Presynaptic Gq-coupled receptors drive biphasic dopamine transporter trafficking that modulates dopamine clearance and motor function

Abstract

Extracellular dopamine (DA) levels are constrained by the presynaptic DA transporter (DAT), a major psychostimulant target. Despite its necessity for DA neurotransmission, DAT regulation in situ is poorly understood, and it is unknown whether regulated DAT trafficking impacts dopaminergic signaling and/or behaviors. Leveraging chemogenetics and conditional gene silencing, we found that activating presynaptic Gq-coupled receptors, either hM3Dq or mGlu5, drove rapid biphasic DAT membrane trafficking in ex vivo striatal slices, with region-specific differences between ventral and dorsal striata. DAT insertion required D2 DA autoreceptors and intact retromer, whereas DAT retrieval required PKC activation and Rit2. Ex vivo voltammetric studies revealed that DAT trafficking impacts DA clearance. Furthermore, dopaminergic mGlu5 silencing elevated DAT surface expression and abolished motor learning, which was rescued by inhibiting DAT with a subthreshold CE-158 dose. We discovered that presynaptic DAT trafficking is complex, multimodal, and region specific, and for the first time, we identified cell autonomous mechanisms that govern presynaptic DAT tone. Importantly, the findings are consistent with a role for regulated DAT trafficking in DA clearance and motor function.

Keywords: dopamine; membrane trafficking; metabotropic glutamate receptor; motor function; striatum.

Figure 1

Gq-coupled DREADD activation drives region-specific and biphasic DAT trafficking. Ex vivo striatal slice surface biotinylation. Acute striatal slices prepared from Pitx3^IRES-tTA;+ or Pitx3^IRES-tTA;TRE-hM3Dq mice were treated ±500 nM CNO for 5, 10, or 30 min, and DAT surface levels were measured by surface biotinylation as described in Experimental procedures. A, total striatum. Total striatal slices (left) were assessed for hM3Dq-mediated DAT trafficking. Right, top, representative immunoblots showing surface (S) and total (T) DAT bands. Right, bottom, mean DAT surface levels are presented as %vehicle-treated contralateral hemisection ±SEM. Two-way ANOVA: interaction: F_(2,15) = 3.82, ∗p = 0.045; genotype: F_(1,15) = 8.53, ∗p = 0.011, time: F_(2,15) = 2.24, p = 0.24. ∗p < 0.05, Tukey’s multiple comparisons test, n = 3 (Pitx3^IRES-tTA) and 4 (Pitx3^IRES-tTA;TRE-hM3Dq). B, subdissected striatum. Left, dorsal and ventral striata were subdissected prior to solubilizing, as described in Experimental procedures. Right, top, representative immunoblots showing surface (S) and total (T) DAT bands. Right bottom, mean DAT surface levels, presented as %vehicle-treated contralateral hemisection ±SEM. Two-way ANOVA: interaction: (F_(2,37) = 0.12, p = 0.89; time: F_(2,37) = 34.65, ∗∗∗∗p < 0.0001, region: F_(1,37) = 30.05, ∗∗∗∗p < 0.0001). ∗∗p = 0.003; ∗p = 0.011. Tukey’s multiple comparisons test. Ventral: n = 6 (5 min), 7 (10 min), and 8 (30 min); dorsal: n = 6 (5 min), 9 (10 min), and 8 (30 min). CNO, clozapine-N-oxide; DAT, dopamine transporter.

Figure 2

DA release and DRD2 activation are required for Gq-stimulated DAT insertion, whereas PKC activity is required for DAT retrieval. Ex vivo striatal slice surface biotinylation. Acute striatal slices prepared from Pitx3^IRES-tTA;TRE-hM3Dq mice were treated with the indicated drugs for the indicated times. DAT surface levels were measured by slice biotinylation, and VS and DS were isolated prior to tissue lysis as described in Experimental procedures. Mean DAT surface levels in ventral striata (VS; left) and dorsal striata (DS; right) are presented as %vehicle-treated ±SEM, determined as described in Experimental procedures. Representative blots containing both surface (S) and total (T) DAT are shown above each graph, for all treatments and were taken from a single immunoblot exposure, cropped for presentation purposes, with molecular weight markers indicating kilodaltons (kDa). A, reserpine treatment. Mice were injected (I.P.) with either saline (Sal) or 5.0 mg/kg reserpine (Res) 16 h prior to preparing slices, and slices were treated ±1.0 μM reserpine throughout the experiment. Slices were treated ±500 nM CNO, 5 min, 37 °C. Ventral: ∗∗p = 0.007, one-tailed, unpaired Student’s t test, n = 8 (saline) and 6 (reserpine). Dorsal: ∗∗∗∗p < 0.0001, one-tailed, unpaired Student’s t test, n = 7 (saline) and 5 (reserpine). B, DRD2 antagonist pretreatment. Striatal slices were pretreated ±DRD2 antagonist (L-741,626, 25 nM, 15 min, 37 °C) and then treated ±500 nM CNO (5 min, 37 °C). DRD2 blockade abolished hM3Dq-stimulated DAT membrane insertion but had no effect alone in either VS (left) or DS (right). Ventral: Kruskal–Wallis test, 8.82. ∗p < 0.05, Dunn’s multiple comparisons test, n = 5. Dorsal: one-way ANOVA, F_(2,6) = 20.17, ∗∗p = 0.002. ∗∗p < 0.01, Bonferroni’s multiple comparisons test, n = 3. C, PKC inhibition. Slices were pretreated ±500 nM CNO (5 min, 37 °C), followed by treatment ±1.0 μM BIM I (25 min, 37 °C), and DAT surface levels were measured by slice biotinylation at either 5 or 30 min post-CNO treatment. BIM I significantly blocked DAT retrieval following CNO-stimulated membrane insertion in both VS and DS. Ventral: one-way ANOVA: F_(3,19) = 9.33, ∗∗∗p = 0.0005. ∗∗∗p < 0.002, Bonferroni’s multiple comparisons test, n = 5 to 7. Dorsal: one-way ANOVA: F_(3,14) = 9.41, ∗∗p = 0.001. ∗p = 0.04, ∗∗p = 0.004, Bonferroni’s multiple comparisons test, n = 6 to 7. CNO, clozapine-N-oxide; DA, dopamine; DAT, dopamine transporter; DRD2, D2 DA receptor.

Figure 3

Striatal mGlu5 activation drives biphasic DAT trafficking. Ex vivo striatal slice surface biotinylation. Acute striatal slices prepared from male C57Bl/6J mice were treated with the indicated drugs for the indicated times. DAT surface levels were measured by slice biotinylation, dorsal and ventral striata were subdissected, and surface DAT was quantified as described in Experimental procedures. Representative blots containing both surface (S) and total (T) DAT are shown above each graph, for all treatments. Average surface DAT values are expressed as % vehicle-treated contralateral hemisection ±SEM. A and B, DHPG treatment. Slices were treated ±10 μM DHPG for 5, 10, or 30 min. A, ventral: two-way ANOVA: interaction: F_(2,22) = 4.95, ∗p = 0.02; time: F_(2,22) = 4.95, ∗p = 0.02, drug: F_(1,22) = 2.24, p = 0.29. ∗p < 0.05, ∗∗p = 0.006, Tukey’s multiple comparisons test, n = 4 to 5. B, dorsal: two-way ANOVA: interaction: F_(2,20) = 3.65, ∗p = 0.04; time: F_(2,20) = 3.65, ∗p = 0.04, drug: F_(1,20) = 8.80, ∗∗p = 0.008. ∗p < 0.05, Tukey’s multiple comparisons test, n = 4 to 5. C and D, mGlu5 antagonist pretreatment. Slices were pretreated ±MTEP (50 nM, 15 min, 37 °C), a selective mGlu5 antagonist, prior to stimulating DAT insertion with DHPG (10 μM, 5 min, 37 °C). MTEP pretreatment abolished DHPG-stimulated DAT membrane insertion in both ventral (C) and dorsal (D) striata. Ventral: one-way ANOVA: F_(2,9) = 13.42, ∗∗p = 0.002; ∗∗p < 0.01, Bonferroni’s multiple comparisons test, n = 4. Dorsal: one-way ANOVA: F_(2,8) = 6.92, ∗p = 0.02. ∗p < 0.05, Bonferroni’s multiple comparisons test, n = 3 to 4. DAT, dopamine transporter; DHPG, (RS)-3,5-dihydroxyphenylglycine; MTEP, 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]-pyridine.

Figure 4

mGlu5-mediated DAT trafficking is mediated presynaptically and impacts basal DAT surface expression. A and B, conditional mGlu5 silencing in DA neurons. Pitx3^IRES-tTA;mGlu5^fl/fl mouse VTA were bilaterally injected with either AAV9-TRE-eGFP (n = 7) or AAV9-TRE-Cre (n = 10). Midbrain tissue punches were obtained 4 to 5 weeks postinjection and assessed for either mGlu5 mRNA (A) or protein (B). A, Left, viral injection schematic. Right, RT–qPCR ∗∗∗p = 0.0005, one-tailed, unpaired Student’s t test, n = 8 (Cre) and 12 (eGFP). B, midbrain mGlu5 protein levels. Left, representative immunoblot showing mGlu5 and actin signals from four independent mouse midbrains. Right, average data. mGlu5 protein levels were normalized to actin loading controls. ∗∗p < 0.01, Student’s t test, n = 4 to 5. C–E, Ex vivo striatal slice surface biotinylation. Pitx3^IRES-tTA;mGlu5^fl/fl mouse VTA were bilaterally injected with either AAV9-TRE-eGFP (n = 4) or AAV9-TRE-Cre (n = 5). Acute striatal slices were prepared from the indicated mice, treated ±10 μM DHPG (5 min, 37 °C), and DAT surface levels in ventral and dorsal striata were measured by slice biotinylation as described in Experimental procedures. C, representative immunoblots: surface (S) and total (T) DAT bands are presented for each of the indicated treatment conditions, in both ventral and dorsal striatum. D, DHPG-stimulated DAT membrane insertion: mean DAT surface levels are presented as %vehicle-treated contralateral hemisection. DAergic mGlu5 silencing significantly abolished DHPG-stimulated DAT insertion in both ventral (∗∗∗p = 0.0002) and dorsal (∗∗p = 0.003) striata, one-tailed, unpaired Student’s t test. E, basal DAT surface expression: mean surface DAT values are presented as %total DAT on the surface. DAergic mGlu5 silencing significantly increased basal DAT surface expression in ventral (∗∗∗p = 0.001, n = 5) dorsal (∗p = 0.035, n = 4–6) striata, one-tailed, unpaired Student’s t test. AAV, adeno-associated virus; DAT, dopamine transporter; DHPG, (RS)-3,5-dihydroxyphenylglycine; eGFP, enhanced GFP; qPCR, quantitative PCR; VTA, ventral tegmental area.

Figure 5

mGlu5-mediated DAT insertion requires DRD2_autoand intact retromer.A, virally induced DRD2_autosilencing. Left, Pitx3^IRES-tTA;DRD2^fl/fl mouse VTA were bilaterally injected with either AAV9-TRE-eGFP or AAV9-TRE-Cre. Right, midbrain DRD2 mRNA levels were measured by RT–qPCR from tissue punches obtained 4 to 5 weeks postinjection. ∗∗p = 0.002, one-tailed, unpaired Student’s t test, n = 3 to 7. B, ex vivo striatal slice surface biotinylation. Acute striatal slices prepared from the indicated mice were treated ±10 μM DHPG (5 min, 37 °C), and DAT surface levels were measured by slice biotinylation as described in Experimental procedures. Representative immunoblots containing both surface (S) and total (T) DAT are shown above each graph, for all treatments. Average surface DAT values are expressed as % vehicle-treated contralateral hemisection ±SEM. DRD2_auto silencing abolished mGlu5-stimulated DAT membrane delivery in both ventral (∗p = 0.01) and dorsal (∗∗p = 0.004) striata, one-tailed, unpaired Student’s t test, n = 4 (ventral) and 4 to 5 (dorsal). C–E, conditional Vps35 silencing in DA neurons. (C, left) Pitx3^IRES-tTA mouse VTA were bilaterally injected with either AAV9-TRE-eGFP or AAV9-TRE-shVps35. C, right, midbrain Vps35 mRNA levels were measured by RT–qPCR from tissue punches obtained 4 to 5 weeks postinjection and were normalized to TH mRNA levels to account for varied DA neuron enrichment amongst tissue punches. ∗∗p = 0.005, one-tailed, unpaired Student’s t test, n = 4 to 5. D and E, ex vivo striatal slice surface biotinylation. Acute striatal slices were prepared from the indicated mice, treated with the indicated drugs (5 min, 37 °C), and DAT surface levels were measured by slice biotinylation as described inExperimental procedures. Surface DAT is expressed as % contralateral vehicle-treated hemisection ±SEM. D, DRD2-stimulated DAT membrane delivery: slices were treated ±170 nM sumanirole (SUMAN, 5 min, 37 °C). Vps35 silencing abolished DRD2-stimulated DAT membrane delivery in both ventral (∗p = 0.049) and dorsal (∗p = 0.02) striata, one-tailed, unpaired Student’s t test, n = 4 to 5. E, mGlu5-stimulated DAT membrane delivery. Slices were treated ±10 μM DHPG (5 min, 37 °C). Vps35 silencing abolished mGlu5-stimulated DAT membrane delivery in both ventral (∗p = 0.03) and dorsal (∗∗p = 0.003) striata, one-tailed, unpaired Student’s t test, n = 3 to 5. AAV, adeno-associated virus; DAT, dopamine transporter; DHPG, (RS)-3,5-dihydroxyphenylglycine; DRD2_auto, DRD2 autoreceptor; eGFP, enhanced GFP; qPCR, quantitative PCR; TH, tyrosine hydroxylase; VTA, ventral tegmental area.

Figure 6

Rit2 is required for mGlu5-mediated DAT retrieval, but not insertion.A, conditional Rit2 silencing in DA neurons. Left, Pitx3^IRES-tTA mouse VTA were bilaterally injected with either AAV9-TRE-eGFP or AAV9-TRE-Cre. Right, midbrain Rit2 mRNA levels were measured by RT–qPCR from tissue punches obtained 4 to 5 weeks postinjection. ∗∗p = 0.005, one-tailed, unpaired Student’s t test, n = 3 to 4. B and C, ex vivo striatal slice surface biotinylation. Acute striatal slices were prepared from the indicated mice, treated ±10 μM DHPG for the indicated times (37 °C), and DAT surface levels were measured by slice biotinylation as described in Experimental procedures. Surface DAT is expressed as % contralateral vehicle-treated hemisection ±SEM. B, ventral striatum, two-way ANOVA: interaction: F_(2,14) = 3.35, p = 0.065; time: F_(2,14) = 0.29, p = 0.75, virus: F_(1,14) = 30.00, ∗∗∗∗p < 0.0001. Rit2 silencing significantly abolished DAT retrieval at 10 min (∗∗p = 0.002) and 30 min (∗∗p = 0.004), Sidak’s multiple comparisons test, n = 3 to 4. C, dorsal striatum, two-way ANOVA: interaction: F_(2,14) = 1.30, p = 0.30; time: F_(2,14) = 2.19, p = 0.15, virus: F_(1,14) = 11.44, ∗∗p = 0.004. Rit2 silencing significantly abolished DAT retrieval at 10 min (∗p = 0.02), Sidak’s multiple comparisons test, n = 3 to 4. DA, dopamine; DAT, dopamine transporter; DHPG, (RS)-3,5-dihydroxyphenylglycine; eGFP, enhanced GFP; qPCR, quantitative PCR; VTA, ventral tegmental area.

Figure 7

DRD2-stimulated DAT insertion and mGlu5-mediated DAT retrieval impact DA release and clearance in dorsal striatum. Ex vivo fast-scan cyclic voltammetry: Pitx3^IRES-tTA;mGlu5^fl/f mouse VTA were bilaterally injected with either AAV9-TRE-eGFP (n = 7–9) or AAV9-TRE-Cre (n = 9). Acute striatal slices were prepared from the indicated mice, and electrically evoked DA transients were measured in dorsal striatum by FSCV as described in Experimental procedures. A–D, eGFP: (A) Representative voltammograms: voltammograms displaying evoked current over voltage cycles and time, in slices from eGFP-injected in the presence of ACSF alone (top) or supplemented with 25 nM L-741,626 (bottom). Arrowheads indicated delivery of single and squared wave pulse. B, dopamine transients, evoked DA transients in slices from eGFP-injected mice, treated ±L-741,626 (25 nM). Mean traces are presented with SEM indicated by shaded areas. C, mean amplitudes: mean amplitudes are presented in μM ±SEM. ∗Significantly greater than in ACSF alone, p = 0.02. D, mean decay tau, presented in seconds ±S.E.M. ∗Significantly longer clearance time than in ACSF alone, p = 0.03. E–H, Cre: (E) representative voltammograms: Voltammograms displaying evoked current over voltage cycles and time, in slices from eGFP-injected in the presence of ACSF alone (top) or supplemented with 25 nM L-741,626 (bottom). Arrowheads indicated delivery of single squared wave pulse. F, dopamine transients: evoked DA transients in slices from Cre-injected mice, treated ±L-741,626 (25 nM). Average traces are presented with SEM indicated by shaded areas. G, mean amplitudes: mean amplitudes are presented in micrometer ±SEM. DRD2 inhibition did not significantly alter DA release inhibition, p = 0.71. H, mean decay tau, presented in seconds ±SEM. DRD2 inhibition had no significant effect on DA clearance, p > 0.999. See Table 1 for all descriptive statistical analyses. ACSF, artificial cerebrospinal fluid; DA, dopamine; DAT, dopamine transporter; DRD2, D2 DA receptor; eGFP, enhanced GFP; VTA, ventral tegmental area.

Figure 8

DAergic mGlu5 is required for motor learning in a DAT-dependent manner. Pitx3^IRES-tTA;mGlu5^fl/fl mouse VTA were bilaterally injected with either AAV9-TRE-eGFP (n = 7–9) or AAV9-TRE-Cre (n = 9) and assessed by the indicated behavioral assays. A, accelerating rotarod: Mice were assessed over three consecutive trials as described in Experimental procedures and are presented as latency to fall (seconds) in each trial, ±SD. Two-way repeated-measures ANOVA: trial × virus: (F_(2,30) = 17.04, ∗∗∗∗p < 0.0001; trial: F_(2,30) = 38.22, ∗∗∗∗p < 0.0001, virus: F_(1,15) = 6.65, ∗p = 0.02, subject: F_(15,30) = 5.02, ∗∗∗∗p < 0.0001). mGlu5 silencing in DA neurons significantly dampened performance on trials 2 (∗p = 0.049) and 3 (∗∗∗∗p < 0.0001), Bonferroni’s multiple comparisons test. B, fixed speed rotarod: Mice were assessed over the indicated consecutive speeds as described in Experimental procedures and are presented as latency to fall (seconds) at each speed tested, ±SD. Two-way repeated-measures ANOVA: speed × virus: F_(5,75) = 5.06, ∗∗∗∗p < 0.0005; speed: F_(5,75) = 44.91, ∗∗∗∗p < 0.0001, virus: F_(1,15) = 12.23, ∗∗p = 0.003, subject: F_(15,15) = 4.09, ∗∗∗∗p < 0.0001. mGlu5 silencing in DA neurons significantly dampened performance at 40 (∗∗∗∗p < 0.0001) and 45 rpm (∗∗∗p = 0.0002), Bonferroni’s multiple comparisons test. C–E, challenge balance beam. Mean foot fault numbers (C) and beam traversal time (seconds) (D) are presented. mGlu5 silencing in DA neurons did not significantly affect either foot fault number (p = 0.78) or mean traversal times (p = 0.15), two-tailed, unpaired Student’s t test. E, traversal time improvement. eGFP (control, left) mouse traversal times significantly improved between trials 1 and 2 (∗p = 0.02), whereas mice injected with Cre (right) failed to improve their performance (p = 0.77), two-tailed, paired Student’s t test. F, dose-dependent effect of CE-158 on horizontal locomotion in wildtype mice. Wildtype mice were habituated to photobeam activity chambers for 45 min and were injected (I.P.) with either vehicle (on day 1) or the indicated CE-158 dose (on day 2) and their horizontal locomotion was measured for 90 min. Cumulative locomotion is presented as total postinjection beam breaks. Two-way ANOVA: dose × drug: F_(2,54) = 12.01, ∗∗∗∗p < 0.0001; dose: F_(2,54) = 18.70, ∗∗∗∗p < 0.0001, drug: F_(1,54) = 25.36, ∗∗∗∗p < 0.0001. 20 mg/kg CE-158 significantly increased locomotion as compared with vehicle (∗∗∗∗p < 0.0001), 10 mg/kg (∗∗∗∗p < 0.0001), and 5 mg/kg (∗∗∗∗p < 0.0001), Bonferroni’s multiple comparisons test, n = 9 to 10. G, accelerating rotarod rescue studies with CE-158: mice with DAergic mGlu5 silencing were assessed over three trials, injected I.P. with either saline or CE-158 (10 mg/kg), and reassessed over three trials 15 min postinjection. Left, raw rotarod results presented as latency to fall (seconds) in each trial, ±SD. Right, rotarod performance indices, expressed as %maximal latency to fall. Mice injected with CE-158 performed significantly better postinjection as compared with preinjection (∗∗p = 0.0014), whereas saline-injected mice did not perform significantly better (p = 0.11). Two-tailed paired t test, n = 6. H, effect of CE-158 on wildtype mouse performance on accelerating rotarod. Wildtype mice were injected with either saline or CE-158 (10 mg/kg) and assessed on the accelerating rotarod. Data are presented as latency to fall (seconds) in each trial, ±SD. CE-158 had no significant effect on mouse performance over three trials. Two-way ANOVA: trial × drug: F_(2,22) = 0.085, p = 0.92; trial: F_(2,22) = 18.17, p < 0.0001, drug: F_(1,11) = 3.136, p = 0.11, n = 6 (vehicle) or 7 (CE-158). AAV, adeno-associated virus; DA, dopamine; DAT, dopamine transporter; eGFP, enhanced GFP; VTA, ventral tegmental area.

Figure 9

Striatal DAT trafficking model. A presynaptic DAergic terminal and a glutamatergic afferent are depicted. Left, tonic DA neuron firing. During tonic DA release, DRD2_auto activity robustly delivers DAT to the plasma membrane in a PKCβ- and Vps35-dependent manner. Dense DAT surface expression restricts extracellular DA levels. Right, glutamate release during motor learning. When DA demand is higher, such as during motor learning, glutamate release from striatal glutamatergic afferents activates presynaptic mGlu5 (mGR5), which elevates intracellular calcium (Ca²⁺) and enhances DA release and DAT membrane insertion. However, mGlu5-mediated PKC activation concurrently drives DAT membrane retrieval in a Rit2-dependent manner. DAT retrieval decreases DAT surface levels, relative to tonic firing, to sustain extracellular DA levels during a period of increased demand. DA, dopamine; DAT, dopamine transporter; DRD2_auto, DRD2 autoreceptor.