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Dopaminergic and glutamatergic microdomains in a subset of rodent mesoaccumbens axons

Abstract

Mesoaccumbens fibers are thought to co-release dopamine and glutamate. However, the mechanism is unclear, and co-release by mesoaccumbens fibers has not been documented. Using electron microcopy, we found that some mesoaccumbens fibers have vesicular transporters for dopamine (VMAT2) in axon segments that are continuous with axon terminals that lack VMAT2, but contain vesicular glutamate transporters type 2 (VGluT2). In vivo overexpression of VMAT2 did not change the segregation of the two vesicular types, suggesting the existence of highly regulated mechanisms for maintaining this segregation. The mesoaccumbens axon terminals containing VGluT2 vesicles make asymmetric synapses, commonly associated with excitatory signaling. Using optogenetics, we found that dopamine and glutamate were released from the same mesoaccumbens fibers. These findings reveal a complex type of signaling by mesoaccumbens fibers in which dopamine and glutamate can be released from the same axons, but are not normally released at the same site or from the same synaptic vesicles.

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Figure 1: Mesoaccumbens neurons establish either VGluT2-asymmetric synapses or TH-symmetric synapses (wild-type rats).
Figure 2: Mesoaccumbens axons from VGluT2-TH neurons segregate dopaminergic markers and VGluT2 to different micro-domains (wild-type rats).
Figure 3: VGluT2 and VMAT2 localize to distinct subpopulations of synaptic vesicles (wild-type rats).
Figure 4: Subcellular segregation of VGluT2-IR and VMAT2(Myc)-IR in the same VGluT2-TH axon from a Th-Cre rat overexpressing VMAT2(Myc) under the regulation of the Th promoter (TH-VMAT2(Myc) rats).
Figure 5: VGluT2-TH neurons segregate VGluT2 and TH to distinct subcellular compartments in the same axon (Th-Cre mice).
Figure 6: Photo-stimulation of mesoaccumbens VGluT2 fibers expressing ChR2 under the control of the Vglut2 promoter elicits EPSCs and dopamine release in nAcc (Vglut2-Cre mice).

References

  1. Stuber, G.D., Hnasko, T.S., Britt, J.P., Edwards, R.H. & Bonci, A. Dopaminergic terminals in the nucleus accumbens but not the dorsal striatum corelease glutamate. J. Neurosci. 30, 8229–8233 (2010).

    Article  CAS  Google Scholar 

  2. Tecuapetla, F. et al. Glutamatergic signaling by mesolimbic dopamine neurons in the nucleus accumbens. J. Neurosci. 30, 7105–7110 (2010).

    Article  CAS  Google Scholar 

  3. Bérubé-Carrière, N. et al. Ultrastructural characterization of the mesostriatal dopamine innervation in mice, including two mouse lines of conditional VGLUT2 knockout in dopamine neurons. Eur. J. Neurosci. 35, 527–538 (2012).

    Article  Google Scholar 

  4. Bérubé-Carrière, N. et al. The dual dopamine-glutamate phenotype of growing mesencephalic neurons regresses in mature rat brain. J. Comp. Neurol. 517, 873–891 (2009).

    Article  Google Scholar 

  5. Moss, J., Ungless, M.A. & Bolam, J.P. Dopaminergic axons in different divisions of the adult rat striatal complex do not express vesicular glutamate transporters. Eur. J. Neurosci. 33, 1205–1211 (2011).

    Article  Google Scholar 

  6. Morales, M. & Root, D.H. Glutamate neurons within the midbrain dopamine regions. Neuroscience 282C, 60–68 (2014).

    Article  Google Scholar 

  7. Yamaguchi, T., Wang, H.L., Li, X., Ng, T.H. & Morales, M. Mesocorticolimbic glutamatergic pathway. J. Neurosci. 31, 8476–8490 (2011).

    Article  CAS  Google Scholar 

  8. Kawano, M. et al. Particular subpopulations of midbrain and hypothalamic dopamine neurons express vesicular glutamate transporter 2 in the rat brain. J. Comp. Neurol. 498, 581–592 (2006).

    Article  CAS  Google Scholar 

  9. Dal Bo, G. et al. Dopamine neurons in culture express VGLUT2 explaining their capacity to release glutamate at synapses in addition to dopamine. J. Neurochem. 88, 1398–1405 (2004).

    Article  CAS  Google Scholar 

  10. Sulzer, D. et al. Dopamine neurons make glutamatergic synapses in vitro. J. Neurosci. 18, 4588–4602 (1998).

    Article  CAS  Google Scholar 

  11. Li, X., Qi, J., Yamaguchi, T., Wang, H.L. & Morales, M. Heterogeneous composition of dopamine neurons of the rat A10 region: molecular evidence for diverse signaling properties. Brain Struct. Funct. 218, 1159–1176 (2013).

    Article  CAS  Google Scholar 

  12. Hnasko, T.S. et al. Vesicular glutamate transport promotes dopamine storage and glutamate corelease in vivo. Neuron 65, 643–656 (2010).

    Article  CAS  Google Scholar 

  13. Hnasko, T.S., Hjelmstad, G.O., Fields, H.L. & Edwards, R.H. Ventral tegmental area glutamate neurons: electrophysiological properties and projections. J. Neurosci. 32, 15076–15085 (2012).

    Article  CAS  Google Scholar 

  14. Descarries, L., Watkins, K.C., Garcia, S., Bosler, O. & Doucet, G. Dual character, asynaptic and synaptic, of the dopamine innervation in adult rat neostriatum: a quantitative autoradiographic and immunocytochemical analysis. J. Comp. Neurol. 375, 167–186 (1996).

    Article  CAS  Google Scholar 

  15. Yamaguchi, T., Qi, J., Wang, H.L., Zhang, S. & Morales, M. Glutamatergic and dopaminergic neurons in the mouse ventral tegmental area. Eur. J. Neurosci. 10.1111/ejn.12818 (9 January 2015).

  16. Swanson, L.W. The projections of the ventral tegmental area and adjacent regions: a combined fluorescent retrograde tracer and immunofluorescence study in the rat. Brain Res. Bull. 9, 321–353 (1982).

    Article  CAS  Google Scholar 

  17. Morales, M. & Pickel, V.M. Insights to drug addiction derived from ultrastructural views of the mesocorticolimbic system. Ann. NY Acad. Sci. 1248, 71–88 (2012).

    Article  CAS  Google Scholar 

  18. Freund, T.F., Powell, J.F. & Smith, A.D. Tyrosine hydroxylase-immunoreactive boutons in synaptic contact with identified striatonigral neurons, with particular reference to dendritic spines. Neuroscience 13, 1189–1215 (1984).

    Article  CAS  Google Scholar 

  19. Adrover, M.F., Shin, J.H. & Alvarez, V.A. Glutamate and dopamine transmission from midbrain dopamine neurons share similar release properties but are differentially affected by cocaine. J. Neurosci. 34, 3183–3192 (2014).

    Article  CAS  Google Scholar 

  20. Foss, S.M., Li, H., Santos, M.S., Edwards, R.H. & Voglmaier, S.M. Multiple dileucine-like motifs direct VGLUT1 trafficking. J. Neurosci. 33, 10647–10660 (2013).

    Article  CAS  Google Scholar 

  21. Root, D.H. et al. Single rodent mesohabenular axons release glutamate and GABA. Nat. Neurosci. 17, 1543–1551 (2014).

    Article  CAS  Google Scholar 

  22. Borgius, L., Restrepo, C.E., Leao, R.N., Saleh, N. & Kiehn, O. A transgenic mouse line for molecular genetic analysis of excitatory glutamatergic neurons. Mol. Cell. Neurosci. 45, 245–257 (2010).

    Article  CAS  Google Scholar 

  23. Witten, I.B. et al. Recombinase-driver rat lines: tools, techniques, and optogenetic application to dopamine-mediated reinforcement. Neuron 72, 721–733 (2011).

    Article  CAS  Google Scholar 

  24. Tagliaferro, P. & Morales, M. Synapses between corticotropin-releasing factor-containing axon terminals and dopaminergic neurons in the ventral tegmental area are predominantly glutamatergic. J. Comp. Neurol. 506, 616–626 (2008).

    Article  CAS  Google Scholar 

  25. Paxinos, G. & Watson, C. The Rat Brain in Stereotaxic Coordinates (Elsevier, 2007).

  26. Paxinos, G. & Franklin, K.B.J. The Mouse Brain in Stereotaxic Coordinates (Academic Press, 2001).

  27. Peters, A., Palay, S.L. & Webster, H.d. The Fine Structure of the Nervous System: Neurons and Their Supporting Cells (Oxford University Press, 1991).

  28. Boulland, J.L. et al. Vesicular glutamate and GABA transporters sort to distinct sets of vesicles in a population of presynaptic terminals. Cereb. Cortex 19, 241–248 (2009).

    Article  Google Scholar 

  29. Tritsch, N.X., Ding, J.B. & Sabatini, B.L. Dopaminergic neurons inhibit striatal output through non-canonical release of GABA. Nature 490, 262–266 (2012).

    Article  CAS  Google Scholar 

  30. Erickson, J.D., Masserano, J.M., Barnes, E.M., Ruth, J.A. & Weiner, N. Chloride ion increases [3H]dopamine accumulation by synaptic vesicles purified from rat striatum: inhibition by thiocyanate ion. Brain Res. 516, 155–160 (1990).

    Article  CAS  Google Scholar 

  31. Teng, L., Crooks, P.A. & Dwoskin, L.P. Lobeline displaces [3H]dihydrotetrabenazine binding and releases [3H]dopamine from rat striatal synaptic vesicles: comparison with d-amphetamine. J. Neurochem. 71, 258–265 (1998).

    Article  CAS  Google Scholar 

  32. Kadota, K. & Kadota, T. Isolation of coated vesicles, plain synaptic vesicles, and flocculent material from a crude synaptosome fraction of guinea pig whole brain. J. Cell Biol. 58, 135–151 (1973).

    Article  CAS  Google Scholar 

  33. Good, C.H. et al. Impaired nigrostriatal function precedes behavioral deficits in a genetic mitochondrial model of Parkinson's disease. FASEB J. 25, 1333–1344 (2011).

    Article  CAS  Google Scholar 

  34. Britt, J.P., McDevitt, R.A. & Bonci, A. Use of channelrhodopsin for activation of CNS neurons. Curr. Protoc. Neurosci. 2.2, 16 (2012).

    Google Scholar 

  35. Qi, J. et al. A glutamatergic reward input from the dorsal raphe to ventral tegmental area dopamine neurons. Nat. Commun. 5, 5390 (2014).

    Article  CAS  Google Scholar 

  36. Root, D.H., Mejias-Aponte, C.A., Qi, J. & Morales, M. Role of glutamatergic projections from ventral tegmental area to lateral habenula in aversive conditioning. J. Neurosci. 34, 13906–13910 (2014).

    Article  Google Scholar 

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Acknowledgements

The viral packaging and in vitro testing of the AAV-DIO-VMAT2(Myc) vector were done by the National Institute on Drug Abuse Intramural Research Program Optogenetics and Transgenic Technology Core (OTTC). We thank O. Kiehn and L. Borgius (Karolinska Institutet) for providing the Vglut2-Cre transgenic mice and K. Deisseroth (Stanford University) for the Th-Cre transgenic rats. Resources for three-dimensional analysis were supported by NS050274. The Intramural Research Program of the National Institute on Drug Abuse supported this work.

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Authors and Affiliations

Authors

Contributions

M.M. and S.Z. designed the experiments. S.Z., J.Q., X.L., H.-L.W., J.P.B. and A.F.H. performed the experiments. S.Z., J.Q., X.L., H.-L.W., J.P.B., A.F.H., A.B., C.R.L. and M.M. analyzed the data. M.M. wrote the paper with contributions from all of the other authors.

Corresponding author

Correspondence to Marisela Morales.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Subcellular segregation of VGluT2-IR and TH-IR within the same VGluT2-TH axon (wild type rats).

(a-e) Serial sections of a dual VGluT2-TH labeled axon. This axon (blue outline) has two distinguishable microdomains: an axon terminal (AT) with VGluT2 signal (scattered dark material) and an adjacent area containing TH signal (gold particles, arrowheads). Both compartments establish synapses with postsynaptic structures (orange outlines). The VGluT2 AT establishes an asymmetric synapse (green arrows) with the head of a dendritic spine (sp1), which contains a spine apparatus (sa). The segment of the axon containing TH establishes a symmetric synapse (blue arrows) with a dendritic spine (sp2). Scale bars represent 200 nm.

Supplementary Figure 2 Specificity evaluation of anti-VMAT2 antibodies by western blot analysis.

(a) Graphic map of pAAV-EF1a-DIO-VMAT2(Myc)-WPRE-pA. A Myc epitope was inserted in-frame with VMAT2 at the N-terminus for immunodetection of transgenic VMAT2. (b) VMAT2 immunodetection by different anti-VMAT2 antibodies. Western blots in lanes 1-8 from a gel loaded with the same amount of total protein from rat nAcc homogenate, and tested with eight different anti-VMAT2 antibodies as follows: (1) ab371945 from Abcam; (2) H-V008 from Phoenix Pharmaceuticals, Inc; (3) ab81855 from Abcam; (4) VMAT2-Rb-Af720 from Frontier Institute Co. ltd; (5) 138302 from Synaptic Systems; (6) AB1598P from EMD Millipore; (7) SAB2501101 from Sigma-Aldrich; (8) EB06558 from Everest Biotech. Seven out of the 8 tested anti-VMAT2 antibodies recognize multiple bands with exception of the EB06558 that recognizes two bands with a molecular weight 55 kDa (arrow), expected for VMAT2. Lanes 9 and 10 correspond to western blot prepared from total protein from transfected cell homogenates expressing VMAT2 with Myc tag, and probed with anti-VMAT2 antibody (EB06558) or anti-Myc antibody from EMD Millipore (05-724). Each anti-VMAT2 and anti-Myc antibodies recognize a single band with similar molecular weight (55 kDa), as detected in the nAcc homogenate.

Supplementary Figure 3 VGluT2 and VMAT2 localize to distinct subpopulations of synaptic vesicles (wild type rats).

(a) Full length western blots of proteins from isolated vesicles prior to immunoprecipitation, IP (T), and after IP with antibodies against VGluT2 (IP:VGluT2; B1) or VMAT2 (IP:VMAT2; B2). Western blots were immunolabeled (IB) with antibodies against VGluT2, VMAT2 or the vesicular marker synaptophysin. The vesicular nature of each fraction was confirmed by the detection of synaptophysin. VGluT2 and VMAT2 are present in the total pool of vesicles (T). In contrast, VGluT2 is detected only in the sample IP with anti-VGluT2 antibody (IP:VGluT2), and VMAT2 is detected only in the sample IP with anti-VMAT2 antibody (IP:VMAT2). (b) A model showing accumulation of dopamine by VMAT2 in a vesicle different from the one that accumulates glutamate by VGluT2.

Supplementary Figure 4 TH neurons expressing mCherry under the regulation of the TH promoter (TH::Cre mice).

(a) Schematic representation of AAV5-DIO-ChR2-mCherry injection into the VTA of TH::Cre mice. (b) Low magnification of a coronal section, which was incubated with an anti-mCherry antibody and hybridized with a TH antisense radioactive riboprobe. Section is seen under bright field (for visualization of mCherry-IR), and under epiluminescence microscope (for visualization of silver grains indicating expression of TH mRNA). Bregma -3.64 mm. Rectangular area in b is shown at higher magnification in (c-e) to display two neurons co-expressing TH-mRNA and mCherry-IR. (f) The bars indicate the frequency (mean + s.e.m.) of neurons containing mCherry-IR or TH mRNA from a total of 576 mCherry-IR neurons. Out of these neurons, 97.85 ± 0.83% have mCherry-IR and TH mRNA (t(2) = 17.07, P = 0.0034, n = 3). Scale bars represent 100 µm (b) and 10 µm (e).

Supplementary Figure 5 Mesoaccumbens axons from VGluT2 neurons expressing mCherry under the regulation of the VGluT2 promoter (VGluT2::Cre mice).

(a) Schematic representation of AAV5-DIO-ChR2-mCherry injection into the VTA of VGluT2::Cre mice. (b) Double labeled VTA cryosections for the detection of mCherry- IR or TH-IR. From this preparation, three different classes of neurons were laser micro-dissected: cells expressing only mCherry-IR (mCherry-IR+/TH-IR (arrows)), cells co-expressing mCherry-IR and TH-IR (mCherry-IR +/TH-IR+ (arrowheads)) and cells expressing only TH-IR (mCherry-IR/TH-IR+ (stars)). (c) Percentage of neurons containing detectable levels of VGluT2 mRNA or TH mRNA in each of the three classes of laser micro-dissected neurons: mCherry-IR+/TH-IR (n = 30 neurons), mCherry-IR+/TH-IR+ (n = 35 neurons), and mCherry-IR/TH-IR+ (n = 40 neurons). Each mCherry-IR+/TH-IR neuron was confirmed to have VGluT2 mRNA and lack TH mRNA. Each mCherry-IR/TH-IR+ neuron was confirmed to have TH mRNA and lack VGluT2 mRNA. The majority of dissected dual mCherry-IR+/TH-IR+ neurons found in the medial VTA have both VGluT2 mRNA and TH mRNA (97%, 34 out of 35 neurons). (d-g) Subcellular Segregation of TH and VGluT2 in the nAcc of VGluT2-ChR2-mCherry mice. Detection of mCherry (red) under the VGluT2 promoter, VGluT2-IR (green), and TH-IR (blue) in the VTA and nAcc. (d) Low magnification of the VTA showing cells with mCherry within the midline aspects of this structure. (e) High magnification of the area delimited with white box in (d) showing a dual labeled mCherry/TH neuron (1), and single labeled mCherry (2) and single labeled TH (3) neurons. (f) Within the nAcc, mCherry-IR under the VGluT2 promoter is observed along an axon (arrows) and within terminal-like structures (arrowheads), which contain VGluT2-IR. TH-IR is adjacent to the VGluT2-IR axon terminal. (g) Segregation among the VGluT2 terminal-like structures and the TH-axon segment within the same nAcc axon from a VGluT2-TH neuron is better seen in this 3-D reconstruction from Z-stack confocal microscopy images of triple labeled nAcc. Scale bars represent 10 µm (b), 100 µm (d), 5 µm (e) and 2 µm (f).

Supplementary Figure 6 Mouse mesoaccumbens neurons establish VGluT2-asymmetric synapses or TH-symmetric synapses (TH::Cre mice).

(a) Schematic representation of AAV5-DIO-ChR2-mCherry injection into the VTA of TH::Cre mice. (b) Electron micrograph of a nAcc AT from a VTA neuron co-expressing mCherry-IR (scattered dark material) and VGluT2-IR (gold particles, green arrowheads) making an asymmetric synapse (green arrow) with a dendrite (De). (c) Electron micrograph of an AT from a VTA neuron co-expressing mCherry-IR (scattered dark material) and TH-IR (gold particles, blue arrowhead) making a symmetric synapse (blue arrow) on a dendrite (De). (d) Schematic representation of nAcc inputs from VTA neurons expressing mCherry-IR under the regulation of TH promoter (TH-ChR2-mCherry mice). (e-g) A single AT from a VTA-VGluT2 neuron synapses on several postsynaptic structures. Serial sections of a single AT derived from a VTA neuron expressing mCherry-IR (scattered dark material) and containing VGluT2-IR (gold particles, green arrowheads) establishes asymmetric synapses (green arrows) with several postsynaptic structures, two dendritic spines (sp1 and sp2) and one dendrite (De). Scale bars represent 200 nm (b,c,e,f,g).

Supplementary Figure 7 TH immunoreactivity (TH-IR) is distributed in the entire axon and axon terminal lacking VGluT2 (TH::Cre mice).

(a) Schematic representation of nAcc inputs from VTA neurons expressing mCherry immunoreactivity (mCherry-IR) under the regulation of the TH promoter (TH-ChR2-mCherry mice). (b,c) Immunofluorescence detection of mCherry (red), TH (blue), and VGluT2 (green) in the nAcc. Note mCherry detection throughout the axon (red in b), and VGluT2-IR restricted to a neighboring terminal-like structure lacking mCherry-IR (arrowheads in b). Under 2-D view, TH-IR appears to be present in segments within the mCherry-IR axon and terminal (arrow in b). However, the TH distribution throughout the axon and axon terminal is better seen after 3-D reconstruction from Z-stack of confocal microscopy images of triple labeled nAcc (c). (d,e) Electron micrographs of serial sections of a TH-only axon containing mCherry-IR (scattered dark material) and TH-IR (gold particles, arrowheads). One axon terminal (AT) from this axon made a symmetric synapse (arrow) on a dendrite (De, orange outline). Scale bars represent 2 µm (b) and 200 nm (d,e).

Supplementary Figure 8 Compartmentalization of the storage and release of glutamate to axon terminals and the storage and release of dopamine to the adjacent axonal segment.

Mesoaccumbens synapses established by 3 different classes of mesocorticolimbic neurons (TH-only, VGluT2-only and VGluT2-TH neurons), and subcellular segregation for dopaminergic and glutamatergic signaling. Axon terminals containing dopamine vesicles (blue circles) and establishing symmetric synapses on the side of dendritic spines (sp1) are originated from either TH-only or VGluT2-TH neurons. In contrast, axon terminals containing glutamate vesicles (green circles) derived from VGluT2-only or VGluT2-TH neurons make asymmetric synapses on the head of dendritic spines (or dendrites, no representation in this diagram). A single axon terminal from these glutamatergic terminals can establish asymmetric synapses with several postsynaptic targets (sp2 and sp3). A dual glutamatergic-dopaminergic axon from a VGluT2-TH neuron illustrates the compartmentalization of the storage and release of glutamate to axon terminals and the storage and release of dopamine to the adjacent axonal segment.

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Zhang, S., Qi, J., Li, X. et al. Dopaminergic and glutamatergic microdomains in a subset of rodent mesoaccumbens axons. Nat Neurosci 18, 386–392 (2015). https://doi.org/10.1038/nn.3945

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