defeat stress (SDS) regulates 5-HT(1B) mRNA in dorsal and ventral striatum, regions that are critical for integrating the effects of environmental stressors on reward motivated behavior. In addition, 5-HT(1B) mRNA regulation in response to another acute stressor, inescapable tailshock, was measured. Our results indicate that intermittent and daily SDS procedures attenuated body weight gain, induced adrenal hypertrophy, and reduced the preference for saccharin, a sweet solution preferred by normal rats. There was a trend for daily, but not intermittent SDS to increase 5-HT(1B) receptor mRNA levels in nucleus accumbens. Therefore, in the next experiment, we examined daily SDS in greater detail and found that it increased 5-HT(1B) receptor mRNA expression in rostral nucleus accumbens shell, an area especially associated with reward functions. Neither acute SDS, nor acute tailshock stress had a significant impact on 5-HT(1B) mRNA expression in the striatum. Since increased 5-HT(1B) receptor expression in nucleus accumbens shell neurons can facilitate cocaine and alcohol reward mechanisms, this adaptation in endogenous 5-HT(1B) mRNA may be involved in the SDS-associated increase in vulnerability for developing addiction., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Although many studies assert that the serotonin (5-HT) transporter (SERT) is the predominant mechanism controlling extracellular 5-HT concentrations, accumulating evidence suggests that low affinity, high capacity transport mechanisms may contribute more to 5-HT clearance than previously thought. The goal of this study was to quantify the contributions of SERT relative to other mechanisms in clearing extracellular 5-HT concentrations ranging from 50 nM to 1 μM in synaptosomes prepared from wild-type and SERT knockout mice using rotating disk electrode voltammetry. SERT inhibitors combined with decynium-22 (D-22), a blocker of several low-affinity transporters, blocked all uptake of 5-HT into synaptosomes. We found that SERT is responsible for the majority of synaptosomal uptake only at relatively low 5-HT concentrations, but comprises a diminishing proportion of 5-HT clearance when extracellular 5-HT increases above 100 nM. The effect of D-22 was similar in wild-type and SERT knockout synaptosomes. Thus, there was no evidence of upregulation of low-affinity mechanisms in knockout mice across the concentrations of 5-HT tested. These are surprising results, in light of the prevailing view that SERT is the primary uptake mechanism for extracellular 5-HT at physiological concentrations. We conclude that non-SERT mediated 5-HT uptake is substantial even at modest 5-HT concentrations. These findings, in conjunction with other studies, have important implications for understanding serotonergic disorders and may explain the variable efficacy and stability of patients' responses to antidepressants, such as the selective serotonin reuptake inhibitors., (Copyright © 2011 Wiley-Liss, Inc.)

Context: Serotonergic dysfunction is implicated in the pathogenesis of posttraumatic stress disorder (PTSD), and recent animal models suggest that disturbances in serotonin type 1B receptor function, in particular, may contribute to chronic anxiety. However, the specific role of the serotonin type 1B receptor has not been studied in patients with PTSD., Objective: To investigate in vivo serotonin type 1B receptor expression in individuals with PTSD, trauma-exposed control participants without PTSD (TC), and healthy (non-trauma-exposed) control participants (HC) using positron emission tomography and the recently developed serotonin type 1B receptor selective radiotracer [(11)C]P943., Design: Cross-sectional positron emission tomography study under resting conditions., Setting: Academic and Veterans Affairs medical centers., Participants: Ninety-six individuals in 3 study groups: PTSD (n = 49), TC (n = 20), and HC (n = 27). Main Outcome Measure  Regional [(11)C]P943 binding potential (BP(ND)) values in an a priori-defined limbic corticostriatal circuit investigated using multivariate analysis of variance and multiple regression analysis., Results: A history of severe trauma exposure in the PTSD and TC groups was associated with marked reductions in [(11)C]P943 BP(ND) in the caudate, the amygdala, and the anterior cingulate cortex. Participant age at first trauma exposure was strongly associated with low [(11)C]P943 BP(ND). Developmentally earlier trauma exposure also was associated with greater PTSD symptom severity and major depression comorbidity., Conclusions: These data suggest an enduring effect of trauma history on brain function and the phenotype of PTSD. The association of early age at first trauma and more pronounced neurobiological and behavioral alterations in PTSD suggests a developmental component in the cause of PTSD.

Maladaptive responses to stress adversely affect human behavior, yet the signaling mechanisms underlying stress-responsive behaviors remain poorly understood. Using a conditional gene knockout approach, the α isoform of p38 mitogen-activated protein kinase (MAPK) was selectively inactivated by AAV1-Cre-recombinase infection in specific brain regions or by promoter-driven excision of p38α MAPK in serotonergic neurons (by Slc6a4-Cre or ePet1-Cre) or astrocytes (by Gfap-CreERT2). Social defeat stress produced social avoidance (a model of depression-like behaviors) and reinstatement of cocaine preference (a measure of addiction risk) in wild-type mice, but not in mice having p38α MAPK selectively deleted in serotonin-producing neurons of the dorsal raphe nucleus. Stress-induced activation of p38α MAPK translocated the serotonin transporter to the plasma membrane and increased the rate of transmitter uptake at serotonergic nerve terminals. These findings suggest that stress initiates a cascade of molecular and cellular events in which p38α MAPK induces a hyposerotonergic state underlying depression-like and drug-seeking behaviors., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Neurotransmission by serotonin (5-HT) is tightly regulated by several autoreceptors that fine-tune serotonergic neurotransmission through negative feedback inhibition at the cell bodies (predominantly 5-HT(1A)) or at the axon terminals (predominantly 5-HT(1B)); however, more subtle roles for 5-HT(1D) and 5-HT(2B) autoreceptors have also been detected. This review provides an overview of 5-HT autoreceptors, focusing on their contribution in animal behavioral models of stress and emotion. Experiments targeting 5-HT autoreceptors in awake, behaving animals have generally shown that increasing autoreceptor feedback is anxiolytic and rewarding, while enhanced 5-HT function is aversive and anxiogenic; however, the role of serotonergic activity in behavioral models of helplessness is more complex. The prevailing model suggests that 5-HT autoreceptors become desensitized in response to stress exposure and antidepressant administration, two seemingly opposite manipulations. Thus there are still unresolved questions regarding the role of these receptors-and serotonin in general-in normal and pathological states., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Serotonin-6 (5-HT(6)) receptors are densely expressed in the dorsolateral striatum (DLS), a brain region linked to habits. Medications acting on the serotonergic system, including 5-HT(6) receptors, can diminish habitual and repetitive behaviors associated with clinical syndromes such as obsessive-compulsive disorder, and may have implications for addiction as well. To examine the role of 5-HT(6) receptors in the acquisition and persistence of habitual behavior, we manipulated 5-HT(6) receptor expression in the DLS with herpes simplex virus vectors in combination with different behavioral procedures; control rats received a vector expressing enhanced green fluorescent protein. In one set of experiments, rats were tested under conditions that favor the acquisition of either discrete action-outcome responding or repetitive responding; increased 5-HT(6) receptor expression in DLS did not alter learning in either paradigm. In the next experiment, rats were over-trained on fixed- then variable-interval schedules, resulting in an escalation of lever pressing over sessions far in excess of that necessary to receive sucrose pellets. After training, rats received viral vector infusion into the DLS. Subsequently, half of each group underwent an omission contingency training session in which they received reinforcement for refraining from pressing the lever, while the other half served as yoked controls. A probe session under extinction conditions was performed the following day. Only rats that received both the 5-HT(6) vector and omission contingency training showed reduced lever pressing during the probe session. These results suggest that increasing 5-HT(6) receptor signaling in the DLS facilitates behavioral flexibility in the face of changing contingencies., (© 2011 The Authors. European Journal of Neuroscience © 2011 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.)

Background: Serotonin 1B (5-HT(1B)) autoreceptors regulate release of serotonin from terminals of dorsal raphe nucleus (DRN) projections. Expression of 5-HT(1B) in the DRN inversely correlates with behavioral measures of emotion, and viral-mediated overexpression of 5-HT(1B) receptors in the middle DRN inversely reduces measures of fear and anxiety in unstressed rats. Because the caudal subregion of the DRN is important in translating stress into emotional dysregulation, we explored behavioral functions of 5-HT(1B) autoreceptors in the caudal DRN., Methods: We manipulated 5-HT(1B) autoreceptor function in rats using either viral-mediated gene transfer into the caudal DRN or systemic injections of the 5-HT(1B) agonist 3-(1,2,5,6-tetrahydro-4-pyridyl)-5-propoxypyrrolo[3,2-b]pyridine (CP-94253). Rats were tested in forced swim test, open field test, and contextual fear conditioning., Results: Overexpression of 5-HT(1B) in the caudal DRN increased swimming in the forced swim test. It did not alter locomotion or thigmotaxis in the open field test but did reduce conditioned freezing. Freezing was reduced when 5-HT(1B) overexpression was present only during testing but not training. The CP-94253 exerted an inverted U-shaped dose response curve on conditioned freezing, with most pronounced effects seen at 1 mg/kg. At this dose, CP-94253 administered before a fear retention test reduced freezing both during that session and in subsequent drug-free testing, but only when drug was paired with re-exposure to the fear context., Conclusions: The 5-HT(1B) autoreceptors originating in the caudal DRN regulate behavioral expression of helplessness and fear. Because systemic pharmacologic treatment with a 5-HT(1B) agonist facilitates reductions in fear, 5-HT(1B) receptors may be a target for the treatment of certain anxiety disorders., (Copyright © 2011 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.)