William J. Giardino

Activation of the kappa opioid receptor in the dorsal raphe nucleus mediates the aversive effects of stress and reinstates drug seeking

Although stress has profound effects on motivated behavior, the underlying mechanisms responsible are incompletely understood. In this study we elucidate a functional pathway in mouse brain that encodes the aversive effects of stress and mediates stress-induced reinstatement of cocaine place preference (CPP). Activation of the dynorphin/kappa opioid receptor (KOR) system by either repeated stress or agonist produces conditioned place aversion (CPA). Because KOR inhibition of dopamine release in the mesolimbic pathway has been proposed to mediate the dysphoria underlying this response, we tested dopamine-deficient mice in this study and found that KOR agonist in these mice still produced CPA. However, inactivation of serotonergic KORs by injection of the KOR antagonist norBNI into the dorsal raphe nucleus (DRN), blocked aversive responses to the KOR agonist U50,488 and blocked stress-induced reinstatement of CPP. KOR knockout (KO) mice did not develop CPA to U50,488; however, lentiviral re-expression of KOR in the DRN of KOR KO mice restored place aversion. In contrast, lentiviral expression in DRN of a mutated form of KOR that fails to activate p38 MAPK required for KOR-dependent aversion, did not restore place aversion. DRN serotonergic neurons project broadly throughout the brain, but the inactivation of KOR in the nucleus accumbens (NAc) coupled with viral re-expression in the DRN of KOR KO mice demonstrated that aversion was encoded by a DRN to NAc projection. These results suggest that the adverse effects of stress may converge on the serotonergic system and offers an approach to controlling stress-induced dysphoria and relapse.

VTA dopaminergic neurons regulate ethologically relevant sleep–wake behaviors

Dopaminergic ventral tegmental area (VTA) neurons are critically involved in a variety of behaviors that rely on heightened arousal, but whether they directly and causally control the generation and maintenance of wakefulness is unknown. We recorded calcium activity using fiber photometry in freely behaving mice and found arousal-state-dependent alterations in VTA dopaminergic neurons. We used chemogenetic and optogenetic manipulations together with polysomnographic recordings to demonstrate that VTA dopaminergic neurons are necessary for arousal and that their inhibition suppresses wakefulness, even in the face of ethologically relevant salient stimuli. Nevertheless, before inducing sleep, inhibition of VTA dopaminergic neurons promoted goal-directed and sleep-related nesting behavior. Optogenetic stimulation, in contrast, initiated and maintained wakefulness and suppressed sleep and sleep-related nesting behavior. We further found that different projections of VTA dopaminergic neurons differentially modulate arousal. Collectively, our findings uncover a fundamental role for VTA dopaminergic circuitry in the maintenance of the awake state and ethologically relevant sleep-related behaviors.

Stress-related neuropeptides and addictive behaviors: beyond the usual suspects.

Addictive disorders are chronic, relapsing conditions that cause extensive disease burden. Genetic factors partly account for susceptibility to addiction, but environmental factors such as stressful experiences and prolonged exposure of the brain to addictive drugs promote its development. Progression to addiction involves neuroadaptations within neurocircuitry that mediates stress responses and is influenced by several peptidergic neuromodulators. While corticotrophin releasing factor is the prototypic member of this class, recent work has identified several additional stress-related neuropeptides that play an important role in regulation of drug intake and relapse, including the urocortins, nociceptin, substance P, and neuropeptide S. Here, we review this emerging literature, discussing to what extent the properties of these neuromodulators are shared or distinct and considering their potential as drug targets.

Hypocretin (orexin) neuromodulation of stress and reward pathways

Hypocretin (also known as orexin) is a peptide neuromodulator that is expressed exclusively in the lateral hypothalamic area and plays a fundamental role in wakefulness and arousal. Chronic stress and compulsive drug-seeking are two examples of dysregulated states of hyperarousal that are influenced by hypocretin transmission throughout hypothalamic, extended amygdala, brainstem, and mesolimbic pathways. Here, we review current advances in the understanding of hypocretins modulatory actions underlying conditions of negative and positive emotional valence, focusing particularly on mechanisms that facilitate adaptive (and maladaptive) responses to stressful or rewarding environmental stimuli. We conclude by discussing progress toward integrated theories for hypocretin modulation of divergent behavioral domains.

Urocortin-1 within the centrally-projecting Edinger-Westphal nucleus is critical for ethanol preference.

Converging lines of evidence point to the involvement of neurons of the centrally projecting Edinger-Westphal nucleus (EWcp) containing the neuropeptide Urocortin-1 (Ucn1) in excessive ethanol (EtOH) intake and EtOH sensitivity. Here, we expanded these previous findings by using a continuous-access, two-bottle choice drinking paradigm (3%, 6%, and 10% EtOH vs. tap water) to compare EtOH intake and EtOH preference in Ucn1 genetic knockout (KO) and wild-type (WT) mice. Based on previous studies demonstrating that electrolytic lesion of the EWcp attenuated EtOH intake and preference in high-drinking C57BL/6J mice, we also set out to determine whether EWcp lesion would differentially alter EtOH consumption in Ucn1 KO and WT mice. Finally, we implemented well-established place conditioning procedures in KO and WT mice to determine whether Ucn1 and the corticotropin-releasing factor type-2 receptor (CRF-R2) were involved in the rewarding and aversive effects of EtOH (2 g/kg, i.p.). Results from these studies revealed that (1) genetic deletion of Ucn1 dampened EtOH preference only in mice with an intact EWcp, but not in mice that received lesion of the EWcp, (2) lesion of the EWcp dampened EtOH intake in Ucn1 KO and WT mice, but dampened EtOH preference only in WT mice expressing Ucn1, and (3) genetic deletion of Ucn1 or CRF-R2 abolished the conditioned rewarding effects of EtOH, but deletion of Ucn1 had no effect on the conditioned aversive effects of EtOH. The current findings provide strong support for the hypothesis that EWcp-Ucn1 neurons play an important role in EtOH intake, preference, and reward.

Dissection of corticotropin-releasing factor system involvement in locomotor sensitivity to methamphetamine.

Sensitivity to the euphoric and locomotor‐activating effects of drugs of abuse may contribute to risk for excessive use and addiction. Repeated administration of psychostimulants such as methamphetamine (MA) can result in neuroadaptive consequences that manifest behaviorally as a progressive escalation of locomotor activation, termed psychomotor sensitization. The present studies addressed the involvement of specific components of the corticotropin‐releasing factor (CRF) system in locomotor activation and psychomotor sensitization induced by MA (1, 2 mg/kg) by utilizing pharmacological approaches, as well as a series of genetic knockout (KO) mice, each deficient for a single component of the CRF system: CRF‐R1, CRF‐R2, CRF, or the CRF‐related peptide Urocortin 1 (Ucn1). CRF‐R1 KO mice did not differ from wild‐type mice in sensitization to MA, and pharmacological blockade of CRF‐R1 with CP‐154,526 (15, 30 mg/kg) in DBA/2J mice did not selectively attenuate either the acquisition or expression of MA‐induced sensitization. Deletion of either of the endogenous ligands of CRF‐R1 (CRF, Ucn1) either enhanced or had no effect on MA‐induced sensitization, providing further evidence against a role for CRF‐R1 signaling. Interestingly, deletion of CRF‐R2 attenuated MA‐induced locomotor activation, elucidating a novel contribution of the CRF system to MA sensitivity, and suggesting the participation of the endogenous urocortin peptides Ucn2 and Ucn3. Immunohistochemistry for Fos was used to visualize neural activation underlying CRF‐R2‐dependent sensitivity to MA, identifying the basolateral and central nuclei of the amygdala as neural substrates involved in this response. Our results support further examination of CRF‐R2 involvement in neural processes associated with MA addiction.

Characterization of Genetic Differences within the Centrally Projecting Edinger–Westphal Nucleus of C57BL/6J and DBA/2J Mice by Expression Profiling

Detailed examination of the midbrain Edinger–Westphal (EW) nucleus revealed the existence of two distinct nuclei. One population of EW preganglionic (EWpg) neurons was found to control oculomotor functions, and a separate population of EW centrally projecting (EWcp) neurons was found to contain stress- and feeding-related neuropeptides. Although it has been shown that EWcp neurons are highly responsive to drugs of abuse and behavioral stress, a genetic characterization of the EWcp was needed. To identify genetic differences in the EWcp of inbred mouse strains that differ in behaviors relevant to EWcp function, we used publicly available tools from the Allen Brain Atlas to identify 68 transcripts that were selectively expressed in the EWcp, and examined their expression within tissue punch microdissection samples containing the EWcp of adult male C57BL/6J (B6) and DBA/2J (D2) mice. Using 96-well quantitative real-time PCR (qPCR) arrays that included the EWcp-specific genes, several other genes of interest, and five housekeeping genes, we identified strain differences in expression of 11 EWcp-specific genes (BC023892, Btg3, Bves, Cart, Cck, Ghsr, Neto1, Postn, Ptprn, Rcn1, and Ucn), two immediate early genes (Egr1 and Fos), and one dopamine-related gene (Drd5). All significant expression differences were greater in B6 vs. D2 mice, and several of these were verified either at the protein level using immunohistochemistry (IHC) or in silico using microarray data sets from whole brain and other brain areas. These results demonstrate a significant advance in our understanding of the EWcp on three levels. First, we generated a list of EWcp-specific genes (most of which had not yet been reported within the EWcp in the literature) that will be informative for future studies of EWcp function. Second, due to similarity in results from qPCR and IHC, we revealed that strain differences in basal EWcp neuropeptide content are accounted for by differential transcription and number of peptidergic neurons, rather than by differential rates of peptide release. And third, our identification of differentially expressed EWcp-specific genes between B6 and D2 mice may hold powerful insight into the neurogenetic contributions of the EWcp to stress- and addiction-related behaviors.

To sleep or not to sleep: neuronal and ecological insights

Daily, animals need to decide when to stop engaging in cognitive processes and behavioral responses to the environment, and go to sleep. The main processes regulating the daily organization of sleep and wakefulness are circadian rhythms and homeostatic sleep pressure. In addition, motivational processes such as food seeking and predator evasion can modulate sleep/wake behaviors. Here, we discuss the principal processes regulating the propensity to stay awake or go to sleep-focusing on neuronal and behavioral aspects. We first introduce the neuronal populations involved in sleep/wake regulation. Next, we describe the circadian and homeostatic drives for sleep. Then, we highlight studies demonstrating various effects of motivational processes on sleep/wake behaviors, and discuss possible neuronal mechanisms underlying their control.

Hypocretins and Arousal

How the brain controls vigilance state transitions remains to be fully understood. The discovery of hypocretins, also known as orexins, and their link to narcolepsy has undoubtedly allowed us to advance our knowledge on key mechanisms controlling the boundaries and transitions between sleep and wakefulness. Lack of function of hypocretin neurons (a relatively simple and non-redundant neuronal system) results in inappropriate control of sleep states without affecting the total amount of sleep or homeostatic mechanisms. Anatomical and functional evidence shows that the hypothalamic neurons that produce hypocretins/orexins project widely throughout the entire brain and interact with major neuromodulator systems in order to regulate physiological processes underlying wakefulness, attention, and emotions. Here, we review the role of hypocretins/orexins in arousal state transitions, and discuss possible mechanisms by which such a relatively small population of neurons controls fundamental brain state dynamics.

Corticotropin-releasing factor: innocent until proven guilty

, but also implied that the underlying ligand must be!CRF.This example illustrates a broader prob-lem in the literature. Our analysis of the most recent 120 items retrieved by a PubMed search for CRF (or CRH) system involvement in alcoholism or addiction (excluding articles that focused solely on the hypothalamic–pitu-itary–adrenal axis) found that only 34.7% of these articles acknowledged UCN peptides. Furthermore, 53.3% of these articles implied a role for CRF without providing evidence against a role for!UCNs.We also identified several cases in which authors applied a ligand exogenously and inferred that the same ligand must medi-ate the effect endogenously. For example, the 2002 study discussed above found that intra-MRN CRF infusions reinstated alco-hol-seeking, and the authors inferred that this mechanism mediated alcohol-seeking endogenously