Probes for INS

We recently developed a mouse model of appetitive operant aggression and reported that adult male outbred CD-1 mice lever-press for the opportunity to attack subordinate male mice and relapse to aggression seeking during abstinence. Here we studied the role of nucleus accumbens (NAc) dopamine D1- and D2-receptor (Drd1 and Drd2) expressing neurons in aggression self-administration and aggression seeking. We trained CD-1 mice to self-administer intruders (9 d, 12 trials/d) and tested them for aggression self-administration and aggression seeking on abstinence day 1. We used immunohistochemistry and in situ hybridization to measure the neuronal activity marker Fos in the NAc, and cell-type specific colocalization of Fos with Drd1- and Drd2-expressing neurons. To test the causal role of Drd1- and Drd2-expressing neurons, we validated a transgenic hybrid breeding strategy crossing inbred Drd1-Cre and Drd2-Cre transgenic mice with outbred CD-1 mice and used cell-type specific Cre-DREADD (hM4Di) to inhibit NAc Drd1- and Drd2-expressing neuron activity. We found that that aggression self-administration and aggression seeking induced higher Fos expression in NAc shell than in core, that Fos colocalized with Drd1 and Drd2 in both subregions, and that chemogenetic inhibition of Drd1-, but not Drd2-, expressing neurons decreased aggression self-administration and aggression seeking. Results indicate a cell-type specific role of Drd1-expressing neurons that is critical for both aggression self-administration and aggression seeking. Our study also validates a simple breeding strategy between outbred CD-1 mice and inbred C57-based Cre lines that can be used to study cell-type and circuit mechanisms of aggression reward and relapse.SIGNIFICANCE STATEMENTAggression is often comorbid with neuropsychiatric diseases, including drug addiction. One form, appetitive aggression, exhibits symptomatology that mimics that of drug addiction and is hypothesized to be due to dysregulation of addiction-related reward circuits. However, our mechanistic understanding of the circuitry modulating appetitive operant aggression is limited. Here we use a novel mouse model of aggression self-administration and relapse, in combination with immunohistochemistry, in situ hybridization, and chemogenetic manipulations to examine how cell-types in the nucleus accumbens are recruited for, and control, operant aggression self-administration and aggression seeking on abstinence day 1. We found that one population, dopamine receptor 1-expressing neurons, act as a critical modulator of operant aggression reward and aggression seeking.

Palmoplantar skin is structurally and functionally unique, but the transcriptional programs driving this specialization are unclear. Here, we use bulk and single-cell RNA sequencing of human palm, sole, and hip skin to describe the distinguishing characteristics of palmoplantar and non-palmoplantar skin while also uncovering differences between palmar and plantar sites. Our approach reveals an altered immune environment in palmoplantar skin, with downregulation of diverse immunological processes and decreased immune cell populations. Further, we identify specific fibroblast populations that appear to orchestrate key differences in cell-cell communication in palm, sole, and hip. Dedicated keratinocyte analysis highlights major differences in basal cell fraction among the three sites and demonstrates the existence of two spinous keratinocyte populations constituting parallel, site-selective epidermal differentiation trajectories. In summary, this deep characterization of highly adapted palmoplantar skin contributes key insights into the fundamental biology of human skin and provides a valuable data resource for further investigation.

In albinism, aberrations in the ipsi-/contralateral retinal ganglion cell (RGC) ratio compromise the functional integrity of the binocular circuit. Here, we focus on the mouse ciliary margin zone (CMZ), a neurogenic niche at the embryonic peripheral retina, to investigate developmental processes regulating RGC neurogenesis and identity acquisition. We found that the mouse ventral CMZ generates predominantly ipsilaterally projecting RGCs, but this output is altered in the albino visual system because of CyclinD2 downregulation and disturbed timing of the cell cycle. Consequently, albino as well as CyclinD2-deficient pigmented mice exhibit diminished ipsilateral retinogeniculate projection and poor depth perception. In albino mice, pharmacological stimulation of calcium channels, known to upregulate CyclinD2 in other cell types, augmented CyclinD2-dependent neurogenesis of ipsilateral RGCs and improved stereopsis. Together, these results implicate CMZ neurogenesis and its regulators as critical for the formation and function of the mammalian binocular circuit.

Sleep disturbances are strongly associated with cardiovascular diseases. Baroreflex, a basic cardiovascular regulation mechanism, is modulated by sleep-wake states. Here, we show that neurons at key stages of baroreflex pathways also promote sleep. Using activity-dependent genetic labeling, we tagged neurons in the nucleus of the solitary tract (NST) activated by blood pressure elevation and confirmed their barosensitivity with optrode recording and calcium imaging. Chemogenetic or optogenetic activation of these neurons promoted non-REM sleep in addition to decreasing blood pressure and heart rate. GABAergic neurons in the caudal ventrolateral medulla (CVLM)-a downstream target of the NST for vasomotor baroreflex-also promote non-REM sleep, partly by inhibiting the sympathoexcitatory and wake-promoting adrenergic neurons in the rostral ventrolateral medulla (RVLM). Cholinergic neurons in the nucleus ambiguous-a target of the NST for cardiac baroreflex-promoted non-REM sleep as well. Thus, key components of the cardiovascular baroreflex circuit are also integral to sleep-wake brain-state regulation.

mTOR signaling, involving mTORC1 and mTORC2 complexes, critically regulates neural development and is implicated in various brain disorders. However, we do not fully understand all of the upstream signaling components that can regulate mTOR signaling, especially in neurons. Here, we show a direct, regulated inhibition of mTOR by Tanc2, an adaptor/scaffolding protein with strong neurodevelopmental and psychiatric implications. While Tanc2-null mice show embryonic lethality, Tanc2-haploinsufficient mice survive but display mTORC1/2 hyperactivity accompanying synaptic and behavioral deficits reversed by mTOR-inhibiting rapamycin. Tanc2 interacts with and inhibits mTOR, which is suppressed by mTOR-activating serum or ketamine, a fast-acting antidepressant. Tanc2 and Deptor, also known to inhibit mTORC1/2 minimally affecting neurodevelopment, distinctly inhibit mTOR in early- and late-stage neurons. Lastly, Tanc2 inhibits mTORC1/2 in human neural progenitor cells and neurons. In summary, our findings show that Tanc2 is a mTORC1/2 inhibitor affecting neurodevelopment.

Sevoflurane has been the most widely used inhaled anesthetics with a favorable recovery profile; however, the precise mechanisms underlying its anesthetic action are still not completely understood. Here the authors show that sevoflurane activates a cluster of urocortin 1 (UCN1+ )/cocaine- and amphetamine-regulated transcript (CART+ ) neurons in the midbrain involved in its anesthesia. Furthermore, growth hormone secretagogue receptor (GHSR) is highly enriched in sevoflurane-activated UCN1+ /CART+ cells and is necessary for sleep induction. Blockade of GHSR abolishes the excitatory effect of sevoflurane on UCN1+ /CART+ neurons and attenuates its anesthetic effect. Collectively, their data suggest that anesthetic action of sevoflurane necessitates the GHSR activation in midbrain UCN1+ /CART+ neurons, which provides a novel target including the nucleus and receptor in the field of anesthesia.

During infection, animals exhibit adaptive changes in physiology and behaviour aimed at increasing survival. Although many causes of infection exist, they trigger similar stereotyped symptoms such as fever, warmth-seeking, loss of appetite and fatigue1,2. Yet exactly how the nervous system alters body temperature and triggers sickness behaviours to coordinate responses to infection remains unknown. Here we identify a previously uncharacterized population of neurons in the ventral medial preoptic area (VMPO) of the hypothalamus that are activated after sickness induced by lipopolysaccharide (LPS) or polyinosinic:polycytidylic acid. These neurons are crucial for generating a fever response and other sickness symptoms such as warmth-seeking and loss of appetite. Single-nucleus RNA-sequencing and multiplexed error-robust fluorescence in situ hybridization uncovered the identity and distribution of LPS-activated VMPO (VMPOLPS) neurons and non-neuronal cells. Gene expression and electrophysiological measurements implicate a paracrine mechanism in which the release of immune signals by non-neuronal cells during infection activates nearby VMPOLPS neurons. Finally, we show that VMPOLPS neurons exert a broad influence on the activity of brain areas associated with behavioural and homeostatic functions and are synaptically and functionally connected to circuit nodes controlling body temperature and appetite. Together, these results uncover VMPOLPS neurons as a control hub that integrates immune signals to orchestrate multiple sickness symptoms in response to infection.

Homeostatic thermogenesis is an essential protective feature of endotherms. However, the specific neuronal types involved in cold-induced thermogenesis remain largely unknown. Using functional magnetic resonance imaging and in situ hybridization, we screened for cold-sensitive neurons and found preprodynorphin (PDYN)-expressing cells in the dorsal medial region of the ventromedial hypothalamus (dmVMH) to be a candidate. Subsequent in vivo calcium recording showed that cold temperature activates dmVMHPdyn neurons, whereas hot temperature suppresses them. In addition, optogenetic activation of dmVMHPdyn neurons increases the brown adipose tissue and core body temperature, heart rate, and blood pressure, whereas optogenetic inhibition shows opposite effects, supporting their role in homeostatic thermogenesis. Furthermore, we found that dmVMHPdyn neurons are linked to known thermoregulatory circuits. Importantly, dmVMHPdyn neurons also show activation during mouse social interaction, and optogenetic inhibition suppresses social interaction and associated hyperthermia. Together, our study describes dual functions of dmVMHPdyn neurons that allow coordinated regulation of body temperature and social behaviors.

While apneas are associated with multiple pathological and fatal conditions, the underlying molecular mechanisms remain elusive. We report that a mutated form of the transcription factor Mafa (Mafa4A) that prevents phosphorylation of the Mafa protein leads to an abnormally high incidence of breath holding apneas and death in newborn Mafa4A/4A mutant mice. This apneic breathing is phenocopied by restricting the mutation to central GABAergic inhibitory neurons and by activation of inhibitory Mafa neurons while reversed by inhibiting GABAergic transmission centrally. We find that Mafa activates the Gad2 promoter in vitro and that this activation is enhanced by the mutation that likely results in increased inhibitory drives onto target neurons. We also find that Mafa inhibitory neurons are absent from respiratory, sensory (primary and secondary) and pontine structures but are present in the vicinity of the hypoglossal motor nucleus including premotor neurons that innervate the geniohyoid muscle, to control upper airway patency. Altogether, our data reveal a role for Mafa phosphorylation in regulation of GABAergic drives and suggest a mechanism whereby reduced premotor drives to upper airway muscles may cause apneic breathing at birth.

Vascular integrity is essential for organ homeostasis to prevent edema formation and infiltration of inflammatory cells. Long non-coding RNAs (lncRNAs) are important regulators of gene expression and often expressed in a cell type-specific manner. By screening for endothelial-enriched lncRNAs, we identified the undescribed lncRNA NTRAS to control endothelial cell functions. Silencing of NTRAS induces endothelial cell dysfunction in vitro and increases vascular permeability and lethality in mice. Biochemical analysis revealed that NTRAS, through its CA-dinucleotide repeat motif, sequesters the splicing regulator hnRNPL to control alternative splicing of tight junction protein 1 (TJP1; also named zona occludens 1, ZO-1) pre-mRNA. Deletion of the hnRNPL binding motif in mice (Ntras∆CA/∆CA ) significantly repressed TJP1 exon 20 usage, favoring expression of the TJP1α- isoform, which augments permeability of the endothelial monolayer. Ntras∆CA/∆CA mice further showed reduced retinal vessel growth and increased vascular permeability and myocarditis. In summary, this study demonstrates that NTRAS is an essential gatekeeper of vascular integrity.