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The ventral tegmental area (VTA) contributes to reward and motivation signaling. In addition to the well established populations of dopamine (DA) or gamma-aminobutyric acid (GABA) VTA neurons, glutamatergic neurons were recently discovered in the VTA. These glutamatergic neurons express the vesicular glutamate transporter 2, VGluT2. To investigate whether VTA glutamatergic neurons establish local synapses, we tagged axon terminals from resident VTA neurons by intra-VTA injection of Phaseolus vulgaris- leucoagglutinin (PHAL) or an adeno-associated virus encoding wheat germ agglutinin (WGA), and by immuno electron microscopy determined the presence of VGluT2 in PHAL- or WGA-positive terminals. We found that PHAL- or WGA-positive terminals from tagged VTA cells made asymmetric or symmetric synapses within the VTA. VGluT2 immunoreactivity was detected in the vast majority of PHAL- or WGA-positive terminals forming asymmetric synapses. These results indicate that both VTA glutamatergic and non-glutamatergic (likely GABAergic) neurons establish local synapses. To examine the possible DAergic nature of postsynaptic targets of VTA glutamatergic neurons, we did triple immunolabeling with antibodies against VGluT2, tyrosine hydroxylase (TH), and PHAL. From triple labeled tissue, we found that double labeled PHAL (+)/VGluT2 (+) axon terminals formed synaptic contacts on dendrites of both TH-positive and TH-negative cells. Consistent with these anatomical observations, in whole cell slice recordings of VTA neurons we observed that blocking action potential activity significantly decreased the frequency of synaptic glutamatergic events in DAergic and non-DAergic neurons. These observations indicate that resident VTA glutamatergic neurons are likely to affect both DAergic and non-DAergic neurotransmission arising from the VTA.
The ventral tegmental area (VTA) plays a role in goal-directed behavior and the reward processing of natural reinforcers and several drugs of abuse (Schultz, 2002; Wise, 2004). It is well established that DAergic and GABAergic neurons are present in the VTA, however, recent observations indicate that neurons with glutamatergic phenotype lacking either DAergic (Kawano et al., 2006; Yamaguchi et al., 2007; Nair-Roberts et al., 2008) or GABAergic (Yamaguchi et al., 2007) phenotypes are also present in the VTA.
Glutamatergic neurons can be identified through the detection of mRNA encoding vesicular glutamate transporters (VGluTs), which transport glutamate into synaptic vesicles at presynaptic terminals. Three isoforms of the vesicular glutamate transporter, VGluT1, VGluT2 and VGluT3, are present in the CNS (Bellocchio et al., 1998; Bai et al., 2001; Fremeau et al., 2001; Fujiyama et al., 2001; Hayashi et al., 2001; Herzog et al., 2001; Takamori et al., 2001; Varoqui et al., 2002). Within the VTA, glutamatergic cells express transcripts encoding VGluT2, but not VGluT1 or VGluT3 (Kawano et al., 2006; Yamaguchi et al., 2007).
DAergic and GABAergic VTA neurons communicate locally within the VTA in addition to their projections to other brain structures. DA modulates local neural activity within the VTA through somatodendritic release of DA that can activate postsynaptic D2 receptors (Johnson and North 1992) or presynaptic D1 receptors on GABAergic terminals (Cameron and Williams, 1993). Johnson and North (1992a) provided evidence for local GABAergic connections in the VTA by showing that the frequency of spontaneous inhibitory synaptic events on putative DAergic neurons was decreased by terminating action potential activity with the Na+ channel blocker tetrodotoxin (TTX). Such local signaling is likely to regulate the DAergic and non-DAergic neurotransmission to VTA target structures (Swanson 1982; Fallon et al., 1984; van Bockstaele and Pickel, 1995; Carr and Sesack, 2000; Margolis et al., 2006).
Because both DAergic and GABAergic neurons of the VTA signal locally, we raised the question: do VTA glutamatergic neurons also have both intrinsic and extrinsic connections? While some evidence exists suggesting that VTA glutamatergic neurons project to the prefrontal cortex (Hur and Zaborszky, 2005; Lavin et al., 2005), or to the nucleus accumbens (Chuhma et al., 2004), it is not clear whether or not VTA glutamatergic neurons make local synapses. In the present study we used anatomical and electrophysiological approaches to investigate the local circuitry of VTA glutamatergic neurons. We tagged axon terminals from resident VTA neurons by intra-VTA injection of either the anterograde tracer Phaseolus vulgaris leucoagglutinin (PHAL) or an adeno-associate virus (AAV) vector expressing cDNA for the tracer wheat germ agglutinin (WGA), and by double immunolabeling and electron microscopy determined whether local PHAL- or WGA-positive axon terminals contain or lack VGluT2. We also used triple immunolabeling to determine whether VTA glutamatergic neurons form synapses with DAergic neurons. Finally, we used in vitro electrophysiology to test for functional local glutamatergic synapses from intrinsic VTA neurons in acute brain slices. These experiments all provide evidence for glutamatergic synaptic transmission by local glutamatergic neurons in the VTA.
For anatomical studies, sixty six male Sprague-Dawley rats (250-420 g body weight) were anesthetized with an intraperitoneal injection of 3 ml/kg of chloral hydrate (40 mg/ml) in physiological saline. Anesthetized rats were fixed in a stereotaxic apparatus and either the anterograde axonal tracer PHAL (2.5% in 0.01 M sodium phosphate buffer, pH 7.8; Vector Laboratories, Burlingame, CA) or AAV-WGAtr (see below) was delivered into the VTA (Bregma −5.2, ML +0.8, DV -8.4). Into the VTA of 50 rats PHAL was applied iontophoretically through a stereotaxically positioned glass micropipette (inner tip diameter 10-15 μm) by applying 5μA current, 5 second on/off for 15 minutes. A second group of 16 rats was injected with AAV-WGAtr (25 nl of 4 × 1012 viral genomes/ml of 0.01 M PB containing 0.5 mM magnesium chloride pH 7.4) by pressure through a glass micropipette attached to a Picospritzer III (Parker Hannifin Corporation, Cleveland, OH, USA). Seven days after injection of PHAL or 3 weeks after injection of AAV-WGAtr, the rats were deeply anesthetized with chloral hydrate (35 mg/100g) and perfused transcardially with either 500 ml fixative containing 4% (W/V) paraformaldehyde, 0.05% glutaraldehyde and 15% picric acid in 0.1 M phosphate buffer (PB, pH 7.3) or with 75 ml fixative containing 3.75% acrolein and 2% paraformaldehyde followed with 300 ml of 2% paraformaldehyde. Brains were removed and kept in 2% paraformaldehyde for 2 h. The midbrains were cut into coronal serial sections (50 μm thick) with a vibratome (VT1000S, Leica, Vienna, Austria). The detection of antigens was similar when we compared brain sections of animals perfused with either of the 2 fixatives. However, the best ultrastructural preservation was obtained with the fixative solution containing acrolein. All animal procedures were approved by the NIDA Animal Care and Use Committee.
Using RT-PCR, the cDNA for wheat (T. aestivum) germ agglutinin (kindly provided by Dr. Yoshihara, RIKEN Brain Science Institute, Japan) was truncated after codon 198 (ala) and flanked by KpnI sites. The resulting PCR fragment was digested with KpnI and inserted into the KpnI site of pAAV-GFP (Xiao et al., 1998). The resulting plasmid, pAAV-WGAtr, was sequence verified and used to prepare viral stocks by the triple-transfection method (Xiao et al., 1998; Howard et al., 2008). Briefly, twenty 15 cm dishes containing HEK293 cells at 85-95% confluency were transfected by CaCl2 method with pHelper (Stratagene, La Jolla, CA), pAAV-WGAtr and a plasmid containing rep/cap genes for serotype9, pAAV9 (Gao et al., 2004). Plasmids used for packaging AAV were generously provided by Dr. Xiao Xiao (UNC, Chapel Hill, NC). Approximately 48 hours post-transfection, cells were harvested, lysed by freeze and thaw, and purified by centrifugation on CsCl gradient. Final samples were dialyzed in PBS, aliquoted and stored at -80°C until use. All vectors were titered by quantitative RT-PCR using the CMV promoter as the target sequence. Viral titers were approximately 4×1012 viral genome/ml.
To determine if the injected PHAL or transduced WGA was confined to VTA neurons, every sixth section of the VTA was used to detect PHAL or WGA by immunohistochemistry. Sections were rinsed with phosphate-buffered saline (PBS, pH 7.4) and incubated with a blocking solution [10% normal goat serum (NGS) in PBS supplemented with 0.3% Triton-X-100] for 1 h. Sections were then incubated with either rabbit anti-PHAL antibody (1:2000, Vector Laboratories, Burlingame, CA), rabbit anti-WGA (T 4144; Sigma, Saint Louis, MI, USA, 1: 20 000) or goat anti-WGA antibody (AS-2024; Vector laboratories, Burlingame, CA, USA, 1:500 dilution) in the blocking solution overnight at 4°C. After rinsing 3 × 10 min in PBS, sections were processed with an ABC kit (Vector Laboratories, Burlingame, CA). The sections were incubated for 1 h at room temperature with a 1:200 dilution of the biotinylated anti-rabbit or anti-goat secondary antibody, rinsed with PBS, and incubated with avidin-biotinylated horseradish peroxidase for 1h. Sections were rinsed and the peroxidase reaction was then developed with 0.05% 3, 3-diaminobenzidine-4 HCl (DAB) and 0.003% hydrogen peroxide (H2O2). Following evaluation of PHAL or WGA immunoreactivity in VTA cells, brain sections of rats with PHAL- or WGA-immunoreactivity within the parabrachial pigmental area (PBP) or paranigral nucleus (PN) of the VTA were processed for immunoelectron microscopy. Specificity of primary anti-PHAL antibodies was demonstrated by the lack of PHAL immunolabeling in brain sections from rats injected with saline solution without PHAL.
Coronal brain sections (50 μm) of AAV-WGA injected rats were incubated for 1 h in PB supplemented with 4% BSA and 0.3% Triton X-100. Sections were then incubated with a cocktail of goat anti-WGA antibody (AS-2024; Vector laboratories, Burlingame, CA, USA, 1:500 dilution) and the mouse anti-TH antibody (MAB318; Chemicon, Temecula, CA, USA, 1:500 dilution) overnight at 4°C. After rinsing 3 × 10 min in PB, sections were incubated in a cocktail of Texas Red donkey anti-mouse (715-075-150; Jackson Immunoresearch Laboratories Inc., West Grove, PA, USA, 1;100 dilution) and FITC donkey anti-goat (705-095-147; Jackson Immunoresearch Laboratories Inc., West Grove, PA, USA, 1:100 dilution). After rinsing, sections were mounted on slides and air-dried.
Vibratome tissue sections were rinsed with 50 mM Tris-buffered saline (TBS, pH 7.4), incubated with 1% sodium borohydride in TBS for 30 min to inactivate free aldehyde groups, rinsed in TBS, and then incubated with 0.05% Triton-X for 50 min followed by blocking in TBS containing 20% NGS for 30 min. Sections were then incubated with primary antibodies as follows: rabbit anti-PHAL antibody (single labeling), rabbit anti-WGA (single labeling), rabbit anti-PHAL + guinea pig anti-VGluT2 antibodies (double labeling), rabbit anti-WGA + guinea pig anti-VGluT2 antibodies (double labeling), rabbit anti-PHAL + guinea pig anti-VGluT1 antibodies (double labeling), or rabbit anti-PHAL + guinea pig anti-VGluT2 + mouse anti-TH antibodies (triple labeling), all primary antibodies were diluted in TBS with 2% NGS, incubations were for 48 h at 4°C. The primary antibodies were used at 1:2000 dilution for rabbit anti-PHAL antibody (Vector laboratories, Burlingame, CA, USA), 1:20000 dilution for rabbit anti-WGA, 1:8000 for guinea pig anti-VGluT2 antibody (AB5907; Chemicon, Temecula, CA, USA), 1:5000 for guinea pig anti-VGluT1 antibody (AB5905; Chemicon, Temecula, CA, USA) and 1:2000 for mouse anti-TH antibody (MAB318; Chemicon, Temecula, CA, USA). Sections were rinsed and incubated overnight at 4°C in the corresponding secondary antibodies; biotinylated goat anti-rabbit antibodies (1:100, Vector Laboratories, Burlingame, CA, USA) for single labeling or in a cocktail of secondary antibodies containing biotinylated goat anti-rabbit antibodies and mouse monoclonal anti-guinea pig immunoglobulin G, IgG (1:1000; G0522; Sigma-Aldrich, St. Louis, MO, USA) against VGluT2 primary antibody or a cocktail of secondary antibodies containing biotinylated goat anti-rabbit antibodies and anti-guinea pig IgG (Fab’ fragment, 1:100 dilution) coupled to 1.4-nm gold (Nanoprobes Inc., Stony Brook, NY, USA) against VGluT1 primary antibody in TBS with 2% NGS. After several rinses, sections for VGluT2 and TH labeling were incubated with anti-mouse IgG (Fab’ fragment, 1:100 dilution) coupled to 1.4-nm gold (Nanoprobes Inc., Stony Brook, NY, USA) overnight at 4 °C. Sections for VGluT1, VGluT2 and TH labeling were rinsed in TBS, and then in double-distilled water, followed by silver enhancement of the gold particles with the Nanoprobe Silver Kit (Nanoprobes Inc., Stony Brook, NY, USA) for 7 min. Next, all sections were incubated in avidin-biotinylated horseradish peroxidase complex (ABC, 1: 100 dilution; Vector Labs, Burlingame, CA, USA) in TBS for 2 hr at room temperature and washed in Tris-buffer (TB, pH 7.4). Peroxidase activity was detected by placing the sections in a solution containing DAB (0.025%) and 0.003% H2O2 in PB for 3 min. Peroxidase reaction was stopped by rinsing sections in PBS, sections were fixed with 2% osmium tetroxide in PBS for 25 min, washed in PBS followed by double-distilled water and then contrasted in freshly prepared 1% uranyl acetate for 35 min. Sections were dehydrated through a series of graded alcohols and with propylene oxide. Afterwards, they were flat embedded in Durcupan ACM epoxy resin (Electron Microscopy Sciences, Fort Washington, PA). Resin-embedded sections were polymerized at 60°C for 2 days. Sections of 70 nm were cut from the outer surface of the tissue with an ultramicrotome (Leica, Microsystems, Wetzlar, Germany) using a diamond knife (Diatome, fort Washington, PA). The sections were collected on Formvar-coated single slot grids and counterstained with Reynolds lead citrate (Reynolds, 1963). Sections were examined and photographed using a Tecnai G2 12 transmission electron microscope (Fei Company, Hillsboro, OR) equipped with a digital micrograph 3.4 camera (Gatan, Inc., Pleasanton, CA).
Specificity of primary anti-VGluT2 and anti-VGluT1 antibodies were demonstrated by results showing that pre-incubation of the primary antibodies with the antigenic peptide abolished positive VGluT2 and VGluT1 immunolabeling, as we previously described (Tagliaferro and Morales, 2008). We showed that the anti-TH antibody labels a single protein band in the molecular weight range of 56-60 kDA on Western blots prepared from VTA protein homogenates (Shepard et al., 2006). In addition, we had used this anti-TH antibody to demonstrate that midbrain unilateral lesions with 6-hydroxydopamine (6-OHDA) results in lack of TH-immunolabeling in the lesioned site, while is preserved in the contralateral unlesioned midrain (Sarabi et al., 2001). Omitting one of the primary antibodies during double or triple labeling removed corresponding labeling and confirmed no cross-reaction between different primary antibodies.
Coronal vibratome sections of VTA corresponding to plates 71 - 79 (bregma -4.56 mm to -5.52 mm) of the rat brain atlas of Paxinos and Watson (2007) were used in this study. Four to 6 grids containing thin sections were collected from four to six plastic-embedded sections of the VTA from a total of 6 rats: 3 PHAL- and 3 WGA-injected rats. Synaptic contacts were classified according to their morphology and immunolabel, and photographed at a magnification of 6800-13000X. The morphological criteria used for identification and classification of cellular components observed in these thin sections were as previously described (Peters et al., 1991). Briefly, neuronal cell bodies were identified by the presence of a large and round nucleus as well as rough endoplasmic reticulum and Golgi apparatus. Large and medium sized dendrites were identified by the presence of endoplasmic reticulum, mitochondria and microtubules. Dendritic spines were identified by the absence of mitochondria. Axon terminals were characterized by the presence of synaptic vesicles and mitochondria, whereas axon pre-terminals and unmyelinated axons are smaller in diameter and occasionally contain a small number of synaptic vesicles. Type II synapses referred to here as asymmetric synapses, were characterized by the presence of thick postsynaptic densities while type I synapses are referred to as symmetric synapses and have thin postsynaptic densities. When the identification of a synaptic contact was not clear, contiguous sections were obtained to determine the type of synapse. Pictures were adjusted to match contrast and brightness by using Adobe Photoshop (Adobe Systems Incorporated, Seattle, WA).
Thirty three to forty day old male Sprague-Dawley rats were anesthetized with isofluorane, and their brains were removed. Horizontal slices (250 μm thick) containing the VTA were prepared using a vibratome (Leica Instruments, Germany). Slices were submerged in artificial cerebrospinal fluid (aCSF) containing (in mM) 119 NaCl, 2.5 KCl, 1.3 MgSO4, 1.0 NaH2PO4, 2.5 CaCl2, 26.2 NaHCO3, and 11 glucose saturated with 95% O2-5% CO2 and allowed to recover at 34°C for at least 1 hour. Tissue anterior to the VTA was removed from slices in order to sever any inputs arising from the hypothalamus (supplemental Figure 2). Individual slices were visualized under a Zeiss Axioskop with differential interference contrast optics and infrared illumination. Whole cell patch-clamp recordings were made at 36°C using 2.5-5 μm pipettes containing (in mM) 123 K-gluconate, 10 HEPES, 0.2 EGTA, 8 NaCl, 2 MgATP, and 0.3 Na3GTP (pH 7.2, osmolarity adjusted to 275). Biocytin (0.1%) was also included in the internal solution in order to identify dopaminergic neurons with immunocytochemistry following recordings.
Recordings were made using an Axopatch 1-D (Molecuar Devices), filtered at 2 kHz and collected at 5 kHz using procedures written for IGOR Pro (Wavemetrics, Lake Oswego, OR). All recordings were made in voltage clamp holding the cells at Vm=-60 mV. Ih was recorded at the beginning of each experiment by stepping from -60 to -40, -50, -70, -80, -90, -100, -110, and -120 mV. Series resistance and input resistance were sampled throughout experiments with 4 mV, 200 ms hyperpolarizing steps applied once every 10 seconds. All excitatory events were measured in the presence of the GABA-A receptor antagonist picrotoxin (100 μM) in order to isolate glutamatergic events. Events were identified and quantified using procedures developed for IGOR Pro (Wavemetrics, Lake Oswego, OR). Cells with baseline event frequencies below 0.25 Hz were excluded from this study. In a subset of experiments, the external KCl concentration was raised to 5.0 mM to depolarize the slice, therefore increasing the baseline event frequency in order to better measure the effects of the Na+ channel blocker tetrodotoxin (TTX) on event frequency. In some experiments the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid glutamate receptor (AMPAR) antagonist 6,7-dinitroquinoxaline-2,3(1H-4H)-dione (DNQX) (10 μM) was added after the TTX application to confirm that the measured events were glutamatergic.
Electrophysiological results are presented as means +/- SEM. Summary comparisons of event frequencies and amplitudes were made between the average over the 4 minutes of baseline just preceding TTX application to the last 4 minutes of TTX application using a paired Student’s t-test. All drugs were applied by bath perfusion. Stock solutions were made and diluted in aCSF immediately prior to application. For stock solutions, TTX was diluted in H2O (1 mM); picrotoxin (100 mM) and DNQX (10 mM) were diluted in DMSO. All chemicals were obtained from Sigma Chemical (St. Louis, MO) or Tocris (Ballwin, MO).
Immediately after recording, slices were fixed for 2 hours in 4% formaldehyde. Slices were pre-blocked for 2 hours in PBS with 0.2% BSA and 5% normal goat serum. Rabbit anti-tyrosine hydroxylase antibody (1:100) was then added and the slices agitated at 4°C for 48 hours. Slices were thoroughly rinsed with PBS and agitated with CY5 anti-rabbit secondary antibody (1:100) and fluorescein (DTAF)-conjugated streptavidin (3.25 μL/mL) at 4°C overnight. Cells were visualized with a Zeiss LSM 510 META microscope. Biocytin, BSA, and normal goat serum were obtained from Sigma Chemical (St. Louis, MO). DTAF avidin was obtained from Jackson Immunoresearch (West Grove, PA), and rabbit anti-tyrosine hydroxylase antibody and CY5 anti-rabbit secondary antibody were obtained from Chemicon (Temecula, CA).
We unilaterally injected the anterograde tracer PHAL in the VTA to identify cellular compartments originating from resident neurons. For immunoelectron microscopy (see below), we analyzed 3 cases where the PHAL immunoreactive cell bodies were confined to the parabrachial pigmental area (PBP) and paranigral nucleus (PN) of the VTA (Fig. 1). These cases were selected because we did not find PHAL immunoreactive cell bodies in areas rostral, dorsal or lateral to the VTA, such as the lateral hypothalamic area and substantia nigra. In addition, in these cases we did not find immunoreactive cells bodies in the rostral linear nucleus or interfascicular nucleus, areas known to contain some TH neurons expressing VGluT2 transcripts (Kawano et al., 2006). Within the VTA, PHAL-positive fibers were detected throughout the rostro-caudal and medio-lateral aspects of the VTA. PHAL-positive fibers were occasionally observed in the VTA contralateral to the injection site.
Electron microscopic examination of VTA containing PHAL immunoreactivity confined to the PBP and PN of the VTA showed PHAL immunoreactivity in neuronal cell bodies, dendrites, axon terminals and glia (data not shown). PHAL-positive axon terminals formed asymmetric (Figs. 2A) or symmetric (Figs. 2B) synapses, mostly with dendrites. We observed dense core vesicles in some of the PHAL-positive axon terminals forming symmetric synapses (Fig. 2B). Quantitative analysis of all PHAL-positive terminals making recognizable synaptic contacts (Table 1) indicated that 50% of them established asymmetric synapses with dendrites and 9% made asymmetric synapses with dendritic spines (Table 1). Less than half of all PHAL-positive terminals (41%) made symmetric synapses, all of which were onto dendrites, not on dendritic spines (Table 1).
We performed further neurochemical characterization of PHAL-positive axon terminals to determine whether any of these terminals originated from VTA glutamatergic neurons. Ultrastructural analysis of VTA sections double labeled for the detection of PHAL by immunoperoxidase activity and VGluT2 by immunogold-silver intensification, revealed PHAL-positive terminals containing VGluT2 [PHAL(+)/VGluT2(+)] or lacking detectable levels of VGluT2 immunoreactivity [PHAL(+)/VGluT2(-)] (Fig. 3 A). Quantitative analysis (Table 1) showed that 56% of all PHAL-positive terminals forming synaptic contacts contained VGluT2. As expected of excitatory synapses, the vast majority of PHAL(+)/VGluT2(+) terminals formed asymmetric synapses (90%). The PHAL(+)/VGluT2(+) terminals made synapses primarily on dendrites (Fig. 3), and occasionally on dendritic spines (Fig. 4A). A few PHAL(+)/VGluT2(+) terminals appeared to form symmetric synapses on dendrites, but none were observed synapsing onto dendritic spines.
PHAL-positive terminals that were VGluT2-negative [PHAL(+)/VGluT2(-)] constituted 44% of all PHAL-positive terminals making discernable synaptic contacts. Seventy-nine percent of these PHAL(+)/VGluT2(-) axon terminals formed symmetric synapses with dendrites (Figs. 5A and 5B). The remaining PHAL(+)/VGluT2(-) axon terminals established asymmetric synapses, mostly with dendrites, rarely with dendritic spines.
Because VTA neurons do not manufacture VGluT1, as a control we examined whether or not PHAL(+) axon terminals forming asymmetric synapses contain VGluT1. We performed ultrastructural analysis of double PHAL/VGluT1 immunolabeled VTA, using brain sections from the same 3 rats in which we detected PHAL/VGluT2 immunolabeling. In contrast to the above-described coexistence of PHAL/VGluT2 immunolabeling in axon terminals, VGluT1 labeling was often found in axon terminals making asymmetric synapses, but lacking PHAL immunoreactivity. Of the 60 examined PHAL positive axon terminals making asymmetric synapses on dendrites, none of them contained VGluT1 immunoreactivity.
To address the question of local glutamatergic connections in the VTA utilizing a different anatomical tool, we developed an AAV that causes transduced neurons to express a truncated form of WGA. We then injected the AAV-WGAtr into the VTA of a different group of rats. These injections resulted in expression of WGA in cell bodies and fibers within the VTA (Figs. 6A and 6A’). By intra-VTA delivery of different amounts of viral particles, we varied the number of transduced cells. We analyzed sections from brains showing more than 100 and less than 200 transduced cells confined to the VTA (Figs. 6A, 6A’, 7B and 7B’). We used anti-TH and anti-WGA antibodies in a double immuno fluorescent labeling to determine whether the AAV-WGAtr was able to transduce TH positive cells and TH negative neurons (putative GABAergic or glutamatergic). Within the VTA, WGA was found in cells either containing or lacking TH immunoreacivity (Figs. 7C and 7C’), indicating that under our experimental conditions the transduction is not neurochemically selective and raising the possibility we could utilize the WGA to detect VTA glutamatergic terminals.
WGA-label was not detected in cell bodies outside the VTA, suggesting that transduction was limited to cell bodies local to the injection site and that the virus was not taken up by terminals and transported to cell bodies outside of the VTA (Figs. 6B, 6B’ and supplemental Fig. 1). Surprisingly, we did not detect WGA in VTA projection targets. This lack of detection of WGA immunoreactivity in VTA target areas, such as the nucleus accumbens, was unexpected. We therefore also injected AAV-WGAtr into other brain structures and found that the WGA detection was similarly restricted to local cell bodies and processes. We therefore speculate that the restricted detection of truncated WGA may result from molecular trafficking differences between fibers that terminate proximal to the soma and fibers that project to other brain regions.
To further validate the specificity of the AAV-WGAtr for labeling terminals arising from local neurons, we performed ultrastructural analysis of double WGA/VGluT1 immunolabeled tissue. VGluT1 labeling was seen only in axon terminals lacking WGA immunoreactivity. Further, among the 22 examined WGA immunopositive axon terminals that made asymmetric synapses, none of them contained VGluT1 immunoreactivity. This is consistent with our PHAL/VGluT1 analysis, and suggests that WGA labeling indicates a terminal arising from a transduced VTA neuron.
We analyzed 3 cases where the WGA immunoreactive cell bodies were confined to the PBP and PN of the VTA. Similar to the PHAL- labeled axon terminals, the WGA-positive axon terminals formed symmetric (Fig. 8A) or asymmetric (Fig. 8C) synapses. Ultrastructural analysis of VTA sections double labeled for the detection of WGA by immunoperoxidase activity and VGluT2 by immunogold-silver intensification, revealed WGA-positive terminals that either lacked VGluT2 [WGA(+)/VGluT2(-)] (Fig. 8A) or contained VGluT2 immunoreactivity [WGA(+)/VGluT2(+)] (Fig. 8B,and 8C). Quantitative analysis (Table 2) showed that 55% of all WGA-positive terminals forming synaptic contacts contained VGluT2. In agreement with the putative excitatory nature of asymmetric synapses, the vast majority of WGA(+)/VGluT2(+) terminals formed asymmetric synapses (92%).
WGA-positive terminals that were VGluT2-negative [WGA(+)/VGluT2(-)] constituted 45% of all WGA-positive terminals making recognizable synaptic contacts (Table 2). These included both symmetric and asymmetric synapses.
In order to determine the neurochemical identity of the postsynaptic targets of local VTA glutamatergic terminals, we used tissue from PHAL-injected VTA to perform triple immunocytochemical ultrastructural studies to determine the neurochemical identity of the postsynaptic targets of local VTA glutamatergic terminals. For the triple labeling procedure we took advantage of the minimally overlapping ultrastructural distribution of VGluT2 and TH within the VTA: while VGluT2 protein is confined to presynaptic compartments, TH is almost exclusively localized postsynaptically in dendrites and somata (Bayer and Pickel, 1991; Carr and Sesack, 2000; Omelchenko and Sesack, 2005). This segregation allows the detection of presynaptic VGluT2 simultaneously with postsynaptic TH by immunogold-silver intensification, and PHAL by immunoperoxidase activity.
We observed some double labeled PHAL(+)/VGluT2(+) axon terminals that made asymmetric synapses on dendrites containing detectable levels of TH immunolabeling (Figs. 9A1 and 9A2) and some that made asymmetric synapses on dendrites lacking detectable TH immunoreactivity (Figs. 9B1 and 9B2). In addition, we detected some PHAL(+)/VGluT2(-) terminals that made synaptic contacts on both TH immunopositive and TH immunonegative dendrites (data not shown). Therefore, it is likely that at least some VTA DAergic neurons receive synaptic inputs from VTA glutamatergic neurons.
Given these anatomical results, and because many VTA neurons, including non-DAergic neurons, fire spontaneously in the slice, we hypothesized that some glutamate mediated spontaneous excitatory postsynaptic currents (EPSCs) detected in VTA neurons would be driven by action potential activity of local glutamatergic neurons. We tested for the presence of local, functional glutamatergic synapses using whole cell recordings to detect alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid glutamate receptor (AMPAR) mediated currents in acute horizontal rat brain slices. Care was taken to remove possible sources of glutamate outside of the VTA when blocking the slice preparation (supplemental Fig. 2). Spontaneously occurring events can be voltage and Ca2+ dependent (Hubbard 1961; Boyd and Martin, 1956; Liley 1956; del Castillo and Katz, 1954), however there are also often voltage independent events (Elmqvist and Feldman, 1965; Katz and Miledi, 1963; Hubbard 1961; del Castillo and Katz, 1954). Therefore, spontaneous events can include both action potential driven and action potential independent events. A decrease in event frequency in a slice preparation following blockade of action potential activity suggests that a portion of the spontaneous events were driven by locally firing neurons. Since action potential independent spontaneous glutamatergic events are present in most VTA neurons (Margolis et al., 2005; Deng et al., 2009; Xiao et al., 2009), we should observe a decrease in frequency, but not elimination, of glutamatergic events when action potential activity is blocked by TTX.
All recordings were made in VTA neurons that expressed the hyperpolarization-activated cation current, Ih, many of which were DAergic neurons, as confirmed by TH immunocytochemistry following recording. We recorded spontaneously occurring EPSCs. Excitatory synaptic input was isolated from inhibitory input by pharmacologically blocking GABAA receptors with bath application of the antagonist picrotoxin (100 μM). At the end of a subset of experiments the non-NMDA glutamate receptor antagonist DNQX (10 μM) blocked all remaining synaptic signaling (Fig. 10F), suggesting that the events recorded in the presence of picrotoxin resulted from glutamatergic activation of AMPARs. We then compared the baseline frequency of spontaneous EPSCs to that observed during bath application of the Na+ channel blocker TTX (500 nM) to terminate action potential firing. The baseline frequency of EPSCs in control aCSF was 2.0 ± 0.4 Hz. When TTX was added to the perfusion solution, the frequency of EPSCs decreased by an average of 29% (range: 2-36%; n=6; P<0.05 Student’s t-test; Fig. 10F). Because the baseline frequency of events was low in control aCSF, we conducted additional experiments in which we depolarized the slices by increasing the external concentration of K+ to 5 mM. Depolarizing the slices in this way will increase both the action potential dependent and independent EPSCs by increasing cell firing and depolarizing axon terminals. In these experiments, the baseline EPSC frequency was approximately 3 times greater than in control aCSF, and TTX application similarly decreased the frequency of EPSCs by a mean of 26% (range: 6-64%; n=10; P<0.005, Student’s t-test; Fig. 10F). In immunocytochemically identified neurons, the frequency of spontaneous EPSCs decreased in both TH-immunopositive (in 5 mM K+; range: 12-30%; n=3) and TH-immunonegative (in 5 mM K+; range: 6-34%; n=3) neurons (Fig. 10H). In experiments conducted in elevated K+, there was a very small (5%) but significant decrease in the amplitude of EPSCs with TTX application (n=10; P<0.05, Student’s t-test; Fig. 10E). A trend towards such an amplitude change with TTX application was observed in both TH-immunopositive and TH-immunonegative neurons (Fig. 10G). These data support the conclusion that the anatomically identified synapses onto VTA neurons from VTA glutamatergic neurons are functional.
We found that VTA glutamatergic neurons make local synapses on both DAergic and non-DAergic VTA neurons. We provide both anatomical and functional evidence for these connections by utilizing ultrastructural and electrophysiological techniques. Our ultrastructural findings also indicate that VTA non-glutamatergic neurons make symmetric synaptic contacts with other VTA neurons; these terminals likely arise from VTA GABAergic neurons.
We established the VTA origin of identified glutamatergic synapses using 2 anatomical procedures: a traditional approach (local cellular uptake of PHAL) and a novel procedure (local cellular synthesis and corresponding intracellular accumulation of WGA). PHAL is a sensitive anterograde tracer that is incorporated by neurons at the injection site with an insignificant uptake by fibers of passage and negligible retrograde axonal transport (Gerfen and Sawchenko, 1984; Deller et al., 2000). Since local glutamatergic connections are rarely reported, it was important to replicate observation obtained with PHAL with an alternative method. Thus we used intra-VTA injection of AAV-WGAtr that resulted in expression of WGA in cell bodies and fibers within the VTA.
To support the premises that PHAL was selectively incorporated by local VTA neurons and that WGA synthesis and accumulation were limited to resident VTA neurons, we examined the degree of expression of VGluT1 immunoreactivity within PHAL- or WGA-containing axon terminals. We chose VGluT1 as control because VTA neurons do not express VGluT1 mRNA (Kawano et al., 2006; Yamaguchi et al., 2007), and thus axon terminals containing VGluT1, which are not uncommon in the VTA (Omelchenko and Sesack, 2007), have an exclusively extrinsic origin. In agreement with this notion, we did not detect VGluT1 immunoreactivity in terminals containing either PHAL or WGA.
In the analysis of ultrastructural data, asymmetric and symmetric synapses are generally considered to be excitatory and inhibitory, respectively. However, we found that not all PHAL-positive terminals making asymmetric synapses contained VGluT2 immunolabeling. This lack of VGluT2 signal in these terminals may be due to a failure to detect VGluT2 in some terminals, or the possibility that these terminals may derive from intrinsic GABAergic or DAergic neurons with the capability of making asymmetric synapses. GABAergic axon terminals that form asymmetric synapses have been found in the substantia nigra (Ribak et al., 1976; Nitsch and Riesenberg, 1988; Bolam and Smith, 1990), and a small number of TH positive terminals making asymmetric synapses has been observed in the VTA (Bayer and Pickel, 1990). Although symmetric synaptic structure is usually associated with inhibitory synapses, we occasionally found PHAL(+)/VGluT2(+) terminals forming symmetric synapses on dendrites. This is consistent with a previous report of VGluT2-expressing axon terminals forming symmetric contacts in the VTA (Omelchenko and Sesack, 2007).
We provide electrophysiological evidence that the local glutamatergic terminals release neurotransmitter in an action potential dependent manner by showing that the frequency of spontaneous EPSCs in both DAergic and non-DAergic VTA neurons decreases when action potential activity is blocked. We observed, on average, a 30% decrease in frequency of spontaneous EPSCs in VTA neurons in response to TTX application. The remaining events are likely due to vesicle fusion events that are independent of action potential-induced depolarization of the axon terminal. Such action potential independent events have been observed in many parts of the central nervous system (e.g., Staley and Mody, 1991; Farrant and Cull-Candy, 1991; Hubbard 1961), including at both glutamate and GABA synapses in the VTA (e. g. Deng et al., 2009; Xiao et al., 2008; Xiao et al., 2009; Margolis et al., 2005; Margolis et al., 2008). We observed a slight decrease in the amplitude of spontaneous EPSCs following the application of TTX. Since electrical excitation of the presynaptic terminal, such as by action potential propagation, could evoke the simultaneous release of more than one vesicle, it is possible that the decrease in amplitude is due to the TTX application diminishing the frequency of simultaneous vesicle fusions. It is interesting to note that similar to the ultrastructural analysis, both TH immuno-positive and -negative VTA neurons displayed electrophysiological evidence that they receive inputs from local glutamatergic neurons.
It is generally accepted that intrinsic GABAergic neurons synapse onto projecting DAergic neurons. In agreement with this idea, we found that about half of the total population of either PHAL- or WGA-axon terminals from tagged VTA neurons makes symmetric (putative inhibitory) synapses on local dendrites. We infer that the PHAL- or WGA-axon terminals making local symmetric synapses are GABAergic because they lack VGluT2 or VGluT1, and multiple ultrastructural studies have shown that axon terminals of symmetric synapses are GABAergic in many brain areas, including the VTA (Bayer and Pickel, 1991; Omelchenko and Sesack, 2005). Moreover, a recent ultrastructural study utilizing PHAL tagging of VTA fibers and immunolabeling for GABA demonstrated that VTA GABAergic neurons form synapses on local DAergic neurons (Omelchenko and Sesack, 2009). Functional evidence for local connections arising from VTA GABAergic neurons comes from in vitro electrophysiological studies showing that there are TTX-sensitive inhibitory PSCs in putative DAergic neurons (Johnson and North, 1992). It is not clear whether or not these GABAergic inputs arise from interneurons lacking axonal projections outside of the VTA.
Given the current model of intra-VTA circuitry and the prominent role ascribed to local GABAergic connections in the VTA, it is noteworthy that in the present study we found that only half of the total population of PHAL- or WGA-axon terminals forming synaptic contacts make symmetric synapses on VTA dendrites. Moreover, the vast majority of PHAL- or WGA-axon terminals making local asymmetric synapses are glutamatergic, as they contain VGluT2 immunoreactivity. These anatomical observations raise the possibility that the extent of modulation of VTA neurons by local glutamatergic connections rivals that of intrinsic GABAergic neurons. While we show that VTA neurons containing VGluT2 establish local synapses, it is not clear whether or not these neurons are interneurons lacking axonal projections outside of the VTA. To date, the septum is the only brain region in which synapses arising from local VGluT2 expressing neurons has been documented (Hajszan et al., 2004; Manseau et al., 2005).
Finally, since we found some VGluT2-positive contacts on TH positive cells and others on TH negative cells, and similarly, some VGluT2-negative contacts on TH-positive cells and others on TH negative cells, we propose that resident glutamatergic and GABAergic neurons provide synaptic inputs to DAergic, GABAergic and glutamatergic neurons within the VTA.
The VTA is thought to play a role in goal-directed behavior and in the reward processing of natural rewards and of several drugs of abuse (Schultz, 2002; Wise, 2004). The VTA dopamine neurons have two firing patterns that contribute to mesolimbic DA neurotransmission; a tonic DA release mediated by pacemaker-like DA neural activity, and a phasic DA release mediated by burst firing (Grace, 1991; Floresco et al., 2003; Sombers et al., 2009). The nonlinear increase in synaptic DA release induced by burst firing of DA neurons (Chergui et al., 1994) is considered to be functionally relevant for signaling salient environmental stimuli (Grace 1991; Overton and Clark, 1997; Berridge and Robinson, 1998; Schultz 1998; Cooper 2002). Excitatory glutamatergic inputs to midbrain DAergic neurons are necessary for inducing burst firing (Grace and Bunney, 1984; Cherqui et al., 1994; Overton and Clark, 1997; Paladini et al., 1999; Floresco et al., 2003) and DA release in the nucleus accumbens (Sombers et al., 2009). It has recently been suggested that the laterodorsal tegmental nucleus (LDTg) is essential to allow glutamate to elicit burst firing of VTA neurons, as the inactivation of the LDTg prevents glutamate from inducing burst firing of VTA neurons (Lodge and Grace, 2006). While it remains to be determined if intrinsic glutamategic neurons play a role in VTA neurons burst firing, the local glutamatergic synapses reported here raise this intriguing possibility.
It has recently been suggested that VTA glutamategic and DAergic neurons projecting to the prefrontal cortex (PFC) may provide both transient reward-related information via glutamatergic signaling as well as exert state-dependent control of PFC networks via DA release (Lavin et al., 2005). Anatomical studies provide evidence for reciprocal connectivity between VTA DAergic neurons and principal neurons in the PFC (Carr and Sesack, 2000). In addition, recent observations have shown that VTA neurons expressing VGluT2 mRNA project to the PFC (Hur and Zaborszky, 2005). Therefore, VTA glutamatergic neurons may regulate activity of DA neurons directly through local synaptic connections or indirectly by signaling on PFC neurons that provide feedback to VTA DA neurons.
In conclusion, we demonstrate here that VTA glutamatergic and non-glutamatergic neurons establish local synaptic connections. The targets of these local synapses include both DAergic and non-DAergic VTA neurons. This novel type of local communication expands the modalities of local signaling in the VTA to now include glutamatergic, GABA and DA, each of which, likely modulate both DAergic and non-DAergic projection neurons.
This work was supported by the Intramural Research Program of the National Institute on Drug Abuse. EBM was supported by funds from the State of California for medical research on alcohol and substance abuse through the University of California, San Francisco. We thank Mr. Doug Howard for assistance with viral vector preparation.