The dynorphin (DYN)/kappa-opioid receptor (KOR) system plays a conserved role in stress-induced reinstatement of drug seeking for prototypical substances of abuse. Due to nicotine's high propensity for stress-induced relapse, we hypothesized that stress would induce reinstatement of nicotine seeking-like behavior in a KOR-dependent manner. Using a conditioned place preference (CPP) reinstatement procedure in mice, we show that both foot-shock stress and the pharmacological stressor yohimbine (2 mg/kg, i.p.) induce reinstatement of nicotine CPP in a norbinaltorphimine (norBNI, a KOR antagonist)-sensitive manner, indicating that KOR activity is necessary for stress-induced nicotine CPP reinstatement. After reinstatement testing, we visualized robust c-fos expression in the basolateral amygdala (BLA), which was reduced in mice pretreated with norBNI. We then used several distinct but complementary approaches of locally disrupting BLA KOR activity to assess the role of KORs and KOR-coupled intracellular signaling cascades on reinstatement of nicotine CPP. norBNI injected locally into the BLA prevented yohimbine-induced nicotine CPP reinstatement without affecting CPP acquisition. Similarly, selective deletion of BLA KORs in KOR conditional knock-out mice prevented foot-shock-induced CPP reinstatement. Together, these findings strongly implicate BLA KORs in stress-induced nicotine seeking-like behavior. In addition, we found that chemogenetic activation of Gαi signaling within CaMKIIα BLA neurons was sufficient to induce nicotine CPP reinstatement, identifying an anatomically specific intracellular mechanism by which stress leads to reinstatement. Considered together, our findings suggest that activation of the DYN/KOR system and Gαi signaling within the BLA is both necessary and sufficient to produce reinstatement of nicotine preference.

Optogenetic methods to modulate cells and signaling pathways via targeted expression and activation of light-sensitive proteins have greatly accelerated the process of mapping complex neural circuits and defining their roles in physiological and pathological contexts. Recently demonstrated technologies based on injectable, microscale inorganic light-emitting diodes (μ-ILEDs) with wireless control and power delivery strategies offer important functionality in such experiments, by eliminating the external tethers associated with traditional fiber optic approaches. Existing wireless μ-ILED embodiments allow, however, illumination only at a single targeted region of the brain with a single optical wavelength and over spatial ranges of operation that are constrained by the radio frequency power transmission hardware. Here we report stretchable, multiresonance antennas and battery-free schemes for multichannel wireless operation of independently addressable, multicolor μ-ILEDs with fully implantable, miniaturized platforms. This advance, as demonstrated through in vitro and in vivo studies using thin, mechanically soft systems that separately control as many as three different μ-ILEDs, relies on specially designed stretchable antennas in which parallel capacitive coupling circuits yield several independent, well-separated operating frequencies, as verified through experimental and modeling results. When used in combination with active motion-tracking antenna arrays, these devices enable multichannel optogenetic research on complex behavioral responses in groups of animals over large areas at low levels of radio frequency power (<1 W). Studies of the regions of the brain that are involved in sleep arousal (locus coeruleus) and preference/aversion (nucleus accumbens) demonstrate the unique capabilities of these technologies.

In vivo optogenetics provides unique, powerful capabilities in the dissection of neural circuits implicated in neuropsychiatric disorders. Conventional hardware for such studies, however, physically tethers the experimental animal to an external light source, limiting the range of possible experiments. Emerging wireless options offer important capabilities that avoid some of these limitations, but the current size, bulk, weight, and wireless area of coverage is often disadvantageous. Here, we present a simple but powerful setup based on wireless, near-field power transfer and miniaturized, thin, flexible optoelectronic implants, for complete optical control in a variety of behavioral paradigms. The devices combine subdermal magnetic coil antennas connected to microscale, injectable light-emitting diodes (LEDs), with the ability to operate at wavelengths ranging from UV to blue, green-yellow, and red. An external loop antenna allows robust, straightforward application in a multitude of behavioral apparatuses. The result is a readily mass-producible, user-friendly technology with broad potential for optogenetics applications.

Preclinical work has long focused on male animals, though biological sex clearly influences risk for certain diseases, including many psychiatric disorders. Such disorders are often treated by drugs targeting the CNS norepinephrine system. Despite roles for noradrenergic neurons in behavior and neuropsychiatric disease models, their molecular characterization has lagged. We profiled mouse noradrenergic neurons in vivo, defining over 3,000 high-confidence transcripts expressed therein, including druggable receptors. We uncovered remarkable sex differences in gene expression, including elevated expression of the EP3 receptor in females-which we leverage to illustrate the behavioral and pharmacologic relevance of these findings-and of Slc6a15 and Lin28b, both major depressive disorder (MDD)-associated genes. Broadly, we present a means of transcriptionally profiling locus coeruleus under baseline and experimental conditions. Our findings underscore the need for preclinical work to include both sexes and suggest that sex differences in noradrenergic neurons may underlie behavioral differences relevant to disease.

Motivational states consist of cognitive, emotional, and physiological components controlled by multiple brain regions. An integral component of this neural circuitry is the bed nucleus of the stria terminalis (BNST). Here, we identify that neurons within BNST that express the gene prepronociceptin (PnocBNST) modulate rapid changes in physiological arousal that occur upon exposure to motivationally salient stimuli. Using in vivo two-photon calcium imaging, we find that PnocBNST neuronal responses directly correspond with rapid increases in pupillary size when mice are exposed to aversive and rewarding odors. Furthermore, optogenetic activation of these neurons increases pupillary size and anxiety-like behaviors but does not induce approach, avoidance, or locomotion. These findings suggest that excitatory responses in PnocBNST neurons encode rapid arousal responses that modulate anxiety states. Further histological, electrophysiological, and single-cell RNA sequencing data reveal that PnocBNST neurons are composed of genetically and anatomically identifiable subpopulations that may differentially tune rapid arousal responses to motivational stimuli.

Nociceptin and its receptor are widely distributed throughout the brain in regions associated with reward behavior, yet how and when they act is unknown. Here, we dissected the role of a nociceptin peptide circuit in reward seeking. We generated a prepronociceptin (Pnoc)-Cre mouse line that revealed a unique subpopulation of paranigral ventral tegmental area (pnVTA) neurons enriched in prepronociceptin. Fiber photometry recordings during progressive ratio operant behavior revealed pnVTAPnoc neurons become most active when mice stop seeking natural rewards. Selective pnVTAPnoc neuron ablation, inhibition, and conditional VTA nociceptin receptor (NOPR) deletion increased operant responding, revealing that the pnVTAPnoc nucleus and VTA NOPR signaling are necessary for regulating reward motivation. Additionally, optogenetic and chemogenetic activation of this pnVTAPnoc nucleus caused avoidance and decreased motivation for rewards. These findings provide insight into neuromodulatory circuits that regulate motivated behaviors through identification of a previously unknown neuropeptide-containing pnVTA nucleus that limits motivation for rewards.

Injury, inflammation, and nerve damage initiate a wide variety of cellular and molecular processes that culminate in hyperexcitation of sensory nerves, which underlies chronic inflammatory and neuropathic pain. Using behavioral readouts of pain hypersensitivity induced by angiotensin II (Ang II) injection into mouse hindpaws, our study shows that activation of the type 2 Ang II receptor (AT2R) and the cell-damage-sensing ion channel TRPA1 are required for peripheral mechanical pain sensitization induced by Ang II in male and female mice. However, we show that AT2R is not expressed in mouse and human dorsal root ganglia (DRG) sensory neurons. Instead, expression/activation of AT2R on peripheral/skin macrophages (MΦs) constitutes a critical trigger of mouse and human DRG sensory neuron excitation. Ang II-induced peripheral mechanical pain hypersensitivity can be attenuated by chemogenetic depletion of peripheral MΦs. Furthermore, AT2R activation in MΦs triggers production of reactive oxygen/nitrogen species, which trans-activate TRPA1 on mouse and human DRG sensory neurons via cysteine modification of the channel. Our study thus identifies a translatable immune cell-to-sensory neuron signaling crosstalk underlying peripheral nociceptor sensitization. This form of cell-to-cell signaling represents a critical peripheral mechanism for chronic pain and thus identifies multiple druggable analgesic targets.SIGNIFICANCE STATEMENT Pain is a widespread health problem that is undermanaged by currently available analgesics. Findings from a recent clinical trial on a type II angiotensin II receptor (AT2R) antagonist showed effective analgesia for neuropathic pain. AT2R antagonists have been shown to reduce neuropathy-, inflammation- and bone cancer-associated pain in rodents. We report that activation of AT2R in macrophages (MΦs) that infiltrate the site of injury, but not in sensory neurons, triggers an intercellular redox communication with sensory neurons via activation of the cell damage/pain-sensing ion channel TRPA1. This MΦ-to-sensory neuron crosstalk results in peripheral pain sensitization. Our findings provide an evidence-based mechanism underlying the analgesic action of AT2R antagonists, which could accelerate the development of efficacious non-opioid analgesic drugs for multiple pain conditions.

Optical manipulations of genetically defined cell types have generated significant insights into the dynamics of neural circuits. While optogenetic activation has been relatively straightforward, rapid and reversible synaptic inhibition has proven more elusive. Here, we leveraged the natural ability of inhibitory presynaptic GPCRs to suppress synaptic transmission and characterize parapinopsin (PPO) as a GPCR-based opsin for terminal inhibition. PPO is a photoswitchable opsin that couples to Gi/o signaling cascades and is rapidly activated by pulsed blue light, switched off with amber light, and effective for repeated, prolonged, and reversible inhibition. PPO rapidly and reversibly inhibits glutamate, GABA, and dopamine release at presynaptic terminals. Furthermore, PPO alters reward behaviors in a time-locked and reversible manner in vivo. These results demonstrate that PPO fills a significant gap in the neuroscience toolkit for rapid and reversible synaptic inhibition and has broad utility for spatiotemporal control of inhibitory GPCR signaling cascades.

Torpor is an energy-conserving state in which animals dramatically decrease their metabolic rate and body temperature to survive harsh environmental conditions. Here, we report the noninvasive, precise and safe induction of a torpor-like hypothermic and hypometabolic state in rodents by remote transcranial ultrasound stimulation at the hypothalamus preoptic area (POA). We achieve a long-lasting (>24 h) torpor-like state in mice via closed-loop feedback control of ultrasound stimulation with automated detection of body temperature. Ultrasound-induced hypothermia and hypometabolism (UIH) is triggered by activation of POA neurons, involves the dorsomedial hypothalamus as a downstream brain region and subsequent inhibition of thermogenic brown adipose tissue. Single-nucleus RNA-sequencing of POA neurons reveals TRPM2 as an ultrasound-sensitive ion channel, the knockdown of which suppresses UIH. We also demonstrate that UIH is feasible in a non-torpid animal, the rat. Our findings establish UIH as a promising technology for the noninvasive and safe induction of a torpor-like state.

Nociceptin/orphanin-FQ (N/OFQ) is a recently appreciated critical opioid peptide with key regulatory functions in several central behavioral processes including motivation, stress, feeding, and sleep. The functional relevance of N/OFQ action in the mammalian brain remains unclear due to a lack of high-resolution approaches to detect this neuropeptide with appropriate spatial and temporal resolution. Here we develop and characterize NOPLight, a genetically encoded sensor that sensitively reports changes in endogenous N/OFQ release. We characterized the affinity, pharmacological profile, spectral properties, kinetics, ligand selectivity, and potential interaction with intracellular signal transducers of NOPLight in vitro. Its functionality was established in acute brain slices by exogeneous N/OFQ application and chemogenetic induction of endogenous N/OFQ release from PNOC neurons. In vivo studies with fiber photometry enabled a direct recording of binding by N/OFQ receptor ligands, as well as the detection of natural or chemogenetically-evoked endogenous N/OFQ release within the paranigral ventral tegmental area (pnVTA). In summary, we show that NOPLight can be used to detect N/OFQ opioid peptide signal dynamics in tissue and freely-behaving animals.

Though the last decade has seen accelerated advances in techniques and technologies to perturb neuronal circuitry in the brain, we are still poorly equipped to adequately dissect endogenous peptide release in vivo. To this end we developed a system that combines in vivo optogenetics with microdialysis and a highly sensitive mass spectrometry-based assay to measure opioid peptide release in freely moving rodents.

The ventrolateral periaqueductal gray (vlPAG) constitutes a major descending pain modulatory system and is a crucial site for opioid-induced analgesia. A number of previous studies have demonstrated that glutamate and GABA play critical opposing roles in nociceptive processing in the vlPAG. It has been suggested that glutamatergic neurotransmission exerts antinociceptive effects, whereas GABAergic neurotransmission exert pronociceptive effects on pain transmission, through descending pathways. The inability to exclusively manipulate subpopulations of neurons in the PAG has prevented direct testing of this hypothesis. Here, we demonstrate the different contributions of genetically defined glutamatergic and GABAergic vlPAG neurons in nociceptive processing by employing cell type-specific chemogenetic approaches in mice. Global chemogenetic manipulation of vlPAG neuronal activity suggests that vlPAG neural circuits exert tonic suppression of nociception, consistent with previous pharmacological and electrophysiological studies. However, selective modulation of GABAergic or glutamatergic neurons demonstrates an inverse regulation of nociceptive behaviors by these cell populations. Selective chemogenetic activation of glutamatergic neurons, or inhibition of GABAergic neurons, in vlPAG suppresses nociception. In contrast, inhibition of glutamatergic neurons, or activation of GABAergic neurons, in vlPAG facilitates nociception. Our findings provide direct experimental support for a model in which excitatory and inhibitory neurons in the PAG bidirectionally modulate nociception.

Chronic itch or pruritus is a debilitating disorder that is refractory to conventional anti-histamine treatment. Kappa opioid receptor (KOR) agonists have been used to treat chronic itch, but the underlying mechanism remains elusive. Here, we find that KOR and gastrin-releasing peptide receptor (GRPR) overlap in the spinal cord, and KOR activation attenuated GRPR-mediated histamine-independent acute and chronic itch in mice. Notably, canonical KOR-mediated Gαi signaling is not required for desensitizing GRPR function. In vivo and in vitro studies suggest that KOR activation results in the translocation of Ca2+-independent protein kinase C (PKC)δ from the cytosol to the plasma membrane, which in turn phosphorylates and inhibits GRPR activity. A blockade of phospholipase C (PLC) in HEK293 cells prevented KOR-agonist-induced PKCδ translocation and GRPR phosphorylation, suggesting a role of PLC signaling in KOR-mediated GRPR desensitization. These data suggest that a KOR-PLC-PKCδ-GRPR signaling pathway in the spinal cord may underlie KOR-agonists-induced anti-pruritus therapies.

Cultures of Sertoli cells isolated from 20-day-old mice are widely used in research as substitutes for adult Sertoli cell cultures. This practice is based on the fact that Sertoli cells cease to proliferate and become mature in vivo by 16 to 20 days after birth. However, it is important to verify whether cultured Sertoli cells derived from 20-day-old mice do not proliferate ex vivo and whether they have the same properties as cultured adult Sertoli cells. Herein we described an isolation/culture method of Sertoli cells from 10-week-old adult mice with > 90% purity. Properties of these cultured adult Sertoli cells were then compared with those of cultured Sertoli cells derived from 20-day-old mice (also > 90% purity). By cell counting, bromo-2-deoxyuridine incorporation, and metaphase plate detection, we demonstrated that only adult Sertoli cells did not proliferate throughout 12 culture days. In contrast, Sertoli cells derived from 20-day-old mice still proliferated until Day 10 in culture. The morphology and profiles of intracellular lipidomics and spent medium proteomics of the 2 cultures were also different. Cultured adult Sertoli cells were larger in size and contained higher levels of triacylglycerols, cholesteryl esters, and seminolipid, and the proteins in their spent medium were mainly engaged in cellular metabolism. In contrast, proteins involved in cell division, including anti-Mullerian hormone, cell division cycle protein 42 (CDC42), and collagen isoforms, were at higher levels in Sertoli cell cultures derived from 20-day-old mice. Therefore, cultured Sertoli cells derived from 10-week-old mice, rather than those from 20-day-old animals, should be used for studies on properties of adult Sertoli cells.

Head-fixed behavioral experiments in rodents permit unparalleled experimental control, precise measurement of behavior, and concurrent modulation and measurement of neural activity. Here we present OHRBETS (Open-Source Head-fixed Rodent Behavioral Experimental Training System; pronounced 'Orbitz'), a low-cost, open-source ecosystem of hardware and software to flexibly pursue the neural basis of a variety of motivated behaviors. Head-fixed mice tested with OHRBETS displayed operant conditioning for caloric reward that replicates core behavioral phenotypes observed during freely moving conditions. OHRBETS also permits for optogenetic intracranial self-stimulation under positive or negative operant conditioning procedures and real-time place preference behavior, like that observed in freely moving assays. In a multi-spout brief-access consumption task, mice displayed licking as a function of concentration of sucrose, quinine, and sodium chloride, with licking modulated by homeostatic or circadian influences. Finally, to highlight the functionality of OHRBETS, we measured mesolimbic dopamine signals during the multi-spout brief-access task that display strong correlations with relative solution value and magnitude of consumption. All designs, programs, and instructions are provided freely online. This customizable ecosystem enables replicable operant and consummatory behaviors and can be incorporated with methods to perturb and record neural dynamics in vivo .

Itch is an unpleasant sensation that elicits robust scratching and aversive experience. However, the identity of the cells and neural circuits that organize this information remains elusive. Here, we show the necessity and sufficiency of chloroquine-activated neurons in the central amygdala (CeA) for both itch sensation and associated aversion. Further, we show that chloroquine-activated CeA neurons play important roles in itch-related comorbidities, including anxiety-like behaviors, but not in some aversive and appetitive behaviors previously ascribed to CeA neurons. RNA-sequencing of chloroquine-activated CeA neurons identified several differentially expressed genes as well as potential key signaling pathways in regulating pruritis. Finally, viral tracing experiments demonstrate that these neurons send projections to the ventral periaqueductal gray that are critical in modulation of itch. These findings reveal a cellular and circuit signature of CeA neurons orchestrating behavioral and affective responses to pruritus in mice.

Sleep/wake control involves several neurotransmitter and neuromodulatory systems yet the coordination of the behavioral and physiological processes underlying sleep is incompletely understood. Previous studies have suggested that activation of the Nociceptin/orphanin FQ (N/OFQ) receptor (NOPR) reduces locomotor activity and produces a sedation-like effect in rodents. In the present study, we systematically evaluated the efficacy of two NOPR agonists, Ro64-6198 and SR16835, on sleep/wake in rats, mice, and Cynomolgus macaques. We found a profound, dose-related increase in non-Rapid Eye Movement (NREM) sleep and electroencephalogram (EEG) slow wave activity (SWA) and suppression of Rapid Eye Movement sleep (REM) sleep in all three species. At the highest dose tested in rats, the increase in NREM sleep and EEG SWA was accompanied by a prolonged inhibition of REM sleep, hypothermia, and reduced locomotor activity. However, even at the highest dose tested, rats were immediately arousable upon sensory stimulation, suggesting sleep rather than an anesthetic state. NOPR agonism also resulted in increased expression of c-Fos in the anterodorsal preoptic and parastrial nuclei, two GABAergic nuclei that are highly interconnected with brain regions involved in physiological regulation. These results suggest that the N/OFQ-NOPR system may have a previously unrecognized role in sleep/wake control and potential promise as a therapeutic target for the treatment of insomnia.

The dynorphin (DYN)/kappa-opioid receptor (KOR) system is involved in dysphoria and negative emotional states. Dysregulation of KOR function promotes maladaptive behavioral regulation during withdrawal associated with alcohol dependence. Mesolimbic dopaminergic (DA) projections from the ventral tegmental area (VTA) innervate the extended amygdala circuitry and presynaptic KORs attenuate DA in these regions leading to an excessive alcohol consumption and negative affective-like behavior, whereas mesocortical KOR-regulated DA projections have been implicated in executive function and decision-making. Thus, the neuroadaptations occurring in DYN/KOR systems are important aspects to consider for the development of personalized therapeutic solutions. Herein, we study the contribution of the VTA DA neuron Oprk1 (KOR gene) in excessive alcohol consumption, negative emotional state, and executive function. To do so, Oprk1 mRNA expression and KOR function were characterized to confirm alcohol dependence-induced dysregulation in the VTA. Then, a transgenic Cre-Lox rat model (male and female TH::Cre rats) was used to allow for conditional and inducible overexpression of Oprk1 in VTA DA neurons. The effect of this overexpression was evaluated on operant alcohol self-administration, negative emotional states, and executive function. We found that VTA Oprk1 overexpression recapitulates some phenotypes of alcohol dependence including escalated alcohol self-administration and depressive-like behavior. However, working memory performance was not impacted following VTA Oprk1 overexpression in TH::Cre rats. This supports the hypothesis that dysregulated KOR signaling within the mesolimbic DA system is an important contributor to symptoms of alcohol dependence and shows that understanding Oprk1-mediated contributions to alcohol use disorder (AUD) should be an important future goal.

Stress is a complex human experience and having both rewarding and aversive motivational properties. The adverse effects of stress are well documented, yet many of underlying mechanisms remain unclear and controversial. Here we report that the anxiogenic properties of stress are encoded by the endogenous opioid peptide dynorphin acting in the basolateral amygdala. Using pharmacological and genetic approaches, we found that the anxiogenic-like effects of Corticotropin Releasing Factor (CRF) were triggered by CRF(1)-R activation of the dynorphin/kappa opioid receptor (KOR) system. Central CRF administration significantly reduced the percent open-arm time in the elevated plus maze (EPM). The reduction in open-arm time was blocked by pretreatment with the KOR antagonist norbinaltorphimine (norBNI), and was not evident in mice lacking the endogenous KOR ligand dynorphin. The CRF(1)-R agonist stressin 1 also significantly reduced open-arm time in the EPM, and this decrease was blocked by norBNI. In contrast, the selective CRF(2)-R agonist urocortin III did not affect open arm time, and mice lacking CRF(2)-R still showed an increase in anxiety-like behavior in response to CRF injection. However, CRF(2)-R knockout animals did not develop CRF conditioned place aversion, suggesting that CRF(1)-R activation may mediate anxiety and CRF(2)-R may encode aversion. Using a phosphoselective antibody (KORp) to identify sites of dynorphin action, we found that CRF increased KORp-immunoreactivity in the basolateral amygdala (BLA) of wildtype, but not in mice pretreated with the selective CRF(1)-R antagonist, antalarmin. Consistent with the concept that acute stress or CRF injection-induced anxiety was mediated by dynorphin release in the BLA, local injection of norBNI blocked the stress or CRF-induced increase in anxiety-like behavior; whereas norBNI injection in a nearby thalamic nucleus did not. The intersection of stress-induced CRF and the dynorphin/KOR system in the BLA was surprising, and these results suggest that CRF and dynorphin/KOR systems may coordinate stress-induced anxiety behaviors and aversive behaviors via different mechanisms.

Stress increases the risk of drug abuse, causes relapse to drug seeking, and potentiates the rewarding properties of both nicotine and cocaine. Understanding the mechanisms by which stress regulates the rewarding properties of drugs of abuse provides valuable insight into potential treatments for drug abuse. Prior reports have demonstrated that stress causes dynorphin release, activating kappa opioid receptors (KOR) in monoamine circuits resulting in both potentiation and reinstatement of cocaine and nicotine conditioned place preference. Here we report that kappa opioid-dependent reinstatement of cocaine and nicotine place preference is reduced when the mice are exposed to a randomized chronic mild stress (CMS) regime prior to training in a conditioned place preference-reinstatement paradigm. The CMS schedule involves seven different stressors (removal of nesting for 24 h, 5 min forced swim stress at 15°C, 8 h food and water deprivation, damp bedding overnight, white noise, cage tilt, and disrupted home cage lighting) rotated over a 3-week period. This response is KOR-selective, as CMS does not protect against cocaine or nicotine drug-primed reinstatement. This protection from reinstatement is also observed following sub-chronic social defeat stress, where each mouse is placed in an aggressor mouse home cage for a period of 20 min over 5 days. In contrast, a single acute stressor resulted in a potentiation of KOR-induced reinstatement, as previously reported. Prior studies have shown that stress alters sensitivity to opioids and prior stress can influence the pharmacodynamics of the opioid receptor system. Together, these findings suggest that exposure to different forms of stress may cause a dysregulation of kappa opioid circuitry and that changes resulting from mild stress can have protective and adaptive effects against drug relapse.