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      nNOS-Expressing Neurons in the Ventral Tegmental Area and Substantia Nigra Pars Compacta

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          Abstract

          GABA neurons in the VTA and SNc play key roles in reward and aversion through their local inhibitory control of dopamine neuron activity and through long-range projections to several target regions including the nucleus accumbens. It is not clear whether some of these GABA neurons are dedicated local interneurons or if they all collateralize and send projections externally as well as making local synaptic connections. Testing between these possibilities has been challenging in the absence of interneuron-specific molecular markers. We hypothesized that one potential candidate might be neuronal nitric oxide synthase (nNOS), a common interneuronal marker in other brain regions. To test this, we used a combination of immunolabelling (including antibodies for nNOS that we validated in tissue from nNOS-deficient mice) and cell type-specific virus-based anterograde tracing in mice. We found that nNOS-expressing neurons, in the parabrachial pigmented (PBP) part of the VTA and the SNc were GABAergic and did not make detectable projections, suggesting they may be interneurons. In contrast, nNOS-expressing neurons in the rostral linear nucleus (RLi) were mostly glutamatergic and projected to a number of regions, including the lateral hypothalamus (LH), the ventral pallidum (VP), and the median raphe (MnR) nucleus. Taken together, these findings indicate that nNOS is expressed by neurochemically- and anatomically-distinct neuronal sub-groups in a sub-region-specific manner in the VTA and SNc.

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          Petilla terminology: nomenclature of features of GABAergic interneurons of the cerebral cortex.

          Neuroscience produces a vast amount of data from an enormous diversity of neurons. A neuronal classification system is essential to organize such data and the knowledge that is derived from them. Classification depends on the unequivocal identification of the features that distinguish one type of neuron from another. The problems inherent in this are particularly acute when studying cortical interneurons. To tackle this, we convened a representative group of researchers to agree on a set of terms to describe the anatomical, physiological and molecular features of GABAergic interneurons of the cerebral cortex. The resulting terminology might provide a stepping stone towards a future classification of these complex and heterogeneous cells. Consistent adoption will be important for the success of such an initiative, and we also encourage the active involvement of the broader scientific community in the dynamic evolution of this project.
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            Cell-type-specific isolation of ribosome-associated mRNA from complex tissues.

            Gene profiling techniques allow the assay of transcripts from organs, tissues, and cells with an unprecedented level of coverage. However, most of these approaches are still limited by the fact that organs and tissues are composed of multiple cell types that are each unique in their patterns of gene expression. To identify the transcriptome from a single cell type in a complex tissue, investigators have relied upon physical methods to separate cell types or in situ hybridization and immunohistochemistry. Here, we describe a strategy to rapidly and efficiently isolate ribosome-associated mRNA transcripts from any cell type in vivo. We have created a mouse line, called RiboTag, which carries an Rpl22 allele with a floxed wild-type C-terminal exon followed by an identical C-terminal exon that has three copies of the hemagglutinin (HA) epitope inserted before the stop codon. When the RiboTag mouse is crossed to a cell-type-specific Cre recombinase-expressing mouse, Cre recombinase activates the expression of epitope-tagged ribosomal protein RPL22(HA), which is incorporated into actively translating polyribosomes. Immunoprecipitation of polysomes with a monoclonal antibody against HA yields ribosome-associated mRNA transcripts from specific cell types. We demonstrate the application of this technique in brain using neuron-specific Cre recombinase-expressing mice and in testis using a Sertoli cell Cre recombinase-expressing mouse.
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              Stereological estimates of dopaminergic, GABAergic and glutamatergic neurons in the ventral tegmental area, substantia nigra and retrorubral field in the rat

              Midbrain dopamine neurons in the ventral tegmental area, substantia nigra and retrorubral field play key roles in reward processing, learning and memory, and movement. Within these midbrain regions and admixed with the dopamine neurons, are also substantial populations of GABAergic neurons that regulate dopamine neuron activity and have projection targets similar to those of dopamine neurons. Additionally, there is a small group of putative glutamatergic neurons within the ventral tegmental area whose function remains unclear. Although dopamine neurons have been intensively studied and quantified, there is little quantitative information regarding the GABAergic and glutamatergic neurons. We therefore used unbiased stereological methods to estimate the number of dopaminergic, GABAergic and glutamatergic cells in these regions in the rat. Neurons were identified using a combination of immunohistochemistry (tyrosine hydroxylase) and in situ hybridization (glutamic acid decarboxylase mRNA and vesicular glutamate transporter 2 mRNA). In substantia nigra pars compacta 29% of cells were glutamic acid decarboxylase mRNA-positive, 58% in the retrorubral field and 35% in the ventral tegmental area. There were further differences in the relative sizes of the GABAergic populations in subnuclei of the ventral tegmental area. Thus, glutamic acid decarboxylase mRNA-positive neurons represented 12% of cells in the interfascicular nucleus, 30% in the parabrachial nucleus, and 45% in the parainterfascicular nucleus. Vesicular glutamate transporter 2 mRNA-positive neurons were present in the ventral tegmental area, but not substantia nigra or retrorubral field. They were mainly confined to the rostro-medial region of the ventral tegmental area, and represented approximately 2–3% of the total neurons counted (∼1600 cells). These results demonstrate that GABAergic and glutamatergic neurons represent large proportions of the neurons in what are traditionally considered as dopamine nuclei and that there are considerable heterogeneities in the proportions of cell types in the different dopaminergic midbrain regions.
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                Author and article information

                Journal
                eNeuro
                eNeuro
                eneuro
                eneuro
                eNeuro
                eNeuro
                Society for Neuroscience
                2373-2822
                29 October 2018
                16 November 2018
                Sep-Oct 2018
                : 5
                : 5
                : ENEURO.0381-18.2018
                Affiliations
                [1 ]MRC London Institute of Medical Sciences (LMS) , London W12 0NN, United Kingdom
                [2 ]Institute of Clinical Sciences (ICS), Faculty of Medicine, Imperial College London , London W12 0NN, United Kingdom
                [3 ]Department of Life Sciences, Imperial College London , South Kensington, London SW7 2AZ, United Kingdom
                Author notes

                The authors declare no competing financial interests.

                Author contributions: E.J.P., E.K., K.T., E.E.I., N.P.F., W.W., D.J.W., J.L., and M.A.U. designed research; E.J.P., E.K., K.T., and E.E.I. performed research; N.P.F. and W.W. contributed unpublished reagents/analytic tools; E.J.P. and E.K. analyzed data; E.J.P. and M.A.U. wrote the paper.

                This work was supported by United Kingdom Medical Research Council (MRC) Grant s MC-A654-5QB70 (to M.A.U.), MC-A654-5QB60 (to J.L.), and MC-A654-5QB40 (to D.J.W.) and by Wellcome Trust Grants 107839/Z/15/Z (toN.P.F.) and 107841/Z/15/Z (to W.W.).

                Correspondence should be addressed to Mark A. Ungless, Institute of Clinical Sciences (ICS), Faculty of Medicine, Imperial College London, Du Cane Road, London W12 0NN, United Kingdom, E-mail: mark.ungless@ 123456imperial.ac.uk .
                Author information
                http://orcid.org/0000-0003-1183-9285
                http://orcid.org/0000-0001-5508-8140
                http://orcid.org/0000-0003-4874-4212
                http://orcid.org/0000-0003-4743-0334
                http://orcid.org/0000-0002-8009-7521
                http://orcid.org/0000-0002-1730-3353
                Article
                eN-NWR-0381-18
                10.1523/ENEURO.0381-18.2018
                6240015
                30456293
                f4ff85eb-90f0-4b52-b0b6-96cbb5f686e1
                Copyright © 2018 Paul et al.

                This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license, which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.

                History
                : 1 October 2018
                : 2 October 2018
                Page count
                Figures: 10, Tables: 3, Equations: 0, References: 77, Pages: 19, Words: 10109
                Funding
                Funded by: http://doi.org/10.13039/501100000265Medical Research Council (MRC)
                Award ID: MC-A654-5QB70
                Award ID: MC-A654-5QB60
                Award ID: MC-A654-5QB40
                Funded by: http://doi.org/10.13039/100010269Wellcome Trust (Wellcome)
                Award ID: 107839/Z/15/Z
                Award ID: 107841/Z/15/Z
                Categories
                5
                5.1
                New Research
                Integrative Systems
                Custom metadata
                September/October 2018

                gaba,glutamate,nnos,snc,vta
                gaba, glutamate, nnos, snc, vta

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