VGLUT2 localizes almost exclusively to synaptic vesicles and hence to nerve terminals, making it difficult to identify the cell bodies which express VGLUT2 by immunostaining. To circumvent this problem, we used bacterial artificial chromosome (BAC) transgenic mice expressing enhanced green fluorescent protein (EGFP) under the control of VGLUT2 regulatory sequences (Gong et al., 2003
). Since EGFP is a soluble protein, it fills the cell bodies of neurons which express it, and the BAC transgenic mice exhibit appropriate expression of the EGFP reporter in the cell populations well established to express endogenous VGLUT2 (data not shown). We therefore used adult VGLUT2-EGFP mice to assess expression of VGLUT2 by mature midbrain dopamine neurons. shows that although many VTA neurons labeling for tyrosine hydroxylase (TH) do not express EGFP, and some EGFP+
neurons do not contain TH (Yamaguchi et al., 2007
), a substantial number of neurons express both TH and EGFP, consistent with previous reports indicating expression of VGLUT2 mRNA by a subset of VTA dopamine neurons in vivo
(Kawano et al., 2006
; Mendez et al., 2008
Histochemical analysis of VGLUT2-EGFP BAC transgenic mice and the conditional VGLUT2 knockout
To determine the function of VGLUT2 in midbrain dopamine neurons, we used homologous recombination in embryonic stem cells to produce a conditional allele of the mouse Slc17a6
gene encoding VGLUT2, with exon 2 surrounded by loxP sites (Fig. S1A
). After excision of the positive selectable marker at flanking FRT sites by crossing to mice that express germ line flp recombinase (Farley et al., 2000
), the resulting VGLUT2+/lox
animals were bred to mice expressing germ line cre recombinase (Tallquist and Soriano, 2000
), excising exon 2 to cause a frameshift that disrupts translation. Since VGLUT2 knockout mice die immediately after birth from respiratory failure (Moechars et al., 2006
; Wallen-Mackenzie et al., 2006
), we assessed the loss of VGLUT2 using brains from homozygous VGLUT2Δ/Δ
embryos at 14.5–18.5 days gestation. Cre-mediated recombination eliminated VGLUT2 expression, as shown by western blot (Fig. S1C
) and immunohistochemistry (IHC) (). In addition, VGLUT2lox/lox
mice show no difference from wild type (WT) littermates in brain VGLUT2 expression (not shown), indicating that the residual FRT and loxP sites do not interfere with normal expression.
To disrupt VGLUT2 expression specifically in dopamine neurons, we crossed mice containing the conditional VGLUT2+/lox
allele to mice that express cre recombinase under the control of regulatory sequences from the Slc6a3
gene that encodes the plasma membrane dopamine transporter (DAT) (Zhuang et al., 2005
). To assess the specificity of recombination () and identify the dopamine neurons with targeted disruption of the VGLUT2 gene, the mice carried one copy of the cre-inducible floxed-stop yellow fluorescent protein (fsYFP) reporter (Srinivas et al., 2001
). For all experiments, we compared control mice [Slc17a6+/lox
] to cKO littermates [Slc17a6lox/lox
]. cKO mice were born at the expected mendelian ratios and showed no obvious difference from littermate controls.
VGLUT2 Mediates Glutamate Corelease by Dopamine Neurons In Vitro and In Vivo
Previous work has shown that dopamine neurons grown in culture form glutamatergic synapses (Joyce and Rayport, 2000
; Sulzer et al., 1998
) and express VGLUT2 (Dal Bo et al., 2004
). To determine whether the loss of VGLUT2 from dopamine neurons affects glutamate release, we first made postnatal (P1–P3) midbrain cultures, which contain a high proportion of tyrosine hydroxylase (TH)-positive dopamine neurons. Double staining for TH and YFP showed nearly complete colocalization (), indicating that cre-mediated recombination had occurred in all dopamine neurons. We then double stained for YFP as well as the transporter, observing VGLUT2 immunoreactivity at synaptic boutons made by YFP+
dopamine neurons from control, but not cKO mice (). Conditional inactivation of the gene encoding VGLUT2 thus results in the loss of VGLUT2 specifically from dopamine neurons.
The selective deletion of VGLUT2 from dopamine neurons eliminates glutamatergic currents observed in autaptic cultures
To determine whether VGLUT2 is required for glutamate release by dopamine neurons, we produced single-cell cultures of dopamine neurons that form synapses onto themselves (autapses), and examined the glutamatergic EPSCs evoked by brief depolarization. In autapses from control neurons, stimulation evoked EPSCs in 10/11 YFP+ dopamine neurons. However, EPSCs could not be evoked in any of the 8 YFP+ cKO cells examined (). The cKO thus eliminates VGLUT2 from dopamine neurons, and at least in vitro, VGLUT2 is required for the corelease of glutamate by dopamine neurons.
We then used a mesoaccumbens slice preparation to assess a role for VGLUT2 in glutamate release by dopamine neurons in vivo
. In this preparation, direct stimulation of the VTA elicits fast, glutamatergic EPSCs in MSNs of the NAc shell (Chuhma et al., 2004
), enabling a distinction between glutamatergic input originating in the midbrain from abundant glutamatergic projections originating in cortex and thalamus (Fujiyama et al., 2006
). Using this preparation, we observed a dramatic reduction in the glutamatergic EPSCs of postnatal day 9–11 (P9-11) cKO mice relative to control littermates (). Only 2.6% (1/39) of NAc neurons responded to VTA stimulation with an EPSC >2 pA in cKO mice, compared to 28% (19/67) of neurons from controls. Since dopamine neurons from older animals may express lower levels of VGLUT2 (Berube-Carriere et al., 2009
; Mendez et al., 2008
), we also examined mesoaccumbens slices from P21-23 mice. We again observed a major reduction in the evoked EPSC amplitude in cKO slices relative to controls (). VTA stimulation produced an EPSC >2 pA in 33% (15/46) of NAc neurons from control slices, but in only 5.4% (4/74) of cKO neurons. Since the cKO removes VGLUT2 selectively from dopamine neurons, dopamine neurons from wild-type mice must both express VGLUT2 and corelease glutamate in vivo
Mice lacking VGLUT2 in dopamine neurons show reduced glutamatergic currents in NAc neurons in response to VTA stimulation
In this mesoaccumbens slice preparation, only a minority of dopaminergic projections remain intact, so EPSCs <2 pA could reflect either defective glutamate release or the lack of intact dopamine neuron projections to the recorded MSN. Since we could not discriminate between these two possibilities, we included data from all recorded MSNs, including those without any EPSC (). It is also important to note that the littermate controls used for these experiments [Slc17a6+/lox; Slc6a3+/Cre] had undergone recombination of one VGLUT2 allele specifically in dopamine neurons, and were therefore heterozygous for VGLUT2. Consistent with this, the mean evoked EPSC amplitude of NAc neurons from these control animals was ~50% that of age-matched mice wild type at the VGLUT2 locus [Slc17a6+/+; Slc6a3+/Cre] (data not shown).
To assess the specificity of the cKO for VGLUT2 expressed by dopamine neurons, we examined synaptic input to the VTA, much of which depends on VGLUT2 (Geisler et al., 2007
; Omelchenko and Sesack, 2007
). Stimulating afferent projections to the VTA, we found that mean evoked EPSC amplitude in the midbrain does not differ between control and cKO animals (). We also observed no significant difference in the amplitude () or frequency () of spontaneous EPSCs recorded from VTA dopamine neurons. Although the cKO impairs glutamatergic transmission by dopaminergic efferents, it thus does not affect afferent inputs to these same cells.
The Conditional Knockout Reduces Cocaine-Induced Locomotor Activity, Dopamine Storage and Release in the Ventral Striatum
To assess the utility of glutamate corelease, we examined a series of behaviors in cKO mice and littermate controls. Monitoring spontaneous activity, cKO animals showed no significant difference in either novelty-associated locomotion over 4 hours, or in total locomotor activity over the following 3 days (). The cKO mice also exhibited no significant difference in the ability to perform or learn an accelerating rotarod task performed daily for 5 days (). Thus, the loss of VGLUT2 from dopamine neurons does not affect spontaneous activity, coordination or motor learning.
Conditional knockout mice show reduced locomotor response to cocaine
Cocaine increases extracellular dopamine by inhibiting its reuptake, stimulating locomotor activity through a mechanism that depends first on exocytotic dopamine release (Torres et al., 2003
; Uhl et al., 2002
). When challenged with 20 mg/kg cocaine, cKO mice showed significantly less locomotor activity than littermate controls (). However, repeated daily injections with cocaine still elicited behavioral sensitization in cKO mice, and after several days, locomotor responses became indistinguishable from controls (, S2A
). Despite the initially reduced effect of cocaine on locomotion, cKO mice formed a conditioned place preference (CPP) to cocaine, suggesting that at least some forms of reward and associative learning remain intact (Fig. S2B,C
Since cocaine-induced locomotion involves the extracellular accumulation of dopamine released by exocytosis, the initially reduced locomotor response might result from reduced vesicular stores. We thus measured striatal dopamine content, which closely reflects vesicular stores. Although dopamine levels in the dorsal striatum did not differ between cKO and littermate controls (), the levels of dopamine and its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) all showed a 30–40% reduction in the ventral striatum (including olfactory tubercle and NAc core as well as shell) of cKO mice ().
Reduced dopamine tissue content and evoked dopamine release in the ventral striatum of cKO mice
To assess directly the vesicular release of dopamine, we used fast-scan cyclic voltammetry (FSCV) (Wightman and Zimmerman, 1990
) to measure the kinetics and amount of dopamine release evoked by a single action potential in striatal slices. Due to the heterogeneity of dopaminergic innervation, the results from three sites in the ventral striatum (specifically, within the NAc shell) of each slice were measured and averaged. Relative to control littermates, cKO mice showed a 23% reduction in the dopamine release evoked by a single action potential in the ventral striatum, with no change in dorsal striatum ().
To assess a potential effect on dopamine reuptake, we examined the kinetics of release at higher time resolution by amperometry. Similar to FSCV, amperometry showed a reduction in dopamine release from the NAc shell of cKO mice (28.7 ± 5 pA) relative to control (52.7 ± 5.4 pA) (n=20/genotype and p<0.001 by paired two-tailed t-test). The rates of dopamine clearance did not differ significantly between genotypes, but a trend toward slower clearance (τ=407 ± 34 ms for cKO mice and 354 ± 26 ms for control, n = 20/genotype, p=0.057 by paired t-test) argues against increased clearance as a mechanism for reduced release in the slice.
To determine whether changes in dopamine production or terminal density might account for these differences, we quantified expression of the biosynthetic enzyme TH by fluorescent western analysis. The ventral striatum of cKO and control mice showed no difference in the amount of TH protein (Fig. S3A
). We also assessed functional TH activity in vivo
, administering the aromatic acid decarboxylase (AADC) inhibitor NSD-1015 and measuring the accumulation of L-dopa in the ventral striatum. NSD-1015 increased tissue L-dopa levels as anticipated, but cKO mice did not differ from controls in L-dopa accumulation (Fig. S3B
Glutamate Stimulates Monoamine Uptake by VMAT2
Previous work has shown that glutamate uptake increases ΔpH across the synaptic vesicle membrane (Cidon and Sihra, 1989
; Maycox et al., 1988
). Since uptake by the vesicular monoamine transporters (VMATs) depends predominantly on ΔpH (Johnson, 1988
), transport of glutamate into the same vesicles might thus be expected to stimulate monoamine uptake. The loss of glutamate transport in VGLUT2 cKO mice may therefore account for the reduced dopamine content in ventral striatum (). However, this mechanism requires that VMAT2 and VGLUT2 localize to an overlapping population of synaptic vesicles.
To assess the colocalization of VGLUT2 with VMAT2 on synaptic vesicles, we performed immunoisolations using a light membrane fraction from the ventral striatum. We used rats for these experiments due to the greater availability of tissue, and the limited cross-reactivity of several antibodies for mouse proteins. As anticipated, antibodies to the ubiquitous synaptic vesicle protein synaptophysin isolated significant amounts of VMAT2 (). Antibodies to VGLUT2 also precipitated most of the VMAT2 immunoreactivity, indicating that many of the VMAT2+ vesicles also contain VGLUT2. An antibody to Golgi matrix protein GM130, as well as controls lacking a primary antibody, failed to precipitate VMAT2, demonstrating the specificity of the immunoisolation. Reciprocal experiments using antibodies to VGLUT2 and synaptophysin also isolated large amounts of VGLUT2 (). VMAT2 antibodies isolated more VGLUT2 than negative controls, but the efficiency of VGLUT2 immuno-isolation by VMAT2 appears low, presumably because most VGLUT2 in the ventral striatum originates in the thalamus (Fujiyama et al., 2006
), which does not express VMAT2.
Glutamate co-transport stimulates monoamine uptake into synaptic vesicles
To determine whether glutamate co-transport has the potential to stimulate monoamine uptake, we examined the effect of VGLUT2 co-expression on VMAT2 activity. In HEK293 cell membranes expressing both transporters, the addition of 10 mM glutamate stimulated monoamine uptake ~15% relative to 10 mM aspartate (), which is not recognized by VGLUTs (Naito and Ueda, 1985
; Takamori, 2006
). We then examined the effect of glutamate on monoamine uptake by synaptic vesicles from the ventral striatum (including olfactory tubercle, NAc core and shell). Again, glutamate stimulated monoamine uptake into native synaptic vesicles ~20% relative to aspartate (), providing additional, functional evidence for the colocalization of a VGLUT with VMAT2. Since only a fraction of dopamine synapses are likely to contain VGLUT2 (Berube-Carriere et al., 2009
; Kawano et al., 2006
; Mendez et al., 2008
), the effect of glutamate on monoamine storage that we observe may underestimate the full effect on those vesicles that contain both transporters.
Synaptic Vesicles Acidified with Glutamate or Chloride Differ in the Magnitude and Stability of ΔpH
Both glutamate and Cl−
appear to increase ΔpH by dissipating ΔΨ, thereby reducing the efflux of H+
driven by membrane potential and/or secondarily activating the H+
pump. Glutamate may thus stimulate monoamine uptake through a mechanism redundant with Cl−
. Indeed, Cl−
occurs at substantially higher concentrations than glutamate in most cells. To determine whether glutamate and Cl−
differ in their uptake by synaptic vesicles, we monitored vesicle acidification using acridine orange, a dye quenched at low pH (Schuldiner et al., 1972
). Since Cl−
and glutamate have both been shown to acidify synaptic vesicles from the whole brain, we used this mixed population due to the large amount of material required for these studies. Adding ATP to activate the H+
pump, we observed relatively little acidification in the absence of an anion. Because VGLUT activity depends on allosteric activation by low concentrations of Cl−
, we then added 2 mM Cl−
, a concentration that produces relatively little acidification on its own. The subsequent addition of either Cl−
or glutamate caused a greater increase in acidification (). At low concentrations, however, glutamate produced much more acidification than equimolar Cl−
(). Indeed, as little as 0.125 mM glutamate increased ΔpH more than 4 mM Cl−
. On the other hand, increasing concentrations of Cl−
caused progressively greater increases in ΔpH, whereas the effects of glutamate saturated at 3–5 mM (), consistent with the known Km of VGLUTs (~1–3 mM) (Fremeau et al., 2004
; Takamori, 2006
). High concentrations of Cl−
thus increase ΔpH more than high concentrations of glutamate. The kinetics of acidification also differed between the two anions. Although low concentrations of glutamate acidified to a greater extent than equivalent concentrations of Cl−
acidified faster even at these low concentrations, and the difference in rate was more apparent at high concentrations (). Since recent work at the calyx of Held has demonstrated that the concentration of Cl−
at the nerve terminal can reach ~20 mM (Price and Trussell, 2006
), we also tested the ability of 4 mM glutamate to acidify vesicles in the presence of 20 mM Cl−
. We found that glutamate acidifies synaptic vesicles even after the addition of 20 mM Cl−
(). Glutamate also has a clear effect on peak ΔpH when added simultaneously with Cl−
(). However, the decline in ΔpH that followed this peak was less pronounced in vesicles acidified with glutamate than in those acidified with Cl−
(), suggesting that the anion responsible for acidification influences the stability of ΔpH.
Chloride and glutamate acidify synaptic vesicles through distinct mechanisms
To assess specifically the role of the anion in ΔpH stability, we took advantage of the vacuolar H+-ATPase inhibitor bafilomycin. When bafilomycin inhibits the H+ pump, ΔpH immediately starts to collapse, enabling us to compare the rate of collapse for synaptic vesicles acidified with Cl− versus glutamate. Since Cl− and glutamate acidify to differing extents, we used concentrations of each that produce equivalent peak ΔpH (). After acidification with 14 mM Cl−, the addition of bafilomycin rapidly dissipated ΔpH, as anticipated. However, bafilomycin produced a much slower alkalinization of synaptic vesicles acidified with 4 mM glutamate (). For equivalent acidification, glutamate thus produces a more stable ΔpH than Cl−. This effect presumably accounts for the slower decline in ΔpH observed after peak acidification even with an active H+ pump ().
The differential stability of ΔpH in vesicles acidified with Cl− and glutamate might reflect either differences in buffering by the two anions, or differences in the mechanism of anion flux. The much higher pKa of glutamate (4.3) than Cl− (−9) suggests that glutamate may delay alkalinization simply by acting as a buffer for vesicle pH after inhibition of the H+ pump. On the other hand, dissipation of ΔpH requires a ΔΨ sufficiently positive to promote H+ efflux, and since anion efflux produces positive ΔΨ, differences in the mechanism of anion translocation may influence the rate of ΔpH collapse. To distinguish between these possibilities, we determined whether the imposition of a lumen positive ΔΨ accelerates dissipation of ΔpH. Synaptic vesicles were acidified in the presence of 10 mM K+ gluconate with ATP, 2 mM Cl− and then either mM Cl− or 4 mM glutamate (). In vesicles acidified with Cl−, the K+ ionophore valinomycin had no clear effect on the dissipation of ΔpH by bafilomycin (), indicating that the development of ΔΨ and hence anion efflux did not limit the rate of H+ efflux. In contrast, valinomycin greatly accelerated the dissipation of ΔpH triggered by bafilomycin in vesicles acidified with glutamate (). Thus, ΔΨ can limit the efflux of H+ from vesicles acidified with glutamate, suggesting that the mechanism of anion flux contributes to the stability of ΔpH. Under all conditions, however, vesicles acidified with glutamate lost ΔpH more slowly than those acidified with Cl− (), suggesting that glutamate may also act as a buffer to slow the dissipation of ΔpH.
Membrane potential limits proton efflux in vesicles acidified by glutamate
The distinct roles of Cl−
and glutamate in synaptic vesicle acidification suggest that glutamate also has a specific role in the stimulation of monoamine uptake. However, the experiments demonstrating stimulation of monoamine uptake by glutamate were performed first with only 2 mM Cl−
(), a concentration sufficient to provide the well-established allosteric activation of VGLUTs (Hartinger and Jahn, 1993
; Wolosker et al., 1996
) but well below concentrations relevant for the nerve terminal. Consistent with previous results using synaptic vesicles (Erickson et al., 1990
), we indeed found that 20 mM Cl−
produces a substantial increase in monoamine uptake relative to 2 mM (). Nonetheless, glutamate stimulated monoamine uptake at 20 mM Cl−
to the same extent it does at 2 mM (), supporting a role for glutamate distinct from Cl−