Figure 3.

Gestational stress and CIT exposure differentially affect serotonin immunoreactivity in the fetal forebrain. (A) A 3D reconstruction of an iDISCO-cleared E17 brain showing rostral-to-caudal levels (clipping planes) used for analyses of serotonin immunofluorescence distributions. (B–D) Serotonin immunoreactivity was analyzed in coronal sections at three levels of the medial forebrain bundle (mfb; boxed areas: B, rostral, magenta; C, medial, green; D, caudal, light blue; scale bars = 500 jam). (F) Quantification of serotonin-immunopositive axon densities did not reveal differences between treatment groups throughout the mfb (corresponding images above). (E, G–J) The serotonin immunostaining was observed in neuronal cell bodies located in the ventroposteriolateral thalamic nucleus (vpl) in the thalamus (arrows; scale bar = 100 μm). Thalamic neurons and their axons are immunolabeled with the specific marker netrin-G1. The densities of serotonin immunoreactivity were significantly increased in thalamic neurons from embryos exposed to gestational stress (CUS) compared to untreated controls (F, right bar graph). In contrast, serotonin immunoreactivity densities were significantly decreased in thalamic neurons from embryos exposed to CIT compared to controls and in embryos exposed to CUS+CIT compared to CUS. In panel F, data are means ± SEMs for normalized pixel intensities in each region of interest (N = 4 dams per condition, 1 fetal brain per dam). *P < 0.05, **P < 0.01, or ***P < 0.001.