Figure 2. DA neuron selective PAG deletion.

(A) PCR screens for the floxGLS1 allele (left) and ΔGLS1 allele (right) in brain regions from both GLS1^lox/lox and DAT GLS cKO mice. The ΔGLS1 allele was present solely in DAT GLS1 cKO ventral midbrain. dStr, dorsal striatum; HIPP, hippocampus; VMB, ventral midbrain; CTX, cortex. Gel is representative of 3 replications. (B) Single-cell rtPCR analysis of TH expressing cells in the VTA in DAT^IREScre/+ and DAT GLS1 cKO mice. In the VTA of DAT^IREScre/+mice, 11/30 TH cells expressed PAG mRNA, while in DAT GLS1 cKO none did (0/38 cells). (C) Confocal photomicrographs of the VTA from DAT^IREScre/+ and DAT GLS1 cKO mice showing TH⁺ only (thin blue arrow) and PAG⁺ only (blue arrow head) and TH⁺/PAG⁺ cells (thick blue arrow). There were no TH⁺/PAG⁺ cells in the DAT GLS1 cKO ventral midbrain. Expression of dopaminergic markers and amphetamine-induced hyperlocomotion were not affected in DAT^IREScre mice; see Figure 2—figure supplement 1. These mice were control (CTRL) mice in subsequent experiments.

DOI: http://dx.doi.org/10.7554/eLife.27566.006

Figure 2—figure supplement 1. Expression of dopaminergic markers and amphetamine-induced hyperlocomotion were not affected in DAT^IREScre mice.

(A) The relative mRNA expression of dopamine transporter (DAT), tyrosine hydroxylase (TH), vesicular monoamine transporter 2 (VMAT2) and dopamine D2 receptor (D2R) in the ventral midbrain (left), and D1R and D2R in dorsal striatum (dStr, right) of DAT^IREScre/+ and wild-type littermates (CTRL). A multivariate ANOVA showed no genotypic effect for any of the dopaminergic markers (ventral midbrain, DAT, F_(1,8)= 0.061, p=0.811; TH, F_(1,8)= 0.320, p=0.587; VMAT2, F_(1,8)= 1.742, p=0.223; D2, F_(1,8)= 3.903, p=0.084; dStr, D1 = F_(1,10)= 0.384, p=0.549; F_(1,10)= 0.851, p=0.004). (B) Amphetamine (Amph) stimulated locomotion. Total locomotor counts (i.e., beam breaks) in the open field made over 2.5 hr following Vehicle (0 mg/kg) or Amph, 3 or 5 mg/kg (i.p.). A two-way ANOVA showed a main effect of drug (F_(2,28) = 83.1; p<0.001, ES partial η² = 0.86), but no significant main effect of genotype (F_(1,28) = 0.846; p=0.366) or significant interaction (F_(2,28) = 28.2; p=0.973). (C) Time course of Amph-evoked locomotion. There were no genotypic differences for either the 3 mg/kg (top) or 5 mg/kg doses (bottom). The repeated measures (RM) ANOVA showed no significant main effect of genotype (3 mg/kg dose, F_(1,10) = 0.003, p=0.960; 5 mg/kg dose, F_(1,9) = 1.322, p=0.280) or time X genotype interaction (3 mg/kg dose, F_(14,140) = 0.784, p=0.685; 5 mg/kg dose, F_(14,126) = 1.663, p=0.071); there was a main effect of time (3 mg/kg dose, F_(14,140) = 20.5, p<0.0001, ES partial η² = 0.67; 5 mg/kg dose, F_(14,126) = 20.5, p<0.0001, ES partial η² = 0.69). Numbers of cells are shown above each bar in the graphs. See Figure 2—figure supplement 1—source data 1.xlsx for source data and statistical analysis.

DOI: http://dx.doi.org/10.7554/eLife.27566.007