Limits...
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.

Show MeSH

Related in: MedlinePlus

VGluT2 and VMAT2 localize to distinct subpopulations of synaptic vesicles (wild type rats)(a) Electron micrograph showing the purity and integraty of nAcc isolated synaptic vesicles used for either dual detection of VGluT2-IR and VMAT2-IR (b-d) or co-immunoprecipitation of VGluT2 and VMAT2 (f).(b-d) Detection of VGluT2-IR (arrows; 18 nm gold particles) or VMAT2-IR (arrowheads; 12 nm gold particles) associated to purified synaptic vesicles.(e) Bars indicating the frequency (mean + s.e.m.) of vesicles containing VGluT2-IR or VMAT2-IR from a total of 560 labeled vesicles. Out of these vesicles, 66.74 ± 4.10% have VGluT2-IR; 31.71 ± 3.99% have VMAT2-IR and 1.55 ± 0.35% appear to co-label for VGluT2 and VMAT2 (paired t-test, t(2) = 4.333, p = 0.0493). Synaptic vesicles were quantified from 3 different preparations of isolated vesicles from the nAcc of rats (n = 110).(f) 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 antibodies (IP:VGluT2), and VMAT2 is detected only in the sample IP with anti-VMAT2 antibodies. The western blots were successfully repeated at least 3 times. Full-length blots are presented in Supplementary Figure 3.Bars: (a and b) 200 nm; (c and d) 50 nm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4340758&req=5

Figure 3: VGluT2 and VMAT2 localize to distinct subpopulations of synaptic vesicles (wild type rats)(a) Electron micrograph showing the purity and integraty of nAcc isolated synaptic vesicles used for either dual detection of VGluT2-IR and VMAT2-IR (b-d) or co-immunoprecipitation of VGluT2 and VMAT2 (f).(b-d) Detection of VGluT2-IR (arrows; 18 nm gold particles) or VMAT2-IR (arrowheads; 12 nm gold particles) associated to purified synaptic vesicles.(e) Bars indicating the frequency (mean + s.e.m.) of vesicles containing VGluT2-IR or VMAT2-IR from a total of 560 labeled vesicles. Out of these vesicles, 66.74 ± 4.10% have VGluT2-IR; 31.71 ± 3.99% have VMAT2-IR and 1.55 ± 0.35% appear to co-label for VGluT2 and VMAT2 (paired t-test, t(2) = 4.333, p = 0.0493). Synaptic vesicles were quantified from 3 different preparations of isolated vesicles from the nAcc of rats (n = 110).(f) 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 antibodies (IP:VGluT2), and VMAT2 is detected only in the sample IP with anti-VMAT2 antibodies. The western blots were successfully repeated at least 3 times. Full-length blots are presented in Supplementary Figure 3.Bars: (a and b) 200 nm; (c and d) 50 nm.

Mentions: The ultrastructural findings detailed above provide evidence that in the nAcc the storage of dopamine and glutamate takes place in distinct vesicular pools enriched in different axonal micro-domains. However, to discard that our ultrastructural techniques lacked the sensitivity for co-detection of VGluT2 and VMAT2, we next explored the possibility of coexistence of VGluT2 and VMAT2 at the vesicular level by immunolabeling and by co-immunoprecipitation of VGluT2 and VMAT2 from vesicles obtained from rat nAcc synaptomes. After testing several conditions of isolation for synaptic vesicles, we achieved the experimental conditions (see online methods) that allowed the purification of a homogeneous population of synaptic vesicles, the quality and purity of which were confirmed by electron microscopy analysis (Fig. 3a). By immunolabeling of these isolated synaptic vesicles, we detected two distinct pools of vesicles, those containing VGluT2-IR and those containing VMAT2-IR (Fig. 3b-d). We found that from a total of 560 labeled vesicles, 66.74 ± 4.10% had VGluT2; 31.71 ± 3.99% had VMAT2, and a very small proportions (1.55 ± 0.35%) appeared to co-label for VGluT2 and VMAT2 (Fig. 3e). The lack of coexistence of VGluT2 and VMAT2 at the vesicular level was next confirmed by co-immunoprecipitation of VGluT2 and VMAT2 from the isolated vesicles. Consistent with our ultrastructural findings, protein preparations of vesicles immunoprecipitated with anti-VGluT2 antibodies showed vesicular immunodetection of both VGluT2 and the vesicular marker synaptophysin, but lacked evidence of VMAT2 (Fig. 3f and Supplementary Fig. 3a). Moreover, protein preparations of vesicles immunoprecipitated with anti-VMAT2 antibodies showed vesicular immunodetection of both VMAT2 and synaptophysin, but lacked evidence of VGluT2 (Fig. 3f and Supplementary Fig. 3a). Thus, we conclude that accumulation of dopamine by VMAT2 occurs in vesicles different from those that accumulate glutamate by VGluT2 (Supplementary Fig. 3b). In contrast to our findings, a previous study using enriched membrane preparations from nAcc reported co-immunoprecipitation of VMAT2 and VGluT212. The discrepancy between this earlier work and our present results may be due to major differences in the material used for immunoprecipitation. In our studies, we used purified synaptic vesicles instead of enriched membranes. In addition, we utilized sucrose to avoid organelle disruption, as organelle disruption may result in membrane fusion among different types of membranes. Moreover, we characterized the specificity of the antibodies used for detection of VMAT2 (Supplementary Fig. 2), to reduce the likelihood of nonspecific antigen recognition. Together, our ultrastructural findings are consistent with our molecular findings and do not support the previously proposed hypothesis that dopamine and glutamate are packaged and co-released in the nAcc from the same pool of vesicles1, 12, 13.


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 and VMAT2 localize to distinct subpopulations of synaptic vesicles (wild type rats)(a) Electron micrograph showing the purity and integraty of nAcc isolated synaptic vesicles used for either dual detection of VGluT2-IR and VMAT2-IR (b-d) or co-immunoprecipitation of VGluT2 and VMAT2 (f).(b-d) Detection of VGluT2-IR (arrows; 18 nm gold particles) or VMAT2-IR (arrowheads; 12 nm gold particles) associated to purified synaptic vesicles.(e) Bars indicating the frequency (mean + s.e.m.) of vesicles containing VGluT2-IR or VMAT2-IR from a total of 560 labeled vesicles. Out of these vesicles, 66.74 ± 4.10% have VGluT2-IR; 31.71 ± 3.99% have VMAT2-IR and 1.55 ± 0.35% appear to co-label for VGluT2 and VMAT2 (paired t-test, t(2) = 4.333, p = 0.0493). Synaptic vesicles were quantified from 3 different preparations of isolated vesicles from the nAcc of rats (n = 110).(f) 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 antibodies (IP:VGluT2), and VMAT2 is detected only in the sample IP with anti-VMAT2 antibodies. The western blots were successfully repeated at least 3 times. Full-length blots are presented in Supplementary Figure 3.Bars: (a and b) 200 nm; (c and d) 50 nm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4340758&req=5

Figure 3: VGluT2 and VMAT2 localize to distinct subpopulations of synaptic vesicles (wild type rats)(a) Electron micrograph showing the purity and integraty of nAcc isolated synaptic vesicles used for either dual detection of VGluT2-IR and VMAT2-IR (b-d) or co-immunoprecipitation of VGluT2 and VMAT2 (f).(b-d) Detection of VGluT2-IR (arrows; 18 nm gold particles) or VMAT2-IR (arrowheads; 12 nm gold particles) associated to purified synaptic vesicles.(e) Bars indicating the frequency (mean + s.e.m.) of vesicles containing VGluT2-IR or VMAT2-IR from a total of 560 labeled vesicles. Out of these vesicles, 66.74 ± 4.10% have VGluT2-IR; 31.71 ± 3.99% have VMAT2-IR and 1.55 ± 0.35% appear to co-label for VGluT2 and VMAT2 (paired t-test, t(2) = 4.333, p = 0.0493). Synaptic vesicles were quantified from 3 different preparations of isolated vesicles from the nAcc of rats (n = 110).(f) 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 antibodies (IP:VGluT2), and VMAT2 is detected only in the sample IP with anti-VMAT2 antibodies. The western blots were successfully repeated at least 3 times. Full-length blots are presented in Supplementary Figure 3.Bars: (a and b) 200 nm; (c and d) 50 nm.
Mentions: The ultrastructural findings detailed above provide evidence that in the nAcc the storage of dopamine and glutamate takes place in distinct vesicular pools enriched in different axonal micro-domains. However, to discard that our ultrastructural techniques lacked the sensitivity for co-detection of VGluT2 and VMAT2, we next explored the possibility of coexistence of VGluT2 and VMAT2 at the vesicular level by immunolabeling and by co-immunoprecipitation of VGluT2 and VMAT2 from vesicles obtained from rat nAcc synaptomes. After testing several conditions of isolation for synaptic vesicles, we achieved the experimental conditions (see online methods) that allowed the purification of a homogeneous population of synaptic vesicles, the quality and purity of which were confirmed by electron microscopy analysis (Fig. 3a). By immunolabeling of these isolated synaptic vesicles, we detected two distinct pools of vesicles, those containing VGluT2-IR and those containing VMAT2-IR (Fig. 3b-d). We found that from a total of 560 labeled vesicles, 66.74 ± 4.10% had VGluT2; 31.71 ± 3.99% had VMAT2, and a very small proportions (1.55 ± 0.35%) appeared to co-label for VGluT2 and VMAT2 (Fig. 3e). The lack of coexistence of VGluT2 and VMAT2 at the vesicular level was next confirmed by co-immunoprecipitation of VGluT2 and VMAT2 from the isolated vesicles. Consistent with our ultrastructural findings, protein preparations of vesicles immunoprecipitated with anti-VGluT2 antibodies showed vesicular immunodetection of both VGluT2 and the vesicular marker synaptophysin, but lacked evidence of VMAT2 (Fig. 3f and Supplementary Fig. 3a). Moreover, protein preparations of vesicles immunoprecipitated with anti-VMAT2 antibodies showed vesicular immunodetection of both VMAT2 and synaptophysin, but lacked evidence of VGluT2 (Fig. 3f and Supplementary Fig. 3a). Thus, we conclude that accumulation of dopamine by VMAT2 occurs in vesicles different from those that accumulate glutamate by VGluT2 (Supplementary Fig. 3b). In contrast to our findings, a previous study using enriched membrane preparations from nAcc reported co-immunoprecipitation of VMAT2 and VGluT212. The discrepancy between this earlier work and our present results may be due to major differences in the material used for immunoprecipitation. In our studies, we used purified synaptic vesicles instead of enriched membranes. In addition, we utilized sucrose to avoid organelle disruption, as organelle disruption may result in membrane fusion among different types of membranes. Moreover, we characterized the specificity of the antibodies used for detection of VMAT2 (Supplementary Fig. 2), to reduce the likelihood of nonspecific antigen recognition. Together, our ultrastructural findings are consistent with our molecular findings and do not support the previously proposed hypothesis that dopamine and glutamate are packaged and co-released in the nAcc from the same pool of vesicles1, 12, 13.

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