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. 2018 Aug 29:12:64.
doi: 10.3389/fncir.2018.00064. eCollection 2018.

Targeting VGLUT2 in Mature Dopamine Neurons Decreases Mesoaccumbal Glutamatergic Transmission and Identifies a Role for Glutamate Co-release in Synaptic Plasticity by Increasing Baseline AMPA/NMDA Ratio

Maria Papathanou  1 , Meaghan Creed  2 , Matthijs C Dorst  3 , Zisis Bimpisidis  1 , Sylvie Dumas  4 , Hanna Pettersson  1 , Camilla Bellone  2 , Gilad Silberberg  3 , Christian Lüscher  2   5 , Åsa Wallén-Mackenzie  1
Affiliations

Targeting VGLUT2 in Mature Dopamine Neurons Decreases Mesoaccumbal Glutamatergic Transmission and Identifies a Role for Glutamate Co-release in Synaptic Plasticity by Increasing Baseline AMPA/NMDA Ratio

Maria Papathanou et al. Front Neural Circuits. .

Abstract

Expression of the Vglut2/Slc17a6 gene encoding the Vesicular glutamate transporter 2 (VGLUT2) in midbrain dopamine (DA) neurons enables these neurons to co-release glutamate in the nucleus accumbens (NAc), a feature of putative importance to drug addiction. For example, it has been shown that conditional deletion of Vglut2 gene expression within developing DA neurons in mice causes altered locomotor sensitization to addictive drugs, such as amphetamine and cocaine, in adulthood. Alterations in DA neurotransmission in the mesoaccumbal pathway has been proposed to contribute to these behavioral alterations but the underlying molecular mechanism remains largely elusive. Repeated exposure to cocaine is known to cause lasting adaptations of excitatory synaptic transmission onto medium spiny neurons (MSNs) in the NAc, but the putative contribution of VGLUT2-mediated glutamate co-release from the mesoaccumbal projection has never been investigated. In this study, we implemented a tamoxifen-inducible Cre-LoxP strategy to selectively probe VGLUT2 in mature DA neurons of adult mice. Optogenetics-coupled patch clamp analysis in the NAc demonstrated a significant reduction of glutamatergic neurotransmission, whilst behavioral analysis revealed a normal locomotor sensitization to amphetamine and cocaine. When investigating if the reduced level of glutamate co-release from DA neurons caused a detectable post-synaptic effect on MSNs, patch clamp analysis identified an enhanced baseline AMPA/NMDA ratio in DA receptor subtype 1 (DRD1)-expressing accumbal MSNs which occluded the effect of cocaine on synaptic transmission. We conclude that VGLUT2 in mature DA neurons actively contributes to glutamatergic neurotransmission in the NAc, a finding which for the first time highlights VGLUT2-mediated glutamate co-release in the complex mechanisms of synaptic plasticity in drug addiction.

Keywords: addiction; amphetamine; cocaine; medium spiny neurons; striatum; substance use disorder; ventral tegmental area (VTA).

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Figures

Figure 1
Figure 1
Ample Vglut2 mRNA-positive cells throughout dorsal and ventral midbrain with more sparse expression within the dopaminergic area. Colorimetric in situ hybridization showing overview of Vglut2 (A,B) and Th (C,D) mRNA in midbrain section of wildtype adult mouse at two rostro-caudal levels. (A,B) Vglut2 mRNA is abundant throughout the midbrain with strong signal in e.g., the red nucleus (RN), RSG and dentate gyrus and weaker signal in the ventral tegmental area (VTA) and SNc areas. (C,D) Th mRNA is selectively localized in dopaminergic neurons of the VTA and SNc and its mRNA signal implemented to visualize these areas. Dotted square around the VTA and SNc (scale bar 500 μm) presented as closeups in (A′–D′; scale bar 200 μm). (C′,D′) SNc, SNr and subregions of VTA outlined in Th closeups and superimposed on Vglut2 closeups (A′,B′). (C′,D′) Th mRNA was strongly localized in the SNc and within the parabrachial pigmented area (PBP) and paranigral nucleus (PN) of the VTA with weaker signal in the RLi and caudal IF. (A′,B′) Within the VTA, Vglut2 mRNA was detected in the PBP, PN, RLi and IF as well as within the medially located szPBP while no Vglut2 mRNA was detected in the GABAergic SNr area. Abbreviations: Ctx, Cortex; DG, Dentate gyrus; IF, interfascicular nucleus; IPN, interpeducular nucleus; PBP, parabrachial pigmented area; PN, paranigral nuclei; RLi rostral linear nucleus; RN, Red nucleus; RSG, Retrosplenial granular cortex; SNc, Substantia nigra pars compacta; SNr, Substantia nigra pars reticulata; szPBP, subzone of the parabrachial pigmented area; VTA, Ventral tegmental area.
Figure 2
Figure 2
Vglut2, Th and Dat mRNA co-localization within certain VTA dopamine (DA) neurons is sparse at E14.5, peaks around birth and is subsequently down-regulated in adulthood. Double fluorescent in situ hybridization for Th (red), Dat (green) and Vglut2 (red) mRNA, respectively, on wildtype mouse midbrain sections. (A–C) Sagittal sections of E14.5 embryo. Dotted square around the area of developing midbrain DA neurons (A–C) with close-ups in (A′–C′). (A) Th and Dat mRNA show co-localization (yellow) in the ventral midbrain (scale bar 500 μm). (A′) higher magnification of insets (scale bar 100 μm); (B,B′) Th and Vglut2 mRNA expression in the midbrain. (C,C′) Dat and Vglut2 mRNA show sparse detection in the midbrain. (D–F) Coronal sections of ventral midbrain in pups of postnatal day (P) 3. (D) Th and Dat show ample co-localization (yellow) in the lateral VTA and SNc (scale bar 250 μm, inset 100 μm). (E) Th and Vglut2 mRNA and (F) Dat and Vglut2 mRNA prominently co-localize (yellow) at this age in the IF, PBP and PN areas (arrows) but not in the RLi of the VTA. (G–I) Coronal sections of the adult midbrain (10 weeks; scale bar 250 μm, inset 100 μm). (G) Th and Dat mRNA co-localization (yellow) remains strong; whilst the level of co-localization between (H) Th and Vglut2 and (I) Dat and Vglut2 mRNAs is lower than at P3 (arrows). Yellow arrows show co-localization green (Dat) and red (Vglut2) channel, red arrows show red (Vglut2) channel (Postnatal Day (P) 3 n = 3; adult n = 3). Abbreviations: cf, cephalic flexture; Ctx, cortex; IF, interfascicular nucleus; IPN, interpeducular nucleus; LV, lateral ventricle; M, medulla; Mb, midbrain; PBP, parabrachial pigmented area; PN, paranigral nuclei; RLi rostral linear nucleus; SNc, Substantia nigra pars compacta; VTA, Ventral tegmental area. See Supplementary Figure S1 for low-magnification images of entire sections at P3 and adult as well as quantification Vglut2/Dat mRNA co-localization.
Figure 3
Figure 3
Validation of tamoxifen-inducible DAT-Cre-mediated targeting via tdTom reporter. (A) tdTom-immunohistochemical analysis of midbrain and striatal sections from tdTomeDAT-Cre-tg and tdTomtxDAT-Cre-tg mice show similar extent of labeling in both mouse lines, verifying a similar recombination efficiency of LoxP sites (scale bar 2 mm). (B) tdTom-positive labeling (red) within the VTA and SNc co-localizes with tyrosine hydroxylase (TH; green) immuno-labeling verifying selectivity to DA neurons (scale bar 250 μm and 50 μm). (C) Low and high magnification of fluorescent in situ hybridization for Vglut2 (green) and tdTom (red) mRNA in tdTomeDAT-Cre-tg and tdTomtxDAT-Cre-tg mice showing co-localization in the PBP, albeit at low level (scale bar 200 μm; inset 25 μm). Abbreviations: DStr, Dorsal striatum; IF, interfascicular nucleus; IPN, interpeducular nucleus; NAcC, Nucleus accumbens core; NAcSh, Nucleus accumbens shell; PBP, parabrachial pigmented area; PN, paranigral nuclei; RLi, rostral linear nucleus; SN, Substantia nigra; SNc, Substantia nigra pars compacta; SNr, Substantia nigra pars reticulata; VTA, Ventral tegmental area; tdTom, tdTomato.
Figure 4
Figure 4
Confirmation of tamoxifen-induced targeting of the Vglut2 gene using tamoxifen-inducible DAT-Cre transgene and validation of intact midbrain DA system. (A) Schematic illustration of breeding strategy to generate Vglut2lx/lx;txDAT-Cre-wt (txCtrl) and Vglut2lx/lx;txDAT-Cre-wt (txKO). (B) Confirmation of Vglut2 gene targeting using the tamoxifen-inducible DAT-CReERT2 (txDAT-Cre) mouse line. Nested RT-PCR for β-actin, TH and Vglut2 from dissected VTA. Vglut2 wildtype band (500 bp) was observed in both txCtrl and txKO midbrain, while knockout (KO) band (KO; 250 bp) was only found in the gene targeted midbrain. β-actin and TH served as controls. (C) Number of TH positive neurons in the SN and VTA did not differ between txCtrl and txKO.Two-way ANOVA with Sidak post hoc for SN and VTA along three rostro-caudal section (txCtrl n = 3; txKO n = 3). (D) TH immunoreactivity in striatum and midbrain in txCtrl and txKO mice (scale bar 500 μm (striatum) and 50 μm (midbrain). (E,F) Cre-driven tdTom expression in ventral midbrain of eCtrl (E, top), eKO (E, bottom), txCtrl (F, top) and txKO (F, bottom) (scale bar 250 μm). Abbreviations: DStr, Dorsal striatum; NAcC, Nucleus accumbens core; NAcSh, Nucleus accumbens shell; SNc, Substantia nigra pars compacta; SNr, Substantia nigra pars reticulata; VTA, Ventral tegmental area; tdTom, tdTomato.
Figure 5
Figure 5
Optogenetics-driven stimulation of dopaminergic fibers in the NAcSh evokes VGLUT2-dependent responses. (A) Eight-week-oldtxCtrl and txKO mice were tamoxifen-treated and 1 week later stereotaxically injected with of AAV5-EF1a-DIO-hChR2(H123R)-eYFP into the VTA. Glutamate release was recorded in the NAcSh upon optical stimulation. (B) ChR2 expression was restricted to the VTA and colocalized with TH immunoreactivity. Inset: eYFP and TH immunofluorescence showing ample co-localization (scale bar 50 μm). No eYFP expression was detected in the SNc or SNr. ChR2 expression was detected in the projecting fibers to theNAc of the ventral striatum (scale bar 200 μm). (C) Representative example of accumbal cell imaged under IR-DIC and patched with 4–6 MΩ patch pipettes (scale bar 20 μm). (D) Patched neurons were filled with neurobiotin and stained with Cy5-conjugated streptavidin. Blue: DAPI, Green: eYFP, Red: Cy5 (scale bar 200 μm). (E) Representative average traces of light-induced responses in NAc neurons in txCtrl and txKO, before and after bath application of NBQX. (F) Light-evoked response amplitude for txKO and txCtrl mice. txKO exhibited smaller responses compared to txCtrl in control, but not under NBQX conditions. Unpaired Mann-Whitney test, **p < 0.01, ***p < 0.001 (txCtrl n = 25, 3 mice; txKO n = 24, 3 mice). Abbreviation: aca; anterior commissure, anterior part, DStr, Dorsal striatum; NAc, Nucleus accumbens; NAcC, Nucleus accumbens core; NAcSh, Nucleus accumbens shell; SNc, Substantia nigra pars compacta; SNr, Substantia nigra pars reticulata; VTA, Ventral tegmental area.
Figure 6
Figure 6
Amphetamine-induced locomotor sensitization is impeded upon VGLUT2 targeting from developing, but not mature, DA neurons. (A,A′) Schematic illustration of amphetamine-induced sensitization paradigm in (A) eCtrl and eKO or (A′) txCtrl and txKO mice. Animals received a single dose of saline (Day 1) followed by four consecutive injections of amphetamine (3 mg/kg; Days 2–5). Acute dose of amphetamine (Challenge) was administered after a washout period of 14 days. A week later (Day 26), the protocol was repeated and included two acute challenges (Challenge 1 and Challenge 2) on days 37 and day 44, respectively. (B) eKO show blunted response to amphetamine compared to eCtrl during both rounds of sensitization. There was no difference in locomotion between last days of repeated injection (Amp 4) and acute challenges. (B′) Both txCtrl and txKO showed an increase in amphetamine-induced locomotion compared to saline and no difference was observed between genotypes. Two-way repeated measures ANOVA with Sidak post hoc test (ANOVA ###p < 0.001; post hoc between genotype *p < 0.05, **p < 0.01, ***p < 0.001; eCtrl n = 3; eKO n = 3; txCtrl n = 7; txKO n = 11).
Figure 7
Figure 7
VGLUT2 targeting in mature, but not developing, DA results in maintained cocaine-induced sensitization and elevated baseline AMPA/NMDA ratio. (A,A′) Schematic timeline of cocaine-induced behavioral locomotor sensitization and subsequent electrophysiological recordings for (A) eKO-DRD1 and eCtrl-DRD1 and (A′) tamoxifen-induced txKO-DRD1 and txCtrl-DRD1 mice. All mice were habituated to the arena for 3 days during which they received saline injections (i.p.) Starting from “Day 1,” mice received either cocaine (20 mg/kg i.p.) or saline injections (i.p.) before behavioral testing for 5 days, then kept for additionally 10 days in their home cage. On Day 15, mice were sacrificed and whole-cell patch clamp experiments performed on brain slices. In (A) is also shown a fluorescent image of DRD1-expressing cells (EGFP; green) used in whole-cell patch clamp experiments filled with biocytin and stained with streptavidin (red). (B,B′) Cocaine-induced locomotor sensitization measured as distance travelled following saline or cocaine injections, respectively. (B) eKO-DRD1 and eCtr-DRD1. (B′) txKO-DRD1 and txCtrl-DRD1. (C) Rectification index (RI) and raw traces recorded cells from eKO-DRD1 and eCtrl-DRD1 mice treated with saline or cocaine at −70 mV (black), 0 mV (light gray) and +40 mV (dark gray). (C′) RI and raw traces recorded cells from txKO-DRD1 and txCtrl-DRD1 mice treated with saline or cocaine at −70 mV (blue), 0 mV (light gray) and +40 mV (dark gray). (D) AMPA/NMDA ratio and raw traces of cells from eKO-DRD1 and eCtrl-DRD1 mice treated with saline or cocaine for NMDA current (black); AMPA current (light gray). (D′) AMPA/NMDA ratio and raw traces of cells from txKO-DRD1 and txCtrl-DRD1 mice treated with saline or cocaine for NMDA current (blue); AMPA current (light gray). Two-way repeated measures ANOVA Tukey’s post hoc (behavior) and two-way ANOVA Sidak post hoc (electrophysiology) (ANOVA ###p < 0.001; post hoc between genotype *p < 0.05 and post hoc between treatment of same genotype: *p < 0.05, **p < 0.01, ***p < 0.001; Behavior: saline: eCtrl, n = 3; eKO, n = 2, cocaine: eCtrl, n = 5; eKO, n = 5, saline: txCtrl, n = 4; txKO, n = 4, cocaine: txCtrl, n = 9; txKO, n = 8; electrophysiology: RI saline: eCtrl, n = 4, eKO, n = 5; cocaine: eCtrl, n = 12,: eKO, n = 28; saline txCtrl, n = 6, txKO, n = 6; cocaine txCtrl, n = 12: txKO, n = 7; AMPA/NMDA ratio saline: eCtrl, n = 7, eKO, n = 5; cocaine: eCtrl, n = 15; eKO, n = 33; saline: txCtrl, n = 9, txKO, n = 9; cocaine: txCtrl, n = 13, tKO, n = 7). All mice expressed the DRD1-EGFP transgene.
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