Probes for INS

Abstract

Objective

Glucagon-like peptide-1 (GLP-1) neurons in the hindbrain densely innervate the dorsomedial hypothalamus (DMH), a nucleus strongly implicated in body weight regulation and the sympathetic control of brown adipose tissue (BAT) thermogenesis. Therefore, DMH GLP-1 receptors (GLP-1R) are well placed to regulate energy balance by controlling sympathetic outflow and BAT function.

Methods

We investigate this possibility in adult male rats by using direct administration of GLP-1 (0.5 ug) into the DMH, knocking down DMH GLP-1R mRNA with viral-mediated RNA interference, and by examining the neurochemical phenotype of GLP-1R expressing cells in the DMH using in situ hybridization.

Results

GLP-1 administered into the DMH increased BAT thermogenesis and hepatic triglyceride (TG) mobilization. On the other hand, Glp1r knockdown (KD) in the DMH increased body weight gain and adiposity, with a concomitant reduction in energy expenditure (EE), BAT temperature, and uncoupling protein 1 (UCP1) expression. Moreover, DMH Glp1r KD induced hepatic steatosis, increased plasma TG, and elevated liver specific de-novo lipogenesis, effects that collectively contributed to insulin resistance. Interestingly, DMH Glp1r KD increased neuropeptide Y (NPY) mRNA expression in the DMH. GLP-1R mRNA in the DMH, however, was found in GABAergic not NPY neurons, consistent with a GLP-1R-dependent inhibition of NPY neurons that is mediated by local GABAergic neurons. Finally, DMH Glp1r KD attenuated the anorexigenic effects of the GLP-1R agonist exendin-4, highlighting an important role of DMH GLP-1R signaling in GLP-1-based therapies.

Conclusions

Collectively, our data show that DMH GLP-1R signaling plays a key role for BAT thermogenesis and adiposity.

Dysfunction of cholinergic signaling in the brain has long been believed to be associated with depressive disorders. However, the functional impact of habenular cholinergic signaling on the specified depressive behaviors is not well understood. Here, we demonstrated that the expression levels of cholinergic signaling genes (CHAT, VACHT, CHT, CHRNA3, CHRNB3 and CHRNB4) were down-regulated in a chronic restraint stress (CRS) rat model of depression, in which rats display depression-like behaviors such as anhedonia and mood despair. Moreover, knockdown of CHAT in the rat habenula was sufficient to evoke anhedonia-like behavior. The anhedonia-like behavior induced by CHAT knockdown was not reversed by chronic administration of the selective serotonin reuptake inhibitor fluoxetine. To determine whether habenular cholinergic signaling is associated with regulation of dopamine neurons in the ventral tegmental area (VTA) and serotonin neurons in the dorsal raphe nucleus (DRN), we used CHAT::cre transgenic mice expressing the Designer Receptors Exclusively Activated by Designer Drugs (DREADD). Pharmacogenetic activation of habenular cholinergic neurons induces the excitation of dopamine neurons in the VTA and reduces the immunoreactivity of 5-hydroxytryptamine (5-HT) in the DRN. Habenular cholinergic gene down-regulation was recapitulated in the postmortem habenula of suicide victims diagnosed with major depressive disorder (MDD).

Noradrenergic signaling plays a well-established role in promoting the stress response. Here we identify a subpopulation of noradrenergic neurons, defined by developmental expression of Hoxb1, that has a unique role in modulating stress-related behavior. Using an intersectional chemogenetic strategy, in combination with behavioral and physiological analyses, we show that activation of Hoxb1-noradrenergic (Hoxb1-NE) neurons decreases anxiety-like behavior and promotes an active coping strategy in response to acute stressors. In addition, we use cerebral blood volume-weighted functional magnetic resonance imaging to show that chemoactivation of Hoxb1-NE neurons results in reduced activity in stress-related brain regions, including the bed nucleus of the stria terminalis, amygdala, and locus coeruleus. Thus, the actions of Hoxb1-NE neurons are distinct from the well-documented functions of the locus coeruleus in promoting the stress response, demonstrating that the noradrenergic system contains multiple functionally distinct subpopulations.

Neuronal circuit asymmetries are important components of brain circuits, but the molecular pathways leading to their establishment remain unknown. Here we found that the mutation of FRMD7, a gene that is defective in human congenital nystagmus, leads to the selective loss of the horizontal optokinetic reflex in mice, as it does in humans. This is accompanied by the selective loss of horizontal direction selectivity in retinal ganglion cells and the transition from asymmetric to symmetric inhibitory input to horizontal direction-selective ganglion cells. In wild-type retinas, we found FRMD7 specifically expressed in starburst amacrine cells, the interneuron type that provides asymmetric inhibition to direction-selective retinal ganglion cells. This work identifies FRMD7 as a key regulator in establishing a neuronal circuit asymmetry, and it suggests the involvement of a specific inhibitory neuron type in the pathophysiology of a neurological disease.

Central activation of fibroblast growth factor (FGF) receptors regulates peripheral glucose homeostasis and reduces food intake in preclinical models of obesity and diabetes. The current work was undertaken to advance our understanding of the receptor expression, as sites of ligand action by FGF19, FGF21, and FGF1 in the mammalian brain remains unresolved. Recent advances in automated RNAscope in situ hybridization and droplet digital PCR (ddPCR) technology allowed us to interrogate central FGFR/beta klotho (Klb) system at the cellular level in the mouse, with relevant comparisons to nonhuman primate and human brain. FGFR1-3 gene expression was broadly distributed throughout the CNS in Mus musculus, with FGFR1 exhibiting the greatest heterogeneity. FGFR4 expression localized only in the medial habenula and subcommissural organ of mice. Likewise, Klb mRNA was restricted to the suprachiasmatic nucleus (SCh) and select midbrain and hindbrain nuclei. ddPCR in the rodent hypothalamus confirmed that, although expression levels are indeed low for Klb, there is nonetheless a bonafide subpopulation of Klb+ cells in the hypothalamus. In NHP and human midbrain and hindbrain, Klb + cells are quite rare, as is expression of FGFR4. Collectively, these data provide the most robust central map of the FGFR/Klb system to date and highlight central regions that may be of critical importance to assess central ligand effects with pharmacological dosing, such as the putative interactions between the endocrine FGFs and FGFR1/Klb, or FGF19 with FGFR4.

The medial septum (MS) complex modulates hippocampal function and related behaviors. Septohippocampal projections promote and control different forms of hippocampal synchronization. Specifically, GABAergic and cholinergic projections targeting the hippocampal formation from the MS provide bursting discharges to promote theta rhythm, or tonic activity to promote gamma oscillations. In turn, the MS is targeted by ascending projections from the hypothalamus and brainstem. One of these projections arises from the nucleus incertus in the pontine tegmentum, which contains GABA neurons that co-express the neuropeptide relaxin-3 (Rln3). Both stimulation of the nucleus incertus and septal infusion of Rln3 receptor agonist peptides promotes hippocampal theta rhythm. The Gi/o-protein-coupled receptor, relaxin-family peptide receptor 3 (RXFP3), is the cognate receptor for Rln3 and identification of the transmitter phenotype of neurons expressing RXFP3 in the septohippocampal system can provide further insights into the role of Rln3 transmission in the promotion of septohippocampal theta rhythm. Therefore, we used RNAscope multiplex in situ hybridization to characterize the septal neurons expressing Rxfp3mRNA in the rat. Our results demonstrate that Rxfp3 mRNA is abundantly expressed in vesicular GABA transporter (vGAT) mRNA- and parvalbumin (PV) mRNA-positive GABA neurons in MS, whereas ChATmRNA-positive acetylcholine neurons lack Rxfp3 mRNA. Approximately 75% of Rxfp3 mRNA-positive neurons expressed vGAT mRNA (and 22% were PV mRNA-positive), while the remaining 25% expressed Rxfp3 mRNA only, consistent with a potential glutamatergic phenotype. Similar proportions were observed in the posterior septum. The occurrence of RXFP3 in PV-positive GABAergic neurons gives support to a role for the Rln3-RXFP3 system in septohippocampal theta rhythm.

Detailed anatomical tracing and mapping of the viscerotopic organization of the vagal motor nuclei has provided insight into autonomic function in health and disease. To further define specific cellular identities, we paired information based on visceral connectivity with a cell-type specific marker of a subpopulation of neurons in the dorsal motor nucleus of the vagus (DMV) and nucleus ambiguus (nAmb) that express the autism-associated MET receptor tyrosine kinase. As gastrointestinal disturbances are common in children with autism spectrum disorder (ASD), we sought to define the relationship between MET-expressing (MET+) neurons in the DMV and nAmb, and the gastrointestinal tract. Using wholemount tissue staining and clearing, or retrograde tracing in a METEGFP transgenic mouse, we identify three novel subpopulations of EGFP+ vagal brainstem neurons: 1) EGFP+ neurons in the nAmb projecting to the esophagus or laryngeal muscles, 2) EGFP+ neurons in the medial DMV projecting to the stomach, and 3) EGFP+ neurons in the lateral DMV projecting to the cecum and/or proximal colon. Expression of the MET ligand, hepatocyte growth factor (HGF), by tissues innervated by vagal motor neurons during fetal development reveal potential sites of HGF-MET interaction. Furthermore, similar cellular expression patterns of MET in the brainstem of both the mouse and nonhuman primate suggest that MET expression at these sites is evolutionarily conserved. Together, the data suggest that MET+ neurons in the brainstem vagal motor nuclei are anatomically positioned to regulate distinct portions of the gastrointestinal tract, with implications for the pathophysiology of gastrointestinal comorbidities of ASD.