VGLUT2 in dopamine neurons is required for psychostimulant-induced behavioral activation

Abstract

The "One neuron-one neurotransmitter" concept has been challenged frequently during the last three decades, and the coexistence of neurotransmitters in individual neurons is now regarded as a common phenomenon. The functional significance of neurotransmitter coexistence is, however, less well understood. Several studies have shown that a subpopulation of dopamine (DA) neurons in the ventral tegmental area (VTA) expresses the vesicular glutamate transporter 2 (VGLUT2) and has been suggested to use glutamate as a cotransmitter. The VTA dopamine neurons project to limbic structures including the nucleus accumbens, and are involved in mediating the motivational and locomotor activating effects of psychostimulants. To determine the functional role of glutamate cotransmission by these neurons, we deleted VGLUT2 in DA neurons by using a conditional gene-targeting approach in mice. A DAT-Cre/Vglut2Lox mouse line (Vglut2(f/f;DAT-Cre) mice) was produced and analyzed by in vivo amperometry as well as by several behavioral paradigms. Although basal motor function was normal in the Vglut2(f/f;DAT-Cre) mice, their risk-taking behavior was altered. Interestingly, in both home-cage and novel environments, the gene targeted mice showed a greatly blunted locomotor response to the psychostimulant amphetamine, which acts via the midbrain DA system. Our results show that VGLUT2 expression in DA neurons is required for normal emotional reactivity as well as for psychostimulant-mediated behavioral activation.

Fig. 1.

Expression of Vglut2 mRNA and DAT-Cre activity in mDA neurons. (A–D) In situ hybridization for Vglut2 mRNA combined with immunohistochemistry for TH on sagittal sections of E12.5 embryo. Vglut2 mRNA is expressed in multiple regions in the embryo, including the ventral midbrain (MB) where DA neurons develop (A). (B) Arrows indicate Vglut2-expressing cytoplasm. Vglut2 mRNA is expressed in TH-immunopositive (green) DA neurons in the MB (Magnification: B ×300) (C). (D) Arrows indicate Vglut2/TH double-positive cells (Magnification: D ×300). (E) Sagittal section of a Tau^mGFP;DAT-Cre brain at P7 with immunofluorescence for GFP visualizing the mDA neurons in the VTA and SNc and their projection pathway in the median forebrain bundle (MFB) to the target neurons in the striatum (CaPu + NA). GFP projections also are seen in the hippocampus (HC) and the cerebellum (CB). (F and G) Coronal sections of newborn (P0) Tau^mGFP;DAT-Cre mouse showing immunohistochemistry for β-gal (red) and GFP (green) in the VTA and SNc. (H–L) Coronal sections from Tau^mGFP;DAT-Cre P0 mouse showing TH (H, red) and β-gal (I, green) immunohistochemistry within the same cells (merged fluorescence in J), and TH (K, red) and GFP (L, green) within another set of cells (merged in M).

Fig. 2.

Disruption of Vglut2 mRNA expression in the mDA neurons. (A) Schematic of coronal mouse brain section at bregma −3.08 (modified from ref.39). Aq, aqueduct; IF, interfascicular nucleus; MM, medial mammillary nucleus; PAG, periaqueductal gray; PBP, parabrachial pigmented nucleus; SNc, substantia nigra pars compacta; SNr, substantia nigra pars reticulata. (B–E) Vglut2 mRNA expression is seen in control mDA neurons but not in the Vglut2^f/f;Dat-Cre mDA neurons, as shown by in situ hybridization. (Left) Control (B and D), Vglut2^f/f;Dat-Cre (C), and Vglut2^{f/f;DAT-Cre;Tau-mGFP} (E). Gene-targeted TH-positive DA neurons (red in B–E, Middle, and merged in B–E, Right). In D and E, the nucleus is visualized by β-gal immunohistochemistry (green) to show that DAT-Cre is active in these neurons. (F–H) Multiplex RT-PCR on single cells verifies that gene targeting has occurred in the Vglut2^{f/f;DAT-Cre;Tau-mGFP} DA cells. (F) The pipette is sucking up a freshly dissociated GFP-expressing DAT-Cre cell. (G) The gel picture from electrophoresis of RT-PCR products shows the presence of the gene-targeted Vglut2 allele in the Vglut2^{f/f;DAT-Cre;Tau-mGFP} (KO) cell but not in the control cell (CL). (H) When the GFP-expressing cells are left in culture for 1 week, they develop extensive neurites immunopositive for TH, confirming their DA phenotype. KO, Vglut2^{f/f;DAT-Cre;Tau-mGFP} (knockout); CL, Vglut2^{f/+;DAT-Cre;Tau-mGFP} (control).

Fig. 3.

Vglut2^f/f;DAT-Cre mice show blunted behavioral response to amphetamine, as measured in a home-cage environment. (A and B) Male and female knockout mice showed a reduced behavioral response to amphetamine as shown by significantly lower total activity at all three doses of amphetamine. (C and D) Male and female knockout mice display a shift in dose-response to amphetamine-induced locomotion. (E and F) Male and female knockout mice show a different dose-response profile regarding rearing behavior in response to amphetamine. A right shift in dose-response in locomotion is apparent in the Vglut2^f/f;DAT-Cre mice. Data were analyzed with 1-way ANOVA followed by Tukey´s post hoc test when appropriate. Data are presented as mean ± SEM (n = 9). *, P < 0.05; **, P < 0.01; ***, P < 0.001.