The Dopamine Transporter Is a New Target for Ischemic Stroke

Abstract

Aims: Dopamine transporter (DAT) can regulate DA homeostasis and has been implicated in many nervous system diseases. Whether DAT is involved in the protection against ischemic stroke is unclear.

Methods: In vivo microdialysis measurements of DA were recorded in the ischemic penumbral area of mice with middle cerebral artery occlusion (MCAO). DAT coding gene, Slc6a3 mutation, and DAT overexpression animals were performed MCAO. Madopar (compound formulation of levodopa) and nomifensine (DA reuptake inhibitor) were administered in MCAO animals. Brain slices were prepared in Slc6a3 mutation or wild-type (WT) animals with MCAO to record miniature excitatory postsynaptic currents (mEPSCs) and miniature inhibitory postsynaptic currents (mIPSCs). The effects of DA and its dopamine-1 receptor (D₁R) antagonists (SCH-23390) on mEPSCs, mIPSCs, and neurons protection were recorded.

Results: MCAO caused a prominent increase in DA. Slc6a3 mutation significantly attenuated the ischemic injury, whereas DAT overexpression aggravated this injury. Both nomifensine and madopar protected against brain injury. Slc6a3 mutation and DA restored the disturbance of mEPSCs and mIPSC, and protected against neuron death, which was abolished by SCH-23390.

Conclusion: DAT inhibition might be explored as a strategy for ischemic stroke prevention. DA and D₁R involve in the restoration of synaptic dysfunction and neuron protection.

Keywords: D1 receptor; dopamine; dopamine transporter; ischemic stroke; miniature excitatory postsynaptic current; miniature inhibitory postsynaptic current.

FIGURE 1

Microdialysis measurement of dopamine (DA) and glutamate (Glu) in the ischemic penumbra area of mice with MCAO. The figure shows the change in extracellular concentrations of three transmitters at different time after MCAO (n = 6). (A) Location of the probe was in the ischemic penumbra area of hippocampus as indicated. Brain slices were stained with 1% TTC. (B–C) The change in DA and Glu before and after MCAO. Data are shown as mean ± SD. Data are analyzed by ANOVA followed by Dunnett testing. *p < 0.05, **p < 0.01 versus before MCAO (0 h).

FIGURE 2

Slc6a3 mutation increases the extracellular concentration of DA and protects against ischemic cerebral injury. (A) Microdialysis measurement to show the changes of DA levels in ischemic penumbra area of hippocampus of Slc6a3 homozygous mutation and WT mice before and after MCAO (n = 5). (B) The left panel shows representative 1% TTC staining images of coronal brain sections of mice 24 h after MCAO (n = 10). The middle panel shows the infarct area and the right panel shows the neurological deficit scores. Data are shown as mean ± SD, and analyzed by student's t‐test in (B) for infarct size or non‐parametric Kruskal–Wallis test for neurological deficit scores, and two‐way ANOVA in (A). *p < 0.05, **p < 0.01 versus WT. #p < 0.05, ##p < 0.01 versus before MCAO (0 h).

FIGURE 3

Nomifensine and madopar reduced the infarct size and improved neurological function in rats with MCAO. (A) and (B) The left panel shows representative 1% TTC staining images of coronal brain sections of rats 24 h after MCAO. The middle panel shows the infarct area and the right panel shows the neurological deficit scores. (A) Rats were administered vehicle (10 μL, i.c.v. n = 10) or nomifensine (100 μM, 10 μL, i.c.v. n = 10). (B) Rats were administered vehicle (0.5% CMC, i.g. n = 10) or madopar (20 mg/kg, i.g. n = 10). Data are shown as mean ± SD and analyzed by student's t‐test or non‐parametric Kruskal–Wallis test for neurological deficit scores. *p < 0.05 versus vehicle group. n.s, no significance.

FIGURE 4

Effects of lentivirus‐mediated DAT overexpression on cerebral injury induced by MCAO. (A) Lentiviral delivery system efficiently transfected the cultured neurons. Representative image of neurons transfected with GFP‐containing lentivirus (The upper panel from light field, the lower panel from fluorescent field) for 2 days (MOI = 10). Scale bar, 100 μm. (B) Immunoblotting and quantification showing efficient overexpression of DAT in cultured cells. n = 3. (C) Neuron cells transfected with LV‐Slc6a3, LV‐GFP, or vehicle were pretreated by DA (20 μM) for 2 h. The intracellular concentration of DA was assayed by HPLC‐EC methods. (D) Time schedule for lentivirus injection, MCAO operation, and sacrifice. (E) Four sites for injection (yellow asterisk). Lentivirus (2 × 10⁶ TU/site) was injected into the left cortex and hippocampus at four sites. (F) Upper panel: General view of lentivirus transfection in rat brain 1 week after injection (left, the brain was stained by Hoechst; right, GFP‐containing transfection site). Lower panel: LV‐GFP efficiently transfected neurons, as detected by colocalization with the neuron‐specific marker NeuN. Scale bar, 20 μm. (G) Immunoblotting and quantification show efficient overexpression DAT in local brain tissue of rats after 3 weeks, n = 3. (H) The left panel shows representative 1% TTC staining of seven corresponding coronal brain sections. The middle panel shows the infarct size and the right panel shows the neurological deficit scores, n = 10. Data are shown as mean ± SD. Data are analyzed by ANOVA followed by Tukey post hoc testing or by non‐parametric Kruskal–Wallis test for neurological deficit scores. *p < 0.05, **p < 0.01 versus LV‐GFP.

FIGURE 5

Slc6a3 mutation and DA restore the imbalance of synaptic function induced by MCAO. (A) Bright‐field images of a recorded neuron (arrow) in ischemic penumbra area (upper panel, area in black square frame), Scale bar: 100 μm. (B, C) Traces of mEPSCs (B) and mIPSCs (C) and summary graphs showing the frequency (middle panel) and amplitude (right panel) before operation (0 h, n = 12 cells/4 mice both for mEPSCs and mIPSCs), 6, 24, and 72 h after MCAO (n = 15 cells/5 mice) in C57BL mice. (D) Summary graphs showing the frequency and amplitude before operation (0 h) and 6, 24, and 72 h after MCAO in WT and Slc6a3 mutation mice, n = 15 cells/5 mice. (E) The panels show the effect of DA (1 μM), SCH‐23390 (1 μM), and their combination on the frequency and amplitude of mEPSCs and mIPSCs in the brain slices of C57BL mice 24 h after MCAO (n = 15 cells/5 mice in each group). (F) Proposed mechanism underlying DAT against ischemic stroke. Data are shown as mean ± SD. (B) and (C) are analyzed by one‐way ANOVA followed by Dunnet's testing. (D) by two‐way ANOVA, and (E) by one‐way ANOVA followed by Tukey testing. *p < 0.05, **p < 0.01 versus before MCAO (0 h) or vehicle. #p < 0.05, ##p < 0.01 versus WT.

FIGURE 6

Protection of DA against neuron death is related to D₁R. (A) In cultured primary neurons, the cells were treated by DA with (left) or without (right) OGD. The cell number and cell viability were assessed by CCK8. (B, C) The neurons were treated by DA (0.1 μM) or D₁R antagonist SCH‐23390 (0.1 μM) under OGD for 12 h. The cell death (Annexin V staining and PI staining) was analyzed by flow cytometer (B) or by TUNEL staining kit and Hoechst 33342 (C) Scale bar, 20 μm. n = 3 in each group. Data are analyzed by one‐way ANOVA followed by Tukey testing. **p < 0.01 versus normal vehicle control. #p < 0.05, ##p < 0.01 versus vehicle group exposed in OGD. $$p < 0.01 versus DA group exposed in OGD.