The NeuroD6 Subtype of VTA Neurons Contributes to Psychostimulant Sensitization and Behavioral Reinforcement

Conditional ablation of the Vmat2 gene in NEX-Cre neurons, a model for spatially restricted DA deficiency. A, Breeding strategy for generation of mice gene-targeted for Vmat2 in VTA NEX-Cre neurons. NEX-Cre transgenic mice were mated to Vmat2^lox/lox mice to generate NEX-Cre-positive mice homozygous for Vmat2^lox/lox (Vmat2^{lox/lox;NEX-Cre-tg}: cKO mice) and littermate control mice homozygous for Vmat2^lox/lox and negative for the NEX-Cre transgene (Vmat2^{lox/lox;NEX-Cre-wt}: Ctrl mice). B, Two-probe approach for detection of Vmat2 mRNA by ISH. Probe 1 detects exons 6–15 and probe 2 detects exon 2 of the Vmat2 gene. Exon 2 is floxed in Vmat2^lox/lox mice leading to failure of probe 2-binding to Vmat2 mRNA in cKO neurons. C, Implementation of Vmat2 mRNA two-probe approach in Vmat2^{lox/lox;NEX-Cre-wt} (Ctrl, left panel) and Vmat2^{lox/lox;NEX-Cre-tg} (cKO, right panel) brains. Wild-type neurons are positive for both Vmat2 probes, while cKO neurons are only positive for probe 1 due to targeted deletion of exon 2 (detected by probe 2). Probe 1 detected in green and probe 2 detected in blue results in green-blue double-labeling in wild-type cells and green-only labeling in cKO cells. Green arrows point to green-only cells, i.e., VMAT2 cKO cells. D, Vmat2 mRNA two-probe ISH in additional monoaminergic areas. E, TH immunohistochemistry in Ctrl and cKO midbrain and striatum. LC, locus coeruleus; ROB, raphe nucleus obscurus; VMH, ventromedial hypothalamus; VTA, ventral tegmental area; SNc, substantia nigra pars compacta; DStr, dorsal striatum; NAc, nucleus accumbens; OT, olfactory tubercle. TH, Tyrosine hydroxylase; Vmat2/VMAT2, Vesicular monoamine transporter 2; Ctrl, control; cKO, conditional knockout.

Altered responsiveness to psychostimulants upon ablation of Vmat2 gene expression in NeuroD6 VTA neurons. Color coding: Vmat2^{lox/lox;NEX-Cre-wt} (Ctrl) in white; Vmat2^{lox/lox;NEX-Cre-tg} (cKO) in green. A, Weight curve for Ctrl (N = 14) and cKO (N = 23) mice. Data presented as mean weight in grams for each week ± SEM (+p < 0.05 effect of genotype; ###p < 0.001, effect of age). B, Baseline locomotion in novel environment. Ctrl (N = 17) and cKO (N = 17). Data expressed as mean distance moved in 5-min bins ± SEM (###p < 0.001, effect of time). C, Sucrose preference expressed as percentage of preference for sucrose over tap water ± SEM. Ctrl (N = 14) and cKO (N = 21; ###p < 0.001 effect of sucrose concentration). D, Ethanol preference expressed as percentage of preference for ethanol solution over tap water ± SEM. Ctrl (N = 14) and cKO (N = 14; ###p < 0.001 effect of ethanol concentration, §§§p < 0.001 3% vs 6% and 10% in ctrl mice). E, Cocaine-induced locomotion. Top, Administration schedule. Bottom, Average distance moved 1 h after injection of saline and 5, 10, 20 mg/kg of cocaine; Ctrl (N = 14) and cKO (N = 21) mice. Data expressed as total distance moved during the 1-h recording period ± SEM (###p < 0.001 effect of session). F, Amphetamine-induced locomotion. Top, Administration schedule. Bottom, Average distance moved 1.5 h after injection; Ctrl (N = 17) and cKO mice (N = 17). Data presented as mean of total distance moved in cm ± SEM for each session; ++p < 0.01 effect of genotype, ###p < 0.001 effect of session, *p < 0.05 and ***p < 0.001 cKO versus Ctrl. G, CPP. Illustration of setup and administration schedule. H, J, Preference score displayed as Δsec, the difference between time spent in drug-paired compared during pretest and test ± SEM, positive value indicates preference (cocaine: Ctrl N = 12, cKO N = 15; amphetamine: Ctrl N = 13, cKO N = 16). I, K, Cocaine-induced and amphetamine-induced locomotion during conditioning in the CPP setup displayed as distance moved in 30 min ± SEM (cocaine: Ctrl N = 12, cKO N = 15; amphetamine: Ctrl N = 15, cKO N = 17, +p = 0.031 effect of genotype, ##p = 0.006, ###p < 0.001 effect of session). Ctrl, Control; cKO, conditional knockout; CPP, Conditioned place preference.

NeuroD6 mRNA co-localizes partly with Calb2 mRNA, but Calb2 mRNA is abundant throughout the VTA and SNc. Double-labeling FISH in the ventral midbrain of adult wild-type mice detecting the following mRNAs. A, NeuroD6 (red) and Calb2 (green), inset with high magnification of overlap (yellow), pie charts illustrating quantification of overlap between NeuroD6 and Calb2. B, Calb (red) and Th (green), inset with high magnification of overlap (yellow), pie charts illustrating quantification of overlap between Th and Calb2. C, Calb2 (red) and Dat (green), inset with high magnification of Dat/Calb2 mRNA overlap (yellow), pie chart illustrating quantification of overlap between Th and Calb2. D, Calb2 (red) and Vglut2 (green), inset with high magnification of Vglut2/Calb2 mRNA overlap (yellow) in blue square and Vglut2-negative/Calb2-positive (red) in white square, pie chart illustrating quantification of overlap between Th and Calb2. E, Calb2 (red) and Viaat (green), inset with high magnification of Viaat negative or positive (red, yellow, green) in white square. VTA, ventral tegmental area; SNc, substantia nigra pars compacta; PBP, parabrachial pigmented nucleus; PN, paranigral nucleus; PIF, parainterfascicular nucleus; RLi, rostral linear nucleus; IF, interfascicular nucleus. Calb2, Calbindin 2 (Calretinin); Dat, Dopamine transporter; Th, Tyrosine hydroxylase; Vglut2, Vesicular glutamate transporter 2; Viaat, Vesicular inhibitory amino acid transporter; FISH, fluorescent in situ hybridization.

Spatially restricted striatal innervation by NeuroD6 and Calb2 VTA neurons. A, Schematic illustration of stereotaxic injection into VTA of Cre-dependent DIO-ChR2-eYFP DNA construct packaged into AAV. B, Representative VTA neurons immunopositive for TH (red), YFP (green), or both (yellow; DIO-ChR2-eYFP-injected NEX-Cre mice). C–F, Representative pictures of VTA (left panels) and striatal complex (right panels) in DIO-ChR2-eYFP-injected DAT-Cre (C–C’’), Vglut2-Cre (D–D’’), Calb2-Cre (E–E’’), and NEX-Cre (F–F’’) mice. Panel far right, Schematic summary of striatal innervation pattern. Additional target areas listed in Table 2. Quantification of YFP and TH immunofluorescent overlap: schematic illustration of four representative VTA areas selected for counting, shown as squares and labeled VTA 1–4 (G). Results of quantifications shown in histograms for each VTA area and the total sum (H). PBP, parabrachial pigmented nucleus; PN, paranigral nucleus; PIF, parainterfascicular nucleus; RLi, rostral linear nucleus; IF, interfascicular nucleus; SNc, substantia nigra pars compacta; SNr, substantia nigra pars reticulata; IPR, interpeduncular nucleus, rostral subnucleus; IPC, interpeduncular nucleus, caudal subnucleus; DMStr, dorsomedial striatum; NAcC, nucleus accumbens core; NAcSh, nucleus accumbens shell; aca; anterior commissure, anterior part; OT, olfactory tubercle. DAT, Dopamine transporter, Calb2, Calbindin 2 (Calretinin); NEX, NeuroD6; Vglut2; Vesicular glutamate transporter 2; Th, Tyrosine hydroxylase; ChR2; Channelrhodopsin 2; eYFP, enhanced Yellow fluorescent protein.

Optogenetic stimulation in striatal target areas of NeuroD6 and Calb2 VTA neurons verifies DA release. A, Schematic representation of stereotaxic injection into VTA of Cre-dependent DIO-ChR2-eYFP and DIO-eYFP (Ctrl); FSCV recording sites within NAcSh and OT (red dots). B, Illustration of the experimental setup. C, Representative light-evoked DA recordings from injected DAT-Cre (left), NEX-Cre (middle), and Calb2-Cre (right) mice in the NAcSh (top) and the OT (bottom). D, Quantification of photostimulation-evoked DA release in the NAc shell (left) and OT (right); N = 10 recording sites per group for each region. Mice used for the recordings: DAT-Cre/ChR2 N = 2, DAT-Cre/eYFP N = 2, NEX-Cre/ChR2 N = 3, NEX-Cre/eYFP N = 2 Calb2-Cre/ChR2 N = 3, and Calb2-Cre/eYFP N = 2. Box and whisker plots, Center lines indicate medians, box edges represent the interquartile range, whiskers extend to the minimal and maximal values (*p < 0.05, **p < 0.01, ***p < 0.001 ChR2 vs ctrl). DStr, dorsal striatum; NAcSh, nucleus accumbens shell; OT, olfactory tubercle. DAT, Dopamine transporter; Calb2, Calbindin 2 (Calretinin); ChR2; Channelrhodopsin 2; eYFP, enhanced Yellow fluorescent protein; NEX, NeuroD6.

Optogenetic stimulation in striatal target areas of NeuroD6 and Calb2 VTA neurons reveals glutamatergic postsynaptic responses. A, Representative picture from patch-clamp slice electrophysiology in NAcSh of NEX-Cre mice and OT of Calb2-Cre mice injected with DIO-ChR2-eYFP. B, Representative traces of photostimulation-evoked postsynaptic currents recorded from NAcSh cells from NEX-Cre and OT cells from Calb2-Cre mice injected with DIO-ChR2-eYFP. C, Pie charts represent the percentage of cells showing EPSCs (white) versus negative (black) upon photostimulation of terminals in the NAcSh (N = 18 cells from four mice) of NEX-Cre mice, and OT (N = 14 cells from 4 mice) of Calb2-Cre mice. The y-axis shows amplitude in pA; each circle represents one cell, and bold lines the mean amplitude ± SEM. D, Patch-clamp recordings pre-bath (control) and post-bath application of DNQX upon photostimulation in NAcSh of NEX-Cre/ChR2 (left, N = 6 cells from three mice) and OT of Calb2-Cre/ChR2 mice (right, N = 5 cells from three mice). Each circle represents one cell (*p < 0.05 control vs DNQX). NAcSh, nucleus accumbens shell; OT, olfactory tubercle. Calb2, Calbindin 2 (Calretinin); NEX; NeuroD6; ChR2; Channelrhodopsin 2; eYFP, enhanced Yellow fluorescent protein.