Synaptic transmission between midbrain dopamine neurons and target neurons in the striatum is essential for the selection and reinforcement of movements. Recent evidence indicates that nigrostriatal dopamine neurons inhibit striatal projection neurons by releasing a neurotransmitter that activates GABAA receptors. Here, we demonstrate that this phenomenon extends to mesolimbic afferents, and confirm that the released neurotransmitter is GABA. However, the GABA synthetic enzymes GAD65 and GAD67 are not detected in midbrain dopamine neurons. Instead, these cells express the membrane GABA transporters mGAT1 (Slc6a1) and mGAT4 (Slc6a11) and inhibition of these transporters prevents GABA co-release. These findings therefore indicate that GABA co-release is a general feature of midbrain dopaminergic neurons that relies on GABA uptake from the extracellular milieu as opposed to de novo synthesis. This atypical mechanism may confer dopaminergic neurons the flexibility to differentially control GABAergic transmission in a target-dependent manner across their extensive axonal arbors.https://doi.org/10.7554/eLife.01936.001
The electrical signals that are fired along neurons cannot be transmitted across the small gaps, called synapses that are found between neurons. Instead, the neuron sending the signal releases chemicals called neurotransmitters into the synapse. These neurotransmitters bind to receptor proteins on the surface of the second neuron and control how it fires.
A neurotransmitter called dopamine plays a key role in the circuits of the brain that control how we learn certain tasks involving movement. In particular, two populations of neurons from the midbrain that release dopamine target the striatum, an area of the brain that is responsible for motor control. These neurons also release other neurotransmitters, but the identity of these other chemicals is not known, and the details of the interaction between the neurons and the striatum are poorly understood.
Previous research showed that some of the midbrain neurons activate receptors that normally respond to a neurotransmitter called gamma-aminobutyric acid (GABA). However, several different chemicals can trigger this receptor. Using a range of techniques, Tritsch et al. now confirm that dopamine neurons release GABA alongside dopamine, and that this applies to both sets of the dopamine-producing neurons that feed into the striatum.
Some neurons can manufacture GABA from amino acids found in their internal fluid. However, Tritsch et al. could not detect the enzymes needed for this reaction in dopamine-producing neurons. Instead, these neurons contain proteins that can transport GABA across the cell membrane, which suggests that the neurons collect GABA from the extracellular fluid that surrounds them.https://doi.org/10.7554/eLife.01936.002
Dopamine (DA)-releasing neurons in the mammalian midbrain play an important role in fundamental behaviors, including motivation, reinforcement learning and motor control, and their dysfunction is associated with a wide range of neuropsychiatric disorders (Wise, 2004; Costa, 2007; Schultz, 2007; Morris et al., 2009; Redgrave et al., 2010). The major target of midbrain DA neurons is the striatum, a large subcortical structure implicated in the selection and reinforcement of motor actions. DA neurons located in the substantia nigra pars compacta (SNc) project mainly to the dorsal striatum (also known as the caudate and putamen, CPu), whereas those in the ventral tegmental area (VTA) innervate the ventral striatum (or nucleus accumbens, NAc), forming the nigrostriatal and mesolimbic pathways, respectively. The striatum controls motor behavior through two parallel output streams with opposing effects; the so-called ‘direct’ and ‘indirect’ pathways. Each pathway arises from distinct groups of GABAergic striatal projection neurons (SPNs) that differ, amongst other things, in their response to DA (Gerfen and Surmeier, 2011; Tritsch and Sabatini, 2012). By providing DA to the striatum, SNc and VTA neurons are believed to play a pivotal role in balancing the activity of direct- and indirect-pathway SPNs. However, our understanding of the cellular and molecular mechanisms employed by DA neurons to modulate striatal function remains incomplete.
Although the activation of metabotropic receptors following the release of DA from SNc/VTA neurons is undoubtedly central to their function, DA neurons also co-release several other transmitters that shape striatal output (Hnasko et al., 2010; Stuber et al., 2010; Tecuapetla et al., 2010; Tritsch et al., 2012). In particular, we recently showed that DA neurons in SNc potently inhibit action potential firing in SPNs by releasing a transmitter that activates GABAA receptors (Tritsch et al., 2012). Release of this neurotransmitter requires activity of the vesicular monoamine transporter Slc18a2 (VMAT2), which can be replaced by exogenous expression of the vesicular GABA transporter Slc32a1 (also known as VGAT or VIAAT). In addition, VMAT2 expression in SPNs lacking VGAT is sufficient to sustain GABAergic transmission. Collectively, these findings suggest that SNc neurons co-release GABA using VMAT2 for vesicular loading. However, this study raises several important questions. First, does GABAergic transmission generalize to DA neurons in the VTA, or is it limited to nigrostriatal afferents? Second, what is the identity of the transmitter released by DA neurons? GABA exists as a zwitterion at neutral pH and lacks the characteristic molecular structure of VMAT2 substrates, which typically feature an aromatic ring and a positive charge (Yelin and Schuldiner, 1995). GABAA receptors can be activated by several naturally-occurring agonists and allosteric modulators, including β-alanine, taurine, imidazole-4-acetic acid and neurosteroids (Johnston, 1996; Belelli and Lambert, 2005). Although none of these molecules constitute ideal candidates for VMAT2-dependent vesicular transport based on their structure, charge, or both, they nonetheless raise the possibility that DA neurons liberate a transmitter other than GABA to inhibit SPNs. Third, do all DA neurons contribute to GABAergic signaling, or is it reserved to a subpopulation of cells, similar to glutamate co-release from DA neurons (Hnasko and Edwards, 2011). Finally, how is inhibitory synaptic transmission sustained in DA neurons? Addressing this point will help identify molecules required for GABAergic transmission by DA neurons and will permit the development of genetic tools to determine the relative contribution of SPN inhibition by DA neurons in vivo under normal and pathological conditions.
In this study, we address these questions by examining the cellular and molecular mechanisms that underlie the rapid GABAA receptor-mediated inhibition of SPNs upon stimulation of DA axons. Our results provide strong evidence that the inhibitory transmitter released by midbrain DA neurons is GABA, and suggest that GABA co-release is a general feature of all midbrain DA neurons. Moreover, we reveal that DA neurons rely on GABA uptake through the plasma membrane—but not on de novo GABA synthesis—to sustain GABAergic transmission.
We recently reported that activation of SNc axons in dorsal striatum evokes monosynaptic, GABAA receptor-mediated inhibitory postsynaptic currents (IPSCs) in SPNs (Tritsch et al., 2012; Figure 1—figure supplement 1). To determine whether this GABAergic signaling extends to DA neurons projecting to ventral striatum, we expressed channelrhodopsin 2 (ChR2) in the VTA using one of two approaches (‘Materials and methods’). We either injected Slc6a3-ires-Cre mice (Backman et al., 2006) stereotaxically with an adeno-associated virus expressing Cre recombinase-dependent ChR2 (Figure 1A) or drove ChR2 expression genetically by crossing Slc6a3-ires-Cre mice to transgenic mice (Madisen et al., 2012) containing a conditional allele of ChR2 in the Rosa26 locus (Figure 1D). Mice also harbored Drd2-Egfp or Drd1a-tdTomato bacterial artificial chromosome (BAC) transgenes to permit distinction between direct- and indirect-pathway SPNs, respectively (Gong et al., 2003; Ade et al., 2011). We performed whole-cell voltage-clamp recordings from SPNs in sagittal brain slices of NAc in the presence of inhibitors of ionotropic glutamate receptors (NBQX and R-CPP) and metabotropic GABAB receptors (CGP55845) to prevent excitatory synaptic transmission by dopaminergic axons, as well as modulatory effects of GABAB receptors, respectively. Under our recording conditions, optogenetic stimulation of VTA axons with brief (1 ms) flashes of blue light reliably evoked inward IPSCs in direct- and indirect-pathway SPNs that were blocked by the GABAA receptor antagonists picrotoxin (n = 12; Figure 1B,E) and SR95531 (n = 4, not shown). With the exception of synaptic latency, optogenetically-evoked IPSCs (oIPSCs) exhibited similar properties in both experimental systems and were consequently pooled for analysis. They averaged 353 ± 60 pA in peak amplitude (range = 68–905 pA; n = 16) and their kinetics were similar to those observed in dorsal striatum (Tritsch et al., 2012; Figure 1—figure supplement 1), with a 10–90% rise time of 2.4 ± 0.2 ms and a decay time constant of 46.8 ± 7.2 ms. The synaptic latency of oIPSCs in NAc did not differ from that observed in dorsal striatum under similar experimental conditions (Figure 1C,F), indicating that the connection is monosynaptic. Synaptic stimulation of midbrain DA neurons therefore directly engages GABAA receptors on SPNs in both dorsal and ventral striatum.
The chemical identity of a synaptic neurotransmitter is traditionally established by determining whether a synapse fulfills several necessary criteria. They include the presence of (1) presynaptic synthetic enzymes, (2) presynaptic vesicular transporters, (3) postsynaptic receptors, and (4) a biochemical mechanism for inactivation. In the case of GABAergic signaling at dopaminergic synapses, conditions 2 and 3 are respectively satisfied by the presence of VMAT2 in DA neurons and GABAA receptors in SPNs. For many central nervous system transmitters, condition 4 is mediated by diffusion and reuptake through plasma membrane transporters. Of the four genes identified in mice that encode high affinity membrane GABA transporters, Slc6a1 is expressed predominantly throughout the brain (Borden, 1996; Lein et al., 2007). Its gene product, mGAT1, distributes mainly to presynaptic terminals of GABAergic neurons and serves to shape the amplitude and time course of IPSCs as well as regulate extrasynaptic levels of GABA (Isaacson et al., 1993; Jensen et al., 2003; Overstreet and Westbrook, 2003; Conti et al., 2004; Chiu et al., 2005; Bragina et al., 2008; Kirmse et al., 2008; Cepeda et al., 2013). Importantly, mGAT1 is highly selective for GABA and does not transport structurally similar GABAA receptor agonists such as β-alanine, taurine, and muscimol (Johnston et al., 1978; Tamura et al., 1995). Thus, we reasoned that if dopaminergic IPSCs are modulated by inhibition of mGAT1, it would provide compelling evidence that GABA is the neurotransmitter released.
To test this, we recorded dopaminergic oIPSCs from SPNs in dorsal striatum (Figure 2). Stimulation intensity was calibrated to evoke sub-maximal IPSCs (approximately 20% of IPSC peak amplitude at maximum intensity; mean: 232 ± 40 pA, n = 19) in order to minimize spillover of GABA at dopaminergic synapses (Figure 2—figure supplement 1). We compared the amplitude and kinetics of IPSCs recorded before (baseline) and during the first 3–4 min following bath application of control saline (ACSF; n = 9; Figure 2A) or saline containing SKF 89976A, a selective mGAT1 antagonist (n = 10; Figure 2B). In both conditions, the IPSC amplitude progressively declined to ∼70% of baseline (p<0.01 each, Wilcoxon signed-rank test; Figure 2C), whereas the synaptic latency and 10–90% rise time remained unchanged relative to baseline (p>0.1, Wilcoxon signed-rank test). The decrease in amplitude is consistent with previous reports of activity-dependent rundown of GABA and DA release from midbrain DA neurons in vitro (Schmitz et al., 2003; Tritsch et al., 2012; Ishikawa et al., 2013) and did not differ between conditions (p=0.8, Mann–Whitney test; Figure 2C, Figure 1—figure supplement 1G), indicating that acute inhibition of mGAT1 does not interfere with neurotransmitter release or postsynaptic GABAA receptors. By contrast, mGAT1 antagonism significantly affected decay kinetics: whereas oIPSC decay time constants remained stable in control recordings (baseline: 33.6 ± 5.8 ms; 3–4 min after ACSF wash-in: 32.1 ± 5.8 ms; p=0.6, Wilcoxon signed-rank test), they were prolonged by a factor of 3 in the presence of SKF 89976A (baseline: 38.6 ± 6.0 ms; 3–4 min after SKF 89976A wash-in: 115.9 ± 26.3 ms; p=0.002, Wilcoxon signed-rank test; Figure 2D). These results indicate that mGAT1 activity normally shortens the duration of dopaminergic IPSCs, likely by clearing released neurotransmitter from the extracellular space.
In the striatum, mGAT1 controls ambient levels of GABA that evoke a tonic GABAA receptor-mediated conductance in SPNs (Ade et al., 2008; Kirmse et al., 2008; Santhakumar et al., 2010; Cepeda et al., 2013). Consistent with this, acute pharmacological inhibition of mGAT1 was accompanied by a significant increase in holding current caused by a threefold increase in tonic GABA current in both direct- and indirect-pathway SPNs (Figure 2—figure supplement 2). To exclude the possibility that the increase in oIPSC decay time constant stems from changes in recording conditions during bath application of SKF 89976A, we also monitored spontaneous (s) IPSCs as well as IPSCs evoked electrically within the striatum (eIPSCs; Figure 3). Unlike dopaminergic oIPSCs, neither the amplitude nor the kinetics of eIPSCs were significantly affected after acute mGAT1 inhibition (baseline amplitude: 304 ± 70 pA; 3–4 min after SKF 89976A wash-in: 339 ± 80 pA. Baseline 10–90% rise time: 1.3 ± 0.4 ms; 3–4 min after SKF 89976A wash-in: 1.3 ± 0.4 ms. Baseline decay time constant: 14.8 ± 2.6 ms; 3–4 min after SKF 89976A wash-in: 19.6 ± 4.9 ms; n = 9; p>0.1 for all, Wilcoxon signed-rank test; Figure 3A,B), despite a notable increase in holding current during the first few minutes following bath application of SKF 89976A (baseline: −82 ± 16 pA; SKF 89976A: −143 ± 21 pA; n = 9; p<0.001, Wilcoxon signed-rank test). Moreover, although the amplitude of sIPSCs decreased slightly in SKF 89976A relative to baseline (97 ± 9 vs 83 ± 7 pA, n = 14; p=0.01, Wilcoxon signed-rank test), the frequency and kinetics of sIPSCs remained unchanged (baseline frequency: 2.7 ± 0.5 Hz; 3–4 min after SKF 89976A wash-in: 2.5 ± 0.5 Hz. Baseline 10–90% rise time: 0.9 ± 0.1 ms; 3–4 min after SKF 89976A wash-in: 0.8 ± 0.1 ms. Baseline decay time constant: 6.8 ± 0.4 ms; 3–4 min after SKF 89976A wash-in: 6.4 ± 0.5 ms; p>0.05 for all, Wilcoxon signed-rank test; Figure 3C,D). These results are consistent with previous reports in the striatum and hippocampus (Isaacson et al., 1993; Kirmse et al., 2008). Thus, our results indicate that prolongation of light-evoked IPSCs by SKF 89976A is specific to dopaminergic synapses and not secondary to increased GABAergic tone. Together, these data reveal that the duration of dopaminergic IPSCs is critically dependent on mGAT1 function and, therefore, strongly support that GABA is the transmitter co-released by DA neurons.
Previous reports indicate that up to 10% of midbrain DA neurons in rat contain detectable levels of mRNA for the 65 kDa isoform of glutamic acid decarboxylase (GAD65) (Gonzalez-Hernandez et al., 2001; 2004). Together with GAD67, these enzymes constitute the major biosynthetic pathway for GABA in the central nervous system (Soghomonian and Martin, 1998). To determine the fraction of midbrain DA neurons capable of synthesizing (and by extension releasing) GABA in mice, we performed double fluorescence in situ hybridization for Slc18a2 (Vmat2) and Gad1 or Gad2 (which encode GAD67 and GAD65, respectively). We focused our analyses to regions highlighted in Figure 4A, which consist of the SNc and lateral VTA. In agreement with previous findings (Gonzalez-Hernandez et al., 2001, 2004), we did not detect significant overlap between Vmat2 and Gad1, as only 4 out of 958 Vmat2+ neurons (0.4%) showed weak signal for Gad1 in SNc and lateral VTA (Figure 4A,B). To our surprise, we were also unable to detect Gad2 mRNA in these regions (Figure 4D,E): only 7 out of 1200 Vmat2+ cells (0.6%) were weakly Gad2+, despite the presence of numerous brightly-labeled GABAergic neurons in neighboring substantia nigra pars reticulata (SNr) and within the SNc and lateral VTA. We confirmed our ability to detect co-labeling by examining the histaminergic tuberomamillary nucleus (TMN) and dopaminergic A13 cell group (Figure 4C,F), both of which express GADs (Lin et al., 1990; Esclapez et al., 1993).
Levels of mRNA for GAD65 and GAD67 in DA neurons may be below the detection threshold, yet high enough to sustain GABA synthesis. To address this concern, we attempted to directly visualize GAD protein in DA neurons by immunofluorescence using antibodies directed against GAD65/67 and tyrosine hydroxylase (TH) to label catecholaminergic neurons. However, the high density of GABAergic axons converging into the substantia nigra, combined with the low concentration of GADs in cell bodies compared to axon terminals prevented us from clearly identifying DA neurons that expressed GADs (not shown). We instead imaged coronal brain sections from knock-in mice (Tamamaki et al., 2003; Taniguchi et al., 2011) expressing either EGFP or Cre recombinase under transcriptional control of the endogenous promoter for Gad1 or Gad2 (Figure 5). Cre expression was visualized using a sensitive fluorescent reporter allele (Madisen et al., 2010) and TH expression was revealed via immunofluorescence. These mice offer the advantage of having bright somatic labeling including, in the case of Gad2-ires-Cre mice, of cells with very little transcriptional activity. However, we were unable to detect any DA neuron expressing fluorescent reporter protein driven by either Gad1 (0 out of 519 TH+ neurons) or Gad2 (0 out of 526 TH+ neurons) promoters (Figure 5A–D). Collectively, these results indicate that midbrain DA neurons in the SNc and lateral VTA of mice do not express the GABA synthetic enzymes GAD65 and GAD67.
Mammalian cells possess alternative means of synthesizing GABA (Seiler, 1980; Caron et al., 1987; Petroff, 2002). GABA transaminase is an enzyme expressed at high levels in GABAergic neurons, including in the ventral midbrain that can reversibly convert succinate semialdehyde into GABA, and vice versa (Bessman et al., 1953; Roberts and Bregoff, 1953; Medina-Kauwe et al., 1994). Because succinate semialdehyde is readily oxidized to succinate to sustain energy production as part of the Krebs cycle, GABA transaminase is thought to participate in the degradation of GABA in most cells, not its synthesis (Balazs et al., 1970). Nevertheless, we considered the possibility that GABA transaminase might contribute GABA in DA neurons. Double fluorescence in situ hybridization for Vmat2 and Abat (the gene encoding GABA transaminase) revealed strong co-labeling in 701 out of 738 DA neurons (95%) within the SNc and lateral VTA (Figure 6A,B), indicating that DA neurons express genes involved in GABA metabolism. To test whether DA neurons employ GABA transaminase to synthesize GABA, we incubated slices of striatum in control ACSF or vigabatrin (VGT)—an irreversible inhibitor of GABA transaminase—for at least thirty minutes prior to recording oIPSCs in SPNs. In agreement with prior work (Overstreet and Westbrook, 2001), this manipulation significantly elevated extracellular GABA levels (picrotoxin-sensitive tonic current in SPNs in ACSF: 24 ± 3 pA, n = 11; in VGT: 115 ± 23 pA, n = 12; p<0.001, Dunn's Multiple Comparison Test), confirming the effectiveness of the drug. To minimize differences in light-evoked responses within experiments, we obtained maximal oIPSCs using strong ChR2 stimulation and limited comparisons to SPNs located in similar areas of dorsal striatum in adjacent slices. Under these conditions, we did not detect significant differences in oIPSC amplitude between both groups (ACSF: 1.1 ± 0.3 nA; VGT: 1.4 ± 0.2 nA; n = 14; p=0.3, Wilcoxon signed-rank test; Figure 6C,D), indicating that GABA transaminase function is not required for GABAergic signaling by DA neurons.
Our data indicate that inhibitory synaptic transmission from DA neurons does not depend on synthesis of GABA by either GADs or GABA transaminase. In the central nervous system, several synapses rely on neurotransmitter reuptake across the plasma membrane as opposed to de novo synthesis to sustain synaptic transmission (Torres et al., 2003; Edwards, 2007). It is therefore conceivable that DA neurons inhibit SPNs by releasing GABA they acquire from the extracellular environment. mGAT1 and mGAT4 (encoded by Slc6a11) are the two major plasma membrane GABA transporters expressed in the mouse midbrain (Borden, 1996; Lein et al., 2007). We therefore performed double fluorescence in situ hybridization for Vmat2 and mGat1 or mGat4 to establish whether either isoform is present in DA neurons (Figure 7). Surprisingly, we found considerable expression of mGat1 in 667 out of 748 Vmat2+ neurons (89%), particularly within the SNc. mGat4 localized predominantly to glial cells, consistent with previous reports (Borden, 1996), although faint labeling above background was also detected in a substantial fraction (80%) of DA neurons in SNc and lateral VTA (568 out of 713 Vmat2+ neurons).
The expression of mRNA for mGAT1 and mGAT4 in DA neurons raises the possibility that DA neurons acquire GABA not through de novo synthesis, but rather through plasma membrane uptake. Being almost exclusively composed of GABAergic neurons, the striatum represents a rich source of extracellular GABA for dopaminergic terminals (Ade et al., 2008; Kirmse et al., 2008; Janssen et al., 2009; Santhakumar et al., 2010; Cepeda et al., 2013). Inhibition of mGAT1 for a few minutes does not affect the release of GABA from SNc axons (Figure 2C), indicating either that this pharmacological manipulation incompletely blocks GABA reuptake, or that it is too short to deplete presynaptic GABA levels. To determine whether uptake of ambient GABA is necessary for sustaining GABAergic transmission by DA neurons, we therefore inhibited both mGAT1 and mGAT4 in striatal slices using a cocktail of SKF 89976A and SNAP-5114 (a mGAT4 antagonist) for at least 30 min prior to examining oIPSCs in SPNs in the continued presence of GAT antagonists. Whereas oIPSCs obtained under control conditions averaged 1.3 ± 0.2 nA in amplitude (n = 15), oIPSCs recorded in adjacent slices with GABA transport chronically blocked were significantly smaller (0.20 ± 0.07 nA, n = 16; p<0.001 vs ACSF, Dunn's Multiple Comparison Test; Figure 8A,B). Importantly, prolonged exposure to SKF 89976A and SNAP-5114 did not prevent transmitter release non-specifically, as light-evoked dopamine release from SNc axons as well as GABA release from striatopallidal terminals were maintained under these conditions (Figure 8—figure supplement 1). Moreover, the small picrotoxin-sensitive oIPSCs that remained exhibited extremely long decay time constants (>1.5 s), consistent with persistent GABAergic signaling in the absence of plasma membrane reuptake.
Chronic inhibition of GABA reuptake was also accompanied by considerable elevation of ambient GABA in the striatum, as evidenced by a 7.5-fold increase in tonic GABAA receptor-mediated current in SPNs (picrotoxin-evoked tonic current in ACSF: 24 ± 3 pA, n = 11; in GAT antagonists: 182 ± 32 pA, n = 11; p<0.001, Dunn's Multiple Comparison Test; Figure 8C). This increase in extracellular GABA might account for the reduced amplitude of oIPSCs by promoting the desensitization of GABAA receptors, by shunting synaptic currents, or both. To examine these possibilities, we included an additional experimental condition in which slices were pre-incubated in muscimol before evoking dopaminergic oIPSCs (Figure 8A,B). Muscimol is a potent synaptic and extrasynaptic GABAA receptor agonist that promotes GABAA receptor desensitization to the same extent as GABA (Mortensen et al., 2010), but displays minimal affinity for plasma membrane GABA transporters (Johnston et al., 1978). The concentration of muscimol was titrated to evoke tonic GABAA receptor-mediated currents similar those that developed upon persistent GAT inhibition (picrotoxin-sensitive tonic current: 212 ± 46 pA, n = 10; p<0.001 vs ACSF; p>0.05 vs GAT antagonists, Dunn's Multiple Comparison Test; Figure 8C). Strikingly, this manipulation did not reduce the amplitude of dopaminergic oIPSCs (1.3 ± 0.3 nA, n = 12; p>0.05 vs ACSF, p<0.01 vs GAT antagonists, Dunn's Multiple Comparison Test; Figure 8A,B), indicating that the reduction in oIPSC amplitude observed following GAT inhibition is not secondary to elevation of ambient GABAergic tone. In agreement with this conclusion, the amplitude of oIPSCs was not diminished by prolonged treatment with VGT either (Figure 6C,D), despite significant increases in extracellular GABA (picrotoxin-sensitive tonic current in VGT: 115 ± 23 pA, n = 12; p<0.01 vs ACSF, p>0.05 vs GAT antagonists or muscimol, Dunn's Multiple Comparison Test; Figure 8C).
These results collectively indicate that GAT function is necessary for GABAergic transmission from SNc axons. If membrane GABA reuptake is required for supplying GABA for vesicular exocytosis, inhibiting GATs should prevent DA neurons from maintaining GABA release with repeated stimulation. Indeed, chronic inhibition of GATs precipitated the rundown of GABAergic transmission from DA neurons in an activity-dependent manner (Figure 8D, Figure 8—figure supplement 2). A second prediction is that elevating extracellular GABA should attenuate synaptic rundown. To test this, we locally applied GABA (100 μM) to the dorsal striatum for 5–10 min prior to recording oIPSCs from SPNs. Following this manipulation, the amplitude of oIPSCs was maintained for several minutes after break-in (Figure 8E). Importantly, this increase in oIPSC amplitude by exogenous GABA was prevented in slices incubated in GAT antagonists (Figure 8E). These data therefore provide strong evidence that membrane GABA transporters play an important role in sustaining GABAergic transmission from DA neurons by supplying the neurotransmitter GABA to presynaptic terminals for vesicular loading and exocytosis.
Despite the importance of midbrain DA neurons, the mechanisms by which they shape the activity of target neurons in the striatum remain poorly understood. In this study, we combined histochemical and electrophysiological approaches to define the cellular and molecular mechanisms that govern GABAergic signaling by DA neurons. Our findings indicate that the vast majority of midbrain DA neurons have the capacity to co-release GABA and identify presynaptic membrane GABA transporters as the likely mechanism employed by DA neurons to acquire GABA for vesicular exocytosis.
The chemical identity of a synaptic transmitter is typically established only after several independent observations converge on a plausible candidate. In this case, the transmitter released by midbrain DA neurons (1) functions as a GABAA receptor agonist, (2) serves as a substrate for VGAT (Tritsch et al., 2012), and (3) is a substrate for mGAT1. Although VGAT can transport GABA, glycine, and β-alanine into synaptic vesicles (Gasnier, 2000; Wojcik et al., 2006; Juge et al., 2013), glycine does not function as a GABAA receptor agonist, and the low affinity of β-alanine for GABAA receptors would give rise to IPSCs with faster kinetics than the ones we observed (Jones et al., 1998a). In addition, mGAT1 is highly selective for GABA (Borden, 1996), and applying GABA exogenously helped sustain IPSCs from DA neurons in a GAT-dependent fashion. Although we did not detect GABA synthetic enzymes in DA neurons in our experiments, SNc and VTA neurons display a GABAergic phenotype, as they contain mRNA for GABA transaminase and the membrane GABA transporters mGAT1 and mGAT4. Together, these results strongly suggest that the neurotransmitter released by midbrain DA neurons is GABA. In agreement with this conclusion, a recent electron microscopic study detected GABA in close association with VMAT2-containing synaptic vesicles in dopaminergic terminals within striatum (Stensrud et al., 2013). Moreover, other dopaminergic neurons in the central nervous system have been reported to contain and/or release GABA, including amacrine cells in the retina (Hirasawa et al., 2012), periglomerular cells in the olfactory bulb (Maher and Westbrook, 2008; Borisovska et al., 2013; Liu et al., 2013), as well as dopaminergic cell groups throughout the brain (Bjorklund and Dunnett, 2007; Stamatakis et al., 2013), suggesting that GABA co-release may be a general property of dopaminergic cells.
Synaptic transmission requires constant refilling of new and recycled vesicles, which is contingent upon the availability of transmitter in the cytosol. Most GABAergic neurons sustain inhibitory transmission by maintaining high cytosolic concentrations of GABA using de novo synthesis from glutamate by GAD67 and to a lesser extent GAD65 (Asada et al., 1997; Tian et al., 1999; Petroff, 2002). Given that all cells contain glutamate, an amino acid necessary for the production of proteins, the expression of either GAD67 or GAD65 in conjunction with a vesicular GABA transporter is sufficient to mediate vesicular release of GABA. Previous reports have estimated that up to 10% of DA neurons in the SNc and VTA of rats express mRNA for GAD65 (Gonzalez-Hernandez et al., 2001, 2004), suggesting that GABA release may be limited to a subpopulation of DA neurons. We were unable to replicate this observation using two separate approaches, indicating that DA neurons in the SNc and lateral VTA of mice do not rely on de novo synthesis of GABA and have instead adopted other means of obtaining GABA.
Most classical transmitters are transported back into the presynaptic terminal after vesicular release using plasma membrane transporters. This process is important not only for limiting the duration of synaptic currents and preventing extrasynaptic spillover, but also for replenishing presynaptic transmitter levels (Edwards, 2007; Conti et al., 2011). In fact, several synapses, including monoaminergic (Giros et al., 1996; Bengel et al., 1998; Jones et al., 1998b; Xu et al., 2000), glycinergic (Gomeza et al., 2003a, 2003b; Rousseau et al., 2008; Apostolides and Trussell, 2013) as well as some GABAergic terminals (Mathews and Diamond, 2003; Bak et al., 2006; Fricke et al., 2007; Brown and Mathews, 2010; Wang et al., 2013) rely heavily on membrane transporter function to maintain cytosolic transmitter pools available for vesicular loading. Our results show that DA neurons contain mRNA for mGAT1 and mGAT4, indicating that DA neurons might acquire GABA through membrane transport. Indeed, we find that prolonged inhibition of mGAT1 and mGAT4 inhibited GABAergic transmission by DA neurons and precipitated the rundown of oIPSCs, suggesting that DA neurons critically depend on extracellular GABA uptake to maintain cytoplasmic GABA levels and fill synaptic vesicles. By contrast, application of exogenous GABA helped sustain inhibitory transmission from DA neurons. By virtue of the fact that the vast majority of DA neurons in the SNc and lateral VTA contain mRNA for mGATs, our results suggest that GABA co-release is, unlike glutamate co-transmission (Hnasko and Edwards, 2011), a common feature of these cells in the adult nervous system. The reliance on plasma membrane uptake may partially explain why release of both DA and GABA from these neurons is prone to rundown upon repeated stimulation in brain slices (Schmitz et al., 2003; Tritsch et al., 2012; Ishikawa et al., 2013), whereas release of glutamate from dopaminergic neurons or GABA from SPNs (which presumably depend on neurotransmitter synthesis to sustain release) do not suffer from the same shortcoming (Tritsch et al., 2012; Figure 1—figure supplement 1D). Nevertheless, these findings provide evidence that the expression of GABA synthetic enzymes is not required to sustain GABAergic transmission, and identify a GABAergic synapse that instead relies entirely on recycling extracellular GABA. Interestingly, a similar mechanism underlies exocytic release of serotonin from dopaminergic neurons (Zhou et al., 2005) as well as from glutamatergic thalamocortical neurons during development (Lebrand et al., 1996).
Although DA neurons in SNc and VTA differ in the inputs they receive and the signals they convey (Matsumoto and Hikosaka, 2009; Lammel et al., 2011, 2012; Watabe-Uchida et al., 2012; Roeper, 2013), we find that both cell populations are capable of co-releasing GABA with DA, pointing to a fundamental property of dopaminergic signaling. Morphological studies of SPNs at the electron microscopic level have revealed that the majority of dopaminergic synapses terminate on the neck of spines that receive excitatory inputs from cortex and thalamus (Wickens and Arbuthnott, 2005). Although the actions of DA are not believed to be spatially localized (Arbuthnott and Wickens, 2007), this unique synaptic arrangement is indicative of an additional, point-to-point mode of action. An intriguing possibility is that GABA co-release may function to hyperpolarize spines or shunt excitatory cortical and thalamic inputs by activating GABAA receptors. Phasic activation of DA neurons may thereby dampen ongoing cortical and thalamic drive onto SPNs to limit DA receptor-mediated plastic changes to synaptic inputs most strongly activated by salient or rewarding stimuli. In addition, the mechanism adopted by DA neurons to obtain GABA may confer these cells the flexibility to dynamically and locally control GABAergic transmission across their extensive axonal arbors. For instance, VTA neurons projecting to NAc and medial prefrontal cortex may only co-release GABA in the former, where extracellular GABA levels are high compared to cortex (Drasbek and Jensen, 2006; Weitlauf and Woodward, 2008). Alternatively, phasic and chronic changes in mGAT function or extracellular GABA levels resulting from synaptic activity, drugs or disease may alter the amplitude and kinetics of GABAergic currents arising from midbrain dopamine neurons. A greater understanding of the relative effects of DA and GABA on the activity of striatal circuits will help reveal how DA neurons contribute to behavior in health and disease.
All experimental manipulations were performed in accordance with protocols approved by the Harvard Standing Committee on Animal Care following guidelines described in the US National Institutes of Health Guide for the Care and Use of Laboratory Animals. All mice were group-housed and maintained on a 12-hr light cycle with ad libitum access to food and water. Slc6a3-ires-Cre knock-in mice expressing Cre recombinase in DA neurons (Backman et al., 2006) were obtained from Jackson Labs (Bar Harbor, ME; stock # 006660). These mice were crossed with Drd2-Egfp (GENSAT, founder line S118) or Drd1a-tdTomato (stock # 016204; Jackson Labs) BAC transgenic mice to permit distinction between direct- and indirect-pathway SPNs (Gong et al., 2003; Ade et al., 2011). To genetically target indirect pathway SPNs, Adora2a-Cre BAC transgenic mice (GENSAT, founder line KG139) were used. For stereotaxic viral injections, postnatal day 18–25 mice were anesthetized with isoflurane, placed in a small animal stereotaxic frame (David Kopf Instruments, Tujunga, CA) and injected with 1 μl of an adeno-associated virus (∼1012 genome copies per ml; UNC Vector Core Facility, Chapel Hill, NC) encoding Cre-dependent ChR2 (AAV2/8.EF1α.DIO.hChR2(H134R)-mCherry or AAV2/8.EF1α.DIO.hChR2(H134R)-EYFP), as described previously (Tritsch et al., 2012). Injection coordinates were 0.8 mm anterior from Lambda, 1.3 mm lateral and 4.4 mm below pia for SNc, and 0.8 mm anterior from Lambda, 0.6 mm lateral and 4.4 mm below pia for VTA. Alternatively, ChR2(H134R)-EYFP was expressed genetically in Cre-containing cells by crossing Slc6a3-ires-Cre;Drd2-Egfp or Slc6a3-ires-Cre;Drd1a-tdTomato mice with Ai32 mice (stock # 012569; Jackson Labs) (Madisen et al., 2012). The knock-in lines used in Figure 5 include Gad1-Egfp (Tamamaki et al., 2003) and Gad2-ires-Cre mice (Taniguchi et al., 2011). The latter were crossed with mice bearing a Cre-dependent tdTomato reporter transgene (Ai14; stock # 007914; Jackson Labs) to reveal the distribution of Cre+ cells (Madisen et al., 2010). Mice were maintained on a C57BL/6 background. Only mice heterozygous for all transgenes were used for experiments.
Mice were anesthetized with isoflurane and transcardially perfused with phosphate buffered saline followed by 4% (wt/vol) paraformaldehyde in 0.1 M sodium phosphate buffer. 50-micrometer coronal brain sections were subject to immunohistochemical staining for tyrosine hydroxylase (AB152; 1:2000; Millipore, Billerica, MA), as described previously (Tritsch et al., 2012). Endogenous tdTomato and EGFP fluorescence were not immuno-enhanced. Double fluorescence in situ hybridization was performed using a tyramide signal amplification method according to the manufacturer's instructions (NEL753001KT; PerkinElmer, Waltham, MA), as previously described (Kwon et al., 2012). Briefly, brains from 4-week old mice were dissected and immediately frozen in liquid nitrogen. They were then cut in 25-μm-thick sections with a cryostat (Leica, Buffalo Grove, IL), postfixed in 4% PFA, acetylated in 1% triethanolamine and 0.25% acetic anhydride, dehydrated serially in ethanol, bleached in 3% hydrogen peroxide diluted in methanol, prehybridized, and hybridized at 65°C using the following anti-sense probes provided by the Allen Institute for Brain Science (riboprobe ID specified in parentheses): Gad1 (RP_040324_01_F01), Gad2 (RP_071018_02_B07), Vmat2 (RP_071218_02_B08), mouse Gat1 (RP_071204_04_H01), mouse Gat4 (RP_050428_04_D04), and Abat (RP_050301_02_C02). For in vitro transcription, each cDNA was cloned out from a mouse brain cDNA library using the primer sets suggested in the in situ hybridization data portal from the Allen Institute for Brain Science. Two fluorescein- or digoxigenin-labeled riboprobes generated by an in vitro transcription method (Promega, Madison, WI) were hybridized simultaneously and stained by fluorescein or Cy3 chromogens, respectively. After staining, sections were mounted with Prolong Gold antifade reagent with DAPI nuclear stain (Life Technologies, Grand Island, NY).
Acute brain slices and whole-cell voltage-clamp recordings from identified SPNs were obtained using standard methods, as described previously (Tritsch et al., 2012). Briefly, mice (49–178 days old; median = 107 days) were anesthetized and perfused with ice-cold artificial cerebrospinal fluid (ACSF) containing (in mM) 125 NaCl, 2.5 KCl, 25 NaHCO3, 2 CaCl2, 1 MgCl2, 1.25 NaH2PO4 and 11 glucose (295 mOsm·kg−1). Parasagittal slices of striatum (275-μm thick) were subsequently obtained in cold choline-based cutting solution (in mM: 110 choline chloride, 25 NaHCO3, 2.5 KCl, 7 MgCl2, 0.5 CaCl2, 1.25 NaH2PO4, 25 glucose, 11.6 ascorbic acid, and 3.1 pyruvic acid). Following 15 min recovery in ACSF at 34°C, slices were kept at room temperature (20–22°C) until use. All solutions were constantly bubbled with 95% O2/5% CO2. Whole-cell voltage-clamp recordings were established from direct- and indirect-pathway SPNs in dorsal or ventral striatum resting 18–75 μm below the slice surface (median: 40 μm) in ACSF warmed to 32–34°C. Direct-pathway SPNs were identified as tdTomato+ cells in Drd1a-tdTomato mice or EGFP–cells in Drd2-EGFP mice, and indirect-pathway SPNs as tdTomato−or EGFP+ cells in Drd1a-tdTomato and Drd2-EGFP mice, respectively. Patch pipettes (2–4 MΩ) were filled with (in mM) 125 CsCl, 10 TEA-Cl, 10 HEPES, 0.1 Cs-EGTA, 3.3 QX-314 (Cl−salt), 4 Mg-ATP, 0.3 Na-GTP, 8 Na2-Phosphocreatine (pH 7.3 adjusted with CsOH; 295 mOsm·kg−1). The recording perfusate always contained NBQX (10 μM) and R-CPP (10 μM) to block AMPA and NMDA receptor-mediated inward currents, respectively, as well as CGP55845 (2–5 μM) to prevent GABAB receptor-evoked pre- and post-synaptic modulation. For some experiments (Figures 6 and 8, Figure 8—figure supplements 1 and 2), slices were incubated for at least 30 min (and up to two hours) in ACSF containing CGP55845 (2–5 μM) in addition to vigabatrin (100 μM), muscimol (0.1 μM), or SKF 89976A (10 μM) + SNAP-5114 (50 μM) to allow for the depletion of cytosolic GABA levels and synaptic vesicles containing GABA, and to control for the effects of chronically elevated GABAergic signaling. In these cases, drugs continued to be present in the recording chamber for the duration of the experiment. For Figure 8E, GABA (100 μM, in ACSF containing either 5 μM CGP55845 or 5 μM CGP55845 + 10 μM SKF 89976A + 50 μM SNAP-5114) was locally applied to the slice via a gravity-fed 250-μm-wide flow pipe for 5–10 min prior to recording. For all voltage-clamp experiments, errors due to the voltage drop across the series resistance (<20 MΩ) were left uncompensated. Membrane potentials were corrected for a ∼5-mV liquid junction potential. Under these conditions, GABAA receptor-mediated currents appeared inward when SPNs were held at negative membrane potentials (Vhold = −70 mV). To activate ChR2-expressing fibers, light from a 473-nm laser (Optoengine, Midvale, UT) was focused on the back aperture of the microscope objective to produce wide-field illumination of the recorded cell. Brief pulses of light (1-ms duration; 5 mW·mm−2 under the objective for maximal stimulation, 0.3–2 mW·mm−2, for sub-maximal stimulation) were delivered at the recording site at 30 s intervals under control of the acquisition software. Epifluorescence illumination was used sparingly to minimize ChR2 activation prior to recording. eIPSCs were evoked using constant-current pulses (0.1 ms, 7–90 μA) delivered every 15 s through a bipolar tungsten stimulating electrodes positioned within striatum, 100–200 μm away from the recorded cell. Constant-potential amperometry was performed as before (Tritsch et al., 2012) using commercial glass-encased carbon-fiber microelectrodes (Carbostar-1, Kation Scientific, Minneapolis, MN) placed within dorsal striatum slices and held at 600 mV. All recordings were obtained within 4 hr of slicing. All pharmacological agents were obtained from Tocris (Minneapolis, MN).
Brain sections processed for in situ hybridization or immunofluorescence were imaged with an Olympus VS110 slide-scanning microscope. High-resolution images of regions of interest were subsequently acquired with a Zeiss LSM 510 META confocal microscope (Harvard NeuroDiscovery Center). Individual imaging planes were overlaid and quantified for colocalization in ImageJ (NIH) and thresholded for display in Photoshop (Adobe). Confocal images in figures represent maximum intensity projections of 3-µm confocal stacks. Membrane currents were amplified and low-pass filtered at 3 kHz using a Multiclamp 700B amplifier (Molecular Devices, Sunnyvale, CA), digitized at 10 kHz and acquired using National Instruments acquisition boards and a custom version of ScanImage (Pologruto et al., 2003; available upon request or from https://openwiki.janelia.org/wiki/display/ephus/ScanImage) written in MATLAB (Mathworks). Electrophysiology data were analyzed offline using Igor Pro (Wavemetrics, Natick, MA). In figures, voltage-clamp traces represent the averaged waveform of 3–5 consecutive acquisitions. Averaged waveforms were used to obtain current latency, peak amplitude, 10–90% rise time and decay time constant. The latter was estimated by measuring the time elapsed from the peak of the IPSC to 1/e (36.8%) of the peak amplitude. Detection threshold for sIPSCs was set at 40 pA to facilitate event detection in the presence of large and noisy tonic GABA currents. For pharmacological analyses in Figures 2 and 3, the peak amplitude of IPSCs measured 3–4 min following the onset of drug perfusion were averaged, normalized to baseline averages obtained immediately prior to drug application and compared statistically to values obtained at corresponding times in control preparations bathed in ACSF. Data (reported in text and figures as mean ± SEM) were compared using Prism 6 (GraphPad, La Jolla, CA) with the following non-parametric statistical tests (as indicated in the text): Mann–Whitney for group comparisons, Wilcoxon signed-rank for paired comparisons, and Kruskal–Wallis analysis of variance (ANOVA) followed by Dunn's Multiple Comparison Test for multiple group comparisons. In experiments characterizing the time course of synaptic transmission rundown, two-way ANOVAs were used followed by the Sidak's and Tukey's multiple comparison tests for comparisons between two and more conditions, respectively. p values smaller than 0.05 were considered statistically significant. N values represent the number of recorded cells. For most experiments, a single cell was recorded from in each slice, with each animal contributing fewer than five recordings to individual data sets.
Differential tonic GABA conductances in striatal medium spiny neuronsThe Journal of Neuroscience: the Official Journal of the Society for Neuroscience 28:1185–1197.https://doi.org/10.1523/JNEUROSCI.3908-07.2008
Rapid, activity-independent turnover of vesicular transmitter content at a mixed glycine/GABA synapseThe Journal of Neuroscience: the Official Journal of the Society for Neuroscience 33:4768–4781.https://doi.org/10.1523/JNEUROSCI.5555-12.2013
Cleft palate and decreased brain gamma-aminobutyric acid in mice lacking the 67-kDa isoform of glutamic acid decarboxylaseProceedings of the National Academy of Sciences of the United States of America 94:6496–6499.https://doi.org/10.1073/pnas.94.12.6496
Characterization of a mouse strain expressing Cre recombinase from the 3' untranslated region of the dopamine transporter locusGenesis: the Journal of Genetics and Development 44:383–390.
The operation of the gamma-aminobutyrate bypath of the tricarboxylic acid cycle in brain tissue in vitroThe Biochemical Journal 116:445–461.
Neurosteroids: endogenous regulators of the GABA(A) receptorNature Reviews Neuroscience 6:565–575.https://doi.org/10.1038/nrn1703
Altered brain serotonin homeostasis and locomotor insensitivity to 3, 4-methylenedioxymethamphetamine (“Ecstasy”) in serotonin transporter-deficient miceMolecular Pharmacology 53:649–655.
Gamma-Aminobutyric acid-glutamic acid transamination in brainThe Journal of Biological Chemistry 201:385–391.
Dopamine neuron systems in the brain: an updateTrends in Neurosciences 30:194–202.
GABA transporter heterogeneity: pharmacology and cellular localizationNeurochemistry International 29:335–356.
Distinct modes of dopamine and GABA release in a dual transmitter neuronThe Journal of Neuroscience: the Official Journal of the Society for Neuroscience 33:1790–1796.https://doi.org/10.1523/JNEUROSCI.4342-12.2013
GAT-1 regulates both tonic and phasic GABA(A) receptor-mediated inhibition in the cerebral cortexJournal of Neurochemistry 105:1781–1793.https://doi.org/10.1111/j.1471-4159.2008.05273.x
GABA and its relationship to putrescine metabolism in the rat brain and pancreasNeurochemistry International 10:219–229.
Multiple sources of striatal inhibition are differentially affected in Huntington's disease mouse modelsThe Journal of Neuroscience: the Official Journal of the Society for Neuroscience 33:7393–7406.https://doi.org/10.1523/JNEUROSCI.2137-12.2013
GABA transporter deficiency causes tremor, ataxia, nervousness, and increased GABA-induced tonic conductance in cerebellumThe Journal of Neuroscience: the Official Journal of the Society for Neuroscience 25:3234–3245.https://doi.org/10.1523/JNEUROSCI.3364-04.2005
GABA transporters in the mammalian cerebral cortex: localization, development and pathological implicationsBrain Research Brain Research Reviews 45:196–212.https://doi.org/10.1016/j.brainresrev.2004.03.003
Plastic corticostriatal circuits for action learning: what's dopamine got to do with it?Annals of the New York Academy of Sciences 1104:172–191.https://doi.org/10.1196/annals.1390.015
Comparative localization of mRNAs encoding two forms of glutamic acid decarboxylase with nonradioactive in situ hybridization methodsThe Journal of Comparative Neurology 331:339–362.https://doi.org/10.1002/cne.903310305
Modulation of striatal projection systems by dopamineAnnual Review of Neuroscience 34:441–466.https://doi.org/10.1146/annurev-neuro-061010-113641
Colocalization of tyrosine hydroxylase and GAD65 mRNA in mesostriatal neuronsThe European Journal of Neuroscience 13:57–67.https://doi.org/10.1046/j.1460-9568.2001.01371.x
Response of the GABAergic and dopaminergic mesostriatal projections to the lesion of the contralateral dopaminergic mesostriatal pathway in the ratMovement Disorders: official Journal of the Movement Disorder Society 19:1029–1042.
Corelease of dopamine and GABA by a retinal dopaminergic neuronThe Journal of Neuroscience: the Official Journal of the Society for Neuroscience 32:13281–13291.https://doi.org/10.1523/JNEUROSCI.2213-12.2012
Neurotransmitter corelease: mechanism and physiological roleAnnual Review of Physiology 74:225–243.https://doi.org/10.1146/annurev-physiol-020911-153315
Dopamine modulation of GABA tonic conductance in striatal output neuronsThe Journal of Neuroscience: the Official Journal of the Society for Neuroscience 29:5116–5126.https://doi.org/10.1523/JNEUROSCI.4737-08.2009
Muscimol uptake, release and binding in rat brain slicesJournal of Neurochemistry 31:1519–1523.https://doi.org/10.1111/j.1471-4159.1978.tb06579.x
Defining affinity with the GABAA receptorThe Journal of Neuroscience: the Official Journal of the Society for Neuroscience 18:8590–8604.
Profound neuronal plasticity in response to inactivation of the dopamine transporterProceedings of the National Academy of Sciences of the United States of America 95:4029–4034.https://doi.org/10.1073/pnas.95.7.4029
Vesicular GABA transporter (VGAT) transports beta-alanineJournal of Neurochemistry 127:482–486.https://doi.org/10.1111/jnc.12393
GABA transporter 1 tunes GABAergic synaptic transmission at output neurons of the mouse neostriatumThe Journal of Physiology 586:5665–5678.https://doi.org/10.1113/jphysiol.2008.161943
Neuroligin-1-dependent competition regulates cortical synaptogenesis and synapse numberNature Neuroscience 15:1667–1674.https://doi.org/10.1038/nn.3256
Olfactory bulb short axon cell release of GABA and dopamine produces a temporally biphasic inhibition-excitation response in external tufted cellsThe Journal of Neuroscience: the Official Journal of the Society for Neuroscience 33:2916–2926.https://doi.org/10.1523/JNEUROSCI.3607-12.2013
Co-transmission of dopamine and GABA in periglomerular cellsJournal of Neurophysiology 99:1559–1564.https://doi.org/10.1152/jn.00636.2007
Neuronal glutamate uptake Contributes to GABA synthesis and inhibitory synaptic strengthThe Journal of Neuroscience: the Official Journal of the Society for Neuroscience 23:2040–2048.
Striatal action-learning based on dopamine concentrationExperimental Brain Research 200:307–317.https://doi.org/10.1007/s00221-009-2060-6
Distinct activities of GABA agonists at synaptic- and extrasynaptic-type GABAA receptorsThe Journal of Physiology 588:1251–1268.https://doi.org/10.1113/jphysiol.2009.182444
Paradoxical reduction of synaptic inhibition by vigabatrinJournal of Neurophysiology 86:596–603.
Synapse density regulates independence at unitary inhibitory synapsesThe Journal of Neuroscience: the Official Journal of the Society for Neuroscience 23:2618–2626.
ScanImage: flexible software for operating laser scanning microscopesBiomedical Engineering Online 2:13.https://doi.org/10.1186/1475-925X-2-13
Goal-directed and habitual control in the basal ganglia: implications for Parkinson's diseaseNature Reviews Neuroscience 11:760–772.https://doi.org/10.1038/nrn2915
Transamination of gamma-aminobutyric acid and beta-alanine in brain and liverThe Journal of Biological Chemistry 201:393–398.
Dissecting the diversity of midbrain dopamine neuronsTrends in Neurosciences 36:336–342.https://doi.org/10.1016/j.tins.2013.03.003
The glycine transporter GlyT2 controls the dynamics of synaptic vesicle refilling in inhibitory spinal cord neuronsThe Journal of Neuroscience: the Official Journal of the Society for Neuroscience 28:9755–9768.https://doi.org/10.1523/JNEUROSCI.0509-08.2008
Presynaptic regulation of dopaminergic neurotransmissionJournal of Neurochemistry 87:273–289.https://doi.org/10.1046/j.1471-4159.2003.02050.x
Multiple dopamine functions at different time coursesAnnual Review of Neuroscience 30:259–288.https://doi.org/10.1146/annurev.neuro.28.061604.135722
On the role of GABA in vertebrate polyamine metabolismPhysiological Chemistry and Physics 12:411–429.
GABA is localized in dopaminergic synaptic vesicles in the rodent striatumBrain Structure & Function, 10.1007/s00429-013-0609-4.
Dopaminergic terminals in the nucleus accumbens but not the dorsal striatum corelease glutamateThe Journal of Neuroscience: the Official Journal of the Society for Neuroscience 30:8229–8233.https://doi.org/10.1523/JNEUROSCI.1754-10.2010
Green fluorescent protein expression and colocalization with calretinin, parvalbumin, and somatostatin in the GAD67-GFP knock-in mouseThe Journal of Comparative Neurology 467:60–79.https://doi.org/10.1002/cne.10905
Short external loops as potential substrate binding site of gamma-aminobutyric acid transportersThe Journal of Biological Chemistry 270:28712–28715.
Glutamatergic signaling by mesolimbic dopamine neurons in the nucleus accumbensThe Journal of Neuroscience: the Official Journal of the Society for Neuroscience 30:7105–7110.https://doi.org/10.1523/JNEUROSCI.0265-10.2010
The role of the synthetic enzyme GAD65 in the control of neuronal gamma-aminobutyric acid releaseProceedings of the National Academy of Sciences of the United States of America 96:12911–12916.https://doi.org/10.1073/pnas.96.22.12911
Plasma membrane monoamine transporters: structure, regulation and functionNature Reviews Neuroscience 4:13–25.https://doi.org/10.1038/nrn1008
Ethanol selectively attenuates NMDAR-mediated synaptic transmission in the prefrontal cortexAlcoholism, Clinical and Experimental Research 32:690–698.https://doi.org/10.1111/j.1530-0277.2008.00625.x
Structural and functional interactions in the striatum at the receptor levelIn: SB Dunnett et al., editors. Structural and functional interactions in the striatum at the receptor level, vol 21. San Diego: Elsevier. pp. 199–236.
Mice lacking the norepinephrine transporter are supersensitive to psychostimulantsNature Neuroscience 3:465–471.https://doi.org/10.1038/74839
Gary L WestbrookReviewing Editor; Vollum Institute, United States
eLife posts the editorial decision letter and author response on a selection of the published articles (subject to the approval of the authors). An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent (see review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.
Thank you for sending your work entitled “Midbrain dopamine neurons sustain inhibitory transmission using plasma membrane uptake of GABA, not synthesis” for consideration at eLife. Your article has been favorably evaluated by a Senior editor and 3 reviewers, one of whom is a member of our Board of Reviewing Editors. The Reviewing editor and the other reviewers discussed their comments before reaching this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.
This relatively straightforward report follows up on prior work of these authors examining the corelease of GABA from “dopamine” neurons. In this report they extend their studies from the nigrostriatal pathway to the mesolimbic pathway. Interestingly GABA is supplied not by synthetic enzymes in the neurons, but rather by expression of membrane GABA transporters. As previously shown, vesicular uptake of GABA can be performed by the vesicular monamine transporter VMAT2. The questions addressed in this manuscript are whether such corelease occurs in the mesolimbic pathway and whether the agonist is GABA or a related analog.
The authors nicely demonstrate in Figure 1 that VTA inputs to NAc have similar properties with respect to GABA corelease as the previously demonstrated SNc inputs to dorsal striatum. The majority of the figures then focus on the identity and source of GABA in the VTA-NAc pathway. These experiments are carefully conducted and well presented and convincingly demonstrate that GABA is the principal agonist and that the main source is membrane uptake rather than intracellular synthesis. Although these results are somewhat predictable from prior studies and could be further extended, the results are important in understanding the rather novel, albeit still somewhat mysterious, role of a fast-acting transmitter in midbrain dopamine neurons, and thus should be of interest to a broad community of neuroscientists. The substantial comments of the 3 reviewers are numbered below.
1) These data make several specific, testable predictions that would enhance the strength of the main interpretation. For example, increasing extracellular GABA should increase the amplitude of DA-IPSCs through an mGAT-dependent mechanism. Does bath application of GABA, or perhaps extracellular GABA uncaging, increase DA-IPSCs? And is this effect blocked by the acute presence of mGAT blockers during said GABA application? Wang et al. 2013 (cited) showed that GAT activity can rescue transmission in conventional GABAergic synapses when GABA is bath applied in the presence of gabazine. This experiment would reveal the timecourse over which DA neurons take up GABA from the extracellular space and package it into synaptic vesicles, and it would be important to contrast the timecourse of the authors' novel mechanism with that of enzymatic synthesis at more traditional GABA synapses. This experiment would enhance also the claim that GAT supplies the GABA used for synaptic vesicles.
2) A second prediction is that rundown of transmission induced by SKF should be activity dependent. I found it confusing at first that Figure 2 shows little effect of SKF on IPSC amplitude and Figure 7 shows a large effect, and I am sure readers will also be caught off guard. The difference is that with time the synapses in SKF are depleted of GABA. Therefore if the synapses were stimulated one might expect to accelerate the process, confirming that it reflects depletion of vesicular GABA rather than a side effect of the drug.
3) The authors deal with a number of potential confounding factors such as the increased tonic GABA currents produced by GABA transporter inhibition, showing that this does not affect the kinetics of quantal GABA events from sources other than dopamine neurons. On the other hand, it is remarkable that the inhibitor does not affect the kinetics of these other events – is it so selective for GABA release from dopamine neurons because the transporter is only expressed on those terminals? It would be helpful if the authors could address this surprising result – previous work has indicated expression of GAT-1 by SPNs and an effect of a different inhibitor (NO-711) on amplitude and decay time of events evoked by local depolarization (Kirmse et al. 2008).
4) Treatment with GAT-1 inhibitor for longer times effectively eliminates the GABA release by dopamine neurons, as anticipated if it depends on the GABA taken up from other cells. However, it is also possible that dopamine neurons make GABA themselves, and the main argument used here against this is the apparent lack of expression by a variety of criteria. Although these seem reasonable, other work cited by the authors in a different context (Stensrud et al. 2013) along with other work they do cite suggests expression of GAD by a subset of dopamine neurons. Rather than rely on the anatomy, it would help greatly to demonstrate that inactivation of GAD specifically in dopamine neurons does not eliminate the dopaminergic IPSCs. This might require a substantial amount of work that would lie outside the scope of this manuscript (for example a double knockout of both isoforms that is conditional and may not even be available), but it would certainly be helpful. However, showing that expression of a GAD isoform in dopamine neurons makes the dopaminergic IPSCs resistant to inhibition of GAT-1 would be considerably simpler.
5) The authors also suggest that GABA corelease involves both mesostriatal and mesolimbic projections, in contrast to the apparent corelease of glutamate by a subset of VTA neurons. However, Stensrud et al. (2013) also showed that only ∼10 % of striatal terminals apparently contain GABA, raising the question whether the dopaminergic IPSCs derive from a subset of both populations, or all of them. It would be great if the authors could address this point, and one way might be to determine whether GAT-1 inhibition influences dopamine release measured by voltammetry-GABA corelease by many terminals should have some effect on total dopamine release, but involvement of only a subset would produce minimal if any effect. This would also help to address one important functional outcome of corelease that has not yet been examined.https://doi.org/10.7554/eLife.01936.016
- Bernardo L Sabatini
- Nicolas X Tritsch
- Won-Jong Oh
- Won-Jong Oh
- Bernardo L Sabatini
- Chenghua Gu
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
We thank R Shah, N Mulder, and R Pemberton for technical support and members of the Sabatini laboratory for discussions throughout the course of this study.
Animal experimentation: This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All experimental manipulations were performed in accordance with protocols approved by the Harvard Medical Area Standing Committee on Animal Care (#03551).
- Gary L Westbrook, Reviewing Editor, Vollum Institute, United States
© 2014, Tritsch et al.
This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.