Probes for INS

Major depressive disorder (MDD) patients display a common but often variable set of symptoms making successful, sustained treatment difficult to achieve. Separate depressive symptoms may be encoded by differential changes in distinct circuits in the brain, yet how discrete circuits underlie behavioral subsets of depression and how they adapt in response to stress has not been addressed. We identify two discrete circuits of parvalbumin-positive (PV) neurons in the ventral pallidum (VP) projecting to either the lateral habenula or ventral tegmental area contributing to depression. We find that these populations undergo different electrophysiological adaptations in response to social defeat stress, which are normalized by antidepressant treatment. Furthermore, manipulation of each population mediates either social withdrawal or behavioral despair, but not both. We propose that distinct components of the VP PV circuit can subserve related, yet separate depressive-like phenotypes in mice, which could ultimately provide a platform for symptom-specific treatments of depression.

Stress contributes to numerous psychiatric disorders. CRF signaling and CRF neurons in the bed nucleus of the stria terminalis (BNST) drive negative affective behaviors, thus agents that decrease activity of these cells may be of therapeutic interest. Here, we show that acute restraint stress increases cFos expression in CRF neurons in the mouse dorsal BNST, consistent with a role for these neurons in stress-related behaviors. We find that activation of α2A-adrenergic receptors (ARs) by the agonist guanfacine reduced cFos expression in these neurons both in stressed and unstressed conditions. Further, we find that α- and β-ARs differentially regulate excitatory drive onto these neurons. Pharmacological and channelrhodopsin-assisted mapping experiments suggest that α2A-ARs specifically reduce excitatory drive from parabrachial nucleus (PBN) afferents onto CRF neurons. Given that the α2A-AR is a Gi-linked GPCR, we assessed the impact of activating the Gi-coupled DREADD hM4Di in the PBN on restraint stress regulation of BNST CRF neurons. CNO activation of PBN hM4Di reduced stress-induced Fos in BNST Crh neurons. Further, utilizing Prkcd as an additional marker of BNST neuronal identity, we uncovered a female-specific upregulation of the co-expression of Prkcd/Crh in BNST neurons following stress, which was prevented by ovariectomy. These findings show that stress activates BNST CRF neurons, and that α2A-AR activation suppresses the in vivo activity of these cells, at least in part by suppressing excitatory drive from PBN inputs onto CRF neurons.SIGNIFICANCE STATEMENTStress is a major variable contributing to mood disorders. Here, we show that stress increases activation of BNST CRF neurons that drive negative affective behavior. We find that the clinically well-tolerated α2A-AR agonist guanfacine reduces activity of these cells in vivo, and reduces excitatory PBN inputs onto these cells ex vivo Additionally, we uncover a novel sex-dependent co-expression of Prkcd with Crh in female BNST neurons after stress, an effect abolished by ovariectomy. These results demonstrate input-specific interactions between NE and CRF, and point to an action by which guanfacine may reduce negative affective responses.

The central amygdala (CeA) is important for fear responses to discrete cues. Recent findings indicate that the CeA also contributes to states of sustained apprehension that characterize anxiety, although little is known about the neural circuitry involved. The stress neuropeptide corticotropin releasing factor (CRF) is anxiogenic and is produced by subpopulations of neurons in the lateral CeA and the dorsolateral bed nucleus of the stria terminalis (dlBST). Here we investigated the function of these CRF neurons in stress-induced anxiety using chemogenetics in male rats that express Cre recombinase from a Crh promoter. Anxiety-like behavior was mediated by CRF projections from the CeA to the dlBST and depended on activation of CRF1 receptors and CRF neurons within the dlBST. Our findings identify a CRFCeA→CRFdlBST circuit for generating anxiety-like behavior and provide mechanistic support for recent human and primate data suggesting that the CeA and BST act together to generate states of anxiety.SIGNIFICANCE STATEMENT Anxiety is a negative emotional state critical to survival, but persistent, exaggerated apprehension causes substantial morbidity. Identifying brain regions and neurotransmitter systems that drive anxiety can help in developing effective treatment. Much evidence in rodents indicates that neurons in the bed nucleus of the stria terminalis (BST) generate anxiety-like behaviors, but more recent findings also implicate neurons of the CeA. The neuronal subpopulations and circuitry that generate anxiety are currently subjects of intense investigation. Here we show that CeA neurons that release the stress neuropeptide corticotropin-releasing factor (CRF) drive anxiety-like behaviors in rats via a pathway to dorsal BST that activates local BST CRF neurons. Thus, our findings identify a CeA→BST CRF neuropeptide circuit that generates anxiety-like behavior.

Previous studies revealed that cementum formation is tightly regulated by inorganic pyrophosphate (PPi), a mineralization inhibitor. Local PPiconcentrations are determined by regulators, including ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), which increases PPiconcentrations by adenosine triphosphate hydrolysis. Orthodontic forces stimulate alveolar bone remodelling, leading to orthodontic tooth movement (OTM). To better understand how disturbed mineral metabolism and the resulting altered periodontal structures affect OTM, we employed Enpp1 mutant mice that feature reduced PPi and increased cervical cementum in a model of OTM induced by a stretched closed-coil spring ligated between the maxillary left first molar and maxillary incisors. We analyzed tooth movement, osteoclast/odontoclast response, and tooth root resorption by micro-computed tomography, histology, histomorphometry, and immunohistochemistry. Preoperatively, we noted an altered periodontium in Enpp1 mutant mice, with significantly increased periodontal ligament (PDL) volume and thickness, as well as increased PDL-bone/tooth root surface area, compared to wild-type (WT) controls. After 11 d of orthodontic treatment, Enpp1 mutant mice displayed 38% reduced tooth movement versus WT mice. Molar roots in Enpp1 mutant mice exhibited less change in PDL width in compression and tension zones compared to WT mice. Root resorption was noted in both groups with no difference in average depths, but resorption lacunae in Enpp1 mutant mice were almost entirely limited to cementum, with 150% increased cementum resorption and 92% decreased dentin resorption. Osteoclast/odontoclast cells were reduced by 64% in Enpp1 mutant mice, with a predominance of tartrate-resistant acid phosphatase (TRAP)-positive cells on root surfaces, compared to WT mice. Increased numbers of TRAP-positive cells on root surfaces were associated with robust immunolocalization of osteopontin (OPN) and receptor-activator of NF-κB ligand (RANKL). Collectively, reduced response to orthodontic forces, decreased tooth movement, and altered osteoclast/odontoclast distribution suggests Enpp1 loss of function has direct effects on clastic function/recruitment and/or indirect effects on periodontal remodeling via altered periodontal structure or tissue mineralization.

Voluntary urination ensures that waste is eliminated when safe and socially appropriate, even without a pressing urge. Uncontrolled urination, or incontinence, is a common problem with few treatment options. Normal urine release requires a small region in the brainstem known as Barrington's nucleus (Bar), but specific neurons that relax the urethral sphincter and enable urine flow are unknown. Here we identify a small subset of Bar neurons that control the urethral sphincter in mice. These excitatory neurons express estrogen receptor 1 (BarESR1), project to sphincter-relaxing interneurons in the spinal cord and are active during natural urination. Optogenetic stimulation of BarESR1 neurons rapidly initiates sphincter bursting and efficient voiding in anesthetized and behaving animals. Conversely, optogenetic and chemogenetic inhibition reveals their necessity in motivated urination behavior. The identification of these cells provides an expanded model for the control of urination and its dysfunction.

Understanding the neural framework behind appetite control is fundamental to developing effective therapies to combat the obesity epidemic. The paraventricular hypothalamus (PVH) is critical for appetite regulation, yet, the real-time, physiological response properties of PVH neurons to nutrients are unknown. Using a combination of fiber photometry, electrophysiology, immunohistochemistry, and neural manipulation strategies, we determined the population dynamics of four molecularly delineated PVH subsets implicated in feeding behavior: glucagon-like peptide 1 receptor (PVHGlp1r), melanocortin-4 receptor (PVHMc4r), oxytocin (PVHOxt), and corticotropin-releasing hormone (PVHCrh). We identified both calorie- and state-dependent sustained activity increases and decreases in PVHGlp1r and PVHCrh populations, respectively, while observing transient bulk changes of PVHMc4r, but no response in PVHOxt, neurons to food. Furthermore, we highlight the role of PVHGlp1r neurons in orchestrating acute feeding behavior, independent of the anti-obesity drug liraglutide, and demonstrate the indispensability of PVHGlp1r and PVHMc4r, but not PVHOxt or PVHCrh neurons, in body weight maintenance.