Dopaminergic terminals in the nucleus accumbens but not the dorsal striatum corelease glutamate

Abstract

Coincident signaling by dopamine and glutamate is thought to be crucial for a variety of motivated behaviors. Previous work has suggested that some midbrain dopamine neurons are themselves capable of glutamate corelease, but this phenomenon remains poorly understood. Here, we expressed the light-activated cation channel Channelrhodopsin-2 (ChR2) in genetically defined midbrain dopamine neurons to stimulate exocytosis specifically from dopaminergic terminals in both the nucleus accumbens (NAc) shell and dorsal striatum of brain slices from adult mice. Optical activation resulted in robust glutamate-mediated EPSCs in all medium spiny neurons examined in the NAc shell. In contrast, optically evoked glutamatergic currents were nearly undetectable in the dorsal striatum. Further, we used a conditional knock-out mouse lacking vesicular glutamate transporter 2 (VGLUT2) specifically in dopamine neurons to determine whether VGLUT2 is required for the exocytotic release of glutamate from dopamine neurons. Our data show that conditional knock-out completely abolished all optically evoked glutamate release. These results provide definitive physiological evidence for VGLUT2-mediated glutamate release by mature dopamine neurons projecting to the NAc shell, but not to the dorsal striatum. Thus, the unique ability of NAc-projecting dopamine neurons to synchronously activate both dopamine and glutamate receptors may have crucial implications for the ability to respond to motivationally significant stimuli.

Figure 1.

Expression of ChR2-mcherry in midbrain dopamine neurons and their projections to the striatum. A, Immunostaining for ChR2-mcherry (red) and TH (green) in the VTA/SN ∼30 d after injection of AAV-DIO-ChR2-mcherry in a mouse expressing cre recombinase under the control of the DAT promoter. B, Confocal microscopy shows that nearly all ChR2-mcherry-expressing neurons in the VTA colabel for TH. C, Staining as described above in a coronal plane including both DS and NAc shell. D, Confocal images of dopaminergic fibers in the NAc shell indicate that essentially all ChR2-mcherry-labeled puncta also contain TH.

Figure 2.

Dopaminergic terminals in the nucleus accumbens shell but not dorsal striatum corelease glutamate. A, Example EPSCs recorded in the NAc shell at −70 mV following a 5 ms blue light pulse at maximal intensity. B, Light-evoked EPSC amplitudes recorded in the NAc shell were significantly greater in DAT:VGLUT2 heterozygous mice than in conditional knock-outs (n = 9–24 cells per group; 2-way ANOVA for the interaction of genotype and light intensity: F_(4,128) = 2.56, p < 0.05). C, Example EPSCs recorded in the DS at −70 mV following a 5 ms blue light pulse at maximal intensity. D, Light-evoked EPSC amplitudes recorded in the DS were not significantly different in DAT:VGLUT2 heterozygous mice versus conditional knock-outs as a function of light intensity (2-way ANOVA for the interaction of genotype and light intensity: F_(4,87) = 0.31, p = 0.91). EPSC amplitudes (independent of light intensity) were significantly different across genotypes (F_(1,87) = 12.80, p = 0.006).

Figure 3.

EPSCs evoked by dopamine terminal stimulation in the nucleus accumbens shell are blocked by AMPAR antagonism but unaffected by D₁R/D₂R antagonism. A, Example EPSCs recorded at −70 mV in the NAc shell from DAT:VGLUT2 heterozygous mice before and after bath application of DNQX. B, Example EPSCs recorded at −70 mV in the NAc shell from DAT:VGLUT2 heterozygous mice before and after bath application of SCH23390/raclopride. C, Light-evoked EPSCs from dopamine terminals were significantly reduced by DNQX bath application (n = 5 cells; t₍₄₎ = 2.61; p < 0.05) but not by SCH23390/raclopride (n = 9 cells; t₍₈₎ = 0.40; p = 0.70). All data indicate mean ± SEM.

Figure 4.

Optical stimulation of dopamine terminals in dorsal striatum and nucleus accumbens shell results in dopamine release. A, Example trace of dopamine release resulting from optical stimulation of dopamine terminals in the DS (1 pulse, 5 ms pulse duration). Inset shows background-subtracted cyclic voltammograms taken immediately after optical stimulation. The color plot below shows current measured as the electrode across the entire scanned potential range. B, Example trace of dopamine release resulting from optical stimulation of dopamine terminals in the NAc shell. C, Average dopamine release (solid line; dashed lines indicate ±SEM) in the dorsal striatum following one-pulse optical stimulation (n = 7 recording sites). D, Average dopamine release in the nucleus accumbens shell following one-pulse optical stimulation (n = 5 recording sites).