VTA glutamatergic inputs to nucleus accumbens drive aversion by acting on GABAergic interneurons


The ventral tegmental area (VTA) is best known for its dopamine neurons, some of which project to nucleus accumbens (nAcc). However, the VTA also has glutamatergic neurons that project to nAcc. The function of the mesoaccumbens glutamatergic pathway remains unknown. Here we report that nAcc photoactivation of mesoaccumbens glutamatergic fibers promotes aversion. Although we found that these mesoaccumbens glutamatergic fibers lack GABA, the aversion evoked by their photoactivation depended on glutamate- and GABA-receptor signaling, and not on dopamine-receptor signaling. We found that mesoaccumbens glutamatergic fibers established multiple asymmetric synapses on single parvalbumin GABAergic interneurons and that nAcc photoactivation of these fibers drove AMPA-mediated cellular firing of parvalbumin GABAergic interneurons. These parvalbumin GABAergic interneurons in turn inhibited nAcc medium spiny output neurons, thereby controlling inhibitory neurotransmission in nAcc. To our knowledge, the mesoaccumbens glutamatergic pathway is the first glutamatergic input to nAcc shown to mediate aversion instead of reward, and the first pathway shown to establish excitatory synapses on nAcc parvalbumin GABAergic interneurons.

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Figure 1: VGluT2 neurons of the VTA make asymmetric synapses on nAcc mShell neurons.
Figure 2: Photostimulation of VTA VGluT2 inputs to nAcc mShell results in place aversion and behavioral avoidance in a negative-reinforcement task.
Figure 3: Photoinhibition of nAcc mShell fibers from VTA VGluT2 neurons does not change the conditioning response but attenuates foot-shock-induced learned helplessness.
Figure 4: Photostimulation of nAcc mShell fibers from VTA VGluT2 neurons results in place aversion mediated by glutamate receptors and GABA receptors.
Figure 5: The majority of VTA VGluT2 neurons projecting to nAcc mShell lack Gad65 and Gad67 mRNA.
Figure 6: A single nAcc mShell PV interneuron forms asymmetric synapses with several axon terminals from VTA VGluT2 neurons.
Figure 7: Photostimulation of VTA fibers evokes EPSCs on nAcc mShell PV fast-spiking interneurons.

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We thank O. Kiehn and L. Borgius (Karolinska Institutet, Solna, Sweden) for providing the VGluT2::Cre transgenic mice. We thank R. Wise, M.F. Barbano and D. Root for comments. We thank B. Liu and R. Ye for their help in processing brain tissue. This work was supported by the Intramural Research Program of the National Institute on Drug Abuse, US National Institutes of Health (IRP/NIDA/NIH).

Author information

M.M. conceptualized the project. J.Q. performed behavioral and pharmacological studies. J.Q. and D.J.B. analyzed data from behavioral experiments. J.Q., S.Z. and H.-L.W. performed neuroanatomy and immunolabeling studies. H.-L.W. performed in situ hybridization. J.Q. and S.Z. performed confocal microscopy studies. S.Z. performed electron microscopy studies. J.M.-B. performed electrophysiological studies. All authors participated in conception, experimental design and data interpretation. M.M. wrote the manuscript with the participation of all other authors.

Correspondence to Marisela Morales.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Histological verification of viral infection in the VTA of VGluT2::Cre mice.

(a-b)Virus (AAV-DIO-ChR2-eYFP) infected VGluT2 neurons are restricted to the VTA. Double labeled coronal sections for detection of eYFP (green) and TH (red) from rostral (−2.8 mm) to caudal (−3.9 mm) levels of the midbrain. SNC: substantia nigra compacta.

Supplementary Figure 2 Photostimulation of nAcc mShell fibers from VTA VGluT2 neurons produces place aversion in mice.

(a) Histological verification of optical fiber placements within the nAcc-mShell. Representative image showing VTA-VGluT2-inputs (eYFP-green) in the nAcc-mShell and diagrams corresponding to optical fiber placements in the nAcc-mShell of VGluT2::Cre mice. Sections were counterstained with the nuclear dye DAPI (blue). Optical fibers placements are indicated by blue lines (VGluT2-ChR2-eYFP group) or pink lines (VGluT2-eYFP group). aca, anterior commissure anterior part; CPu, caudate putamen; Core, nucleus accumbens core; mShell, nucleus accumbens medial shell. (b) VGluT2-ChR2-eYFP mice (n = 6) spent significantly more time in the photostimulation-paired chamber than the unpaired chamber at a stimulation frequency of 10 Hz or 20 Hz, but not at 5 Hz or 40 Hz (F6,40 = 0.64, P = 0.70; two-way ANOVA, * P < 0.05). (c) Tracking data show representative traces from a VGluT2-ChR2-eYFP mouse (left panel) and a VGluT2-eYFP mouse (right panel) on test day. On test day when photostimulation was no longer available, VGluT2-ChR2-eYFP mice avoided the chamber previously-paired with photostimulation. (d) Photostimulation does not affect travel distance by VGluT2-ChR2-eYFP mice in the conditioning chambers on conditioning day (main effect group: F1,18 = 0.202, P = 0.659; two-way ANOVA). (e) VGluT2-ChR2-eYFP mice avoid the photostimulation-paired chamber on both the photostimulation conditioning days and on the first test day (as shown in Fig. 2, * P < 0.05, Newman-Keuls post hoc test), but on a second test day (24 h after the first test day) in which mice spent similar time in the photostimulation-paired and photostimulation-unpaired chambers (n.s. = no significant difference, P = 0.997). Data are represented as mean + SEM.

Supplementary Figure 3 Histological verification of cannula placement for pharmacological studies of real-time place aversion induced by photostimulation of nAcc mShell fibers from VTA VGluT2 neurons.

(a) The microinjection needle protruded 1 mm to the microinjection cannula tips in nAcc. The cannula tips are indicated by green triangles (ACSF group, n = 11), brown squares (MK801 + CNQX group, n = 6), pink circles (bicuculline + saclofen group, n = 7), blue circles (SCH23390 group, n = 7), orange squares (eticlopride group, n = 7) or red triangles (SCH23390 + eticlopride group, n = 6). aca, anterior commissure anterior part; CPu, caudate putamen; Core, accumbens nucleus core; mShell, accumbens nucleus medial shell. (b) Travel distance during the conditioning day. The average travel distances for each group of mice are represented as mean + SEM. MK801 + CNQX significantly increased the locomotor activity of VGluT2-ChR2-eYFP mice on training day (F5,38 = 5.49, P = 0.0007, one-way ANOVA; * P = 0.021).

Supplementary Figure 4 FluoroGold (FG) injection sites in nAcc mShell and frequency of seven different phenotypes of VTA neurons projecting to nAcc mShell.

(a) Schematic representation of FG injection sites within the mouse nAcc-mShell (3 mice). Diagrams corresponding to the injection sites arranged rostrocaudally from +1.70 mm to +0.86 mm. (b) Frequency of seven different phenotypes of VTA neurons innervating the nAcc-mShell (mean + SEM). Among the 675 identified VTA neurons projecting to the nAcc-mShell, 69.3% expressed only TH, 18.7% coexpressed VGluT2 mRNA and TH, 7.7% expressed only VGluT2 mRNA, 2.3% coexpressed VGluT2 mRNA and GADs mRNA, 0.8% coexpressed TH and GADs mRNA, 0.5% expressed only GADs mRNA, and 0.7% coexpressed VGluT2 mRNA, TH and GADs mRNA. FG neurons were counted between bregma −3.08 mm and −3.88 mm (n = 3 mice, 12–13 sections per mouse).

Supplementary Figure 5 Injection of 6-OHDA resulted in the loss of TH in the nAcc mShell without eliminating aversion induced by photoactivation of mesoaccumbens glutamatergic fibers, and without eliminating synapses between PV dendrites and terminals from VTA VGluT2 neurons.

(a) TH terminals lesion was induced by unilateral injection of 6-OHDA into the nAcc-mShell. aca, anterior commissure anterior part; Core, nucleus accumbens core; CPu, caudate putamen; LV, lateral ventricle; mShell, nucleus accumbens medial shell. (b) High magnification image shows the loss of TH in the nAcc-mShell after 6-OHDA injection, and the contralateral mShell maintained the normal level of TH after saline injection. (c) nAcc-mShell photostimulation of mesoaccumbens-fibers promoted real-time place aversion, which remained after 6-OHDA lesions (n = 9, 6-OHDA group; n = 13, vehicle group; main effect group: F1,20 = 0.03, P = 0.863; main effect chamber: F2,40 = 30.28, P < 0.001; three-way ANOVA with Newman-Keuls post hoc test). (d-e) 6-OHDA did not affect the morphology of VGluT2 axon terminals (AT) derived from the VTA. Electron micrographs showing the detection of PV (gold particles; blue arrowheads) in dendrites that establish asymmetric synapses (green arrows) with terminals coexpressing mCherry (scattered dark material) and VGluT2 (gold particles; green arrowheads) derived from VTA VGluT2-neurons. (d) A PV-dendrite establishing asymmetric synapses with two VGluT2-mesoaccumbens terminals (AT1 and AT2) (e) A PV-dendrite establishing asymmetric synapses with a VGluT2-mesoaccumbens terminal and containing a long mitochondrion (4.2 µm in length).

Supplementary Figure 6 Photostimulation of nAcc mShell fibers from VTA VGAT neurons does not produce rewarding or aversive effects in place-conditioning tests.

(a) Diagram of virus injection in VTA of VGAT-ires-Cre mice, and nAcc-mShell photostimulation of mesoaccumbens VGAT-fibers. (b) Detection of eYFP (green) under the control of the vgat-promoter within the VTA containing TH (red). (c-e) VTA VGAT-inputs in the nAcc are infrequent. (f) Real-time place conditioning procedure timeline. The nAcc-mShell photostimulation of mesoaccumbens VGAT-fibers was available during conditioning day 1 and day 2. (g) Photostimulation did not have rewarding or aversive effects in real-time place conditioning test. VGAT-ChR2-eYFP mice (n = 6) and VGAT-eYFP mice (n = 6) spent similar time in both chambers during both conditioning and test days (main effect group: F1,10 = 0.95, P = 0.353; group × day × chamber interaction: F6,60 = 0.12, P = 0.993; three-way ANOVA). Relative time spent in each chamber is represented as mean + SEM. Blue rectangles indicate photostimulation available in the photostimulation-paired chamber. (h) Photostimulation has no effects on distance traveled by VGAT-ChR2-eYFP mice in the conditioning chambers on conditioning day (main effect group: F1,10 = 0.831, P = 0.385; two-way ANOVA). The average travel distances for each group of mice are represented as mean + SEM.

Supplementary Figure 7 Photostimulation of nAcc mShell fibers from VTA VGluT2 neurons induces c-Fos expression in PV interneurons, but not in NOS or ChAT interneurons.

(a-d) c-Fos expression was detected in nAcc mShell of photostimulated mice. At high magnification, the PV-immunoreactive neurons coexpressed c-Fos in VGluT2-ChR2-eYFP mice (arrows in b), but not in VGluT2-eYFP mice (arrowheads in d). aca, anterior commissure anterior part; LV, lateral ventricle; Core, nucleus accumbens core; mShell, nucleus accumbens medial shell. (e-h) Photostimulation of VGluT2 inputs in nAcc mShell failed to induce c-Fos expression in NOS neurons (double arrowheads in f), or ChAT neurons (double arrows in h).

Supplementary Figure 8 PV interneurons after 20-Hz nAcc mShell photostimulation of VGluT2 mesoaccumbens fibers.

(a) Response of a PV-fast-spiking-interneuron to current injections (from −50 pA to +350 pA) (left). PV-fast-spiking interneuron fired at a rate of 25 Hz after injection of 250 pA (bottom right), 55 Hz after injection of 300 pA (middle right), and 80 Hz after injection of 350 pA (upper right). (b) EPSCs recorded in a PV-fast-spiking-interneuron after photostimulation of VGluT2-mesoaccumbens fibers at 1 Hz (top left), 10 Hz (top right), 20 Hz (bottom left) or 30 Hz (bottom right). Individual trials are shown in gray and averaged EPSCs are shown in black.

Supplementary Figure 9 nAcc mShell photostimulation of VGluT2 mesoaccumbens fibers evokes local polysynaptic GABA release.

(a) Response of a medium spiny neuron (MSN) to current steps injection (from −60 pA to + 100 pA). (b) EPSCs recorded at −70 mV in a MSN in response to nAcc-mShell photostimulation of VGluT2-mesoaccumbens fibers, before (red) or after bath application of bicuculline (10 µM; blue) or bicuculline (10 µM) + CNQX (10 µM; gray) (control = −35.78 ± 9.06 pA; bicuculline = −36.71 ± 11.04 pA; bicuculline + CNQX = −1.98 ± 0.36 pA; F2,41 = 10.83, *P = 0.0004 repeated measures ANOVA, post hoc Dunnett’s multiple comparison test; n = 14 cells from 8 mice), error bars correspond to SEM. (c) EPSCs and IPSCs recorded at −45 mV in a MSN in response to nAcc-mShell photostimulation of VGluT2-mesoaccumbens fibers, before (red) or after bath application of bicuculline (10 µM; blue) or bicuculline (10 µM) + CNQX (10 µM; gray) (EPSCs control = −35.71 ± 12.73 pA; EPSCs bicuculline = −41.69 ± 15.61 pA; EPSCs bicuculline + CNQX = −3.34 ± 1.09 pA; F2,20 = 3.793, P = 0.0152 repeated measures ANOVA, post hoc Dunnett’s multiple comparison test; IPSCs control = 28.01 ± 10.57 pA; IPSCs bicuculline = 0.51 ± 1.20 pA; IPSCs Bicuculline + CNQX = 0.38 ± 1.05 pA; F2,20 = 0.87, *P = 0.01 repeated measures ANOVA, post hoc Dunnett’s multiple comparison test, n = 7 cells from 5 mice) error bars correspond to SEM. (d) Representative traces from an MSN recording demonstrating an EPSC-IPSC response following nAcc-mShell photostimulation of VGluT2-mesoaccumbens fibers. Note the IPSC was reversibly eliminated by bicuculline (blue) whereas CNQX eliminated both the EPSC and IPSC (gray). (e) Schematic representation of direct excitatory monosynaptic VGluT2-mesoaccumbens inputs onto MSNs in the nAcc mShell (left) or polysynaptic inhibition of MSNs via mesoaccumbens-driven glutamate release onto PV-interneurons (right).

Supplementary Figure 10 Photostimulation of nAcc mShell PV interneurons results in place aversion.

(a) Diagram of virus injection and optical probe in nAcc-mShell of PV::Cre mice. (b) nAcc expression of eYFP (green) under the control of the pv-promoter in PV::Cre mice. (c and d) Detection of eYFP (green) in neurons coexpressing PV (red). (e) Real-time place conditioning procedure timeline. The PV-ChR2-eYFP and PV-eYFP mice received nAcc mShell photostimulation during conditioning days (D1 and D2). (f) During conditioning days, PV-ChR2-eYFP mice (n = 6) but not PV-eYFP (n = 6) spent less time in the chamber where nAcc mShell photostimulation was given. Blue rectangles indicate photostimulation available in the photostimulation-associated chamber. On the test day, PV-ChR2-eYFP mice still spent less time in the chamber previously associated with photostimulation. PV-ChR2-eYFP mice showed aversion to the photostimulation-associated chamber on both conditioning days and test day (main effect chamber: F2,20 = 7.88, P = 0.003; group × day × chamber interaction: F6,60= 0.85, P = 0.54; three-way ANOVA, * P < 0.05, Newman-Keuls post hoc test). (g) Photostimulation had no differences in travel distance between PV-ChR2-eYFP and PV-eYFP mice. Data are represented as mean + SEM.

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Qi, J., Zhang, S., Wang, H. et al. VTA glutamatergic inputs to nucleus accumbens drive aversion by acting on GABAergic interneurons. Nat Neurosci 19, 725–733 (2016). https://doi.org/10.1038/nn.4281

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