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Quantitative analysis of glutamatergic innervation of the mouse dorsal raphe nucleus using array tomography.

Serotonin (5-hydroxytryptamine, 5-HT) containing neurons located in the dorsal raphe nucleus (DR) comprise the main source of forebrain 5-HT and regulate emotional states in normal and pathological conditions including affective disorders. However, there are many features of the local circuit architecture within the DR that remain poorly understood. DR neurons receive glutamatergic innervation from different brain areas that selectively express three different types of the vesicular glutamate transporter (VGLUT). In this study we used a new high-resolution imaging technique, array tomography, to quantitatively analyze the glutamatergic innervation of the mouse DR. In the same volumetric images, we studied the distribution of five antigens: VGLUT1, VGLUT2, VGLUT3, the postsynaptic protein PSD-95, and a marker for 5-HT cells, the enzyme tryptophan hydroxylase (TPOH). We found that all three populations of glutamatergic boutons are present in the DR; however, the density of paired association between VGLUT2 boutons and PSD-95 was ≈2-fold higher than that of either VGLUT1- or VGLUT3-PSD-95 pairs. In addition, VGLUT2-PSD-95 pairs were more commonly found associated with 5-HT cells than the other VGLUT types. These data support a prominent contribution of glutamate axons expressing VGLUT2 to the excitatory drive of DR neurons. The current study also emphasizes the use of array tomography as a quantitative approach to understand the fine molecular architecture of microcircuits in a well-preserved neuroanatomical context.

Pubmed ID: 21800318 RIS Download

Mesh terms: Animals | Imaging, Three-Dimensional | Immunohistochemistry | Mice | Mice, Inbred C57BL | Microscopy, Fluorescence | Neural Pathways | Neuroanatomical Tract-Tracing Techniques | Presynaptic Terminals | Raphe Nuclei | Serotonergic Neurons | Vesicular Glutamate Transport Proteins

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Associated grants

  • Agency: NIDA NIH HHS, Id: R01 DA011386
  • Agency: NIDA NIH HHS, Id: R01 DA021801-04
  • Agency: NIEHS NIH HHS, Id: R21 ES011667
  • Agency: NIDA NIH HHS, Id: R01 DA021801
  • Agency: NIDA NIH HHS, Id: DA021801
  • Agency: NCI NIH HHS, Id: P50 CA090388

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A national mouse monoclonal antibody generating resource for biochemical and immunohistochemical applications in mammalian brain. NeuroMabs are generated from mice immunized with synthetic and recombinant immunogens corresponding to components of the neuronal proteome as predicted from genomic and other large-scale cloning efforts. Comprehensive biochemical and immunohistochemical analyses of human, primate and non-primate mammalian brain are incorporated into the initial NeuroMab screening procedure. This yields a subset of mouse mAbs that are optimized for use in brain (i.e. NeuroMabs): for immunocytochemical-based imaging studies of protein localization in adult, developing and pathological brain samples, for biochemical analyses of subunit composition and post-translational modifications of native brain proteins, and for proteomic analyses of native brain protein networks. The NeuroMab facility was initially funded with a five-year U24 cooperative grant from NINDS and NIMH. The initial goal of the facility for this funding period is to generate a library of novel NeuroMabs against neuronal proteins, initially focusing on membrane proteins (receptors/channels/transporters), synaptic proteins, other neuronal signaling molecules, and proteins with established links to disease states. The scope of the facility was expanded with supplements from the NIH Blueprint for Neuroscience Research to include neurodevelopmental targets, the NIH Roadmap for Medical Research to include epigenetics targets, and NIH Office of Rare Diseases Research to include rare disease targets. These NeuroMabs will then be produced on a large scale and made available to the neuroscience research community on an inexpensive basis as tissue culture supernatants or purified immunoglobulin by Antibodies Inc. The UC Davis/NIH NeuroMab Facility makes NeuroMabs available directly to end users and is unable to accommodate sales to distributors for third party distribution. Note, NeuroMab antibodies are now offered through antibodiesinc.

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