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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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VGluT2-TH neurons segregate VGluT2-IR and TH-IR to distinct subcellular compartments within the same axon (TH::Cre mice)(a) Schematic representation of nAcc inputs from VTA neurons expressing mCherry-IR under the regulation of TH-promoter (TH-ChR2-mCherry mice, n = 4).(b-d) Fluorescence detection of mCherry-IR (red), TH-IR (blue), and VGluT2-IR (green) in the lateral VTA and nAcc. (b) Within the VTA, mCherry-IR is seen in cell bodies (1, 2, and 3) and processes. These mCherry-IR cell bodies lack VGluT2-IR, but have TH-IR. Within the nAcc, mCherry (under the regulation of the TH-promoter) is detected throughout the axon (red in c). In contrast, VGluT2-IR is restricted to a terminal-like structure (arrowheads in c). TH-IR is present in segments within the mCherry-IR axon (arrow in c). (d) Segregation among one VGluT2 terminal-like structures and TH-axon segments 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 .Bars: (b) 5 μm; and (c) 2 μm.
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Figure 5: VGluT2-TH neurons segregate VGluT2-IR and TH-IR to distinct subcellular compartments within the same axon (TH::Cre mice)(a) Schematic representation of nAcc inputs from VTA neurons expressing mCherry-IR under the regulation of TH-promoter (TH-ChR2-mCherry mice, n = 4).(b-d) Fluorescence detection of mCherry-IR (red), TH-IR (blue), and VGluT2-IR (green) in the lateral VTA and nAcc. (b) Within the VTA, mCherry-IR is seen in cell bodies (1, 2, and 3) and processes. These mCherry-IR cell bodies lack VGluT2-IR, but have TH-IR. Within the nAcc, mCherry (under the regulation of the TH-promoter) is detected throughout the axon (red in c). In contrast, VGluT2-IR is restricted to a terminal-like structure (arrowheads in c). TH-IR is present in segments within the mCherry-IR axon (arrow in c). (d) Segregation among one VGluT2 terminal-like structures and TH-axon segments 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 .Bars: (b) 5 μm; and (c) 2 μm.

Mentions: To determine whether in the mouse, as in the rat, the VGluT2 neurons form asymmetric synapses in the nAcc, and segregate TH-IR and VGluT2-IR within the same axons, we injected into the VTA of TH::Cre or of VGluT2::Cre mice an AAV vector encoding Cre-inducible light-activated channelrhodopsin2 (ChR2) tethered to mCherry (Fig. 5a and Supplementary Fig. 4a, 5a). In the VTA of infected TH::Cre mice (TH-ChR2-mCherry mice, n = 3), we detected mCherry confined to neurons expressing TH-mRNA (Supplementary Fig. 4b-e). From a total of 576 mCherry-IR neurons, 97.85 ± 0.83% co-expressed TH mRNA (Supplementary Fig. 4f), indicating the selective expression of mCherry under the TH-promoter. In addition, we detected co-expression of mCherry and TH-IR in neurons located within the VTA (Fig. 5b), corresponding to the location of dopamine neurons innervating the nAcc19. Because VGluT2-protein is undetectable in cell bodies of glutamatergic neurons, we determined the selectivity of mCherry expression under the VGluT2 promoter by the detection of VGluT2 mRNA in mCherry expressing neurons taken from VTA tissue by laser-capture dissection. By qRT-PCR analysis of individual micro-dissected neurons we confirmed the selective expression of mCherry under the VGluT2 promoter (Supplementary Fig. 5b,c). We found that the ultrastructural features of TH- or VGluT2-mesoaccumbens synapses from mice expressing mCherry under the TH promoter (TH-ChR2-mCherry; Supplementary Fig. 6a) or under the VGluT2 promoter (VGluT2-ChR2-mCherry) were indistinguishable; the mCherry terminals making asymmetric synapses contained VGluT2-protein but not TH-protein (Supplementary Fig. 6b), and those making symmetric synapses contained TH-protein, but not VGluT2-protein (Supplementary Fig. 6c). These ultrastructural findings provide evidence that as in the rat, the VGluT2-TH neurons make TH-terminals which are distinct from their VGluT2-terminals, and that their postsynaptic targets on dendritic spines are mutually exclusive (Supplementary Fig. 6d). We observed examples in which a single VGluT2-terminal (from TH-ChR2-mCherry mice or VGluT2-ChR2-mCherry mice) established multiple synapses in the nAcc (Supplementary Fig. 6e-g), indicating that a single VGluT2-terminal has the capacity of affecting multiple postsynaptic neurons.


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)

VGluT2-TH neurons segregate VGluT2-IR and TH-IR to distinct subcellular compartments within the same axon (TH::Cre mice)(a) Schematic representation of nAcc inputs from VTA neurons expressing mCherry-IR under the regulation of TH-promoter (TH-ChR2-mCherry mice, n = 4).(b-d) Fluorescence detection of mCherry-IR (red), TH-IR (blue), and VGluT2-IR (green) in the lateral VTA and nAcc. (b) Within the VTA, mCherry-IR is seen in cell bodies (1, 2, and 3) and processes. These mCherry-IR cell bodies lack VGluT2-IR, but have TH-IR. Within the nAcc, mCherry (under the regulation of the TH-promoter) is detected throughout the axon (red in c). In contrast, VGluT2-IR is restricted to a terminal-like structure (arrowheads in c). TH-IR is present in segments within the mCherry-IR axon (arrow in c). (d) Segregation among one VGluT2 terminal-like structures and TH-axon segments 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 .Bars: (b) 5 μm; and (c) 2 μm.
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Figure 5: VGluT2-TH neurons segregate VGluT2-IR and TH-IR to distinct subcellular compartments within the same axon (TH::Cre mice)(a) Schematic representation of nAcc inputs from VTA neurons expressing mCherry-IR under the regulation of TH-promoter (TH-ChR2-mCherry mice, n = 4).(b-d) Fluorescence detection of mCherry-IR (red), TH-IR (blue), and VGluT2-IR (green) in the lateral VTA and nAcc. (b) Within the VTA, mCherry-IR is seen in cell bodies (1, 2, and 3) and processes. These mCherry-IR cell bodies lack VGluT2-IR, but have TH-IR. Within the nAcc, mCherry (under the regulation of the TH-promoter) is detected throughout the axon (red in c). In contrast, VGluT2-IR is restricted to a terminal-like structure (arrowheads in c). TH-IR is present in segments within the mCherry-IR axon (arrow in c). (d) Segregation among one VGluT2 terminal-like structures and TH-axon segments 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 .Bars: (b) 5 μm; and (c) 2 μm.
Mentions: To determine whether in the mouse, as in the rat, the VGluT2 neurons form asymmetric synapses in the nAcc, and segregate TH-IR and VGluT2-IR within the same axons, we injected into the VTA of TH::Cre or of VGluT2::Cre mice an AAV vector encoding Cre-inducible light-activated channelrhodopsin2 (ChR2) tethered to mCherry (Fig. 5a and Supplementary Fig. 4a, 5a). In the VTA of infected TH::Cre mice (TH-ChR2-mCherry mice, n = 3), we detected mCherry confined to neurons expressing TH-mRNA (Supplementary Fig. 4b-e). From a total of 576 mCherry-IR neurons, 97.85 ± 0.83% co-expressed TH mRNA (Supplementary Fig. 4f), indicating the selective expression of mCherry under the TH-promoter. In addition, we detected co-expression of mCherry and TH-IR in neurons located within the VTA (Fig. 5b), corresponding to the location of dopamine neurons innervating the nAcc19. Because VGluT2-protein is undetectable in cell bodies of glutamatergic neurons, we determined the selectivity of mCherry expression under the VGluT2 promoter by the detection of VGluT2 mRNA in mCherry expressing neurons taken from VTA tissue by laser-capture dissection. By qRT-PCR analysis of individual micro-dissected neurons we confirmed the selective expression of mCherry under the VGluT2 promoter (Supplementary Fig. 5b,c). We found that the ultrastructural features of TH- or VGluT2-mesoaccumbens synapses from mice expressing mCherry under the TH promoter (TH-ChR2-mCherry; Supplementary Fig. 6a) or under the VGluT2 promoter (VGluT2-ChR2-mCherry) were indistinguishable; the mCherry terminals making asymmetric synapses contained VGluT2-protein but not TH-protein (Supplementary Fig. 6b), and those making symmetric synapses contained TH-protein, but not VGluT2-protein (Supplementary Fig. 6c). These ultrastructural findings provide evidence that as in the rat, the VGluT2-TH neurons make TH-terminals which are distinct from their VGluT2-terminals, and that their postsynaptic targets on dendritic spines are mutually exclusive (Supplementary Fig. 6d). We observed examples in which a single VGluT2-terminal (from TH-ChR2-mCherry mice or VGluT2-ChR2-mCherry mice) established multiple synapses in the nAcc (Supplementary Fig. 6e-g), indicating that a single VGluT2-terminal has the capacity of affecting multiple postsynaptic neurons.

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