Probes for INS

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.

Oxytocin receptor (Oxtr) signaling in neural circuits mediating discrimination of social stimuli and affiliation or avoidance behavior is thought to guide social recognition. Remarkably, the physiological functions of Oxtrs in the hippocampus are not known. Here we demonstrate using genetic and pharmacological approaches that Oxtrs in the anterior dentate gyrus (aDG) and anterior CA2/CA3 (aCA2/CA3) of mice are necessary for discrimination of social, but not non-social, stimuli. Further, Oxtrs in aCA2/CA3 neurons recruit a population-based coding mechanism to mediate social stimuli discrimination. Optogenetic terminal-specific attenuation revealed a critical role for aCA2/CA3 outputs to posterior CA1 for discrimination of social stimuli. In contrast, aCA2/CA3 projections to aCA1 mediate discrimination of non-social stimuli. These studies identify a role for an aDG-CA2/CA3 axis of Oxtr expressing cells in discrimination of social stimuli and delineate a pathway relaying social memory computations in the anterior hippocampus to the posterior hippocampus to guide social recognition.

Abstract

KEY POINTS:

Alpha-melanocyte stimulating hormone (α-MSH) is an anorexigenic peptide, and injection of the α-MSH analog MTII into the ventral tegmental area (VTA) decreases food and sucrose intake and food reward. Melanocortin-3 receptors (MC3R) are highly expressed in the VTA, suggesting that the effects of intra-VTA α-MSH may be mediated by α-MSH changing the activity of MC3R-expressing VTA neurons. α-MSH increased the firing rate of MC3R VTA neurons in acute brain slices from mice, but did not affect the firing rate of non-MC3R VTA neurons. The α-MSH induced increase in MC3R neuron firing rate is likely activity dependent, and was independent of fast synaptic transmission and intracellular Ca2+ levels. These results help us to better understand how α-MSH acts in the VTA to affect feeding and other dopamine dependent behaviors.

ABSTRACT:

The mesocorticolimbic dopamine system, the brain's reward system, regulates multiple behaviors including food intake and food reward. There is substantial evidence that the melanocortin system of the hypothalamus, an important neural circuit controlling feeding and body weight, interacts with the mesocorticolimbic dopamine system to affect feeding, food reward, and body weight. For example, melanocortin-3 receptors (MC3Rs) are expressed in the ventral tegmental area (VTA), and our lab previously showed that intra-VTA injection of the MC3R agonist, MTII, decreases home-cage food intake and operant responding for sucrose pellets. The cellular mechanisms underlying the effects of intra-VTA α-MSH on feeding and food reward are unknown, however. To determine how α-MSH acts in the VTA to affect feeding, we performed electrophysiological recordings in acute brain slices from mice expressing EYFP in MC3R neurons to test how α-MSH affects the activity of VTA MC3R neurons. α-MSH significantly increased the firing rate of VTA MC3R neurons without altering the activity of non-MC3R expressing VTA neurons. In addition, the α-MSH-induced increase in MC3R neuron activity was independent of fast synaptic transmission and intracellular Ca2+ levels. Finally, we show that the effect of α-MSH on MC3R neuron firing rate is likely activity dependent. Overall, these studies provide an important advancement in the understanding of how α-MSH acts in the VTA to affect feeding and food reward. 

c-MET and its ligand HGF are frequently overexpressed in colorectal cancer (CRC) and increased c-MET levels are found in CRC liver metastases. This study investigated the role of the HGF/c-MET axis in regulating migration/invasion in CRC, using pre-clinical models and clinical samples. Pre-clinically, we found marked upregulation of c-MET at both protein and mRNA levels in several invasive CRC cells. Down-regulation of c-MET using RNAi suppressed migration/invasion of parental and invasive CRC cells. Stimulation of CRC cells with rh-HGF or co-culture with HGF-expressing colonic myofibroblasts, resulted in significant increases in their migratory/invasive capacity. Importantly, HGF-induced c-MET activation promoted rapid downregulation of c-MET protein levels, while the MET transcript remained unaltered. Using RNA in situ hybridization (RNA ISH), we further showed that MET mRNA, but not protein levels, were significantly upregulated in tumor budding foci at the invasive front of a cohort of stage III CRC tumors (p < 0.001). Taken together, we show for the first time that transcriptional upregulation of MET is a key molecular event associated with CRC invasion and tumor budding. This data also indicates that RNA ISH, but not immunohistochemistry, provides a robust methodology to assess MET levels as a potential driving force of CRC tumor invasion and metastasis.

The central melanocortin system plays a crucial role in the control of energy balance. Although the decreased energy expenditure and increased adiposity of melanocortin-3 receptor (Mc3R)-null mice suggest the importance of Mc3R-regulated neurons in energy homeostasis, the roles for specific subsets of Mc3R neurons in energy balance have yet to be determined. Because the lateral hypothalamic area (LHA) contributes to the control of energy expenditure and feeding, we generated Mc3rcre mice to determine the roles of LHA Mc3R (Mc3RLHA) neurons in energy homeostasis. We found that Mc3RLHA neurons overlap extensively with LHA neuron markers that contribute to the control of energy balance (neurotensin, galanin, and leptin receptor) and project to brain areas involved in the control of feeding, locomotion, and energy expenditure, consistent with potential roles for Mc3RLHA neurons in these processes. Indeed, selective chemogenetic activation of Mc3RLHA neurons increased locomotor activity and augmented refeeding after a fast. Although the ablation of Mc3RLHA neurons did not alter food intake, mice lacking Mc3RLHA neurons displayed decreased energy expenditure and locomotor activity, along with increased body mass and adiposity. Thus, Mc3R neurons lie within LHA neurocircuitry that modulates locomotor activity and energy expenditure and contribute to energy balance control.

Abstract

Background

Atypical synapse development and plasticity are implicated in many neurodevelopmental disorders (NDDs). NDD-associated, high confidence risk genes have been identified, yet little is known about functional relationships at the level of protein-protein interactions, which are the dominant molecular bases responsible for mediating circuit development.

Methods

Proteomics in three independent developing neocortical synaptosomal preparations identified putative interacting proteins of the ligand-activated MET receptor tyrosine kinase, an autism risk gene that mediates synapse development. The candidates were translated into interactome networks and analyzed bioinformatically. Additionally, three independent quantitative proximity ligation assays (PLA) in cultured neurons and four independent immunoprecipitation analyses of synaptosomes validated protein interactions.

Results

Approximately 11% (8/72) of MET-interacting proteins, including SHANK3, SYNGAP1 and GRIN2B, are associated with NDDs. Proteins in the MET interactome were translated into a novel MET interactome network based on human protein-protein interaction databases. High confidence genes from different NDD datasets that encode synaptosomal proteins were analyzed for being enriched in MET interactome proteins. This was found for autism, but not schizophrenia, bipolar disorder, major depressive disorder or attentional deficit hyperactivity disorder. There is correlated gene expression between MET and its interactive partners in developing human temporal and visual neocortices, but not with highly expressed genes that are not in the interactome. PLA and biochemical analyses demonstrate that MET-protein partner interactions are dynamically regulated by receptor activation.

Conclusions

The results provide a novel molecular framework for deciphering the functional relations of key regulators of synaptogenesis that contribute to both typical cortical development and to NDDs.