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Intracellular chloride concentration influences the GABAA receptor subunit composition.

Succol F, Fiumelli H, Benfenati F, Cancedda L, Barberis A - Nat Commun (2012)

Bottom Line: The intracellular concentration of chloride ([Cl(-)](i)), the main ion permeating through GABA(A)Rs, also undergoes considerable changes during maturation, being higher at early neuronal stages with respect to adult neurons.We show that [Cl(-)](i) regulates the expression of α3-1 and δ-containing GABA(A) receptors, responsible for phasic and tonic inhibition, respectively.Our findings highlight the role of [Cl(-)](i) in tuning the strength of GABAergic responses by acting as an intracellular messenger.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience and Brain Technologies, The Italian Institute of Technology, Genova 16163, Italy.

ABSTRACT
GABA(A) receptors (GABA(A)Rs) exist as different subtype variants showing unique functional properties and defined spatio-temporal expression pattern. The molecular mechanisms underlying the developmental expression of different GABA(A)R are largely unknown. The intracellular concentration of chloride ([Cl(-)](i)), the main ion permeating through GABA(A)Rs, also undergoes considerable changes during maturation, being higher at early neuronal stages with respect to adult neurons. Here we investigate the possibility that [Cl(-)](i) could modulate the sequential expression of specific GABA(A)Rs subtypes in primary cerebellar neurons. We show that [Cl(-)](i) regulates the expression of α3-1 and δ-containing GABA(A) receptors, responsible for phasic and tonic inhibition, respectively. Our findings highlight the role of [Cl(-)](i) in tuning the strength of GABAergic responses by acting as an intracellular messenger.

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Related in: MedlinePlus

Pharmacological treatment with DIOA affects GABAergic currents and regulates the expression of α3-α1 and δ subunits.(a) Example of I–V relations of isoguvacine (10 μM) evoked GABAA currents at increasing holding potentials in control neurons. (b) Example of I-V relations of isoguvacine (10 μM) evoked GABAA currents at increasing holding potentials in DIOA treated neurons. (c) Summary of ECl at DIV12 in control (grey bar n=8) and DIOA treated neurons (white filled bar n=8; *P<0.05; unpaired t-test). (d) Representative sIPSCs from control and DIOA (10 μM) treated neurons. (e) Summary of sIPSCs decay kinetics in control (grey bar n=33) and DIOA treated neurons (white filled bar n=35; ***P<0.001; unpaired t-test). (f) Summary of sIPSC frequency in control (grey bar n=33) and DIOA treated neurons (white filled bar n=35; ***P<0.001; unpaired t-test). (g) Summary of tonic current amplitude in control (grey bar n=25) and DIOA treated neurons (white filled bar n=32; ***P<0.001; unpaired t-test). (h) Quantification of immunofluorescence signals for α3 and α1 subunits (α3 control, grey bar n=52; DIOA, white filled bar n=55; *P<0.5; unpaired t-test; α1 control, grey bar n=53; DIOA, white filled bar n=59; ***P<0.001; unpaired t-test). (i) Quantification of immunofluorescence signals for δ subunit in control (grey bar n=71) and DIOA treated neurons (white filled bar n=41; ***P<0.001; unpaired t-test). (j) Somatic immunostaining of immunofluorescence signals for α3 and α1 subunits in control and DIOA treated neurons at DIV12. (k) Somatic immunostaining of immunofluorescence signals for δ subunit in control and DIOA treated neurons. Scale bar: 23 μm. Data is presented as mean±s.e.m.
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f5: Pharmacological treatment with DIOA affects GABAergic currents and regulates the expression of α3-α1 and δ subunits.(a) Example of I–V relations of isoguvacine (10 μM) evoked GABAA currents at increasing holding potentials in control neurons. (b) Example of I-V relations of isoguvacine (10 μM) evoked GABAA currents at increasing holding potentials in DIOA treated neurons. (c) Summary of ECl at DIV12 in control (grey bar n=8) and DIOA treated neurons (white filled bar n=8; *P<0.05; unpaired t-test). (d) Representative sIPSCs from control and DIOA (10 μM) treated neurons. (e) Summary of sIPSCs decay kinetics in control (grey bar n=33) and DIOA treated neurons (white filled bar n=35; ***P<0.001; unpaired t-test). (f) Summary of sIPSC frequency in control (grey bar n=33) and DIOA treated neurons (white filled bar n=35; ***P<0.001; unpaired t-test). (g) Summary of tonic current amplitude in control (grey bar n=25) and DIOA treated neurons (white filled bar n=32; ***P<0.001; unpaired t-test). (h) Quantification of immunofluorescence signals for α3 and α1 subunits (α3 control, grey bar n=52; DIOA, white filled bar n=55; *P<0.5; unpaired t-test; α1 control, grey bar n=53; DIOA, white filled bar n=59; ***P<0.001; unpaired t-test). (i) Quantification of immunofluorescence signals for δ subunit in control (grey bar n=71) and DIOA treated neurons (white filled bar n=41; ***P<0.001; unpaired t-test). (j) Somatic immunostaining of immunofluorescence signals for α3 and α1 subunits in control and DIOA treated neurons at DIV12. (k) Somatic immunostaining of immunofluorescence signals for δ subunit in control and DIOA treated neurons. Scale bar: 23 μm. Data is presented as mean±s.e.m.

Mentions: In order to increase the [Cl−]i without interfering with the expression level of the KCC2 protein, DIV5 neurons were chronically treated with the KCC2 inhibitor DIOA. Although DIOA shows only a partial specificity for KCC2 (refs 16,17) this drug caused a marked shift of ECl in mature neurons, validating its effect in increasing [Cl−]i (−64.96±7.5 and −45.05±8.43 mV in control and DIOA conditions, respectively; P<0.05 unpaired t-test; Fig. 5a–c). Experiments aimed at investigating sIPSCs in the presence of DIOA showed that, in DIV12–13 cultures, this drug was able to induce a slowdown of the sIPSC current relaxation (11.8±0.8 and 24.4±1.3 ms in control and DIOA conditions, respectively; P<0.001 unpaired t-test) (Fig. 5d,e) and significantly decreased the sIPSC frequency (Fig. 5f). Chronic treatment with DIOA also decreased the tonic current at DIV12–13 (Fig. 5g). In addition, DIOA slightly, but significantly increased the expression of α3 subunit, while it markedly decreased the expression of both α1 and δ subunits (Fig. 5h–k). Also in this case, electrophysiological data paralleled the immunocytochemistry results, indicating that differences in phasic and tonic GABAergic currents under conditions of altered [Cl−]i rely on changes in the expression of α3–α1 and δ subunits.


Intracellular chloride concentration influences the GABAA receptor subunit composition.

Succol F, Fiumelli H, Benfenati F, Cancedda L, Barberis A - Nat Commun (2012)

Pharmacological treatment with DIOA affects GABAergic currents and regulates the expression of α3-α1 and δ subunits.(a) Example of I–V relations of isoguvacine (10 μM) evoked GABAA currents at increasing holding potentials in control neurons. (b) Example of I-V relations of isoguvacine (10 μM) evoked GABAA currents at increasing holding potentials in DIOA treated neurons. (c) Summary of ECl at DIV12 in control (grey bar n=8) and DIOA treated neurons (white filled bar n=8; *P<0.05; unpaired t-test). (d) Representative sIPSCs from control and DIOA (10 μM) treated neurons. (e) Summary of sIPSCs decay kinetics in control (grey bar n=33) and DIOA treated neurons (white filled bar n=35; ***P<0.001; unpaired t-test). (f) Summary of sIPSC frequency in control (grey bar n=33) and DIOA treated neurons (white filled bar n=35; ***P<0.001; unpaired t-test). (g) Summary of tonic current amplitude in control (grey bar n=25) and DIOA treated neurons (white filled bar n=32; ***P<0.001; unpaired t-test). (h) Quantification of immunofluorescence signals for α3 and α1 subunits (α3 control, grey bar n=52; DIOA, white filled bar n=55; *P<0.5; unpaired t-test; α1 control, grey bar n=53; DIOA, white filled bar n=59; ***P<0.001; unpaired t-test). (i) Quantification of immunofluorescence signals for δ subunit in control (grey bar n=71) and DIOA treated neurons (white filled bar n=41; ***P<0.001; unpaired t-test). (j) Somatic immunostaining of immunofluorescence signals for α3 and α1 subunits in control and DIOA treated neurons at DIV12. (k) Somatic immunostaining of immunofluorescence signals for δ subunit in control and DIOA treated neurons. Scale bar: 23 μm. Data is presented as mean±s.e.m.
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Related In: Results  -  Collection

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f5: Pharmacological treatment with DIOA affects GABAergic currents and regulates the expression of α3-α1 and δ subunits.(a) Example of I–V relations of isoguvacine (10 μM) evoked GABAA currents at increasing holding potentials in control neurons. (b) Example of I-V relations of isoguvacine (10 μM) evoked GABAA currents at increasing holding potentials in DIOA treated neurons. (c) Summary of ECl at DIV12 in control (grey bar n=8) and DIOA treated neurons (white filled bar n=8; *P<0.05; unpaired t-test). (d) Representative sIPSCs from control and DIOA (10 μM) treated neurons. (e) Summary of sIPSCs decay kinetics in control (grey bar n=33) and DIOA treated neurons (white filled bar n=35; ***P<0.001; unpaired t-test). (f) Summary of sIPSC frequency in control (grey bar n=33) and DIOA treated neurons (white filled bar n=35; ***P<0.001; unpaired t-test). (g) Summary of tonic current amplitude in control (grey bar n=25) and DIOA treated neurons (white filled bar n=32; ***P<0.001; unpaired t-test). (h) Quantification of immunofluorescence signals for α3 and α1 subunits (α3 control, grey bar n=52; DIOA, white filled bar n=55; *P<0.5; unpaired t-test; α1 control, grey bar n=53; DIOA, white filled bar n=59; ***P<0.001; unpaired t-test). (i) Quantification of immunofluorescence signals for δ subunit in control (grey bar n=71) and DIOA treated neurons (white filled bar n=41; ***P<0.001; unpaired t-test). (j) Somatic immunostaining of immunofluorescence signals for α3 and α1 subunits in control and DIOA treated neurons at DIV12. (k) Somatic immunostaining of immunofluorescence signals for δ subunit in control and DIOA treated neurons. Scale bar: 23 μm. Data is presented as mean±s.e.m.
Mentions: In order to increase the [Cl−]i without interfering with the expression level of the KCC2 protein, DIV5 neurons were chronically treated with the KCC2 inhibitor DIOA. Although DIOA shows only a partial specificity for KCC2 (refs 16,17) this drug caused a marked shift of ECl in mature neurons, validating its effect in increasing [Cl−]i (−64.96±7.5 and −45.05±8.43 mV in control and DIOA conditions, respectively; P<0.05 unpaired t-test; Fig. 5a–c). Experiments aimed at investigating sIPSCs in the presence of DIOA showed that, in DIV12–13 cultures, this drug was able to induce a slowdown of the sIPSC current relaxation (11.8±0.8 and 24.4±1.3 ms in control and DIOA conditions, respectively; P<0.001 unpaired t-test) (Fig. 5d,e) and significantly decreased the sIPSC frequency (Fig. 5f). Chronic treatment with DIOA also decreased the tonic current at DIV12–13 (Fig. 5g). In addition, DIOA slightly, but significantly increased the expression of α3 subunit, while it markedly decreased the expression of both α1 and δ subunits (Fig. 5h–k). Also in this case, electrophysiological data paralleled the immunocytochemistry results, indicating that differences in phasic and tonic GABAergic currents under conditions of altered [Cl−]i rely on changes in the expression of α3–α1 and δ subunits.

Bottom Line: The intracellular concentration of chloride ([Cl(-)](i)), the main ion permeating through GABA(A)Rs, also undergoes considerable changes during maturation, being higher at early neuronal stages with respect to adult neurons.We show that [Cl(-)](i) regulates the expression of α3-1 and δ-containing GABA(A) receptors, responsible for phasic and tonic inhibition, respectively.Our findings highlight the role of [Cl(-)](i) in tuning the strength of GABAergic responses by acting as an intracellular messenger.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience and Brain Technologies, The Italian Institute of Technology, Genova 16163, Italy.

ABSTRACT
GABA(A) receptors (GABA(A)Rs) exist as different subtype variants showing unique functional properties and defined spatio-temporal expression pattern. The molecular mechanisms underlying the developmental expression of different GABA(A)R are largely unknown. The intracellular concentration of chloride ([Cl(-)](i)), the main ion permeating through GABA(A)Rs, also undergoes considerable changes during maturation, being higher at early neuronal stages with respect to adult neurons. Here we investigate the possibility that [Cl(-)](i) could modulate the sequential expression of specific GABA(A)Rs subtypes in primary cerebellar neurons. We show that [Cl(-)](i) regulates the expression of α3-1 and δ-containing GABA(A) receptors, responsible for phasic and tonic inhibition, respectively. Our findings highlight the role of [Cl(-)](i) in tuning the strength of GABAergic responses by acting as an intracellular messenger.

Show MeSH
Related in: MedlinePlus