Figure 3

Conditional dcc loss-of-function within DA neurons, exclusively, increases DCC-expressing dopamine synaptic sites in the mPFC and postsynaptic structural changes in mPFC layer V pyramidal neurons. (a) Coronal sections through the pregenual mPFC (plates 14–18;⁶⁴) were immunolabeled with TH for stereological analysis of DA varicosity innervation to the cortical inner layers. Left panel, micrograph illustrating how contours were drawn around the TH-positive fiber innervation to the mPFC inner layers within each subregion of interest (Cg1, PrL; see white tracing). Scale bar=500 μm. Right panel, representative micrograph of TH-positive immunoreactivity in the mPFC inner layers that was used for stereological counting (taken with a × 100 objective). TH-positive varicosities were defined as dilated elements associated with axonal processes. Scale bar=10 μm. Stereological estimates of total TH-positive varicosity number was estimated using a stereological fractionator sampling design, with the optical fractionator probe of the Stereo Investigator software. Conditional dcc mice exhibited an increase in the total number of TH-positive varicosities innervating the inner layers of the mPFC (dcc^lox/lox: n=5; dcc^lox/+DAT^cre: n=4; dcc^lox/loxDAT^cre: n=5; two-way repeated measures ANOVA, main effect of genotype: F_(2,11)=5.362, P=0.0293; main effect of mPFC subregion (repeated measure): F_(1,11)=21.34, P=0.0013; no significant interaction: F_(2,11)=0.898, P=0.4410). Underlying this increase was a larger volume of dense TH-positive innervation of the mPFC inner layers (two-way repeated measures ANOVA, main effect of genotype: F_(2,11)=5.046, P=0.028; main effect of mPFC subregion (repeated measure): F_(1,11)=18.03, P=0.0014; no significant interaction: F_(2,11)=1.653, P=0.236). There were no differences in the density of TH-positive innervation (two-way repeated measures ANOVA, no main effect of genotype: F_(2,11)=1.583, P=0.249; main effect of mPFC subregion (repeated measure): F_(1,11)=9.816, P=0.0095; no significant interaction: F_(2,11)=3.062, P=0.0877). Note: while our two-way ANOVAs revealed main effects of genotype in (1) the total TH-positive varicosity number and (2) volume of TH-positive innervation, the presence of a significant interaction between genotype and mPFC subregion was not detected. Therefore, post hoc analyses were not required. (b) Conditional dcc mice exhibited an increase in DA innervation to the mPFC inner layers by DA varicosities that were double-labeled with DCC. Schematics generated by Neurolucida explorer (Microbrightfield) illustrating the distribution of DCC/TH-positive (green triangles) varicosity counts made within the dense TH-positive projection in one brain section (see red tracing in schematic) in a dcc^lox/lox versus dcc^lox/+DAT^cre mouse. The counts for each brain were performed across five coronal sections through the pregenual mPFC. The mean density of DCC/TH-positive varicosities was increased two- to threefold in the Cg1 and PrL in comparison with wild-type littermates (dcc^lox/lox: n=4; dcc^lox/+DAT^cre: n=4; two-way repeated measures ANOVA, main effect of genotype: F_(1,6)=17.54, P=0.006; main effect of mPFC cortex subregion (repeated measure): F_(1,6)=3.97, P=0.094; no significant interaction: F_(1,6)=1.471, P=0.271). Note: while our two-way ANOVA revealed a main effect of genotype in the density of DCC/TH-positive varicosities in the mPFC, the presence of a significant interaction between genotype and mPFC cortex subregion was not detected. Post hoc analyses were therefore not required. (c) There was no difference between genotypes in the total number of TH-positive varicosities in the NAcc (dcc^lox/lox: n=4; dcc^lox/+DAT^cre: n=4; dcc^lox/loxDAT^cre: n=4; one-way ANOVA, no main effect of genotype, F_(2,9)=0.556, P=0.5918). (d) Conditional dcc loss-of-function within DA neurons, exclusively, produces postsynaptic structural changes in mPFC Layer V pyramidal neurons. The density of Layer V pyramidal neuron basilar dendritic spines is significantly lower in dcc^lox/+DAT^cre and dcc^lox/loxDAT^cre mice relative to control dcc-floxed littermates (dcc^lox/lox: n=10; dcc^lox/+DAT^cre: n=10; dcc^lox/loxDAT^cre: n=8; one-way ANOVA, significant main effect of genotype: F_(2,25)=4.724, P=0.0182; Tukey’s multiple comparison test: (1) dcc^lox/lox and dcc^lox/+DAT^cre, P=0.05), (2) dcc^lox/lox and dcc^lox/loxDAT^cre, P=0.0276)). Representative micrographs of Golg–Cox-labeled layer V pyramidal neuron basilar dendritic spines in dcc^lox/lox, dcc^lox/+DAT^cre, and dcc^lox/loxDAT^cre mice. Scale bar=5 μm. Structural changes were not observed in NAcc medium spiny neurons (dcc^lox/lox: n=7; dcc^lox/+DAT^cre: n=8; dcc^lox/loxDAT^cre: n=7; one-way ANOVA, no significant main effect of genotype: F_(2,19)=0.086, P=0.915). Representative micrographs of Golgi–Cox-labeled medium spiny neurons in dcc^lox/lox, dcc^lox/+DAT^cre, and dcc^lox/loxDAT^cre mice. Scale bar=5 μm.