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Co-release of glutamate and GABA from single vesicles in GABAergic neurons exogenously expressing VGLUT3.

Zimmermann J, Herman MA, Rosenmund C - Front Synaptic Neurosci (2015)

Bottom Line: Though a functional role for glutamate release from these non-glutamatergic neurons has been demonstrated, the interplay between VGLUT3 and the neuron's characteristic neurotransmitter transporter, particularly in the case of GABAergic neurons, at the synaptic and vesicular level is less clear.We found that VGLUT3 expression in isolated, autaptic GABAergic neurons leads to action potential evoked release of glutamate.Finally, we found postsynaptic detection of glutamate released from GABAergic terminals difficult when bona fide glutamatergic synapses were present, suggesting that co-released glutamate cannot induce postsynaptic glutamate receptor clustering.

View Article: PubMed Central - PubMed

Affiliation: Neurowissenschaftliches Forschungszentrum (NWFZ), NeuroCure Exzellenzcluster, CCO Charité Universitätsmedizin Berlin, Germany.

ABSTRACT
The identity of the vesicle neurotransmitter transporter expressed by a neuron largely corresponds with the primary neurotransmitter that cell releases. However, the vesicular glutamate transporter subtype 3 (VGLUT3) is mainly expressed in non-glutamatergic neurons, including cholinergic, serotonergic, or GABAergic neurons. Though a functional role for glutamate release from these non-glutamatergic neurons has been demonstrated, the interplay between VGLUT3 and the neuron's characteristic neurotransmitter transporter, particularly in the case of GABAergic neurons, at the synaptic and vesicular level is less clear. In this study, we explore how exogenous expression of VGLUT3 in striatal GABAergic neurons affects the packaging and release of glutamate and GABA in synaptic vesicles (SVs). We found that VGLUT3 expression in isolated, autaptic GABAergic neurons leads to action potential evoked release of glutamate. Under these conditions, glutamate and GABA could be packaged together in single vesicles release either spontaneously or asynchronously. However, the presence of glutamate in GABAergic vesicles did not affect uptake of GABA itself, suggesting a lack of synergy in vesicle filling for these transmitters. Finally, we found postsynaptic detection of glutamate released from GABAergic terminals difficult when bona fide glutamatergic synapses were present, suggesting that co-released glutamate cannot induce postsynaptic glutamate receptor clustering.

No MeSH data available.


Related in: MedlinePlus

Amount of detectable glutamate co-release varies between different culture systems. (A) Plot of PSC amplitudes in GABAergic striatal neurons expressing VGLUT3. Recordings in three different culture systems: mass culture (red), autaptic culture (black/gray) and 2 or more cells on an astrocyte feeder island (blue). Depicted are the PSC sizes in control ECS (darker colors) and the glutamatergic component in the presence of Bic (lighter colors). Each data point represents a cell. Horizontal lines show the average response size. (B) Plot of PSC amplitude in ECS (“total amplitude”) against amplitude in Bic (“amplitude glu component”) of the same cell compared over the three different culture systems.
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Figure 5: Amount of detectable glutamate co-release varies between different culture systems. (A) Plot of PSC amplitudes in GABAergic striatal neurons expressing VGLUT3. Recordings in three different culture systems: mass culture (red), autaptic culture (black/gray) and 2 or more cells on an astrocyte feeder island (blue). Depicted are the PSC sizes in control ECS (darker colors) and the glutamatergic component in the presence of Bic (lighter colors). Each data point represents a cell. Horizontal lines show the average response size. (B) Plot of PSC amplitude in ECS (“total amplitude”) against amplitude in Bic (“amplitude glu component”) of the same cell compared over the three different culture systems.

Mentions: Our findings show that glutamate can be both released and detected at GABAergic synapses from autapses expressing VGLUT3. However, in neural circuits in the brain, GABAergic synapses co-releasing glutamate must exist on the same postsynaptic target cells as bona fide glutamatergic synapses. Therefore, we examined whether we could still detect glutamate released from GABAergic striatal neurons expressing VGLUT3 when they were cultured in the presence of glutamatergic neurons from hippocampus. We performed paired whole cells recordings from mixed cultures of striatal and hippocampal neurons infected with VGLUT3, and pharmacologically isolated the PSCs as previously described (Figures 1, 3–4). On average, the responses from VGLUT3-expressing GABAergic presynaptic neurons were almost completely blocked by application of Bic, indicating that glutamatergic co-release could not be detected. To test whether the lack of detection of glutamate co-release from GABAergic neurons was due to a low expression level of VGLUT3 in mixed striatal/hippocampal cultures, we performed sister-culture experiments with pure striatal neurons or mixed striatal/hippocampal neurons plated on astrocyte microislands, and again recorded from autaptic striatal neurons expressing VGLUT3 or from paired neurons in multi-cell microisland circuits. We found, again, that the majority of autaptic GABAergic neurons expressing VGLUT3 had an NBQX-sensitive component of their evoked release (Figure 5A). On the other hand, the PSCs detected in paired recordings from multi-cell microislands where striatal GABAergic neurons expressing VGLUT3 were co-cultured with hippocampal neurons were almost entirely blocked by Bic application. Plotting the total amplitude of each cell against its amplitude in the presence of Bic (“glutamatergic component”) demonstrates the influence of the culturing system on the size of the glutamatergic component compared to the influence of the total response size (Figure 5B). While this could indicate that VGLUT3 is actively excluded from GABAergic vesicles in mixed cultures, a more likely explanation could be that even though glutamate is co-released from GABAergic terminals expressing VGLUT3, the detection could be limited by the availability of postsynaptic AMPA receptors, which may be sequestered by bona fide glutamatergic synapses in a network environment.


Co-release of glutamate and GABA from single vesicles in GABAergic neurons exogenously expressing VGLUT3.

Zimmermann J, Herman MA, Rosenmund C - Front Synaptic Neurosci (2015)

Amount of detectable glutamate co-release varies between different culture systems. (A) Plot of PSC amplitudes in GABAergic striatal neurons expressing VGLUT3. Recordings in three different culture systems: mass culture (red), autaptic culture (black/gray) and 2 or more cells on an astrocyte feeder island (blue). Depicted are the PSC sizes in control ECS (darker colors) and the glutamatergic component in the presence of Bic (lighter colors). Each data point represents a cell. Horizontal lines show the average response size. (B) Plot of PSC amplitude in ECS (“total amplitude”) against amplitude in Bic (“amplitude glu component”) of the same cell compared over the three different culture systems.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4585203&req=5

Figure 5: Amount of detectable glutamate co-release varies between different culture systems. (A) Plot of PSC amplitudes in GABAergic striatal neurons expressing VGLUT3. Recordings in three different culture systems: mass culture (red), autaptic culture (black/gray) and 2 or more cells on an astrocyte feeder island (blue). Depicted are the PSC sizes in control ECS (darker colors) and the glutamatergic component in the presence of Bic (lighter colors). Each data point represents a cell. Horizontal lines show the average response size. (B) Plot of PSC amplitude in ECS (“total amplitude”) against amplitude in Bic (“amplitude glu component”) of the same cell compared over the three different culture systems.
Mentions: Our findings show that glutamate can be both released and detected at GABAergic synapses from autapses expressing VGLUT3. However, in neural circuits in the brain, GABAergic synapses co-releasing glutamate must exist on the same postsynaptic target cells as bona fide glutamatergic synapses. Therefore, we examined whether we could still detect glutamate released from GABAergic striatal neurons expressing VGLUT3 when they were cultured in the presence of glutamatergic neurons from hippocampus. We performed paired whole cells recordings from mixed cultures of striatal and hippocampal neurons infected with VGLUT3, and pharmacologically isolated the PSCs as previously described (Figures 1, 3–4). On average, the responses from VGLUT3-expressing GABAergic presynaptic neurons were almost completely blocked by application of Bic, indicating that glutamatergic co-release could not be detected. To test whether the lack of detection of glutamate co-release from GABAergic neurons was due to a low expression level of VGLUT3 in mixed striatal/hippocampal cultures, we performed sister-culture experiments with pure striatal neurons or mixed striatal/hippocampal neurons plated on astrocyte microislands, and again recorded from autaptic striatal neurons expressing VGLUT3 or from paired neurons in multi-cell microisland circuits. We found, again, that the majority of autaptic GABAergic neurons expressing VGLUT3 had an NBQX-sensitive component of their evoked release (Figure 5A). On the other hand, the PSCs detected in paired recordings from multi-cell microislands where striatal GABAergic neurons expressing VGLUT3 were co-cultured with hippocampal neurons were almost entirely blocked by Bic application. Plotting the total amplitude of each cell against its amplitude in the presence of Bic (“glutamatergic component”) demonstrates the influence of the culturing system on the size of the glutamatergic component compared to the influence of the total response size (Figure 5B). While this could indicate that VGLUT3 is actively excluded from GABAergic vesicles in mixed cultures, a more likely explanation could be that even though glutamate is co-released from GABAergic terminals expressing VGLUT3, the detection could be limited by the availability of postsynaptic AMPA receptors, which may be sequestered by bona fide glutamatergic synapses in a network environment.

Bottom Line: Though a functional role for glutamate release from these non-glutamatergic neurons has been demonstrated, the interplay between VGLUT3 and the neuron's characteristic neurotransmitter transporter, particularly in the case of GABAergic neurons, at the synaptic and vesicular level is less clear.We found that VGLUT3 expression in isolated, autaptic GABAergic neurons leads to action potential evoked release of glutamate.Finally, we found postsynaptic detection of glutamate released from GABAergic terminals difficult when bona fide glutamatergic synapses were present, suggesting that co-released glutamate cannot induce postsynaptic glutamate receptor clustering.

View Article: PubMed Central - PubMed

Affiliation: Neurowissenschaftliches Forschungszentrum (NWFZ), NeuroCure Exzellenzcluster, CCO Charité Universitätsmedizin Berlin, Germany.

ABSTRACT
The identity of the vesicle neurotransmitter transporter expressed by a neuron largely corresponds with the primary neurotransmitter that cell releases. However, the vesicular glutamate transporter subtype 3 (VGLUT3) is mainly expressed in non-glutamatergic neurons, including cholinergic, serotonergic, or GABAergic neurons. Though a functional role for glutamate release from these non-glutamatergic neurons has been demonstrated, the interplay between VGLUT3 and the neuron's characteristic neurotransmitter transporter, particularly in the case of GABAergic neurons, at the synaptic and vesicular level is less clear. In this study, we explore how exogenous expression of VGLUT3 in striatal GABAergic neurons affects the packaging and release of glutamate and GABA in synaptic vesicles (SVs). We found that VGLUT3 expression in isolated, autaptic GABAergic neurons leads to action potential evoked release of glutamate. Under these conditions, glutamate and GABA could be packaged together in single vesicles release either spontaneously or asynchronously. However, the presence of glutamate in GABAergic vesicles did not affect uptake of GABA itself, suggesting a lack of synergy in vesicle filling for these transmitters. Finally, we found postsynaptic detection of glutamate released from GABAergic terminals difficult when bona fide glutamatergic synapses were present, suggesting that co-released glutamate cannot induce postsynaptic glutamate receptor clustering.

No MeSH data available.


Related in: MedlinePlus