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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.

No MeSH data available.


Related in: MedlinePlus

Developmental changes in gephyrin clustering at GABAergic synapses of MSNs. (A,B) Immunolabeling of gephyrin clusters (i–iv; red) and α1- (ii; pink), α2- (iii; green) and α1/α2- (iv; white) subunit-containing clusters along the primary dendrites of 7 and 14 DIV MSNs, respectively. (i) Scale bar = 10 μm. (ii–iv) Scale bar = 5 μm. (C,D) Increase in the number and size, respectively, of gephyrin clusters along the primary dendrites of MSNs from 7 to 14 DIV. (E,F,G) Increase in the percentage of gephyrin clusters colocalized with GABAAR α1-, α2- and α1/α2- clusters, respectively, along the primary dendrites of MSNs from 7 to 14 DIV. The box plots display the median and IQRs of indicated synaptic parameters measured along the first 20 μm of primary dendrites (n = 78 dendrites at 7 DIV, n = 70 dendrites at 14 DIV) of total of n = 16 neurons from two independent experiments. Statistical analysis was performed using Mann Whitney test, *p < 0.05.
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Figure 6: Developmental changes in gephyrin clustering at GABAergic synapses of MSNs. (A,B) Immunolabeling of gephyrin clusters (i–iv; red) and α1- (ii; pink), α2- (iii; green) and α1/α2- (iv; white) subunit-containing clusters along the primary dendrites of 7 and 14 DIV MSNs, respectively. (i) Scale bar = 10 μm. (ii–iv) Scale bar = 5 μm. (C,D) Increase in the number and size, respectively, of gephyrin clusters along the primary dendrites of MSNs from 7 to 14 DIV. (E,F,G) Increase in the percentage of gephyrin clusters colocalized with GABAAR α1-, α2- and α1/α2- clusters, respectively, along the primary dendrites of MSNs from 7 to 14 DIV. The box plots display the median and IQRs of indicated synaptic parameters measured along the first 20 μm of primary dendrites (n = 78 dendrites at 7 DIV, n = 70 dendrites at 14 DIV) of total of n = 16 neurons from two independent experiments. Statistical analysis was performed using Mann Whitney test, *p < 0.05.

Mentions: It is now well established that the scaffolding protein gephyrin plays a central role in the maintenance and stability of GABAA receptor clusters (Tyagarajan and Fritschy, 2014). To characterize changes in gephyrin association with GABAA receptors α1-, α2- or α1/α2-clusters during development, we have performed immunolabeling with specific antibodies and confocal imaging (Figures 6A,B). Initially, we have estimated the number and the size of gephyrin clusters along the first 20 μm of primary dendrites. At 7 DIV, the median number of gephyrin clusters was significantly increased from 10 (IQR = 6–16; n = 78 dendrites) to 24 (IQR = 18–3; n = 70 dendrites) at 14 DIV (p value < 0.001, Mann Whitney test; Figure 6C). The size of gephyrin clusters was also significantly increased from 0.4 μm2 (IQR = 0.3–0.5; n = 914 clusters, n = 78 dendrites) at 7 DIV to 0.5 μm2 (IQR = 0.3–0.7; n = 1706 clusters, n = 70 dendrites) at 14 DIV (p value < 0.001, Mann Whitney test; Figure 6D). These changes were accompanied with a significant increase in the percentage of α1-, α2- or α1/α2-clusters associated with gephyrin during development. Thus, at 7 DIV, 13.7% (IQR = 0–30.6; n = 72 dendrites) of all α1-clusters was associated with gephyrin, compared with 37.5% (IQR = 20–72; n = 65 dendrites) at 14 DIV (p value < 0.00001, Mann Whitney test; Figure 6E). A substantially larger number of α2-clusters associated with gephyrin at 7 DIV, i.e., 33.3% (IQR = 8.2–54.8; n = 74 dendrites) of all α2-clusters, was increased to 67.5% (IQR = 46.6–83.3; n = 68 dendrites) at 14 DIV (p value < 0.000001, Mann Whitney test; Figure 6F). Finally, the percentage of α1/α2-clusters associated with gephyrin was also significantly increased (7 DIV: 60% (IQR = 20–81.8; n = 71 dendrites), 14 DIV: 87.5% (IQR = 61.1–100; n = 66 dendrites), p value < 0.001, Mann Whitney test; Figure 6G). Together, the data indicates that gephyrin association with GABAergic synapses is gradually increased during MSN development. However, this does not correlate tightly with changes observed in either the number or the size of postsynaptic GABAA receptor clusters, as α1-clusters underwent a significant reduction in size despite the fact that their association with gephyrin was increased from 7 to 14 DIV.


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 changes in gephyrin clustering at GABAergic synapses of MSNs. (A,B) Immunolabeling of gephyrin clusters (i–iv; red) and α1- (ii; pink), α2- (iii; green) and α1/α2- (iv; white) subunit-containing clusters along the primary dendrites of 7 and 14 DIV MSNs, respectively. (i) Scale bar = 10 μm. (ii–iv) Scale bar = 5 μm. (C,D) Increase in the number and size, respectively, of gephyrin clusters along the primary dendrites of MSNs from 7 to 14 DIV. (E,F,G) Increase in the percentage of gephyrin clusters colocalized with GABAAR α1-, α2- and α1/α2- clusters, respectively, along the primary dendrites of MSNs from 7 to 14 DIV. The box plots display the median and IQRs of indicated synaptic parameters measured along the first 20 μm of primary dendrites (n = 78 dendrites at 7 DIV, n = 70 dendrites at 14 DIV) of total of n = 16 neurons from two independent experiments. Statistical analysis was performed using Mann Whitney test, *p < 0.05.
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Figure 6: Developmental changes in gephyrin clustering at GABAergic synapses of MSNs. (A,B) Immunolabeling of gephyrin clusters (i–iv; red) and α1- (ii; pink), α2- (iii; green) and α1/α2- (iv; white) subunit-containing clusters along the primary dendrites of 7 and 14 DIV MSNs, respectively. (i) Scale bar = 10 μm. (ii–iv) Scale bar = 5 μm. (C,D) Increase in the number and size, respectively, of gephyrin clusters along the primary dendrites of MSNs from 7 to 14 DIV. (E,F,G) Increase in the percentage of gephyrin clusters colocalized with GABAAR α1-, α2- and α1/α2- clusters, respectively, along the primary dendrites of MSNs from 7 to 14 DIV. The box plots display the median and IQRs of indicated synaptic parameters measured along the first 20 μm of primary dendrites (n = 78 dendrites at 7 DIV, n = 70 dendrites at 14 DIV) of total of n = 16 neurons from two independent experiments. Statistical analysis was performed using Mann Whitney test, *p < 0.05.
Mentions: It is now well established that the scaffolding protein gephyrin plays a central role in the maintenance and stability of GABAA receptor clusters (Tyagarajan and Fritschy, 2014). To characterize changes in gephyrin association with GABAA receptors α1-, α2- or α1/α2-clusters during development, we have performed immunolabeling with specific antibodies and confocal imaging (Figures 6A,B). Initially, we have estimated the number and the size of gephyrin clusters along the first 20 μm of primary dendrites. At 7 DIV, the median number of gephyrin clusters was significantly increased from 10 (IQR = 6–16; n = 78 dendrites) to 24 (IQR = 18–3; n = 70 dendrites) at 14 DIV (p value < 0.001, Mann Whitney test; Figure 6C). The size of gephyrin clusters was also significantly increased from 0.4 μm2 (IQR = 0.3–0.5; n = 914 clusters, n = 78 dendrites) at 7 DIV to 0.5 μm2 (IQR = 0.3–0.7; n = 1706 clusters, n = 70 dendrites) at 14 DIV (p value < 0.001, Mann Whitney test; Figure 6D). These changes were accompanied with a significant increase in the percentage of α1-, α2- or α1/α2-clusters associated with gephyrin during development. Thus, at 7 DIV, 13.7% (IQR = 0–30.6; n = 72 dendrites) of all α1-clusters was associated with gephyrin, compared with 37.5% (IQR = 20–72; n = 65 dendrites) at 14 DIV (p value < 0.00001, Mann Whitney test; Figure 6E). A substantially larger number of α2-clusters associated with gephyrin at 7 DIV, i.e., 33.3% (IQR = 8.2–54.8; n = 74 dendrites) of all α2-clusters, was increased to 67.5% (IQR = 46.6–83.3; n = 68 dendrites) at 14 DIV (p value < 0.000001, Mann Whitney test; Figure 6F). Finally, the percentage of α1/α2-clusters associated with gephyrin was also significantly increased (7 DIV: 60% (IQR = 20–81.8; n = 71 dendrites), 14 DIV: 87.5% (IQR = 61.1–100; n = 66 dendrites), p value < 0.001, Mann Whitney test; Figure 6G). Together, the data indicates that gephyrin association with GABAergic synapses is gradually increased during MSN development. However, this does not correlate tightly with changes observed in either the number or the size of postsynaptic GABAA receptor clusters, as α1-clusters underwent a significant reduction in size despite the fact that their association with gephyrin was increased from 7 to 14 DIV.

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