Probes for INS

Little is known about the targets in the CNS that mediate ethanol analgesia. This study explores the role of spinal astrocyte aldehyde dehydrogenase-2 (ALDH2), a key ethanol-metabolising enzyme, in the analgesic effects of ethanol in mice. Astrocyte and hepatocyte ALHD2-deficient mice were generated and tested in acute and chronic pain models. Cell-type-specific distribution of ALDH2 was analysed by RNA in situ hybridisation in spinal slices from astrocytic ALDH2-deficient mice and their wild-type littermates. Spinal ethanol metabolites and γ-aminobutyric acid (GABA) content were measured using gas chromatography/mass spectrometry and liquid chromatography/mass spectrometry. ALDH2 mRNA was expressed in both astrocytes and neurones in spinal cord slices. Astrocyte ALDH2-deficient mice had decreased expression of ALDH2 mRNA in astrocytes, but not in neurones. Astrocyte ALDH2 deficiency inhibited ethanol-derived acetate, but not acetaldehyde content in spinal cord tissues. Depletion of spinal astrocyte ALDH2 selectively inhibited ethanol-induced anti-nociceptive effect, but not the effect of ethanol, on motor function. Astrocyte ALDH2 deficiency abolished ethanol-induced GABA elevation. The ethanol metabolite acetate produced anti-nociception and increased GABA synthesis in a manner similar to ethanol. I.T. delivery of either GABAA or GABAB receptor antagonists prevented ethanol and acetate-induced analgesia. These findings provide evidence that ALDH2 in spinal astrocytes mediates spinal ethanol metabolism and ethanol-induced analgesic effects by promoting GABA synthesis and GABAergic transmission in spinal cord.

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.

Microtubule-associated protein tau (MAPT) aggregates in neurons, astrocytes and oligodendrocytes in a number of neurodegenerative diseases, including progressive supranuclear palsy (PSP). Tau is a target of therapy and the strategy includes either the elimination of pathological tau aggregates or reducing MAPT expression, and thus the amount of tau protein made to prevent its aggregation. Disease-associated tau affects brain regions in a sequential manner that includes cell-to-cell spreading. Involvement of glial cells that show tau aggregates is interpreted as glial cells taking up misfolded tau assuming that glial cells do not express enough MAPT. Although studies have evaluated MAPT expression in human brain tissue homogenates, it is not clear whether MAPT expression is compromised in cells accumulating pathological tau. To address these perplexing aspects of disease pathogenesis, this study used RNAscope combined with immunofluorescence (AT8), and single-nuclear(sn) RNAseq to systematically map and quantify MAPT expression dynamics across different cell types and brain regions in controls (n = 3) and evaluated whether tau cytopathology affects MAPT expression in PSP (n = 3). MAPT transcripts were detected in neurons, astrocytes and oligodendrocytes, and varied between brain regions and within each cell type, and were preserved in all cell types with tau aggregates in PSP. These results propose a complex scenario in all cell types, where, in addition to the ingested misfolded tau, the preserved cellular MAPT expression provides a pool for local protein production that can (1) be phosphorylated and aggregated, or (2) feed the seeding of ingested misfolded tau by providing physiological tau, both accentuating the pathological process. Since tau cytopathology does not compromise MAPT gene expression in PSP, a complete loss of tau protein expression as an early pathogenic component is less likely. These observations provide rationale for a dual approach to therapy by decreasing cellular MAPT expression and targeting removal of misfolded tau.

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.

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.

Previous studies identify a role for hypothalamic glia in energy balance regulation; however, a narrow hypothalamic focus provides an incomplete understanding of how glia throughout the brain respond to and regulate energy homeostasis. We examined the responses of glia in the dorsal vagal complex (DVC) to the adipokine leptin and high fat diet-induced obesity. DVC astrocytes functionally express the leptin receptor; in vivo pharmacological studies suggest that DVC astrocytes partly mediate the anorectic effects of leptin in lean but not diet-induced obese rats. Ex vivo calcium imaging indicated that these changes were related to a lower proportion of leptin-responsive cells in the DVC of obese versus lean animals. Finally, we investigated DVC microglia and astroglia responses to leptin and energy balance dysregulation in vivo: obesity decreased DVC astrogliosis, whereas the absence of leptin signaling in Zucker rats was associated with extensive astrogliosis in the DVC and decreased hypothalamic micro- and astrogliosis. These data uncover a novel functional heterogeneity of astrocytes in different brain nuclei of relevance to leptin signaling and energy balance regulation

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.

Shank3 is an excitatory postsynaptic scaffolding protein implicated in multiple brain disorders, including autism spectrum disorders (ASD) and Phelan-McDermid syndrome (PMS). Although previous neurobiological studies on Shank3 and Shank3-mutant mice have revealed diverse roles of Shank3 in the regulation of synaptic, neuronal and brain functions, whether Shank3 expression in specific cell types distinctly contributes to mouse phenotypes remains largely unclear. In the present study, we generated two Shank3-mutant mouse lines (exons 14-16) carrying global and GABA neuron-specific deletions and characterized their electrophysiological and behavioral phenotypes. These mouse lines show similar decreases in excitatory synaptic input onto dorsolateral striatal neurons. In addition, the abnormal social and locomotor behaviors observed in global Shank3-mutant mice are strongly mimicked by GABA neuron-specific Shank3-mutant mice, whereas the repetitive and anxiety-like behaviors are only partially mimicked. These results suggest that GABAergic Shank3 (exons 14-16) deletion has strong influences on striatal excitatory synaptic transmission and social and locomotor behaviors in mice.

Astrocytes express mu/µ opioid receptors, but the function of these receptors remains poorly understood. We evaluated the effects of astrocyte-restricted knockout of µ opioid receptors on reward- and aversion-associated behaviors in mice chronically exposed to morphine. Specifically, one of the floxed alleles of the Oprm1 gene encoding µ opioid receptor 1 was selectively deleted from brain astrocytes in Oprm1 inducible conditional knockout (icKO) mice. These mice did not exhibit changes in locomotor activity, anxiety, or novel object recognition, or in their responses to the acute analgesic effects of morphine. Oprm1 icKO mice displayed increased locomotor activity in response to acute morphine administration but unaltered locomotor sensitization. Oprm1 icKO mice showed normal morphine-induced conditioned place preference but exhibited stronger conditioned place aversion associated with naloxone-precipitated morphine withdrawal. Notably, elevated conditioned place aversion lasted up to 6 weeks in Oprm1 icKO mice. Astrocytes isolated from the brains of Oprm1 icKO mice had unchanged levels of glycolysis but had elevated oxidative phosphorylation. The basal augmentation of oxidative phosphorylation in Oprm1 icKO mice was further exacerbated by naloxone-precipitated withdrawal from morphine and, similar to that for conditioned place aversion, was still present 6 weeks later. Our findings suggest that µ opioid receptors in astrocytes are linked to oxidative phosphorylation and they contribute to long-term changes associated with opioid withdrawal.

Dopamine (DA) neurons in the ventral tier of the substantia nigra pars compacta (SNc) degenerate prominently in Parkinson's disease, while those in the dorsal tier are relatively spared. Defining the molecular, functional, and developmental characteristics of each SNc tier is crucial to understand their distinct susceptibility. We demonstrate that Sox6 expression distinguishes ventrally and dorsally biased DA neuron populations in the SNc. The Sox6+ population in the ventral SNc includes an Aldh1a1+ subset and is enriched in gene pathways that underpin vulnerability. Sox6+ neurons project to the dorsal striatum and show activity correlated with acceleration. Sox6- neurons project to the medial, ventral, and caudal striatum and respond to rewards. Moreover, we show that this adult division is encoded early in development. Overall, our work demonstrates a dual origin of the SNc that results in DA neuron cohorts with distinct molecular profiles, projections, and functions.

Neurons in the superficial dorsal horn that express the gastrin-releasing peptide receptor (GRPR) are strongly implicated in spinal itch pathways. However, a recent study reported that many of these correspond to vertical cells, a population of interneurons that are thought to transmit nociceptive information. In this study, we have used a GRPRCreERT2 mouse line to identify and target cells that possess Grpr mRNA. We find that the GRPR cells are highly concentrated in lamina I and the outer part of lamina II, that they are all glutamatergic, and that they account for ∼15% of the excitatory neurons in the superficial dorsal horn. We had previously identified 6 neurochemically distinct excitatory interneuron populations in this region based on neuropeptide expression and the GRPR cells are largely separate from these, although they show some overlap with cells that express substance P. Anatomical analysis revealed that the GRPR neurons are indeed vertical cells, and that their axons target each other, as well as arborising in regions that contain projection neurons: lamina I, the lateral spinal nucleus and the lateral part of lamina V. Surprisingly, given the proposed role of GRPR cells in itch, we found that most of the cells received monosynaptic input from Trpv1-expressing (nociceptive) afferents, that the great majority responded to noxious and pruritic stimuli, and that chemogenetically activating them resulted in pain- and itch-related behaviours. Together, these findings suggest that the GRPR cells are involved in spinal cord circuits that underlie both pain and itch.