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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

VGLUT3 expression promotes glutamate release in GABAergic neurons. (A) Exemplary traces of current responses to an unclamped AP of a striatal GABAergic neuron exogenously expressing VGLUT3 in control extracellular solution (ECS; black), the AMPA receptor antagonist, NBQX (blue), the GABAA receptor antagonist, bicuculline (Bic; red), or both antagonists combined (green). (B) Example traces in the same conditions as (A) from a striatal GABAergic neuron expressing only GFP. Stimulations are indicated by an open square; stimulation artifacts and action potentials have been blanked for illustrative purposes. (C) Plot of mean amplitudes of evoked response in VGLUT3-expressing cells (left; n = 13) and control cells expressing GFP (right; n = 6) in presence of NBQX, Bic or no drug (control). Significance was assessed by comparing responses in control conditions to each pharmacological treatment using an ANOVA repeated measures with Dunnett’s multiple comparison tests. Error bars = SEM. **p ≤ 0.01. ***p ≤ 0.001. (D) Plot of mean paired-pulse ratio (PPR) in the presence of NBQX for expressing VGLUT3 or GFP only. Significance was assessed by Mann-Whitney test. Error bars = SEM.
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Figure 1: VGLUT3 expression promotes glutamate release in GABAergic neurons. (A) Exemplary traces of current responses to an unclamped AP of a striatal GABAergic neuron exogenously expressing VGLUT3 in control extracellular solution (ECS; black), the AMPA receptor antagonist, NBQX (blue), the GABAA receptor antagonist, bicuculline (Bic; red), or both antagonists combined (green). (B) Example traces in the same conditions as (A) from a striatal GABAergic neuron expressing only GFP. Stimulations are indicated by an open square; stimulation artifacts and action potentials have been blanked for illustrative purposes. (C) Plot of mean amplitudes of evoked response in VGLUT3-expressing cells (left; n = 13) and control cells expressing GFP (right; n = 6) in presence of NBQX, Bic or no drug (control). Significance was assessed by comparing responses in control conditions to each pharmacological treatment using an ANOVA repeated measures with Dunnett’s multiple comparison tests. Error bars = SEM. **p ≤ 0.01. ***p ≤ 0.001. (D) Plot of mean paired-pulse ratio (PPR) in the presence of NBQX for expressing VGLUT3 or GFP only. Significance was assessed by Mann-Whitney test. Error bars = SEM.

Mentions: Borosilicate glass pipettes (Science Products, Hofheim, Germany) had a resistance of 2–3.5 MΩ. All recordings were performed with a Multiclamp 700B amplifier and a Digidata 1440A digitizer under control of Clampex 10.0 (Molecular Devices, Sunnyvale, USA). Data was acquired at 10 kHz and filtered at 3 kHz. In most of the experiments, membrane capacitance and 70% of the series resistance were compensated while changes in series resistance were monitored frequently throughout the experiments. Only cells with a series resistance <10 MΩ were used for analysis. PSCs were elicited by a 2 ms somatic depolarization from −70 mV to 0 mV, which resulted in an unclamped action potential. Response amplitudes were measured from baseline. Quantal PSC (qPSC) were recorded as either spontaneously released events (miniature) or evoked in an ECS in which CaCl2 was replaced by SrCl2 (asynchronous). In both cases, GABAergic and glutamatergic components were pharmacologically isolated with NBQX or Bic, respectively. In order to optimize detection condition of the mPSCs, and because voltage escape poses less of a problem for single vesicle events, series resistance was uncompensated for analysis of mPSCs decay analysis (Figure 3). For analysis of asynchronous PSCs evoked in SrCl2 (Figure 4) series resistance was compensated to assure voltage control during the evoked component of the response.


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)

VGLUT3 expression promotes glutamate release in GABAergic neurons. (A) Exemplary traces of current responses to an unclamped AP of a striatal GABAergic neuron exogenously expressing VGLUT3 in control extracellular solution (ECS; black), the AMPA receptor antagonist, NBQX (blue), the GABAA receptor antagonist, bicuculline (Bic; red), or both antagonists combined (green). (B) Example traces in the same conditions as (A) from a striatal GABAergic neuron expressing only GFP. Stimulations are indicated by an open square; stimulation artifacts and action potentials have been blanked for illustrative purposes. (C) Plot of mean amplitudes of evoked response in VGLUT3-expressing cells (left; n = 13) and control cells expressing GFP (right; n = 6) in presence of NBQX, Bic or no drug (control). Significance was assessed by comparing responses in control conditions to each pharmacological treatment using an ANOVA repeated measures with Dunnett’s multiple comparison tests. Error bars = SEM. **p ≤ 0.01. ***p ≤ 0.001. (D) Plot of mean paired-pulse ratio (PPR) in the presence of NBQX for expressing VGLUT3 or GFP only. Significance was assessed by Mann-Whitney test. Error bars = SEM.
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Related In: Results  -  Collection

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Figure 1: VGLUT3 expression promotes glutamate release in GABAergic neurons. (A) Exemplary traces of current responses to an unclamped AP of a striatal GABAergic neuron exogenously expressing VGLUT3 in control extracellular solution (ECS; black), the AMPA receptor antagonist, NBQX (blue), the GABAA receptor antagonist, bicuculline (Bic; red), or both antagonists combined (green). (B) Example traces in the same conditions as (A) from a striatal GABAergic neuron expressing only GFP. Stimulations are indicated by an open square; stimulation artifacts and action potentials have been blanked for illustrative purposes. (C) Plot of mean amplitudes of evoked response in VGLUT3-expressing cells (left; n = 13) and control cells expressing GFP (right; n = 6) in presence of NBQX, Bic or no drug (control). Significance was assessed by comparing responses in control conditions to each pharmacological treatment using an ANOVA repeated measures with Dunnett’s multiple comparison tests. Error bars = SEM. **p ≤ 0.01. ***p ≤ 0.001. (D) Plot of mean paired-pulse ratio (PPR) in the presence of NBQX for expressing VGLUT3 or GFP only. Significance was assessed by Mann-Whitney test. Error bars = SEM.
Mentions: Borosilicate glass pipettes (Science Products, Hofheim, Germany) had a resistance of 2–3.5 MΩ. All recordings were performed with a Multiclamp 700B amplifier and a Digidata 1440A digitizer under control of Clampex 10.0 (Molecular Devices, Sunnyvale, USA). Data was acquired at 10 kHz and filtered at 3 kHz. In most of the experiments, membrane capacitance and 70% of the series resistance were compensated while changes in series resistance were monitored frequently throughout the experiments. Only cells with a series resistance <10 MΩ were used for analysis. PSCs were elicited by a 2 ms somatic depolarization from −70 mV to 0 mV, which resulted in an unclamped action potential. Response amplitudes were measured from baseline. Quantal PSC (qPSC) were recorded as either spontaneously released events (miniature) or evoked in an ECS in which CaCl2 was replaced by SrCl2 (asynchronous). In both cases, GABAergic and glutamatergic components were pharmacologically isolated with NBQX or Bic, respectively. In order to optimize detection condition of the mPSCs, and because voltage escape poses less of a problem for single vesicle events, series resistance was uncompensated for analysis of mPSCs decay analysis (Figure 3). For analysis of asynchronous PSCs evoked in SrCl2 (Figure 4) series resistance was compensated to assure voltage control during the evoked component of the response.

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