Figure 2

Conditional deletion of exon 23 of the dcc gene produces a loss-of-function mutation in DA neurons. Mice were engineered to carry a mutation in the dcc gene exclusively within dopamine neurons using the Cre-loxP recombination system. (a) The schematic illustrates how Cre-mediated recombination of the dcc gene will be triggered in mice from this line if they carry (1) LoxP sequences flanking exon 23 of dcc and (2) a Cre-recombinase insertion that is under transcriptional control by the DAT promoter. A PCR performed on genomic DNA obtained from tissue punches taken from the ventral tegmental area (VTA) using the primer pair represented in the schematic did not yield a PCR product in dcc-floxed (dcc^lox/lox) control littermates. In this case, the predicted size of the PCR product in the non-recombined gene is of ~5700 bp, which is too large to amplify. However, a 374 bp product was amplified from the DNA obtained from a dcc^lox/loxDAT^cre mouse, confirming successful recombination of the dcc gene. (b) The recombined dcc gene does not produce detectable levels of mRNA. An RT–PCR performed on cDNA of VTA tissue punches of dcc^lox/lox, dcc^lox/+DAT^cre and dcc^lox/loxDAT^cre produced two bands: (1) a larger product corresponding to the wild-type allele (arrowhead), amplified from DCC-expressing cells in the VTA that are not DAT+ DA neurons and (2) a smaller product corresponding to the recombined mRNA (arrow), which was barely above detectable levels. (c) DCC expression in the VTA of dcc^lox/loxDAT^cre mice was markedly reduced in comparison with dcc^lox/lox control mice. A cluster of DA neurons in the ventrolateral VTA continue to express DCC. This is a region of robust DAT expression. The reason why DCC expression persists in this grouping of cells is unknown. Scale bars=250 μm. (d) Western analysis of DCC levels in the NAcc confirmed a marked reduction in DCC expression in dcc conditional mice (dcc^lox/+DAT^cre and dcc^lox/loxDAT^cre) compared with dcc^lox/lox control littermates. Schematic, tissue punches (1 mm in diameter) were taken from the NAcc (core and shell; plates 15–20.⁶⁴ A significantly lower level of DCC expression was observed in both dcc^lox/+DAT^cre (~54% reduction) and dcc^lox/loxDAT^cre (~77% reduction) mice (dcc^lox/lox: n=4; dcc^lox/+DAT^cre: n=4; dcc^lox/loxDAT^cre: n=3; one-way ANOVA, significant main effect of genotype: F_(2,8)=24.94, P=0.0004. Bonferroni’s multiple comparison test (1) dcc-floxed and dcc^lox/+DAT^cre, P=0.0014 and (2) dcc-floxed and dcc^lox/loxDAT^cre, P=0.0003). (e) Loss of DCC expression within DA neurons does not affect their survival. Stereological estimates of total DA neuron number in the VTA and substantia nigra pars compacta (SNc). Sections spanning plates 53–63 were analyzed. ⁶⁴ Representative micrograph of a TH-labeled coronal section through the VTA and SNc (see schematic) used in the stereological analysis. No differences were found across genotypes in the stereological counts of DA neurons in the VTA and SNc (dcc^lox/lox: n=6; dcc^lox/+DAT^cre: n=7; dcc^lox/loxDAT^cre: n=7; two-way repeated measures ANOVA: no significant main effect of genotype, F_(2,17)=1.129, P=0.3463; no significant interaction, F_(2,17), P=0.72).