Probes for INS

Hevin/Sparcl1 is an astrocyte-secreted protein and regulates synapse formation. Here we show that astrocytic hevin signaling plays a critical role in maintaining chronic pain. Compared to wild-type mice, hevin-null mice exhibited normal mechanical and heat sensitivity but reduced inflammatory pain. Interestingly, hevin-null mice have faster recovery than wild-type mice from neuropathic pain after nerve injury. Intrathecal injection of wild-type hevin was sufficient to induce persistent mechanical allodynia in naïve mice. In hevin-null mice with nerve injury, AAV-mediated re-expression of hevin in GFAP-expressing spinal cord astrocytes could reinstate neuropathic pain. Mechanistically, hevin is crucial for spinal cord NMDA receptor (NMDAR) signaling. Hevin potentiated NMDA currents mediated by the GluN2B-containing NMDARs. Furthermore, intrathecal injection of a neutralizing antibody against hevin alleviated acute and persistent inflammatory pain, postoperative pain, and neuropathic pain. Secreted hevin was detected in mouse cerebrospinal fluid (CSF) and nerve injury significantly increased CSF hevin abundance. Finally, neurosurgery caused rapid and substantial increases in SPARCL1/HEVIN levels in human CSF. Collectively, our findings support a critical role of hevin and astrocytes in the maintenance of chronic pain. Neutralizing of secreted hevin with monoclonal antibody may provide a new therapeutic strategy for treating acute and chronic pain and NMDAR-medicated neurodegeneration.

The cerebellum, a primary brain structure involved in the control of sensorimotor tasks, also contributes to higher cognitive functions including reward, emotion and social interaction. Although the regulation of these behaviors has been largely ascribed to the monoaminergic system in limbic regions, the contribution of cerebellar dopamine signaling in the modulation of these functions remains largely unknown. By combining cell-type-specific transcriptomics, histological analyses, three-dimensional imaging and patch-clamp recordings, we demonstrate that cerebellar dopamine D2 receptors (D2Rs) in mice are preferentially expressed in Purkinje cells (PCs) and regulate synaptic efficacy onto PCs. Moreover, we found that changes in D2R levels in PCs of male mice during adulthood alter sociability and preference for social novelty without affecting motor functions. Altogether, these findings demonstrate novel roles for D2R in PC function and causally link cerebellar D2R levels of expression to social behaviors.

Hypothalamic melanin-concentrating hormone (MCH) polypeptide contributes to regulating energy homeostasis, sleep and memory, although the mechanistic bases of its effects are unknown. In this study, in mice, we uncovered the physiological mechanism underlying the functional role of MCH signaling in projections to the dorsolateral septum (dLS), a region involved in routing hippocampal firing rhythms and encoding spatial memory based on such rhythms. Firing activity within the dLS in response to dorsal CA3 (dCA3) excitation is limited by strong feed-forward inhibition (FFI). We found that MCH synchronizes dLS neuronal firing with its dCA3 inputs by enhancing GABA release, which subsequently reduces the FFI and augments dCA3 excitatory input strength, both via pre-synaptic mechanisms. At the functional level, our data reveal a role for MCH signaling in the dLS in facilitating spatial memory. These findings support a model in which peptidergic signaling within the dLS modulates dorsal hippocampal output and supports memory encoding.

Anxiety disorders are highly prevalent in modern society and better treatments are required. Key brain areas and signaling systems underlying anxiety include prefrontal cortex, hippocampus, and amygdala, and monoaminergic and peptidergic systems, respectively. Hindbrain GABAergic projection neurons that express the peptide, relaxin-3, broadly innervate the forebrain, particularly the septum and hippocampus, and relaxin-3 acts via a Gi/o -protein-coupled receptor known as the relaxin-family peptide 3 receptor (RXFP3). Thus, relaxin-3/RXFP3 signaling is implicated in modulation of arousal, motivation, mood, memory, and anxiety. Ventral hippocampus (vHip) is central to affective and cognitive processing and displays a high density of relaxin-3-positive nerve fibers and RXFP3 binding sites, but the identity of target neurons and associated effects on behavior are unknown. Therefore, in adult, male rats, we assessed the neurochemical nature of hippocampal RXFP3 mRNA-expressing neurons and anxiety-like and social behavior following chronic RXFP3 activation in vHip by viral vector expression of an RXFP3-selective agonist peptide, R3/I5. RXFP3 mRNA detected by fluorescent in situ hybridization was topographically distributed across the hippocampus in somatostatin- and parvalbumin-mRNA expressing GABA neurons. Chronic RXFP3 activation in vHip increased anxiety-like behavior in the light-dark box and elevated-plus maze, but not the large open-field test, and reduced social interaction with a conspecific stranger. Our data reveal disruptive effects of persistent RXFP3 signaling on hippocampal GABA networks important in anxiety; and identify a potential therapeutic target for anxiety disorders that warrants further investigation in relevant preclinical models.

Supraspinal brain regions modify nociceptive signals in response to various stressors including stimuli that elevate pain thresholds. The medulla oblongata has previously been implicated in this type of pain control, but the neurons and molecular circuits involved have remained elusive. Here we identify catecholaminergic neurons in the caudal ventrolateral medulla that are activated by noxious stimuli in mice. Upon activation, these neurons produce bilateral feed-forward inhibition that attenuates nociceptive responses through a pathway involving the locus coeruleus and norepinephrine in the spinal cord. This pathway is sufficient to attenuate injury-induced heat allodynia and is required for counter-stimulus induced analgesia to noxious heat. Our findings define a component of the pain modulatory system that regulates nociceptive responses.

Anxiety-like behaviors in mice include social avoidance and avoidance of bright spaces. Whether these features are distinctly regulated is unclear. We demonstrate that in mice, social and anxiogenic stimuli, respectively, increase and decrease serotonin (5-HT) levels in basal amygdala (BA). In dorsal raphe nucleus (DRN), 5-HT∩vGluT3 neurons projecting to BA parvalbumin (DRN5-HT∩vGluT3-BAPV) and pyramidal (DRN5-HT∩vGluT3-BAPyr) neurons have distinct intrinsic properties and gene expression and respond to anxiogenic and social stimuli, respectively. Activation of DRN5-HT∩vGluT3→BAPV inhibits 5-HT release via GABAB receptors on serotonergic terminals in BA, inducing social avoidance and avoidance of bright spaces. Activation of DRN5-HT∩vGluT3→BA neurons inhibits two subsets of BAPyr neurons via 5-HT1A receptors (HTR1A) and 5-HT1B receptors (HTR1B). Pharmacological inhibition of HTR1A and HTR1B in BA induces avoidance of bright spaces and social avoidance, respectively. These findings highlight the functional significance of heterogenic inputs from DRN to BA subpopulations in the regulation of separate anxiety-related behaviors.

Background Substance use disorders (SUDs) are associated with disruptions in circadian rhythms. Both human and animal work has shown the integral role for circadian clocks in the modulation of reward behaviors. Interestingly, astrocytes have emerged as key regulators of circadian rhythmicity. However, no studies to date have identified the role of circadian astrocyte function in the nucleus accumbens (NAc), a hub for reward regulation, or determined the importance of these rhythms for reward-related behavior. Methods Using astrocyte-specific RNA-sequencing across time-of-day, we first characterized diurnal variation of the NAc astrocyte transcriptome. We then investigated the functional significance of this circadian regulation through viral-mediated disruption of molecular clock function in NAc astrocytes, followed by assessment of reward-related behaviors, metabolic-related molecular assays, and whole-cell electrophysiology in the NAc. Results Strikingly, ∼43% of the astrocyte transcriptome has a diurnal rhythm and key metabolic pathways were enriched among the top rhythmic genes. Moreover, mice with a viral-mediated loss of molecular clock function in NAc astrocytes show a significant increase in locomotor response to novelty, exploratory drive, operant food self-administration and motivation. At the molecular level, these animals also show disrupted metabolic gene expression, along with significant downregulation of both lactate and glutathione levels in the NAc. Importantly, loss of NAc astrocyte clock function also significantly altered glutamatergic signaling onto neighboring medium spiny neurons, alongside upregulated glutamate-related gene expression. Conclusions Taken together, these findings demonstrate a novel role for astrocyte circadian molecular clock function in the regulation of the NAc and reward-related behaviors.

Hypidone hydrochloride (YL-0919) is a novel antidepressant in clinical phase II trial. Previous studies show that YL-0919 is a selective 5-HT (serotonin) reuptake inhibitor, 5-HT1A receptor partial agonist, and 5-HT6 receptor agonist, which exerts antidepressant effects in various animal models, but its effects on neural function remain unclear. Medial prefrontal cortex (mPFC), a highly evolved brain region, controls highest order cognitive functions and emotion regulation. In this study we investigated the effects of YL-0919 on the mPFC function, including the changes in neuronal activities using electrophysiological recordings. Extracellular recording (in vivo) showed that chronic administration of YL-0919 significantly increased the spontaneous discharges of mPFC neurons. In mouse mPFC slices, whole-cell recording revealed that perfusion of YL-0919 significantly increased the frequency of sEPSCs, but decreased the frequency of sIPSCs. Then we conducted whole-cell recording in mPFC slices of GAD67-GFP transgenic mice, and demonstrated that YL-0919 significantly inhibited the excitability of GABAergic neurons. In contrast, it did not alter the excitability of pyramidal neurons in mPFC slices of normal mice. Moreover, the inhibition of GABAergic neurons by YL-0919 was prevented by pre-treatment with 5-HT1A receptor antagonist WAY 100635. Finally, chronic administration of YL-0919 significantly increased the phosphorylation levels of mTOR and GSK-3β in the mPFC as compared with vehicle. Taken together, our results demonstrate that YL-0919 enhances the excitability of mPFC via a disinhibition mechanism to fulfill its rapid antidepressant neural mechanism, which was accomplished by 5-HT1A receptor-mediated inhibition of inhibitory GABAergic interneurons.

Background The neuropeptide PACAP is a master regulator of central and peripheral stress responses, yet it is not clear how PACAP projections throughout the brain execute endocrine and behavioral stress responses. Methods We used AAV neuronal tracing, an acute restraint stress (ARS) paradigm, and intersectional genetics, in C57Bl6 mice, to identify PACAP-containing circuits controlling stress-induced behavior and endocrine activation. Results PACAP deletion from forebrain excitatory neurons, including a projection directly from medial prefrontal cortex (mPFC) to hypothalamus, impairs c-fos activation and CRH mRNA elevation in PVN after 2 hr of restraint, without affecting ARS-induced hypophagia, or c-fos elevation in non-hypothalamic brain. Elimination of PACAP within projections from lateral parabrachial nucleus to extended amygdala (EA), on the other hand, attenuates ARS-induced hypophagia, along with EA fos induction, without affecting ARS-induced CRH mRNA elevation in PVN. PACAP projections to EA terminate at PKCδ neurons in both central amygdala (CeA) and oval nuclei of bed nucleus of stria terminalis (BNSTov). Silencing of PKCδ neurons in CeA, but not in BNSTov, attenuates ARS-induced hypophagia. Experiments were carried out in mice of both sexes with n>5 per group. Conclusions A frontocortical descending PACAP projection controls PVN CRH mRNA production, to maintain hypothalamo-pituitary adrenal (HPA) axis activation, and regulate the endocrine response to stress. An ascending PACAPergic projection from eLPBn to PKCδ neurons in central amygdala regulates behavioral responses to stress. Defining two separate limbs of the acute stress response provides broader insight into the specific brain circuitry engaged by the psychogenic stress response.

The lateral habenula (LHb) sends complex projections to several areas of the mesopontine tegmentum, the raphe, and the hypothalamus. However, few markers have been available to distinguish subsets of LHb neurons that may serve these pathways. In order to address this complexity, we examined the mouse and rat LHb for neurons that express the GABA biosynthesis enzymes glutamate decarboxylase 1 and 2 (GAD1, GAD2), and the vesicular GABA transporter (VGAT). The mouse LHb contains a population of neurons that express GAD2, while the rat LHb contains discrete populations of neurons that express GAD1 and VGAT. However, we could not detect single neurons in either species that co-express a GABA synthetic enzyme and VGAT, suggesting that these LHb neurons do not use GABA for conventional synaptic transmission. Instead, all of the neuronal types expressing a GABAergic marker in both species showed co-expression of the glutamate transporter VGluT2. Anterograde tract-tracing of the projections of GAD2-expressing LHb neurons in Gad2Cre mice, combined with retrograde tracing from selected downstream nuclei, show that LHb-GAD2 neurons project selectively to the midline structures in the mesopontine tegmentum, including the median raphe and nucleus incertus, and only sparsely innervate the hypothalamus, rostromedial tegmental nucleus, and ventral tegmental area. Postsynaptic recording of LHb-GAD2 neuronal input to tegmental neurons confirms that glutamate, not GABA, is the fast neurotransmitter in this circuit. Thus GAD2 expression can serve as a marker for functional studies of excitatory neurons serving specific LHb output pathways in mice.SIGNFICANCE STATEMENT The lateral habenula provides a major link between subcortical forebrain areas and the dopamine (DA) and serotonin (5HT) systems of the midbrain and pons, and it has been implicated in reward mechanisms and the regulation of mood states. Few markers have been available for the specific cell types and complex output pathways of the lateral habenula. Here we examined the expression of genes mediating GABAergic and glutamatergic transmission in the mouse and rat LHb, where no neurons in either species expressed a full complement of GABAergic markers, and all expressed the glutamatergic marker VGluT2. Consistent with this, in mice the LHb GAD2 neurons are excitatory and appear to use only glutamate for fast synaptic transmission.

The endocannabinoids have been shown to target the afferents of hypothalamic neurons via cannabinoid 1 receptor (CB1) and thereby to influence their excitability at various physiological and/or pathological processes. Kisspeptin (KP) neurons form afferents of multiple neuroendocrine cells and influence their activity via signaling through a variation of co-expressed classical neurotransmitters and neuropeptides. The differential potency of endocannabinoids to influence the release of classical transmitters or neuropeptides, and the ovarian cycle-dependent functioning of the endocannabinoid signaling in the gonadotropin-releasing hormone (GnRH) neurons initiated us to study whether (a) the different subpopulations of KP neurons express CB1 mRNAs, (b) the expression is influenced by estrogen, and (c) CB1-immunoreactivity is present in the KP afferents to GnRH neurons. The aim of the study was to investigate the site- and cell-specific expression of CB1 in female mice using multiple labeling in situ hybridization and immunofluorescent histochemical techniques. The results support that CB1 mRNAs are expressed by both the GABAergic and glutamatergic subpopulations of KP neurons, the receptor protein is detectable in two-thirds of the KP afferents to GnRH neurons, and the expression of CB1 mRNA shows an estrogen-dependency. The applied estrogen-treatment, known to induce proestrus, reduced the level of CB1 transcripts in the rostral periventricular area of the third ventricle and arcuate nucleus, and differently influenced its co-localization with vesicular GABA transporter or vesicular glutamate transporter-2 in KP neurons. This indicates a gonadal cycle-dependent role of endocannabinoid signaling in the neuronal circuits involving KP neurons.

Neuropathic pain, an intractable pain symptom that occurs after nerve damage, is caused by the aberrant excitability of spinal dorsal horn (SDH) neurons. Gabapentinoids, the most commonly used drugs for neuropathic pain, inhibit spinal calcium-mediated neurotransmitter release by binding to α2δ-1, a subunit of voltage-gated calcium channels, and alleviate neuropathic pain. However, the exact contribution of α2δ-1 expressed in SDH neurons to the altered synaptic transmission and mechanical hypersensitivity following nerve injury is not fully understood. In this study, we investigated which types of SDH neurons express α2δ-1 and how α2δ-1 in SDH neurons contributes to the mechanical hypersensitivity and altered spinal synaptic transmission after nerve injury. Using in situ hybridization technique, we found that Cacna2d1, mRNA coding α2δ-1, was mainly colocalized with Slc17a6, an excitatory neuronal marker, but not with Slc32a1, an inhibitory neuronal marker in the SDH. To investigate the role of α2δ-1 in SDH neurons, we used clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system and showed that SDH neuron-specific ablation of Cacna2d1 alleviated mechanical hypersensitivity following nerve injury. We further found that excitatory post-synaptic responses evoked by electrical stimulation applied to the SDH were significantly enhanced after nerve injury, and that these enhanced responses were significantly decreased by application of mirogabalin, a potent α2δ-1 inhibitor, and by SDH neuron-specific ablation of Cacna2d1. These results suggest that α2δ-1 expressed in SDH excitatory neurons facilitates spinal nociceptive synaptic transmission and contributes to the development of mechanical hypersensitivity after nerve injury.