Figure 5

Viral-mediated recombination during adolescence produces the same anatomical and behavioral adult phenotypes exhibited by dcc conditional mice. (a) Schematics, AAV-CreGFP or control AAV-GFP viruses were microinjected bilaterally into the VTA of juvenile (PND 21) heterozygous and homozygous dcc-floxed mice (dcc^lox/+ and dcc^lox/lox). In adulthood (PND 60-15), (1) amphetamine-induced locomotor activity and (2) the extent of DA innervation to the mPFC were measured. Micrographs show the distribution of the control AAV-GFP virus (green) in the TH-positive fibers (red) in the NAcc and mPFC, providing validation of successful infection of mesocorticolimbic projecting VTA DA neurons. Scale bar=500 μm. (b) Stereological analysis of VTA and SNc DA neurons showed that, as expected, more DA neurons were infected with the AAV-CreGFP virus in the targeted VTA than in the SNc (VTA: n=11 and SNc: n=11, t₍₂₀₎=6.775, P<0.0001). Consistent with previous reports, an average of ~70% of DA neurons were infected.¹⁹ Stereological estimates of total DA cell number were the same across virus treatment groups and were comparable to the numbers reported in the wild-type mouse brain (AAV-GFP: n=4 and AAV-CreGFP: n=11; t₍₁₃₎=0.902, P=0.383). This indicates that neither the viral infection procedure nor the loss of DCC expression affected DA neuron survival. Brain sections through the VTA of adult dcc^lox/lox mice injected with the AAV-CreGFP or control AAV-GFP viruses in adolescence were triple immunolabeled with TH (blue), GFP (green) and DCC (red). A robust decrease in DCC immunoreactivity was observed in TH-positive DA neurons infected with the AAV-CreGFP virus but not with the control AAV-GFP. Scale bars=500 μm. (c) Reduction or complete removal of DCC from individual VTA neurons from early adolescence onward is sufficient to reproduce the adult phenotypes observed in dcc conditional mice. dcc^lox/+ and dcc^lox/lox mice that received injections of AAV-CreGFP in early adolescence had an enlarged volume of TH-positive dense fiber innervation to the cortical inner layers in adulthood relative to dcc-floxed mice that received injections of AAV-GFP (control AAV-GFP virus group: n=3, dcc^lox/+-CreGPF virus: n=5, dcc^lox/lox-CreGPF virus: n=4; Two-way repeated measures ANOVA, main effect of genotype-virus group: F_(2,9)=5.498, P=0.0275; main effect of mPFC cortex subregion (repeated measure): F_(1,9)=47.53, P<0.0001; no significant interaction: F_(2,9)=0.686, P=0.528). Note: while our two-way ANOVA revealed a main effect of genotype-virus group in the volume of TH-positive innervation to the mPFC inner layers, the presence of a significant interaction between genotype-virus and mPFC cortex subregion was not detected. Post hoc analyses were therefore not required. (d) Compromising DCC expression in VTA neurons from early adolescence also reproduced the blunted behavioral responses to amphetamine observed in the adult dcc conditional mice. Both dcc^lox/+-CreGFP and dcc^lox/lox-CreGPF mice exhibited reduced locomotor activity in the first hour after an amphetamine challenged (2.5 mg kg⁻¹), in comparison with dcc-floxed mice injected with the control virus (control AAV-GFP virus group: n=6, dcc^lox/+-CreGPF: n=7, dcc^lox/lox-CreGPF: n=6; two-way repeated measures ANOVA, significant main effect of genotype: F_{(2, 16)}=4.089, P=0.0368; significant interaction: F_{(28, 224)}=1.952, P=0.0042). (e–g) To assess the effects of reducing dcc in adult dcc-floxed mice that developed normally, virus microinfusions were done at PND 60. Behavioral and stereological experiments were performed 4–5 weeks after surgery. (e) Representative micrographs of sections through the VTA of dcc^lox/+ mice injected with the AAV-CreGFP or control AAV-GFP viruses in adulthood. Sections double-immunolabeled with TH (blue) and GFP (green) show TH-positive DA neurons in the VTA infected with virus. Scale bars=500 μm. Stereological analysis of VTA and SNc DA neurons infected with the AAV-CreGFP virus shows a greater percentage of DA neurons infected in the targeted VTA than in the SNc (VTA: n=3 and SNc: n=3, t₍₄₎=3.343, P<0.029). Estimates of total DA neuron number in both virus treatment groups are consistent with previous reports on DA cell number in the normal adult mouse brain (AAV-GFP: n=3 and AAV-CreGFP: n=3; t₍₄₎=1.141, P=0.317). (f) dcc^lox/+ mice that received injections of AAV-CreGFP in adulthood did not exhibit an increased volume of DA fiber innervation to the cortical inner layers in comparison with mice that received the control AAV-GFP virus (control AAV-GFP virus: n=3, dcc^lox/+-CreGPF virus: n=3; two-way repeated measures ANOVA, no main effect of genotype: F_(1,4)=0.004, P=0.954; main effect of region (repeated measure): F_(1,4)=99.73, P=0.0006; no significant interaction: F_(1,4)=7.418, P=0.053). (g) In heterozygous dcc-floxed mice, compromising DCC expression in VTA neurons in adulthood did not lead to blunted behavioral responses to amphetamine compared with mice that received the control AAV-GFP virus. dcc^lox/+-CreGFP mice exhibited a similar level of locomotor activity as control dcc^lox/+-GFP mice in the first hour after a 2.5 mg kg⁻¹ amphetamine challenge (control AAV-GFP virus group: n=6, dcc^lox/+-CreGFP: n=7; two-way repeated measures ANOVA, no main effect of genotype: F_{(1, 6)}=0.070, P=0.7996; no interaction: F_{(20, 120)}=0.263, P=0.999). (h) Brain expression of DCC is elevated in depressed suicide completers. DCC mRNA levels in prefrontal cortex tissue (BA44) obtained from the Quebec Suicide Brain Bank were assessed using qRT–PCR. Brains were matched for individual age and gender, brain pH and postmortem interval (PMI). Mean RNA integrity numbers (RIN) were between 6 and 7. Remarkably, DCC levels were 48% higher in suicide completers in comparison with control subjects (t₍₆₃₎=2.287, *P<0.026). Group mean±s.e.m. of AQ values from qRT–PCR were normalized to GAPDH.