How omics is revealing new roles for glia in addiction.

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.

International Union of Basic and Clinical Pharmacology. CX. Classification of Receptors for 5-hydroxytryptamine; Pharmacology and Function.

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.

Rapid appearance of negative emotion during oral fentanyl self-administration in male and female rats.

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.

Sequencing the serotonergic neuron translatome reveals a new role for Fkbp5 in stress.

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.

Striatal Rgs4 regulates feeding and susceptibility to diet-induced obesity.

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.

Effect of chemogenetic inhibition of lateral habenula neuronal activity on cocaine- and food-seeking behaviors in the rat.

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 hM4 Di (a Gi/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 hM4 Di 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. hM4 Di -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 hM4 Di - 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.

Restoration of Physiological Expression of 5-HT6 Receptor into the Primary Cilia of Null Mutant Neurons Lengthens Both Primary Cilia and Dendrites.

5-HT6 (serotonin) receptors are promising targets for a variety of neuropsychiatric disorders and have been linked to several cellular signaling cascades. Endogenous 5-HT6 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-HT6 receptors decreases cilia length in primary neuronal cultures, but the signaling mechanisms involved are still unclear. Intense overexpression of exogenous 5-HT6 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-HT6R rescue on neuronal morphology in primary neuronal cultures from 5-HT6R-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-HT6R localization). We found that rescue of 5-HT6R 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-HT6R 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-HT6R function and localization, particularly with the use of exogenous overexpression, and provide greater understanding and potential mechanisms for 5-HT6R drug therapies.

Chemogenetic inhibition reveals midline thalamic nuclei and thalamo-accumbens projections mediate cocaine-seeking in rats.

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, Gi/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.

Direct-pathway striatal neurons regulate the retention of decision-making strategies.

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.

Stress produces aversion and potentiates cocaine reward by releasing endogenous dynorphins in the ventral striatum to locally stimulate serotonin reuptake.

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.

Rapid appearance of negative emotion during oral fentanyl self-administration in male and female rats.

Opioid use disorder has become an epidemic in the United States, fueled 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 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 self-administration also revealed individual and sex differences that are essential to studying substance use risk propensity. During a behavioral 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 analyzed thousands of 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 oral fentanyl taking, reflecting a transition to negative reinforcement. Using fiber 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.

5-HT(1B) autoreceptor regulation of serotonin transporter activity in synaptosomes.

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.

A cAMP-Related Gene Network in Microglia Is Inversely Regulated by Morphine Tolerance and Withdrawal.

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 hM4Di 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.

Stress decreases serotonin tone in the nucleus accumbens in male mice to promote aversion and potentiate cocaine preference via decreased stimulation of 5-HT1B receptors.

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-HT1B receptors potentiated subsequent cocaine CPP. 5-HT1B is known to be expressed on 5-HT terminals in NAc, and 5-HT1B transcript was also detected in Pdyn+, Adora2a+ and ChAT+ (markers for direct pathway, indirect pathway, and cholinergic interneurons, respectively). Following stress exposure, 5-HT1B transcript was selectively elevated in Pdyn+ cells of the mNAc. These findings suggest that Dyn/KOR regulates serotonin activation of 5HT1B receptors within the mNAc and dynamically controls stress response, affect, and drug reward.

PACAP-expressing neurons in the lateral habenula diminish negative emotional valence.

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 hM3 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.

Increased expression of 5-HT6 receptors in dorsolateral striatum decreases habitual lever pressing, but does not affect learning acquisition of simple operant tasks in rats.

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.

The contribution of low-affinity transport mechanisms to serotonin clearance in synaptosomes.

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.

Nuclear factor kappa B signaling regulates neuronal morphology and cocaine reward.

Although chronic cocaine-induced changes in dendritic spines on nucleus accumbens (NAc) neurons have been correlated with behavioral sensitization, the molecular pathways governing these structural changes, and their resulting behavioral effects, are poorly understood. The transcription factor, nuclear factor kappa B (NFkappaB), is rapidly activated by diverse stimuli and regulates expression of many genes known to maintain cell structure. Therefore, we evaluated the role of NFkappaB in regulating cocaine-induced dendritic spine changes on medium spiny neurons of the NAc and the rewarding effects of cocaine. We show that chronic cocaine induces NFkappaB-dependent transcription in the NAc of NFkappaB-Lac transgenic mice. This induction of NFkappaB activity is accompanied by increased expression of several NFkappaB genes, the promoters of which show chromatin modifications after chronic cocaine exposure consistent with their transcriptional activation. To study the functional significance of this induction, we used viral-mediated gene transfer to express either a constitutively active or dominant-negative mutant of Inhibitor of kappa B kinase (IKKca or IKKdn), which normally activates NFkappaB signaling, in the NAc. We found that activation of NFkappaB by IKKca increases the number of dendritic spines on NAc neurons, whereas inhibition of NFkappaB by IKKdn decreases basal dendritic spine number and blocks the increase in dendritic spines after chronic cocaine. Moreover, inhibition of NFkappaB blocks the rewarding effects of cocaine and the ability of previous cocaine exposure to increase an animal's preference for cocaine. Together, these studies establish a direct role for NFkappaB pathways in the NAc to regulate structural and behavioral plasticity to cocaine.

Chemogenetic inhibition of lateral habenula projections to the dorsal raphe nucleus reduces passive coping and perseverative reward seeking in rats.

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-hM4Di 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.

Stress induces divergent gene expression among lateral habenula efferent pathways.

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.