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Aldehyde dehydrogenase 1a1 mediates a GABA synthesis pathway in midbrain dopaminergic neurons

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Science  02 Oct 2015:
Vol. 350, Issue 6256, pp. 102-106
DOI: 10.1126/science.aac4690
  • Fig. 1 GABA co-release by midbrain DA neurons does not require GAD.

    (A to J) Expression of Gad1 and Gad2 mRNA in DA neurons of the SNc. Immunolabeled TH-positive dopaminergic neurons (brown) combined with chromogenic in situ hybridization (ISH) for Gad1 (A and B) and Gad2 (F and G) mRNA. Confocal fluorescence images of ISH for Gad1 (D) and Gad2 (I) mRNA (red) combined with TH immunostaining (green) (C) to (E) and (H) to (J) show limited expression of Gad in TH+ cells. (K) Quantification of TH/Gad colocalization in SNc (left) and VTA (right). (L) (Left) Representative oIPSC traces in control (top); 3-MPA–treated (500 μM) (middle); and Gad1fl/fl;Gad2fl/fl (bottom) A2A-Cre;Ai32;Drd1a-tdTomato mice. (Right) Summary statistics for oIPSC recordings. Representative traces (M) and summary statistics (N) for oIPSC and oEPSC recorded from DAT-Cre;Ai32 mice treated with ACSF (control) and 3-MPA, respectively. Representative traces (O) and summary statistics (P) for oIPSC and oEPSC recorded from DAT-Cre;Ai32;Gad1+/+;Gad2+/+ and DAT-Cre;Ai32;Gad1fl/fl;Gad2fl/fl mice. Blue bar indicates 450-nm light stimulation. Scale bars: 200 μm for (A) and (F), 50 μm for (B) to (E) and (G) to (J); current of 400 pA and time of 100 ms for oIPSC and 50 pA and 100 ms for oEPSC. Error bars indicate means ± SEM. **P < 0.01, ***P < 0.001.

  • Fig. 2 ALDH1a1-mediated noncanonical GABA synthesis in DA neurons.

    (A and B) Confocal images depicting double immunostaining for TH (left, red) and ALDH1a1 (middle, green) in SNc (A) and VTA (B). Scale bar, 40 μm. (C) Quantification of ALDH1a1 expression in TH+ DA neurons in SNc and VTA. (D) Confocal images depicting double immunostaining for TH (red), ALDH1a1-GFP (green), and 4′,6′-diamidino-2-phenylindole (DAPI) (blue) in the dorsal striatum (DStri). Scale bar, 10 μm. Representative oIPSC and oEPSC traces (E) and summary statistics (F) recorded from DAT-Cre;Ai32 mice treated with ACSF (control, left) and DEAB (10 μM, right). Representative oIPSC and oEPSC traces (G) and summary statistics (H) recorded from DAT-Cre;Ai32 mice treated with ACSF (control, left) and AG (100 μM, right). Blue bar indicates 450-nm light stimulation. Scale bars: 400 pA and 100 ms for oIPSC and 50 pA and 100 ms for oEPSC. Error bars indicate means ± SEM. ***P < 0.001. The arrows in (A), (B), and (D) highlight the colocalization of TH and ALDH1a1.

  • Fig. 3 Aldh1a1 knockdown and genetic deletion reduce dopaminergic oIPSCs and are rescued by Aldh1a1 overexpression.

    (A) Schematic illustration depicting viral shRNA constructs (Aldh1a1* indicates an shRNA-resistant WT Aldh1a1) and experimental configuration. Stri, striatum. (B) Confocal image showing expression of shRNA (green) in TH+ (red) neurons. Arrowheads indicate absence of ALDH1a1 (blue) in GFP+/TH+ neurons. Scale bar, 20 μm. Representative oIPSC and oEPSC traces (C) and summary statistics (D) in DAT-Cre;Ai32 mice injected with Aldh1a1 knockdown and rescue viruses. Representative oIPSC and oEPSC traces (E) and summary statistics for oIPSC (F) and oEPSC (G) in Aldh1a1+/+;DAT-Cre;Ai32 or Aldh1a1–/–;DAT-Cre;Ai32 mice, or loss-of-function mice treated with DEAB (10 μM) or AG (100 μM). Representative oIPSC and oEPSC traces (H) and summary statistics (I) recorded from Aldh1a1–/–;DAT-Cre;Ai32 mice injected with control and rescue viruses. Blue bar indicates 450-nm light stimulation. Scale bars: 400 pA and 100 ms for oIPSC and 50 pA and 100 ms for oEPSC. Error bars indicate means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 4 Altered GABA co-release in conditions related to alcohol binge drinking.

    Representative oIPSC and oEPSC traces (A) and summary statistics (B) in DAT-Cre;Ai32 mice in control or after repeated in vivo administration of EtOH. Representative oIPSC and oEPSC traces (C) and summary statistics (D) in DAT-Cre;Ai32 mice in control mice or mice treated with EtOH (17 to 50 mM). (E) Shematic illustration depicting the timeline of the two-bottle-choice behavioral assay. (F) Quantification of average daily EtOH intake, (G) average daily water intake, and (H) average EtOH preference. (I) Quantification of average daily EtOH intake in Aldh1a1 KD or Aldh1a1 overexpression mice. (J) Average daily water intake. (K) Average EtOH preference. Blue bar indicates 450-nm light stimulation. Scale bars: 400 pA and 100 ms for oIPSC and 50 pA and 100 ms for oEPSC. Error bars indicate means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

Supplementary Materials

  • Aldehyde dehydrogenase 1a1 mediates a GABA synthesis pathway in midbrain dopaminergic neurons

    Jae-Ick Kim, Subhashree Ganesan, Sarah X. Luo, Yu-Wei Wu, Esther Park, Eric J. Huang, Lu Chen, Jun B. Ding

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Materials and Methods
    • Figs. S1 to S14
    • Table S1

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