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Dopaminergic and glutamatergic microdomains in a subset of rodent mesoaccumbens axons.

Zhang S, Qi J, Li X, Wang HL, Britt JP, Hoffman AF, Bonci A, Lupica CR, Morales M - Nat. Neurosci. (2015)

Bottom Line: However, the mechanism is unclear, and co-release by mesoaccumbens fibers has not been documented.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.Using optogenetics, we found that dopamine and glutamate were released from the same mesoaccumbens fibers.

View Article: PubMed Central - PubMed

Affiliation: National Institute on Drug Abuse, Neuronal Networks Section, US National Institutes of Health, Baltimore, Maryland, USA.

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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Subcellular segregation of VGluT2-IR and VMAT2(Myc)-IR within the same VGluT2-TH axon from a TH::Cre rat overexpressing VMAT2(Myc) under the regulation of the TH-promoter [TH-VMAT2(Myc) rats](a) Schematic representation of VTA inputs (identified by Myc and VMAT2 content) to nAcc. VGluT2-TH and TH-only neurons were infected by injections of AAV-EF1a-DIO-VMAT2(Myc)-WPRE-pA into the VTA of TH::Cre rats [TH-VMAT2(Myc) rats, n = 4].(b) Immunofluorescence detection of Myc-IR (red), VMAT2-IR (blue), and TH-IR (green) in the medial VTA. Myc-IR, VMAT2-IR, and TH-IR are seen in the same cell bodies and processes.(c-d) Immunofluorescence detection of Myc-IR (red), VMAT2-IR (blue), and VGluT2-IR (green) in the nAcc. (c) Myc-IR and VMAT2-IR are present in the same axon, but VGluT2-IR is restricted to a terminal-like structure lacking both Myc-IR and VMAT2-IR. (d) The VGluT2-IR terminal-like structure in continuum to an axon co-expressing Myc-IR and VMAT2-IR is better seen in this 3-D reconstruction from Z-stack confocal microscopy images.(e-f) Electron micrographs of ATs containing VGluT2-IR (scattered dark material), but lacking both VMAT2-IR and Myc-IR (gold particles). The VGluT2-IR axon-terminal is adjacent to an axon segment that contains VMAT2-IR (blue arrow, gold particles in e) or Myc-IR (red arrows, gold particles in f). Note in e that the axon-terminal containing VGluT2-IR establishes an asymmetric synapse (green arrow).Bars: (b) 10 μm; (c) 2 μm; and (e and f) 200 nm.
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Figure 4: Subcellular segregation of VGluT2-IR and VMAT2(Myc)-IR within the same VGluT2-TH axon from a TH::Cre rat overexpressing VMAT2(Myc) under the regulation of the TH-promoter [TH-VMAT2(Myc) rats](a) Schematic representation of VTA inputs (identified by Myc and VMAT2 content) to nAcc. VGluT2-TH and TH-only neurons were infected by injections of AAV-EF1a-DIO-VMAT2(Myc)-WPRE-pA into the VTA of TH::Cre rats [TH-VMAT2(Myc) rats, n = 4].(b) Immunofluorescence detection of Myc-IR (red), VMAT2-IR (blue), and TH-IR (green) in the medial VTA. Myc-IR, VMAT2-IR, and TH-IR are seen in the same cell bodies and processes.(c-d) Immunofluorescence detection of Myc-IR (red), VMAT2-IR (blue), and VGluT2-IR (green) in the nAcc. (c) Myc-IR and VMAT2-IR are present in the same axon, but VGluT2-IR is restricted to a terminal-like structure lacking both Myc-IR and VMAT2-IR. (d) The VGluT2-IR terminal-like structure in continuum to an axon co-expressing Myc-IR and VMAT2-IR is better seen in this 3-D reconstruction from Z-stack confocal microscopy images.(e-f) Electron micrographs of ATs containing VGluT2-IR (scattered dark material), but lacking both VMAT2-IR and Myc-IR (gold particles). The VGluT2-IR axon-terminal is adjacent to an axon segment that contains VMAT2-IR (blue arrow, gold particles in e) or Myc-IR (red arrows, gold particles in f). Note in e that the axon-terminal containing VGluT2-IR establishes an asymmetric synapse (green arrow).Bars: (b) 10 μm; (c) 2 μm; and (e and f) 200 nm.

Mentions: To further validate the detection of subcellular segregation between VGluT2 and VMAT2 within mesoaccumbens axons, we selectively induced in vivo overexpression of VMAT2 with the Myc tag at the amino-terminus [VMAT2(Myc)] in dopamine neurons by injecting an AAV vector encoding Cre-inducible VMAT2(Myc) in the VTA of TH::Cre rats (Fig. 4a). By triple immunofluorescence (TH-VMAT2-Myc), we confirmed Myc expression confined to VTA-TH neurons co-expressing VMAT2 (Fig. 4b). In the nAcc, we observed axons with dual VMAT2-Myc immunolabel contiguous with VGluT2-IR terminals (Fig. 4c,d). By 3-D analysis, we found that the vast majority of mesoaccumbens VGluT2-terminals lacked VMAT2-Myc (99.45 ± 0.08%; 8548 out of 8593 ATs) (t(3) = 527.00, p = 0.0001). In addition, we confirmed by electron microscopy the ultrastructural presence of VGluT2 in ATs (making asymmetric synapses) that were adjacent to axon segments containing VMAT2 (Fig. 4e) or Myc (Fig. 4f). We determined that the majority of ATs expressing VGluT2 lacked VMAT2 or Myc (98.84 ± 0.64%;160 out of 162 ATs) (t(3) = 75.94, p = 0.0002). These findings suggest that VMAT2(Myc) overexpression in mesoaccumbens axons from VGluT2-TH neurons does not alter the segregation between VGluT2 and VMAT2. Segregation between VGluT2-vesicles and VMAT2-vesicles may be conferred by specific sorting signals determined by selective motifs within each of these transporters, as previously documented for other vesicular transporters18.


Dopaminergic and glutamatergic microdomains in a subset of rodent mesoaccumbens axons.

Zhang S, Qi J, Li X, Wang HL, Britt JP, Hoffman AF, Bonci A, Lupica CR, Morales M - Nat. Neurosci. (2015)

Subcellular segregation of VGluT2-IR and VMAT2(Myc)-IR within the same VGluT2-TH axon from a TH::Cre rat overexpressing VMAT2(Myc) under the regulation of the TH-promoter [TH-VMAT2(Myc) rats](a) Schematic representation of VTA inputs (identified by Myc and VMAT2 content) to nAcc. VGluT2-TH and TH-only neurons were infected by injections of AAV-EF1a-DIO-VMAT2(Myc)-WPRE-pA into the VTA of TH::Cre rats [TH-VMAT2(Myc) rats, n = 4].(b) Immunofluorescence detection of Myc-IR (red), VMAT2-IR (blue), and TH-IR (green) in the medial VTA. Myc-IR, VMAT2-IR, and TH-IR are seen in the same cell bodies and processes.(c-d) Immunofluorescence detection of Myc-IR (red), VMAT2-IR (blue), and VGluT2-IR (green) in the nAcc. (c) Myc-IR and VMAT2-IR are present in the same axon, but VGluT2-IR is restricted to a terminal-like structure lacking both Myc-IR and VMAT2-IR. (d) The VGluT2-IR terminal-like structure in continuum to an axon co-expressing Myc-IR and VMAT2-IR is better seen in this 3-D reconstruction from Z-stack confocal microscopy images.(e-f) Electron micrographs of ATs containing VGluT2-IR (scattered dark material), but lacking both VMAT2-IR and Myc-IR (gold particles). The VGluT2-IR axon-terminal is adjacent to an axon segment that contains VMAT2-IR (blue arrow, gold particles in e) or Myc-IR (red arrows, gold particles in f). Note in e that the axon-terminal containing VGluT2-IR establishes an asymmetric synapse (green arrow).Bars: (b) 10 μm; (c) 2 μm; and (e and f) 200 nm.
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Figure 4: Subcellular segregation of VGluT2-IR and VMAT2(Myc)-IR within the same VGluT2-TH axon from a TH::Cre rat overexpressing VMAT2(Myc) under the regulation of the TH-promoter [TH-VMAT2(Myc) rats](a) Schematic representation of VTA inputs (identified by Myc and VMAT2 content) to nAcc. VGluT2-TH and TH-only neurons were infected by injections of AAV-EF1a-DIO-VMAT2(Myc)-WPRE-pA into the VTA of TH::Cre rats [TH-VMAT2(Myc) rats, n = 4].(b) Immunofluorescence detection of Myc-IR (red), VMAT2-IR (blue), and TH-IR (green) in the medial VTA. Myc-IR, VMAT2-IR, and TH-IR are seen in the same cell bodies and processes.(c-d) Immunofluorescence detection of Myc-IR (red), VMAT2-IR (blue), and VGluT2-IR (green) in the nAcc. (c) Myc-IR and VMAT2-IR are present in the same axon, but VGluT2-IR is restricted to a terminal-like structure lacking both Myc-IR and VMAT2-IR. (d) The VGluT2-IR terminal-like structure in continuum to an axon co-expressing Myc-IR and VMAT2-IR is better seen in this 3-D reconstruction from Z-stack confocal microscopy images.(e-f) Electron micrographs of ATs containing VGluT2-IR (scattered dark material), but lacking both VMAT2-IR and Myc-IR (gold particles). The VGluT2-IR axon-terminal is adjacent to an axon segment that contains VMAT2-IR (blue arrow, gold particles in e) or Myc-IR (red arrows, gold particles in f). Note in e that the axon-terminal containing VGluT2-IR establishes an asymmetric synapse (green arrow).Bars: (b) 10 μm; (c) 2 μm; and (e and f) 200 nm.
Mentions: To further validate the detection of subcellular segregation between VGluT2 and VMAT2 within mesoaccumbens axons, we selectively induced in vivo overexpression of VMAT2 with the Myc tag at the amino-terminus [VMAT2(Myc)] in dopamine neurons by injecting an AAV vector encoding Cre-inducible VMAT2(Myc) in the VTA of TH::Cre rats (Fig. 4a). By triple immunofluorescence (TH-VMAT2-Myc), we confirmed Myc expression confined to VTA-TH neurons co-expressing VMAT2 (Fig. 4b). In the nAcc, we observed axons with dual VMAT2-Myc immunolabel contiguous with VGluT2-IR terminals (Fig. 4c,d). By 3-D analysis, we found that the vast majority of mesoaccumbens VGluT2-terminals lacked VMAT2-Myc (99.45 ± 0.08%; 8548 out of 8593 ATs) (t(3) = 527.00, p = 0.0001). In addition, we confirmed by electron microscopy the ultrastructural presence of VGluT2 in ATs (making asymmetric synapses) that were adjacent to axon segments containing VMAT2 (Fig. 4e) or Myc (Fig. 4f). We determined that the majority of ATs expressing VGluT2 lacked VMAT2 or Myc (98.84 ± 0.64%;160 out of 162 ATs) (t(3) = 75.94, p = 0.0002). These findings suggest that VMAT2(Myc) overexpression in mesoaccumbens axons from VGluT2-TH neurons does not alter the segregation between VGluT2 and VMAT2. Segregation between VGluT2-vesicles and VMAT2-vesicles may be conferred by specific sorting signals determined by selective motifs within each of these transporters, as previously documented for other vesicular transporters18.

Bottom Line: However, the mechanism is unclear, and co-release by mesoaccumbens fibers has not been documented.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.Using optogenetics, we found that dopamine and glutamate were released from the same mesoaccumbens fibers.

View Article: PubMed Central - PubMed

Affiliation: National Institute on Drug Abuse, Neuronal Networks Section, US National Institutes of Health, Baltimore, Maryland, USA.

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.

Show MeSH
Related in: MedlinePlus