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GABAA receptor activity shapes the formation of inhibitory synapses between developing medium spiny neurons.

Arama J, Abitbol K, Goffin D, Fuchs C, Sihra TS, Thomson AM, Jovanovic JN - Front Cell Neurosci (2015)

Bottom Line: When activity of GABAARs was under chronic blockade between 4-7 DIV, the structural properties of these synapses remained unchanged.In contrast, chronic inhibition of GABAARs between 7-14 DIV led to reduction in size of α1- and α1/α2-postsynaptic clusters and a concomitant increase in number and size of α2-postsynaptic clusters.Thus, the main subtypes of GABAergic synapses formed by MSNs are regulated by GABAAR activity, but in opposite directions, and thus appear to be driven by different molecular mechanisms.

View Article: PubMed Central - PubMed

Affiliation: UCL School of Pharmacy, University College London London, UK.

ABSTRACT
Basal ganglia play an essential role in motor coordination and cognitive functions. The GABAergic medium spiny neurons (MSNs) account for ~95% of all the neurons in this brain region. Central to the normal functioning of MSNs is integration of synaptic activity arriving from the glutamatergic corticostriatal and thalamostriatal afferents, with synaptic inhibition mediated by local interneurons and MSN axon collaterals. In this study we have investigated how the specific types of GABAergic synapses between the MSNs develop over time, and how the activity of GABAA receptors (GABAARs) influences this development. Isolated embryonic (E17) MSNs form a homogenous population in vitro and display spontaneous synaptic activity and functional properties similar to their in vivo counterparts. In dual whole-cell recordings of synaptically connected pairs of MSNs, action potential (AP)-activated synaptic events were detected between 7 and 14 days in vitro (DIV), which coincided with the shift in GABAAR operation from depolarization to hyperpolarization, as detected indirectly by intracellular calcium imaging. In parallel, the predominant subtypes of inhibitory synapses, which innervate dendrites of MSNs and contain GABAAR α1 or α2 subunits, underwent distinct changes in the size of postsynaptic clusters, with α1 becoming smaller and α2 larger over time, while both the percentage and the size of mixed α1/α2-postsynaptic clusters were increased. When activity of GABAARs was under chronic blockade between 4-7 DIV, the structural properties of these synapses remained unchanged. In contrast, chronic inhibition of GABAARs between 7-14 DIV led to reduction in size of α1- and α1/α2-postsynaptic clusters and a concomitant increase in number and size of α2-postsynaptic clusters. Thus, the main subtypes of GABAergic synapses formed by MSNs are regulated by GABAAR activity, but in opposite directions, and thus appear to be driven by different molecular mechanisms.

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Related in: MedlinePlus

Developmental switch in GABAA receptor signaling in MSNs. (A,B) Changes in intracellular Ca2+ in response to muscimol (50 μM), KCl (60 mM), ionomycin (5 μM) and EGTA (1 mM) in 5–7 DIV and 12–14 DIV MSNs, respectively, measured by fluorescent microscopy. (C) The histogram shows the maximum increase in ΔF for control and muscimol alone or in the presence of bicuculline (+ Bic) or picrotoxin (+ Pic), normalized to the peak response to KCl application. Bars represent mean ± s.e.m. Statistical analysis was performed using ANOVA, *p < 0.01, **p < 0.001.
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Figure 7: Developmental switch in GABAA receptor signaling in MSNs. (A,B) Changes in intracellular Ca2+ in response to muscimol (50 μM), KCl (60 mM), ionomycin (5 μM) and EGTA (1 mM) in 5–7 DIV and 12–14 DIV MSNs, respectively, measured by fluorescent microscopy. (C) The histogram shows the maximum increase in ΔF for control and muscimol alone or in the presence of bicuculline (+ Bic) or picrotoxin (+ Pic), normalized to the peak response to KCl application. Bars represent mean ± s.e.m. Statistical analysis was performed using ANOVA, *p < 0.01, **p < 0.001.

Mentions: The role of GABA as a developmental signal has been well established in many brain regions, where activation of GABAA receptors triggers depolarization and influx of Ca2+ (Cancedda et al., 2007; Porcher et al., 2011; Ben-Ari et al., 2012; Deidda et al., 2015). To assess whether GABAA receptor activation leads to plasma membrane depolarization and Ca2+ influx in MSNs at 7 DIV, a membrane-permeable Ca2+ indicator fluo-4-AM was applied to the cultures. Ca2+ levels were monitored in response to muscimol (50 μM), a specific GABAA receptor agonist, using fluorescence microscopy as described in “Materials and Methods” Section. A muscimol-evoked increase in [Ca2+]i defined as fluorescence intensity at least 50% above the baseline fluorescence, was observed in 50% of cells tested (23 of 46 cells tested, P < 0.05, paired t-test vs. vehicle; n = 23; Figure 7A). All cells examined exhibited [Ca2+]i responses to depolarization evoked by high extracellular K+(60 mM) with no significant difference in the maximal change in fluorescence between those cells insensitive or sensitive to muscimol (P > 0.05, Paired t-test; n = 23). Total Ca2+ levels were assessed by the addition of ionomycin (5 μM) followed by the addition of the Ca2+ chelator EGTA (Figure 7A). Muscimol-induced Ca2+ responses were significantly attenuated in the presence of the GABAA receptor competitive antagonist Bic or a channel blocker Pic (both at 50 μM; P < 0.01, paired t-test; n = 20 (Figure 7C), but not abolished, as it is possible that some GABAA receptors were not completely blocked at these concentrations of antagonists. Bic had no significant effect on basal fluorescence when added alone (P > 0.05, paired t-test, n = 20; data not shown).


GABAA receptor activity shapes the formation of inhibitory synapses between developing medium spiny neurons.

Arama J, Abitbol K, Goffin D, Fuchs C, Sihra TS, Thomson AM, Jovanovic JN - Front Cell Neurosci (2015)

Developmental switch in GABAA receptor signaling in MSNs. (A,B) Changes in intracellular Ca2+ in response to muscimol (50 μM), KCl (60 mM), ionomycin (5 μM) and EGTA (1 mM) in 5–7 DIV and 12–14 DIV MSNs, respectively, measured by fluorescent microscopy. (C) The histogram shows the maximum increase in ΔF for control and muscimol alone or in the presence of bicuculline (+ Bic) or picrotoxin (+ Pic), normalized to the peak response to KCl application. Bars represent mean ± s.e.m. Statistical analysis was performed using ANOVA, *p < 0.01, **p < 0.001.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4526800&req=5

Figure 7: Developmental switch in GABAA receptor signaling in MSNs. (A,B) Changes in intracellular Ca2+ in response to muscimol (50 μM), KCl (60 mM), ionomycin (5 μM) and EGTA (1 mM) in 5–7 DIV and 12–14 DIV MSNs, respectively, measured by fluorescent microscopy. (C) The histogram shows the maximum increase in ΔF for control and muscimol alone or in the presence of bicuculline (+ Bic) or picrotoxin (+ Pic), normalized to the peak response to KCl application. Bars represent mean ± s.e.m. Statistical analysis was performed using ANOVA, *p < 0.01, **p < 0.001.
Mentions: The role of GABA as a developmental signal has been well established in many brain regions, where activation of GABAA receptors triggers depolarization and influx of Ca2+ (Cancedda et al., 2007; Porcher et al., 2011; Ben-Ari et al., 2012; Deidda et al., 2015). To assess whether GABAA receptor activation leads to plasma membrane depolarization and Ca2+ influx in MSNs at 7 DIV, a membrane-permeable Ca2+ indicator fluo-4-AM was applied to the cultures. Ca2+ levels were monitored in response to muscimol (50 μM), a specific GABAA receptor agonist, using fluorescence microscopy as described in “Materials and Methods” Section. A muscimol-evoked increase in [Ca2+]i defined as fluorescence intensity at least 50% above the baseline fluorescence, was observed in 50% of cells tested (23 of 46 cells tested, P < 0.05, paired t-test vs. vehicle; n = 23; Figure 7A). All cells examined exhibited [Ca2+]i responses to depolarization evoked by high extracellular K+(60 mM) with no significant difference in the maximal change in fluorescence between those cells insensitive or sensitive to muscimol (P > 0.05, Paired t-test; n = 23). Total Ca2+ levels were assessed by the addition of ionomycin (5 μM) followed by the addition of the Ca2+ chelator EGTA (Figure 7A). Muscimol-induced Ca2+ responses were significantly attenuated in the presence of the GABAA receptor competitive antagonist Bic or a channel blocker Pic (both at 50 μM; P < 0.01, paired t-test; n = 20 (Figure 7C), but not abolished, as it is possible that some GABAA receptors were not completely blocked at these concentrations of antagonists. Bic had no significant effect on basal fluorescence when added alone (P > 0.05, paired t-test, n = 20; data not shown).

Bottom Line: When activity of GABAARs was under chronic blockade between 4-7 DIV, the structural properties of these synapses remained unchanged.In contrast, chronic inhibition of GABAARs between 7-14 DIV led to reduction in size of α1- and α1/α2-postsynaptic clusters and a concomitant increase in number and size of α2-postsynaptic clusters.Thus, the main subtypes of GABAergic synapses formed by MSNs are regulated by GABAAR activity, but in opposite directions, and thus appear to be driven by different molecular mechanisms.

View Article: PubMed Central - PubMed

Affiliation: UCL School of Pharmacy, University College London London, UK.

ABSTRACT
Basal ganglia play an essential role in motor coordination and cognitive functions. The GABAergic medium spiny neurons (MSNs) account for ~95% of all the neurons in this brain region. Central to the normal functioning of MSNs is integration of synaptic activity arriving from the glutamatergic corticostriatal and thalamostriatal afferents, with synaptic inhibition mediated by local interneurons and MSN axon collaterals. In this study we have investigated how the specific types of GABAergic synapses between the MSNs develop over time, and how the activity of GABAA receptors (GABAARs) influences this development. Isolated embryonic (E17) MSNs form a homogenous population in vitro and display spontaneous synaptic activity and functional properties similar to their in vivo counterparts. In dual whole-cell recordings of synaptically connected pairs of MSNs, action potential (AP)-activated synaptic events were detected between 7 and 14 days in vitro (DIV), which coincided with the shift in GABAAR operation from depolarization to hyperpolarization, as detected indirectly by intracellular calcium imaging. In parallel, the predominant subtypes of inhibitory synapses, which innervate dendrites of MSNs and contain GABAAR α1 or α2 subunits, underwent distinct changes in the size of postsynaptic clusters, with α1 becoming smaller and α2 larger over time, while both the percentage and the size of mixed α1/α2-postsynaptic clusters were increased. When activity of GABAARs was under chronic blockade between 4-7 DIV, the structural properties of these synapses remained unchanged. In contrast, chronic inhibition of GABAARs between 7-14 DIV led to reduction in size of α1- and α1/α2-postsynaptic clusters and a concomitant increase in number and size of α2-postsynaptic clusters. Thus, the main subtypes of GABAergic synapses formed by MSNs are regulated by GABAAR activity, but in opposite directions, and thus appear to be driven by different molecular mechanisms.

No MeSH data available.


Related in: MedlinePlus