Hypocretin (orexin) facilitates reward by attenuating the antireward effects of its cotransmitter dynorphin in ventral tegmental area.

Hypocretin (orexin) and dynorphin are neuropeptides with opposing actions on motivated behavior. Orexin is implicated in states of arousal and reward, whereas dynorphin is implicated in depressive-like states. We show that, despite their opposing actions, these peptides are packaged in the same synaptic vesicles within the hypothalamus. Disruption of orexin function blunts the rewarding effects of lateral hypothalamic (LH) stimulation, eliminates cocaine-induced impulsivity, and reduces cocaine self-administration. Concomitant disruption of dynorphin function reverses these behavioral changes. We also show that orexin and dynorphin have opposing actions on excitability of ventral tegmental area (VTA) dopamine neurons, a prominent target of orexin-containing neurons, and that intra-VTA orexin antagonism causes decreases in cocaine self-administration and LH self-stimulation that are reversed by dynorphin antagonism. Our findings identify a unique cellular process by which orexin can occlude the reward threshold-elevating effects of coreleased dynorphin and thereby act in a permissive fashion to facilitate reward.

Striatal microRNA controls cocaine intake through CREB signalling.

Cocaine addiction is characterized by a gradual loss of control over drug use, but the molecular mechanisms regulating vulnerability to this process remain unclear. Here we report that microRNA-212 (miR-212) is upregulated in the dorsal striatum of rats with a history of extended access to cocaine. Striatal miR-212 decreases responsiveness to the motivational properties of cocaine by markedly amplifying the stimulatory effects of the drug on cAMP response element binding protein (CREB) signalling. This action occurs through miR-212-enhanced Raf1 activity, resulting in adenylyl cyclase sensitization and increased expression of the essential CREB co-activator TORC (transducer of regulated CREB; also known as CRTC). Our findings indicate that striatal miR-212 signalling has a key role in determining vulnerability to cocaine addiction, reveal new molecular regulators that control the complex actions of cocaine in brain reward circuitries and provide an entirely new direction for the development of anti-addiction therapeutics based on the modulation of noncoding RNAs.

Seminar: Extracellular Vesicles as Mediators of Environmental Stress in Human Disease.

BACKGROUND: Extracellular vesicles (EVs), membrane-bound particles containing a variety of RNA types, DNA, proteins, and other macromolecules, are now appreciated as an important means of communication between cells and tissues, both in normal cellular physiology and as a potential indicator of cellular stress, environmental exposures, and early disease pathogenesis. Extracellular signaling through EVs is a growing field of research for understanding fundamental mechanisms of health and disease and for the potential for biomarker discovery and therapy development. EVs are also known to play important roles in mediating the effects of exposure to environmental stress. OBJECTIVES: This seminar addresses the application of new tools and approaches for EV research, developed in part through the National Institutes of Health (NIH) Extracellular RNA Communication Program, and reflects presentations and discussions from a workshop held 27-28 September 2021 by the National Institute of Environmental Health Sciences (NIEHS) and the National Center for Advancing Translational Sciences (NCATS) on "Extracellular Vesicles, Exosomes, and Cell-Cell Signaling in Response to Environmental Stress." The panel of experts discussed current research on EVs and environmental exposures, highlighted recent advances in EV isolation and characterization, and considered research gaps and opportunities toward identifying and characterizing the roles for EVs in environmentally related diseases, as well as the current challenges and opportunities in this field. DISCUSSION: The authors discuss the application of new experimental models, particularly organ-on-chip (OOC) systems and in vitro approaches and how these have the potential to extend findings in population-based studies of EVs in exposure-related diseases. Given the complex challenges of identifying cell-specific EVs related to environmental exposures, as well as the general heterogeneity and variability in EVs in blood and other accessible biological samples, there is a critical need for rigorous reporting of experimental methods and validation studies. The authors note that these efforts, combined with cross-disciplinary approaches, would ensure that future research efforts in environmental health studies on EV biomarkers are rigorous and reproducible. https://doi.org/10.1289/EHP12980.

Insular hypocretin transmission regulates nicotine reward.

Damage to the insular cortex can profoundly disrupt tobacco addiction in human smokers, reflected in spontaneous cessation of the tobacco habit and persistently decreased urge to smoke. Little is known concerning the neurobiological mechanisms through which the insula may control the maintenance of the tobacco habit. Emerging evidence suggests that hypocretin (orexin) transmission may play an important role in drug reinforcement processes, but its role in the rewarding actions of nicotine, considered the key addictive component of tobacco smoke, remains largely unexplored. Here we show that blockade of hypocretin transmission at hypocretin-1 (Hcrt-1; orexin-1) receptors decreases i.v. nicotine self-administration in rats and the motivation to obtain the drug. Blockade of Hcrt-1 receptors also abolished the stimulatory effects of nicotine on brain reward circuitries, as measured by reversal of nicotine-induced lowering of intracranial self-stimulation thresholds. In addition, we show that hypocretin-containing fibers innervate the insula, Hcrt-1 receptors are located on insular cells, and blockade of Hcrt-1 receptors in the insula but not in the adjacent somatosensory cortex decreases nicotine self-administration. These data demonstrate that insular hypocretin transmission plays a permissive role in the motivational properties of nicotine, and therefore may be a key neurobiological substrate necessary for maintaining tobacco addiction in human smokers.

Beyond the looking glass: recent advances in understanding the impact of environmental exposures on neuropsychiatric disease.

The etiologic pathways leading to neuropsychiatric diseases remain poorly defined. As genomic technologies have advanced over the past several decades, considerable progress has been made linking neuropsychiatric disorders to genetic underpinnings. Interest and consideration of nongenetic risk factors (e.g., lead exposure and schizophrenia) have, in contrast, lagged behind heritable frameworks of explanation. Thus, the association of neuropsychiatric illness to environmental chemical exposure, and their potential interactions with genetic susceptibility, are largely unexplored. In this review, we describe emerging approaches for considering the impact of chemical risk factors acting alone and in concert with genetic risk, and point to the potential role of epigenetics in mediating exposure effects on transcription of genes implicated in mental disorders. We highlight recent examples of research in nongenetic risk factors in psychiatric disorders that point to potential shared biological mechanisms-synaptic dysfunction, immune alterations, and gut-brain interactions. We outline new tools and resources that can be harnessed for the study of environmental factors in psychiatric disorders. These tools, combined with emerging experimental evidence, suggest that there is a need to broadly incorporate environmental exposures in psychiatric research, with the ultimate goal of identifying modifiable risk factors and informing new treatment strategies for neuropsychiatric disease.

Cocaine-associated stimuli increase cocaine seeking and activate accumbens core neurons after abstinence.

Electrophysiological recordings were completed in rats (n = 14) trained to self-administer cocaine to determine whether activation of nucleus accumbens (Acb) neurons (core vs shell) by cocaine-associated stimuli is enhanced after 1 month of cocaine abstinence. After self-administration training, 170 cells were recorded during a single test session conducted either the next day or 1 month later. The test session consisted of three phases during which (1) the cocaine cue was presented unexpectedly to rats, (2) rats responded for the same cue in the absence of the drug (extinction), and (3) the cocaine cue was presented randomly between cocaine-reinforced responding during resumption of self-administration. The cocaine stimulus significantly increased activation of Acb core (not shell) neurons after 1 month of cocaine abstinence (compared with 1 d); this finding occurred regardless of contingency of cue presentation or cocaine availability. Acb core activation was not observed in other rats (n = 7) presented with the same stimulus never paired with cocaine. The results reflect a cellular neuroadaptation in the Acb core related to cocaine-associated cues that is observed during initial cue exposure and sustained during extinction and resumption of self-administration after prolonged drug abstinence.

Decreased brain reward function during nicotine withdrawal in C57BL6 mice: evidence from intracranial self-stimulation (ICSS) studies.

Deficits in brain reward function during nicotine withdrawal may serve as an important substrate for negative reinforcement that contributes to the persistence of the tobacco habit in human smokers. The ability to assess withdrawal-associated reward deficits in genetically modified mice may facilitate understanding of the neurobiological mechanisms of nicotine dependence. Here, we assessed the effects of nicotine withdrawal on brain reward function in mice, as measured by intracranial self-stimulation (ICSS) thresholds. Male C57BL6 mice were trained in a discrete-trial current-threshold ICSS procedure until stable reward thresholds were obtained. Mice then received experimenter-administered saline or nicotine (2 mg/kg/injection salt; x4 daily) injections for 7 consecutive days, and ICSS thresholds assessed for 3 days after cessation of injections. Thresholds were unaltered in nicotine- and saline-treated mice after cessation of injections, indicating that this treatment regimen was not sufficient to induce withdrawal-associated reward deficits. Next, mice were implanted subcutaneously with osmotic minipumps delivering a constant daily amount of saline or nicotine (24 mg/kg/day; free-base), with pumps surgically removed 13 days later. The nicotinic receptor antagonist mecamylamine (2 mg/kg) elevated ICSS thresholds in nicotine- but not saline-treated mice when administered 8-10 days after pump implantation. Similarly, reward thresholds were elevated in nicotine-treated mice 12-72 h after minipump removal. These data demonstrate that antagonist-precipitated or spontaneous withdrawal from nicotine delivered via osmotic minipumps induced reward deficits in mice. Further, these findings highlight the potential utility of the ICSS procedure for assessing this important affective component of nicotine withdrawal in genetically modified mice.

Basolateral amygdala neurons encode cocaine self-administration and cocaine-associated cues.

Electrophysiological recording procedures were used to examine basolateral amygdala (BLA) cell firing during cocaine self-administration and relative to response-independent presentations of cocaine-associated stimuli. Of 72 neurons (n = 10 rats), 31 cells (43%) were classified as phasically active, exhibiting one of three types of patterned discharges relative to the drug-reinforced response, similar to that previously described for nucleus accumbens (Acb) neurons (Carelli, 2002). Briefly, neurons exhibited increased firing rates within seconds preceding the response [termed preresponse (PR)], increased activity within seconds after the response [termed reinforcement excitation (RFe)] or an inhibition in cell firing before and/or after the response for intravenous cocaine [termed reinforcement inhibition (RFi)]. To examine the responsiveness of these same neurons to cocaine-associated stimuli, the stimulus "probe" procedure was used. Specifically, probe trials (18-20) were presented in which the audiovisual (tone-house light) stimulus associated with intravenous cocaine delivery during self-administration was randomly presented by the computer, interspersed between reinforced lever press responses. Neurons classified as type PR or type RFi were not activated by the stimulus. In contrast, neurons that exhibited increased firing immediately after the response (type RFe neurons) were significantly activated by the audiovisual cue. These findings are discussed with respect to the role of the BLA in cocaine addiction as well as previous studies characterizing Acb cell firing during cocaine self-administration.

Abstinence from cocaine self-administration heightens neural encoding of goal-directed behaviors in the accumbens.

Cocaine addiction in humans is characterized by cycles of abstinence from drug-taking and relapse. Here, electrophysiological recording procedures were used to determine whether nucleus accumbens (Acb) neuronal firing properties are altered following interruption and resumption of cocaine self-administration. Rats (n = 12) were trained to self-administer cocaine (2 h daily sessions) then divided into two groups. Acb activity was recorded for Group 1 (controls) during two additional self-administration sessions completed over the next 2 days (test sessions 1 and 2). Acb activity was recorded for Group 2 (1-month) during one self-administration session completed the next day (test 1), and during a second self-administration session 1 month later (test 2). As in prior reports, a subset of Acb neurons exhibited patterned discharges (short duration and/or long-term cyclic alterations, termed 'phasically active') relative to cocaine-reinforced responding during test session 1. Remarkably, the percentage of phasically active cells dramatically increased (nearly two-fold) following 1-month abstinence, in the core but not the shell of the Acb. Likewise, the strength of the neural correlates (determined via signal-to-baseline ratios) also increased as a function of abstinence. Extinction experiments in another set of rats (n = 12) revealed an increased motivational state for the drug following abstinence. The results show that abstinence from cocaine self-administration causes a dramatic increase in the number and strength of Acb neurons that encode cocaine-related information, thus representing the first neurophysiological correlate of heightened activation of the 'brain reward system' following abstinence and resumption (relapse) of cocaine consumption.

An examination of nucleus accumbens cell firing during extinction and reinstatement of water reinforcement behavior in rats.

Electrophysiological recording procedures were used to examine nucleus accumbens (Acb) cell firing in rats (n = 13) during water reinforcement sessions consisting of three phases. During phase one (maintenance), a lever press resulted in water reinforcement (fixed ratio 1; 0.05 ml/press) paired with an auditory stimulus (0.5 s). Of 128 Acb neurons recorded during maintenance, 40 cells (31%) exhibited one of three types of neuronal firing patterns described previously [J. Neurosci. 14 (12) (1994) 7735-7746; J. Neurosci. 20 (11) (2000) 4255-4266]. Briefly, Acb neurons exhibited increases in firing rate within seconds preceding the reinforced response (type PR) or increases (type RFe) or decreases (type RFi) in activity seconds following response completion. In phase two (extinction), subsequent lever pressing had no programmed consequences (i.e., water reinforcement and the auditory stimulus were not presented). After 30 min of no responding, animals were given water reinforcement/auditory stimulus 'primes' to reestablish lever pressing behavior during the third phase (reinstatement). Results indicated that all types of phasic neurons (PR, RFe and RFi) exhibited an attenuated firing rate during extinction, and in some cases recovery of patterned discharges were observed during reinstatement. No significant changes in cell firing were observed for any cell type during presentation of the stimulus prime used to reestablish operant responding following extinction. These findings are discussed in terms of how Acb neurons process information related to 'natural' reinforcers versus drugs of abuse.

Hypocretin-1 receptors regulate the reinforcing and reward-enhancing effects of cocaine: pharmacological and behavioral genetics evidence.

Considerable evidence suggests that transmission at hypocretin-1 (orexin-1) receptors (Hcrt-R1) plays an important role in the reinstatement of extinguished cocaine-seeking behaviors in rodents. However, far less is known about the role for hypocretin transmission in regulating ongoing cocaine-taking behavior. Here, we investigated the effects of the selective Hcrt-R1 antagonist SB-334867 on cocaine intake, as measured by intravenous (IV) cocaine self-administration in rats. The stimulatory effects of cocaine on brain reward systems contribute to the establishment and maintenance of cocaine-taking behaviors. Therefore, we also assessed the effects of SB-334867 on the reward-enhancing properties of cocaine, as measured by cocaine-induced lowering of intracranial self-stimulation (ICSS) thresholds. Finally, to definitively establish a role for Hcrt-R1 in regulating cocaine intake, we assessed IV cocaine self-administration in Hcrt-R1 knockout mice. We found that SB-334867 (1-4 mg/kg) dose-dependently decreased cocaine (0.5 mg/kg/infusion) self-administration in rats but did not alter responding for food rewards under the same schedule of reinforcement. This suggests that SB-334867 decreased cocaine reinforcement without negatively impacting operant performance. SB-334867 (1-4 mg/kg) also dose-dependently attenuated the stimulatory effects of cocaine (10 mg/kg) on brain reward systems, as measured by reversal of cocaine-induced lowering of ICSS thresholds in rats. Finally, we found that Hcrt-R1 knockout mice self-administered far less cocaine than wildtype mice across the entire dose-response function. These data demonstrate that Hcrt-R1 play an important role in regulating the reinforcing and reward-enhancing properties of cocaine and suggest that hypocretin transmission is likely essential for establishing and maintaining the cocaine habit in human addicts.

MeCP2 controls BDNF expression and cocaine intake through homeostatic interactions with microRNA-212.

The X-linked transcriptional repressor methyl CpG binding protein 2 (MeCP2), known for its role in the neurodevelopmental disorder Rett syndrome, is emerging as an important regulator of neuroplasticity in postmitotic neurons. Cocaine addiction is commonly viewed as a disorder of neuroplasticity, but the potential involvement of MeCP2 has not been explored. Here we identify a key role for MeCP2 in the dorsal striatum in the escalating cocaine intake seen in rats with extended access to the drug, a process that mimics the increasingly uncontrolled cocaine use seen in addicted humans. MeCP2 regulates cocaine intake through homeostatic interactions with microRNA-212 (miR-212) to control the effects of cocaine on striatal brain-derived neurotrophic factor (BDNF) levels. These data suggest that homeostatic interactions between MeCP2 and miR-212 in dorsal striatum may be important in regulating vulnerability to cocaine addiction.

Accumbens activity during a multiple schedule for water and sucrose reinforcement in rats.

Electrophysiological recording procedures were used to examine nucleus accumbens (Acb) cell firing during operant responding for water reinforcement vs. a highly palatable sweet solution (0.6 M sucrose). Rats (n = 8) were trained on a multiple schedule to press one lever for water (fixed ratio 1, FR1; 15 min) followed by a 20-sec timeout period (chamber dark, levers retracted), and extension of a second spatially distinct lever that the animals pressed for sucrose reinforcement (FR1; 15 min). Of 84 cells, 55 neurons (65%) displayed one of three types of patterned discharges (increases or decreases in firing rate) immediately before or following the sucrose- or water-reinforced response. The major finding of this report was that the majority of these neurons (36/55 cells; 65%) showed similar types of neuronal firing patterns across the two reinforcer conditions. The remaining cells (19/55; 35%) exhibited patterned activity specific to responding for one reinforcer only (water or sucrose). These findings are discussed with respect to how Acb neurons encode goal-directed behaviors for "natural" reinforcers including food, water, and a palatable sweet solution.