Figure 2. Lack of Vglut3 impairs 5-HT metabolism in the brainstem, and the RRG response to 5-HT.

See Supplemental Fig. 1 for schematic representation of the brainstem sections. A, immunolabelling showing that Vglut3 (green) colocalizes poorly with VMAT2 (red) in control mice at the level of the dorsal raphe (DR) (a), raphe pontis (RPn) (b), raphe magnus (RMg) (c) and raphe pallidus nuclei (Rpa) (d). B and C, immunodetection of 5-HT (red) and VGLUT3 (green) expression in coronal sections of control mice at the level of the raphe obscurus nucleus (ROb). Arrows in the upper right corner of panels B and C point toward the ventral medullary surface (B) and RMg (C). VGLUT3 was found in soma of cells at the ROb (Bb) and RMg (Cc) levels, and is co-expressed with 5-HT in cells of ROb and at RMg level (stars, yellow cells in Bc and Cc, respectively). D, 5-HT turnover in Vglut3^−/− pups (n = 12) differs from that in controls (n = 6). Endogenous levels of 5-HT (a), its main metabolite 5-hydroxyindoleacetic acid (5-HIAA) (b) and its precursor l-tryptophan (l-Trp) (c) in the brainstem of Vglut3^−/− and control pups, measured by HPLC. The 5-HT/5-HIAA ratio was significantly higher in Vglut3^−/− than in control pups (d). E and F, the spinal response to exogenous 5-HT is impaired in Vglut3^−/− pups. E, examples of phrenic bursts recorded from the C4 root of in vitro brainstem preparations from control (a) and Vglut3^−/− (b) pups, superfused with normal aCSF and aCSF containing 5-HT (25 μm, 5 min). Int C4: integrated C4 activity. F, tonic discharges in response to 5-HT appeared with the same latency in Vglut3^−/− (n = 11) and control (n = 14) preparations (a) but were shorter (b) and of smaller amplitude (c) in Vglut3^−/− preparations compared to control preparations (*P < 0.05; Student's unpaired t test, ns: non significant). a.u.: arbitrary units. Values given are means ± SEM. See Table 1 for full statistical analyses.