No change in the number of glutamate decarboxylase (GAD67)-positive cells was observed in the SN pars reticulata of Shh-nLZC/C/Dat-Cre mice at 16 months of age ( Figure S3A). Striatal Th+-fiber density was normal at 1 month of age, increased at 8 months and decreased at 12 months of age

in Shh-nLZC/C/Dat-Cre mice compared to controls ( Figure 2F). Gene expression analysis of DA markers in the ventral midbrain (vMB) revealed a downregulation of Th, Dat, and DA receptor-2 (DaR2) at 5 weeks of age, which then returned to normal levels by 12 months in Shh-nLZC/C/Dat-Cre compared to controls ( Figure 2G; all genes probed herein are listed selleck products in Table S2). The expression of the vesicular monoamine transporter-2 (vMat2) appeared normal at 5 weeks, but was diminished at 12 months. The activator of endoplasmic reticulum stress, Xbp1, and the antioxidant enzyme, glutathione-peroxidase-1 (Gpx1), were upregulated in Shh-nLZC/C/Dat-Cre animals at 5 weeks but not at 12 months of age ( Figure 2G) indicative of the activation of physiological cell stress responses in the vMB in the absence of Shh expression in young adult mutant mice. In further support of a protracted dopaminergic cell syndrome in which neuronal degeneration is only the final step, we found progressive alterations in somato-dendritic and

striatal Imatinib price DA content and deficits in amphetamine elicited DA release in Shh-nLZC/C/Dat-Cre mice ( Supplemental Results C and Figures S3B–S3D). Is the observed dopaminergic phenotype new a cell autonomous effect of the interruption of Shh signaling? Shh can bind to several coreceptors which in turn facilitate the relief of repression of the serpentine transmembrane protein Smo by Ptc1 or Ptc2 (Izzi et al., 2011). To distinguish autocrine from paracrine Shh signaling, we analyzed the expression of the Shh coreceptors Ptc1 and Ptc2 and the phenotype of animals with a tissue restricted ablation of Smo from DA neurons, which we produced using the same Dat-Cre

allele with which we also achieved the tissue specific ablation of Shh. We did not find evidence for the expression of Ptc1 in DA neurons utilizing a gene expression tracer mouse line (Ptc1-nLZ) or Ptc2 by in situ hybridization, consistent with public gene expression data information (Gensat, http://www.gensat.org; data not shown). SmoC/C/Dat-Cre mutant animals were born alive and mobile with expected Mendelian frequency and no overt structural or motor signs through adulthood compared to SmoC/+/Dat-Cre control littermates (data not shown). Unbiased stereological cell counting of Th+ and Th− neurons in the SNpc and VTA of 18-month-old SmoC/C/Dat-Cre mutants and SmoC/+/Dat-Cre littermate controls did not reveal DA neuron loss in the SNpc or VTA ( Figures 2H and 2I).