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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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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)(a-c) Photo-stimulation of nAcc fibers from VGluT2 VTA neurons (n = 6 VGluT2-ChR2-mCherry mice). (b) Optically-evoked EPSCs recorded in nAcc shell. (c) Paired pulse ratios (P2/P1) in nAcc neurons (n = 9 per time point).(d) Electrically- or optically-evoked dopamine were measured by voltammetry. (e) Optically-evoked dopamine release was increased in response to the increment of the number of pulses; 5 ms pulses (blue box) at 25 Hz. (f) dopamine voltammogram in response to 10 pulses of photo-stimulation. (g) Electrically-evoked dopamine release (10 × 1 ms pulses at 25 Hz; arrow) at the same location of photo-stimulation. (h) Summary of dopamine release in response to the number of optical pulses (n = 6 slices from 3 mice). (i) Bath application of the VMAT2 inhibitor tetrabenazine (TBZ, 10 μM) significantly reduced optically-evoked dopamine release (p = 0.02 vs control, paired t-test, n = 4 slices from 2 mice). (j) optically-evoked dopamine responses from VGluT2-TH neurons are not dependent on glutamate receptors. Traces show optically-evoked (25 Hz, 5 pulses, 5 ms) nAcc voltammetric currents measured prior to and following application of the glutamate receptor antagonists NBQX (5μM) and AP-5 (40 μM). Graph shows the time course summary (mean ± s.e.m., n = 4 recordings from 2 mice); NBQX and AP-5 were applied during the time indicated by the bar. No significant effect on the dopamine signals was observed (two-tailed paired t-test, t(3) = 0.1239, p = 0.9093).
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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)(a-c) Photo-stimulation of nAcc fibers from VGluT2 VTA neurons (n = 6 VGluT2-ChR2-mCherry mice). (b) Optically-evoked EPSCs recorded in nAcc shell. (c) Paired pulse ratios (P2/P1) in nAcc neurons (n = 9 per time point).(d) Electrically- or optically-evoked dopamine were measured by voltammetry. (e) Optically-evoked dopamine release was increased in response to the increment of the number of pulses; 5 ms pulses (blue box) at 25 Hz. (f) dopamine voltammogram in response to 10 pulses of photo-stimulation. (g) Electrically-evoked dopamine release (10 × 1 ms pulses at 25 Hz; arrow) at the same location of photo-stimulation. (h) Summary of dopamine release in response to the number of optical pulses (n = 6 slices from 3 mice). (i) Bath application of the VMAT2 inhibitor tetrabenazine (TBZ, 10 μM) significantly reduced optically-evoked dopamine release (p = 0.02 vs control, paired t-test, n = 4 slices from 2 mice). (j) optically-evoked dopamine responses from VGluT2-TH neurons are not dependent on glutamate receptors. Traces show optically-evoked (25 Hz, 5 pulses, 5 ms) nAcc voltammetric currents measured prior to and following application of the glutamate receptor antagonists NBQX (5μM) and AP-5 (40 μM). Graph shows the time course summary (mean ± s.e.m., n = 4 recordings from 2 mice); NBQX and AP-5 were applied during the time indicated by the bar. No significant effect on the dopamine signals was observed (two-tailed paired t-test, t(3) = 0.1239, p = 0.9093).

Mentions: The ultrastructural findings demonstrating that VGluT2-axon terminals from both VGluT2-only and VGluT2-TH neurons establish asymmetric terminals in the nAcc suggest that these VGluT2 terminals use glutamate as a signaling molecule. To evaluate glutamatergic signaling by VGluT2 mesoaccumbens terminals, VGluT2-ChR2-mCherry fibers were stimulated using 473 nm light pulses delivered into nAcc slices (Fig. 6a-c). As previously reported, we found by whole-cell recordings of NAcc medium spiny neurons that local photo-stimulation of VGluT2-ChR2-mCherry fibers evokes EPSCs (47 ± 14 pA; n = 16 neurons from 3 mice) that depends on both AMPA and NMDA receptors (AMPA/NMDA ratio 1.9 ± 0.4; n = 5 neurons from 3 mice)13. As detailed above, there is electrophysiological evidence indicating that some VTA-TH neurons have the capacity to release glutamate in the nAcc1, 2. However, it remains to be determined whether some of the same neurons also release dopamine. Here, we applied fast-scan cyclic voltammetry to evaluate dopamine release by VGluT2-TH neurons following photo-stimulation of VGluT2-ChR2-eYFP fibers in nAcc slices. We found that selective photo-stimulation of fibers from VTA VGluT2-expressing neurons evoked nAcc phasic release of dopamine (Fig. 6d-j), which was elevated in response to incremental numbers of light pulses (Fig. 6h), and was significantly reduced by bath application of the VMAT2 inhibitor tetrabenazine (Fig. 6i; p = 0.02 vs control). To determine whether light-evoked dopamine responses from VGluT2-TH neurons were dependent on glutamate receptors, we measured dopamine release in the nAcc prior to and following application of the glutamate receptor antagonists NBQX (5μM) and AP-5 (40 μM). These antagonists did not significantly alter light-evoked dopamine signals (Fig. 6j). These findings indicate that dopamine is synthesized, stored via a VMAT2-dependent process, and released directly by mesoaccumbens fibers arising from VGluT2-TH 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)

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)(a-c) Photo-stimulation of nAcc fibers from VGluT2 VTA neurons (n = 6 VGluT2-ChR2-mCherry mice). (b) Optically-evoked EPSCs recorded in nAcc shell. (c) Paired pulse ratios (P2/P1) in nAcc neurons (n = 9 per time point).(d) Electrically- or optically-evoked dopamine were measured by voltammetry. (e) Optically-evoked dopamine release was increased in response to the increment of the number of pulses; 5 ms pulses (blue box) at 25 Hz. (f) dopamine voltammogram in response to 10 pulses of photo-stimulation. (g) Electrically-evoked dopamine release (10 × 1 ms pulses at 25 Hz; arrow) at the same location of photo-stimulation. (h) Summary of dopamine release in response to the number of optical pulses (n = 6 slices from 3 mice). (i) Bath application of the VMAT2 inhibitor tetrabenazine (TBZ, 10 μM) significantly reduced optically-evoked dopamine release (p = 0.02 vs control, paired t-test, n = 4 slices from 2 mice). (j) optically-evoked dopamine responses from VGluT2-TH neurons are not dependent on glutamate receptors. Traces show optically-evoked (25 Hz, 5 pulses, 5 ms) nAcc voltammetric currents measured prior to and following application of the glutamate receptor antagonists NBQX (5μM) and AP-5 (40 μM). Graph shows the time course summary (mean ± s.e.m., n = 4 recordings from 2 mice); NBQX and AP-5 were applied during the time indicated by the bar. No significant effect on the dopamine signals was observed (two-tailed paired t-test, t(3) = 0.1239, p = 0.9093).
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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)(a-c) Photo-stimulation of nAcc fibers from VGluT2 VTA neurons (n = 6 VGluT2-ChR2-mCherry mice). (b) Optically-evoked EPSCs recorded in nAcc shell. (c) Paired pulse ratios (P2/P1) in nAcc neurons (n = 9 per time point).(d) Electrically- or optically-evoked dopamine were measured by voltammetry. (e) Optically-evoked dopamine release was increased in response to the increment of the number of pulses; 5 ms pulses (blue box) at 25 Hz. (f) dopamine voltammogram in response to 10 pulses of photo-stimulation. (g) Electrically-evoked dopamine release (10 × 1 ms pulses at 25 Hz; arrow) at the same location of photo-stimulation. (h) Summary of dopamine release in response to the number of optical pulses (n = 6 slices from 3 mice). (i) Bath application of the VMAT2 inhibitor tetrabenazine (TBZ, 10 μM) significantly reduced optically-evoked dopamine release (p = 0.02 vs control, paired t-test, n = 4 slices from 2 mice). (j) optically-evoked dopamine responses from VGluT2-TH neurons are not dependent on glutamate receptors. Traces show optically-evoked (25 Hz, 5 pulses, 5 ms) nAcc voltammetric currents measured prior to and following application of the glutamate receptor antagonists NBQX (5μM) and AP-5 (40 μM). Graph shows the time course summary (mean ± s.e.m., n = 4 recordings from 2 mice); NBQX and AP-5 were applied during the time indicated by the bar. No significant effect on the dopamine signals was observed (two-tailed paired t-test, t(3) = 0.1239, p = 0.9093).
Mentions: The ultrastructural findings demonstrating that VGluT2-axon terminals from both VGluT2-only and VGluT2-TH neurons establish asymmetric terminals in the nAcc suggest that these VGluT2 terminals use glutamate as a signaling molecule. To evaluate glutamatergic signaling by VGluT2 mesoaccumbens terminals, VGluT2-ChR2-mCherry fibers were stimulated using 473 nm light pulses delivered into nAcc slices (Fig. 6a-c). As previously reported, we found by whole-cell recordings of NAcc medium spiny neurons that local photo-stimulation of VGluT2-ChR2-mCherry fibers evokes EPSCs (47 ± 14 pA; n = 16 neurons from 3 mice) that depends on both AMPA and NMDA receptors (AMPA/NMDA ratio 1.9 ± 0.4; n = 5 neurons from 3 mice)13. As detailed above, there is electrophysiological evidence indicating that some VTA-TH neurons have the capacity to release glutamate in the nAcc1, 2. However, it remains to be determined whether some of the same neurons also release dopamine. Here, we applied fast-scan cyclic voltammetry to evaluate dopamine release by VGluT2-TH neurons following photo-stimulation of VGluT2-ChR2-eYFP fibers in nAcc slices. We found that selective photo-stimulation of fibers from VTA VGluT2-expressing neurons evoked nAcc phasic release of dopamine (Fig. 6d-j), which was elevated in response to incremental numbers of light pulses (Fig. 6h), and was significantly reduced by bath application of the VMAT2 inhibitor tetrabenazine (Fig. 6i; p = 0.02 vs control). To determine whether light-evoked dopamine responses from VGluT2-TH neurons were dependent on glutamate receptors, we measured dopamine release in the nAcc prior to and following application of the glutamate receptor antagonists NBQX (5μM) and AP-5 (40 μM). These antagonists did not significantly alter light-evoked dopamine signals (Fig. 6j). These findings indicate that dopamine is synthesized, stored via a VMAT2-dependent process, and released directly by mesoaccumbens fibers arising from VGluT2-TH 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