Figure 3. Histological characterization of serotonergic neurons.

Detection of serotonergic-specific markers was performed on coronal brain sections of adult wt control (left panel) and Tph2−/− mice (right panel). Protein labeling was obtained by light immunohistochemistry (a-c) and immunohistofluorescence (e-g). (a) Labeling of Tph2 demonstrated its complete absence in the raphe of Tph2−/− mice. (b) The serotonin transporter (Sert) could be detected in both wt and Tph2−/− mice, in the raphe as well as along fibers in projection areas, e.g. in the frontal cortex (FC) as shown in (c). (d) Detection of the serotonergic-specific transcription factor Pet1 in the raphe by in situ hybridization occurred similarly in wt and Tph2−/− mice. (e) Detection of serotonin (5-HT) in the raphe showed the absence of specific 5-HT immunoreactivity in Tph2−/− mice. Cell nuclei were also labeled by DAPI staining. (f) The vesicular monoamine transporter-2 (Vmat2) could be detected similarly in the raphe of both wt and Tph2−/− mice. (g) Merged images from (e-f) showed the colocalization of 5-HT and Vmat2 in the serotonergic neurons of wt (yellow in g) while Tph2−/− neurons were only labeled with Vmat2 (red in g). Taken together these results demonstrate that despite 5-HT synthesis deficiency, serotonergic neurons of Tph2−/− mice can develop and be maintained. Moreover, except Tph2 and 5-HT, they possess all known 5-HT-specific markers showing that their serotonergic specification took place. Bars represent 100 µm in (c) and 200 µm in (a), (b), (d), (e-g).