Phenotypic characterization of chemosensitive RTN neurons in rat and mouse

  • Published 2007

Abstract

A. Location of Phox2b-immunoreactive (-ir) neurons in RTN of neonatal rat and mouse medulla (P8). B. Combined non-isotopic in situ hybridization (VGlut2) and immunohistochemistry (Phox2b) in rat and mouse brainstem (P8); Phox2b and VGlut2 are expressed in the same neurons (scale bar represents 100 µm). C. Firing rate responses to bath alkalization and acidification typical of RTN chemosensitive neurons in rat (upper) and mouse (lower); composite maps and camera lucida reconstructions of representative labeled cells show the location and morphology of chemosensitive RTN neurons. D. Averaged firing rate at different extracellular pH of RTN neurons from rat (n=10) and mouse (n=8). E. Agarose gel of single cell RT-PCR for Phox2b and GAPDH; the chemosensitive RTN neuron expresses Phox2b but the Purkinje cell does not. Control reactions performed in the absence of template were included for all PCR reactions (data not shown). Supplementary Methods: Phox2b and VGlut2 histochemistry in neonatal rat and mouse RTN Immunohistochemistry for Phox2b and in situ hybridization for VGlut2 was performed on perfusion-fixed (4% paraformaldehyde) free-floating coronal brainstem sections (30-50 µm) obtained from neonatal rats and mice, essentially as described (Stornetta et al., 2006). We detected VGlut2 mRNA by non-radioactive in situ hybridization using digoxigenin-labeled cRNA probes; Phox2b was detected using a rabbit polyclonal antibody (Pattyn et al., 1997). The distribution of neurons with densely-labeled Phox2b-ir cell nuclei was similar in the RTN of neonatal rats and mice (Fig. S1A); by comparison to the rat, neurons in the mouse RTN were less well represented in the dorsal cap region and tended to be located primarily within the marginal layer. By combining immunohistochemistry for Phox2b and in situ hybridization for VGlut2 on brainstem tissue sections (Fig. S1B), we found extensive co-localization in individual RTN neurons, demonstrating that this population of Phox2b neurons is glutamatergic (see also ref #(Stornetta et al., 2006). Distribution and morphology of functionally identified rat and mouse RTN neurons Cell-attached recordings were obtained from RTN neurons from rat and mouse brainstem slices as described in the main text (see also (Mulkey et al., 2004). For both rat and mouse, RTN neurons responded strongly to changes in bath pH. As illustrated for representative rat and mouse cells (Fig. S1C), bath alkalization (from pH 7.3 to pH 7.5) effectively silenced the neurons whereas bath acidification (to pH 6.9) caused a marked increase in discharge. For both species, effects of pH on neuronal discharge were remarkably similar; spontaneous …

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@inproceedings{2007PhenotypicCO, title={Phenotypic characterization of chemosensitive RTN neurons in rat and mouse}, author={}, year={2007} }