Probes for INS

Where the gene is expressed determines the function of the gene. Neuregulin 1 (Nrg1) encodes a tropic factor and is genetically linked with several neuropsychiatry diseases such as schizophrenia, bipolar disorder and depression. Nrg1 has broad functions ranging from regulating neurodevelopment to neurotransmission in the nervous system. However, the expression pattern of Nrg1 at the cellular and circuit levels in rodent brain is not full addressed.Here we used CRISPR/Cas9 techniques to generate a knockin mouse line (Nrg1Cre/+) that expresses a P2A-Cre cassette right before the stop codon of Nrg1 gene. Since Cre recombinase and Nrg1 are expressed in the same types of cells in Nrg1Cre/+ mice, the Nrg1 expression pattern can be revealed through the Cre-reporting mice or adeno-associated virus (AAV) that express fluorescent proteins in a Cre-dependent way. Using unbiased stereology and fluorescence imaging, the cellular expression pattern of Nrg1 and axon projections of Nrg1-positive neurons were investigated.In the olfactory bulb (OB), Nrg1 is expressed in GABAergic interneurons including periglomerular (PG) and granule cells. In the cerebral cortex, Nrg1 is mainly expressed in the pyramidal neurons of superficial layers that mediate intercortical communications. In the striatum, Nrg1 is highly expressed in the Drd1-positive medium spiny neurons (MSNs) in the shell of nucleus accumbens (NAc) that project to substantia nigra pars reticulata (SNr). In the hippocampus, Nrg1 is mainly expressed in granule neurons in the dentate gyrus and pyramidal neurons in the subiculum. The Nrg1-expressing neurons in the subiculum project to retrosplenial granular cortex (RSG) and mammillary nucleus (MM). Nrg1 is highly expressed in the median eminence (ME) of hypothalamus and Purkinje cells in the cerebellum.Nrg1 is broadly expressed in mouse brain, mainly in neurons, but has unique expression patterns in different brain regions.

Neurons in the hypothalamic preoptic area (POA) regulate multiple homeostatic processes, including thermoregulation and sleep, by sensing afferent input and modulating sympathetic nervous system output. The POA has an autonomous circadian clock and may also receive circadian signals indirectly from the suprachiasmatic nucleus. We have previously defined a subset of neurons in the POA termed QPLOT neurons that are identified by the expression of molecular markers (Qrfp, Ptger3, LepR, Opn5, Tacr3) that suggest receptivity to multiple stimuli. Because Ptger3, Opn5, and Tacr3 encode G-protein coupled receptors (GPCRs), we hypothesized that elucidating the G-protein signaling in these neurons is essential to understanding the interplay of inputs in the regulation of metabolism. Here, we describe how the stimulatory Gs-alpha subunit (Gnas) in QPLOT neurons regulates metabolism in mice. We analyzed Opn5cre; Gnasfl/fl mice using indirect calorimetry at ambient temperatures of 22°C (a historical standard), 10°C (a cold challenge), and 28°C (thermoneutrality) to assess the ability of QPLOT neurons to regulate metabolism. We observed a marked decrease in nocturnal locomotion of Opn5cre; Gnasfl/fl mice at both 28°C and 22°C, but no overall differences in energy expenditure, respiratory exchange, or food and water consumption. To analyze daily rhythmic patterns of metabolism, we assessed circadian parameters including amplitude, phase, and MESOR. Loss-of-function GNAS in QPLOT neurons resulted in several subtle rhythmic changes in multiple metabolic parameters. We observed that Opn5cre; Gnasfl/fl mice show a higher rhythm-adjusted mean energy expenditure at 22°C and 10°C, and an exaggerated respiratory exchange shift with temperature. At 28°C, Opn5cre; Gnasfl/fl mice have a significant delay in the phase of energy expenditure and respiratory exchange. Rhythmic analysis also showed limited increases in rhythm-adjusted means of food and water intake at 22°C and 28°C. Together, these data advance our understanding of Gαs-signaling in preoptic QPLOT neurons in regulating daily patterns of metabolism.

The wake-active orexin system plays a central role in the dynamic regulation of glucose homeostasis. Here we show orexin receptor type 1 and 2 are predominantly expressed in dorsal raphe nucleus-dorsal and -ventral, respectively. Serotonergic neurons in ventral median raphe nucleus and raphe pallidus selectively express orexin receptor type 1. Inactivation of orexin receptor type 1 in serotonin transporter-expressing cells of mice reduced insulin sensitivity in diet-induced obesity, mainly by decreasing glucose utilization in brown adipose tissue and skeletal muscle. Selective inactivation of orexin receptor type 2 improved glucose tolerance and insulin sensitivity in obese mice, mainly through a decrease in hepatic gluconeogenesis. Optogenetic activation of orexin neurons in lateral hypothalamus or orexinergic fibers innervating raphe pallidus impaired or improved glucose tolerance, respectively. Collectively, the present study assigns orexin signaling in serotonergic neurons critical, yet differential orexin receptor type 1- and 2-dependent functions in the regulation of systemic glucose homeostasis.

Galanin receptor1 (GalR1) transcript levels are elevated in the rat ventral periaqueductal gray (vPAG) after chronic mild stress (CMS) and are related to depression-like behavior. To explore the mechanisms underlying the elevated GalR1 expression, we carried out molecular biological experiments in vitro and in animal behavioral experiments in vivo. It was found that a restricted upstream region of the GalR1 gene, from -250 to -220, harbors an E-box and plays a negative role in the GalR1 promoter activity. The transcription factor Scratch2 bound to the E-box to down-regulate GalR1 promoter activity and lower expression levels of the GalR1 gene. The expression of Scratch2 was significantly decreased in the vPAG of CMS rats. Importantly, local knockdown of Scratch2 in the vPAG caused elevated expression of GalR1 in the same region, as well as depression-like behaviors. RNAscope analysis revealed that GalR1 mRNA is expressed together with Scratch2 in both GABA and glutamate neurons. Taking these data together, our study further supports the involvement of GalR1 in mood control and suggests a role for Scratch2 as a regulator of depression-like behavior by repressing the GalR1 gene in the vPAG.

Tissue injury induces a long-lasting latent sensitization (LS) of spinal nociceptive signaling that is kept in remission by an opposing µ-opioid receptor (MOR) constitutive activity. To test the hypothesis that supraspinal sites become engaged, we induced hindpaw inflammation, waited 3 weeks for mechanical hypersensitivity to resolve, and then injected the opioid receptor inhibitors naltrexone, CTOP or β-funaltrexamine subcutaneously, and/or into the cerebral ventricles. Intracerebroventricular injection of each inhibitor reinstated hypersensitivity and produced somatic signs of withdrawal, indicative of LS and endogenous opioid dependence, respectively. In naïve or sham controls, systemic naloxone (3 mg/kg) produced conditioned place aversion, and systemic naltrexone (3 mg/kg) increased Fos expression in the central nucleus of the amygdala (CeA). In LS animals tested 3 weeks after plantar incision, systemic naltrexone reinstated mechanical hypersensitivity and produced an even greater increase in Fos than in sham controls, particularly in the capsular subdivision of the right CeA. One third of Fos+ profiles co-expressed protein kinase C delta (PKCδ), and 35% of PKCδ neurons co-expressed tdTomato+ in Oprm1Cre ::tdTomato transgenic mice. CeA microinjection of naltrexone (1 µg) reinstated mechanical hypersensitivity only in male mice and did not produce signs of somatic withdrawal. Intra-CeA injection of the MOR-selective inhibitor CTAP (300 ng) reinstated hypersensitivity in both male and female mice. We conclude that MORs in the capsular subdivision of the right CeA prevent the transition from acute to chronic postoperative pain.

The lateral septum (LS) is a basal forebrain GABAergic region that is implicated in social novelty. However, the neural circuits and cell signaling pathways that converge on the LS to mediate social behaviors aren't well understood. Multiple lines of evidence suggest that signaling of brain-derived neurotrophic factor (BDNF) through its receptor TrkB plays important roles in social behavior. BDNF is not locally produced in LS, but we demonstrate that nearly all LS GABAergic neurons express TrkB. Local TrkB knock-down in LS neurons decreased social novelty recognition and reduced recruitment of neural activity in LS neurons in response to social novelty. Since BDNF is not synthesized in LS, we investigated which inputs to LS could serve as potential BDNF sources for controlling social novelty recognition. We demonstrate that selectively ablating inputs to LS from the basolateral amygdala (BLA), but not from ventral CA1 (vCA1), impairs social novelty recognition. Moreover, depleting BDNF selectively in BLA-LS projection neurons phenocopied the decrease in social novelty recognition caused by either local LS TrkB knockdown or ablation of BLA-LS inputs. These data support the hypothesis that BLA-LS projection neurons serve as a critical source of BDNF for activating TrkB signaling in LS neurons to control social novelty recognition.

In addition to its renal and cardiovascular functions, angiotensin signalling is thought to be responsible for the increases in salt and water intake caused by hypovolaemia. However, it remains unclear whether these behaviours require angiotensin production in the brain or liver. Here, we use in situ hybridization to identify tissue-specific expression of the genes required for producing angiotensin peptides, and then use conditional genetic deletion of the angiotensinogen gene (Agt) to test whether production in the brain or liver is necessary for sodium appetite and thirst. In the mouse brain, we identified expression of Agt (the precursor for all angiotensin peptides) in a large subset of astrocytes. We also identified Ren1 and Ace (encoding enzymes required to produce angiotensin II) expression in the choroid plexus, and Ren1 expression in neurons within the nucleus ambiguus compact formation. In the liver, we confirmed that Agt is widely expressed in hepatocytes. We next tested whether thirst and sodium appetite require angiotensinogen production in astrocytes or hepatocytes. Despite virtually eliminating expression in the brain, deleting astrocytic Agt did not reduce thirst or sodium appetite. Despite markedly reducing angiotensinogen in the blood, eliminating Agt from hepatocytes did not reduce thirst or sodium appetite, and in fact, these mice consumed the largest amounts of salt and water after sodium deprivation. Deleting Agt from both astrocytes and hepatocytes also did not prevent thirst or sodium appetite. Our findings suggest that angiotensin signalling is not required for sodium appetite or thirst and highlight the need to identify alternative signalling mechanisms. KEY POINTS: Angiotensin signalling is thought to be responsible for the increased thirst and sodium appetite caused by hypovolaemia, producing elevated water and sodium intake. Specific cells in separate brain regions express the three genes needed to produce angiotensin peptides, but brain-specific deletion of the angiotensinogen gene (Agt), which encodes the lone precursor for all angiotensin peptides, did not reduce thirst or sodium appetite. Double-deletion of Agt from brain and liver also did not reduce thirst or sodium appetite. Liver-specific deletion of Agt reduced circulating angiotensinogen levels without reducing thirst or sodium appetite. Instead, these angiotensin-deficient mice exhibited an enhanced sodium appetite. Because the physiological mechanisms controlling thirst and sodium appetite continued functioning without angiotensin production in the brain and liver, understanding these mechanisms requires a renewed search for the hypovolaemic signals necessary for activating each behaviour.

Neuropeptide FF (NPFF) group peptides belong to the evolutionary conserved RF-amide peptide family. While they have been assigned a role as pain modulators, their roles in other aspects of physiology have received much less attention. NPFF peptides and their receptor NPFFR2 have strong and localized expression within the dorsal vagal complex that has emerged as the key centre for regulating glucose homeostasis. Therefore, we investigated the role of the NPFF system in the control of glucose metabolism and the histochemical and molecular identities of NPFF and NPFFR2 neurons.We examined glucose metabolism in Npff-/- and wild type (WT) mice using intraperitoneal (i.p.) glucose tolerance and insulin tolerance tests. Body composition and glucose tolerance was further examined in mice after 1-week and 3-week of high-fat diet (HFD). Using RNAScope double ISH, we investigated the neurochemical identity of NPFF and NPFFR2 neurons in the caudal brainstem, and the expression of receptors for peripheral factors in NPFF neurons.Lack of NPFF signalling in mice leads to improved glucose tolerance without significant impact on insulin excursion after the i.p. glucose challenge. In response to an i.p. bolus of insulin, Npff-/- mice have lower glucose excursions than WT mice, indicating an enhanced insulin action. Moreover, while HFD has rapid and potent detrimental effects on glucose tolerance, this diet-induced glucose intolerance is ameliorated in mice lacking NPFF signalling. This occurs in the absence of any significant impact of NPFF deletion on lean or fat masses, suggesting a direct effect of NPFF signalling on glucose metabolism. We further reveal that NPFF neurons in the subpostrema area (SubP) co-express receptors for peripheral factors involved in glucose homeostasis regulation such as insulin and GLP1. Furthermore, Npffr2 is expressed in the glutamatergic NPFF neurons in the SubP, and in cholinergic neurons of the dorsal motor nucleus of the vagus (DMV), indicating that central NPFF signalling is likely modulating vagal output to innervated peripheral tissues including those important for glucose metabolic control.NPFF signalling plays an important role in the regulation of glucose metabolism. NPFF neurons in the SubP are likely to receive peripheral signals and mediate the control of whole-body glucose homeostasis via centrally vagal pathways. Targeting NPFF and NPFFR2 signalling may provide a new avenue for treating type 2 diabetes and obesity.

A growing body of experimental evidence shows that glycinergic inhibition plays vital roles in spinal pain processing. In spite of this, however, our knowledge about the morphology, neurochemical characteristics, and synaptic relations of glycinergic neurons in the spinal dorsal horn is very limited. The lack of this knowledge makes our understanding about the specific contribution of glycinergic neurons to spinal pain processing quite vague. Here we investigated the morphology and neurochemical characteristics of glycinergic neurons in laminae I-IV of the spinal dorsal horn using a GlyT2::CreERT2-tdTomato transgenic mouse line. Confirming previous reports, we show that glycinergic neurons are sparsely distributed in laminae I-II, but their densities are much higher in lamina III and especially in lamina IV. First in the literature, we provide experimental evidence indicating that in addition to neurons in which glycine colocalizes with GABA, there are glycinergic neurons in laminae I-II that do not express GABA and can thus be referred to as glycine-only neurons. According to the shape and size of cell bodies and dendritic morphology, we divided the tdTomato-labeled glycinergic neurons into three and six morphological groups in laminae I-II and laminae III-IV, respectively. We also demonstrate that most of the glycinergic neurons co-express neuronal nitric oxide synthase, parvalbumin, the receptor tyrosine kinase RET, and the retinoic acid-related orphan nuclear receptor β (RORβ), but there might be others that need further neurochemical characterization. The present findings may foster our understanding about the contribution of glycinergic inhibition to spinal pain processing.

KCNT1 encodes the sodium-activated potassium channel KNa1.1, expressed preferentially in the frontal cortex, hippocampus, cerebellum, and brainstem. Pathogenic missense variants in KCNT1 are associated with intractable epilepsy, namely epilepsy of infancy with migrating focal seizures (EIMFS), and sleep-related hypermotor epilepsy (SHE). In vitro studies of pathogenic KCNT1 variants support predominantly a gain-of-function molecular mechanism, but how these variants behave in a neuron or ultimately drive formation of an epileptogenic circuit is an important and timely question. Using CRISPR/Cas9 gene editing, we introduced a gain-of-function variant into the endogenous mouse Kcnt1 gene. Compared to wild-type (WT) littermates, heterozygous and homozygous knock-in mice displayed greater seizure susceptibility to the chemoconvulsants kainate and pentylenetetrazole (PTZ), but not to flurothyl. Using acute slice electrophysiology in heterozygous and homozygous Kcnt1 knock-in and WT littermates, we demonstrated that CA1 hippocampal pyramidal neurons exhibit greater amplitude of miniature inhibitory postsynaptic currents in mutant mice with no difference in frequency, suggesting greater inhibitory tone associated with the Kcnt1 mutation. To address alterations in GABAergic signaling, we bred Kcnt1 knock-in mice to a parvalbumin-tdTomato reporter line, and found that parvalbumin-expressing (PV+) interneurons failed to fire repetitively with large amplitude current injections and were more prone to depolarization block. These alterations in firing can be recapitulated by direct application of the KNa1.1 channel activator loxapine in WT but are occluded in knock-in littermates, supporting a direct channel gain-of-function mechanism. Taken together, these results suggest that KNa1.1 gain-of-function dampens interneuron excitability to a greater extent than it impacts pyramidal neuron excitability, driving seizure susceptibility in a mouse model of KCNT1-associated epilepsy.

Leigh syndrome is a mitochondrial disease characterized by neurodegeneration, neuroinflammation, and early death. Mice lacking NDUFS4, a mitochondrial complex I subunit (Ndufs4 KO mice), have been established as a good animal model for studying human pathology associated with Leigh syndrome. As the disease progresses, there is an increase in neurodegeneration and neuroinflammation, thereby leading to deteriorating neurological symptoms, including motor deficits, breathing alterations, and eventually, death of the animal. However, despite the magnitude of neuroinflammation associated with brain lesions, the role of neuroinflammatory pathways and their main cellular components have not been addressed directly as relevant players in the disease pathology. Here, we investigate the role of microglial cells, the main immune cells of the CNS, in Leigh-like syndrome pathology, by pharmacologically depleting them using the colony-stimulating factor 1 receptor antagonist PLX3397. Microglial depletion extended lifespan and delayed motor symptoms in Ndufs4 KO mice, likely by preventing neuronal loss. Next, we investigated the role of the major cytokine interleukin-6 (IL-6) in the disease progression. IL-6 deficiency partially rescued breathing abnormalities and modulated gliosis but did not extend the lifespan or rescue motor decline in Ndufs4 KO mice. The present results show that microglial accumulation is pathogenic, in a process independent of IL-6, and hints toward a contributing role of neuroinflammation in the disease of Ndufs4 KO mice and potentially in patients with Leigh syndrome.

The arcuate nucleus (ArcN) is an integrative hub for the regulation of energy balance, reproduction, and arterial pressure (AP), all of which are influenced by Angiotensin II (AngII); however, the cellular mechanisms and downstream neurocircuitry are unclear. Here we show that ArcN AngII increases AP in female rats via two phases, both of which are mediated via activation of AngII type 1 receptors (AT1aR): initial vasopressin-induced vasoconstriction, followed by slowly developing increases in sympathetic nerve activity (SNA) and heart rate (HR). In male rats, ArcN AngII evoked a similarly slow increase in SNA, but the initial pressor response was variable. In females, the effects of ArcN AngII varied during the estrus cycle, with significant increases in SNA, HR, and AP occurring during diestrus and estrus, but only increased AP during proestrus. Pregnancy markedly increased the expression of AT1aR in the ArcN with parallel substantial AngII-induced increases in SNA and MAP. In both sexes, the sympathoexcitation relied on suppression of tonic ArcN sympathoinhibitory Neuropeptide Y inputs, and activation of pro-opiomelanocortin (POMC) projections, to the paraventricular nucleus (PVN). Few or no NPY or POMC neurons expressed the AT1aR, suggesting that AngII increases AP and SNA at least in part indirectly via local interneurons, which express tyrosine hydroxylase (TH) and VGat (i.e. GABAergic). ArcN TH neurons release GABA locally, and central AT1aR and TH neurons mediate stress responses; therefore, we propose that TH AT1aR neurons are well situated to locally coordinate the regulation of multiple modalities within the ArcN in response to stress.SIGNIFICANCEThe arcuate nucleus (ArcN) is an integrative hub for the regulation of energy balance, reproduction, and arterial pressure (AP), all of which are influenced by Angiotensin II (AngII). Here we show that ArcN AngII activates AT1aR to increase AP in male and female rats by slowly increasing sympathetic nerve activity. In females, ArcN AngII also evoked an initial pressor response mediated by vasopressin-induced vasoconstriction. Pregnant and estrus females responded more than males, in association with higher ArcN AT1aR expression. AT1aR were identified in ArcN interneurons that express tyrosine hydroxylase (TH) and GABA. Since brain AT1aR and TH mediate stress responses, ArcN AT1aR TH neurons are well situated to locally coordinate autonomic, hormonal, and behavioral responses to stress.