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NEUROTRANSMISSION Aldehyde dehydrogenase 1a1 NEUROTRANSMISSION Aldehyde dehydrogenase 1a1 mediates a GABA synthesis pathway in midbrain dopaminergic neurons Jae-Ick Kim, 1Subhashree

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    Aldehyde dehydrogenase 1a1 mediates a GABA synthesis pathway in midbrain dopaminergic neurons Jae-Ick Kim,1 Subhashree Ganesan,1 Sarah X. Luo,2,3 Yu-Wei Wu,1 Esther Park,1

    Eric J. Huang,2,3,4 Lu Chen,1 Jun B. Ding1,5*

    Midbrain dopamine neurons are an essential component of the basal ganglia circuitry, playing key roles in the control of fine movement and reward. Recently, it has been demonstrated that g-aminobutyric acid (GABA), the chief inhibitory neurotransmitter, is co-released by dopamine neurons. Here, we show that GABA co-release in dopamine neurons does not use the conventional GABA-synthesizing enzymes, glutamate decarboxylases GAD65 and GAD67. Our experiments reveal an evolutionarily conserved GABA synthesis pathway mediated by aldehyde dehydrogenase 1a1 (ALDH1a1). Moreover, GABA co-release is modulated by ethanol (EtOH) at concentrations seen in blood alcohol after binge drinking, and diminished ALDH1a1 leads to enhanced alcohol consumption and preference. These findings provide insights into the functional role of GABA co-release in midbrain dopamine neurons, which may be essential for reward-based behavior and addiction.

    M idbrain dopamine (DA) neurons are im- portant for fine-movement control, moti- vation, and reward-based learning (1, 2). Dysfunction of dopaminergic systems leads to movement disorders, such as

    Parkinson’s disease, and various forms of ad- diction and drug abuse (3, 4). DA is the primary neurotransmitter released by DA neurons, and activation of DA receptors in postsynaptic neu- rons can modulate neuronal excitability and cir- cuit output. It has recently been shown thatGABA is copackaged with DA in midbrain DA neurons by the vesicular monoamine transporter 2 and is subsequently co-released in the striatum (5), where it provides direct and potent inhibition to post- synaptic striatal projection neurons through activation of GABA type A (GABAA) receptors. In the mammalian central nervous system

    (CNS), GABA biosynthesis is mediated by two glutamate decarboxylases (GAD65 and GAD67, 65- and 67-kD isoforms, respectively). Expression of either isoform of GAD has traditionally been used to identify GABAergic neurons in the CNS. To identify which subset of midbrain DA neu- rons is capable of GABA synthesis, we examined GAD expression in DA neurons by coupling im- munohistochemistry for tyrosine hydroxylase (TH), the rate-limiting enzyme inDA synthesis, with in situ hybridization for Gad1 or Gad2 (which en- code GAD67 and GAD65, respectively). Only a

    small percentage of midbrain DA neurons ex- press Gad in the substantia nigra pars compacta (SNc, ~9%) (Fig. 1, A to K) and the ventral teg- mental area (VTA, ~15%) (fig. S1) (6, 7). An individual DA neuron can extend elaborate

    axonal arbors covering large portions of the stri- atum (8). Consequently, even though GAD is expressed in only a small subset of DA neurons, it is possible that GAD-expressing neurons can drive sustained GABA co-release throughout the striatum.We thus askedwhether GAD is required for GABAergic transmission in the striatum by recording alterations in dopaminergic inhibitory postsynaptic currents (IPSCs) in spiny projection neurons (SPNs) that resulted from pharmaco- logical inhibition or conditional genetic dele- tion of GAD. The striatum comprises two parallel output pathways arising from distinct groups of “direct” and “indirect” pathway GABAergic SPNs (dSPNs and iSPNs, respectively) that differ in their expression of postsynaptic DA receptors coupled with heterotrimeric guanine nucleotide– binding protein. SPNs also send collateral inhibi- tory projections within the striatum. As SPNs express GAD and are considered conventional GABAergic neurons, we used striatal collateral inhibition as an internal control for our exper- iments.We expressed channelrhodopsin 2 (ChR2) in iSPNs by crossing A2A-Cre mice (in which Cre recombinase is selectively expressed in iSPNs but not inmidbrainDAneurons) with transgenicmice containing a conditional floxed allele of ChR2 in the Rosa26 locus (Ai32 mice). Progeny from this cross were bred to Drd1a-tdTomato–expressing transgenic mice carrying a bacterial artificial chromosome transgene that selectively labels dSPNs. We then performed whole-cell voltage- clamp recordings in dSPNs in brain slices of dorsal striatum prepared from A2A-Cre;Ai32; Drd1a-tdTomato mice, in which ChR2 is selec-

    tively expressed in A2A adenosine receptor– expressing iSPNs and tdTomato expression is restricted to dopamine 1 (D1) receptor–expressing dSPNs. Optogenetic stimulation of iSPN axons with brief pulses (0.5 ms) of blue light (450 nm) reliably evoked IPSCs in dSPNs. Optogenetically evoked IPSCs (oIPSCs) recorded n dSPNs were isignificantly attenuated by GAD inhibitor 3- mercaptopropionic acid (3-MPA, 500 mM) (Fig. 1L), which confirmed that local collateral inhibitory transmission arising from iSPNs is dependent on GAD function.We next selectively deleted GAD in iSPNs (9), usingGad1 andGad2 double condition- al knockout mice (A2A-Cre;Gad1fl/fl;Gad2fl/fl). When recording oIPSCs from dSPNs in A2A-Cre;Gad1fl/fl; Gad2fl/fl;Ai32;Drd1a-tdTomato mice, we found that genetic deletion of both GADs in iSPNs abolished nearly all of the oIPSCs recorded in dSPNs. These data confirmed that GAD-mediated GABA synthesis is necessary for conventional GABAergic transmission within the striatum (Fig. 1L). To test whether GAD is required for functional

    GABA co-release by midbrain DA neurons, whose axon terminals project onto SPNs, we used DAT-Cre;Ai32 mice to selectively express ChR2 in DA neurons (5, 10) and recorded oIPSCs and oEPSCs in postsynaptic dorsal striatum SPNs. Monosynaptic oIPSCs and oEPSCs can be abol- ished by GABAA and glutamate-receptor block- ers, respectively (fig. S2). To our surprise, neither incubating brain slices with 3-MPA (Fig. 1, M and N) nor genetically deleting both GAD iso- forms in midbrain DA neurons using DAT-Cre; Ai32;Gad1fl/fl;Gad2fl/fl mice (Fig. 1, O and P) sig- nificantly altered the amplitudes of oIPSCs and oEPSCs in SPNs, which indicated that midbrain DA neuron GABA co-released does not require canonical GAD-mediated GABA synthesis. GABA co-release from DA neurons was observed in re- cordings obtained throughout the striatum, in both dorsal and ventral regions. Notably, the oIPSCs recorded in dorsal striatum SPNs were significantly larger than those recorded from SPNs in the nucleus accumbens (NAc). Within NAc, oIPSC and oEPSC amplitudes were not significantly different between SPNs in the core or medial shell, and, as with oIPSCs recorded in dorsal striatum, oIPSCs recorded in the NAc were not blocked by application of 3-MPA (fig. S3). Recent work has suggested that DA can directly activate postsynaptic GABAA receptors (11). To exclude this possibility and test whether dopaminergic oIPSCs were caused by direct acti- vation of GABAA receptors by DA, we locally ap- plied DA and GABA onto individual SPNs and recorded IPSCs. Direct application of DA did not evoke IPSCs, whereas GABA successfully evoked IPSCs in the same SPN, which indicated that DA was not likely to be activating GABAA receptors directly inour cells. This ideawas further supported by application of theDA transporter (DAT) blocker GBR12935,which elevates extracellularDAconcen- trations and similarly did not affect oIPSC ampli- tudes in the striatum (fig. S4). Together, these data suggest that dopaminergic oIPSCswere not caused by direct activation of GABAA receptors by DA.

    102 2 OCTOBER 2015 • VOL 350 ISSUE 6256 SCIENCE

    1Department of Neurosurgery, Stanford University School of Medicine, Palo Alto, CA 94304, USA. 2Department of Pathology, University of California San Francisco, San Francisco, CA 94143, USA. 3Neuroscience Graduate Program, University of California San Francisco, San Francisco, CA 94143, USA. 4Pathology Service 113B, San Francisco VA Medical Center, San Francisco, CA 94121, USA. 5Department of Neurology and Neurological Sciences, Stanford University School of Medicine, Palo Alto, CA 94304, USA. *Corresponding author. E-mail: [email protected]


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  • In plants, GABA can be synthesized from pu- trescine (12) by the enzymes diamine oxidase (DAO) and aldehyde dehydrogenase (ALDH) (fig. S5A) (13, 14). GABA production through this alternative evolutionarily conserved pathway also exists in Xenopus tadpole (15) and mammalian cells (16–19). Glial cells can also use putrescine to produce GABA during retinal early development (18, 20). We tested whether ALDH-mediated al- ternative GABA synthesis drives GABA produc- tion in midbrain DA neurons. ALDH1a1 is the most abundant form of cytosolic ALDH (21, 22) and is highly expressed in the ventral midbrain, including the region delineating the SNc (23) (for Aldh1a1 mRNA) (24). We first examined ALDH expression in midbrain DA neurons by double immunostaining for ALDH1a1 and TH. ALDH1a1 is indeed highly expressed in a subset of DA neu- rons, colocalizing with TH in the SNc and VTA (Fig. 2, A to C, and fig. S5B). To examine subcel- lular localization of ALDH1a1, we injected an adeno-associated virus (AAV) carrying green flu- orescent protein (GFP)–tagged ALDH1a1 into the

    midbrain and examined GFP expression in the striatum.We found that GFP strongly colocalizes with TH within axons in the dorsal striatum (Fig. 2D), which suggested that ALDH1a1 is high- ly abundant in dopaminergic terminals (fig. S5, C to F). We then tested the involvement of ALDH1a1 in GABA synthesis in these neurons by blocking its activity with the ALDH inhibitors 4-(diethylamino)-benzaldehyde (DEAB, 10 mM), or disulfiram (10 mM). To ensure that intracel-

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