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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

Dual whole-cell recordings and firing characteristics of synaptically connected MSNs. The MSNs recordings were carried out at 14 DIV. (A) Single sweep inhibitory postsynaptic currents (IPSCs; lower traces; Vm = −70 mV) elicited by single spikes in the presynaptic MSN (upper trace). (B) Average IPSCs elicited by single spikes at 0.3 Hz, or 2 Hz (red), with standard deviation time course (SDTC, gray). The 0.3 Hz and 2 Hz average scaled and superimposed (lower traces; scale bar 8 pA for the 0.3 Hz). The similar time course of average and SDTC, and of averages obtained at different firing rates, indicates that all events included had a similar shape. (C) Average IPSCs elicited by two spike pairs. Average spontaneous IPSCs (sIPSCS, blue) with SDTC (gray) scaled and superimposed (5 pA scale bar; lower traces). (D) A near tonically firing MSN responding to sequential depolarizing current pulses, recorded at 14 DIV. Single sweeps of the responses of the simultaneously recorded postsynaptic MSN are shown below. (E) A MSN recorded at 16 DIV, displays more mature burst-firing behavior than the cell recorded at 14 DIV. Longer depolarizing current pulses elicit repetitive bursts (lower two records). (F) Response to 150 pA current injection in 7 DIV MSN.
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Figure 3: Dual whole-cell recordings and firing characteristics of synaptically connected MSNs. The MSNs recordings were carried out at 14 DIV. (A) Single sweep inhibitory postsynaptic currents (IPSCs; lower traces; Vm = −70 mV) elicited by single spikes in the presynaptic MSN (upper trace). (B) Average IPSCs elicited by single spikes at 0.3 Hz, or 2 Hz (red), with standard deviation time course (SDTC, gray). The 0.3 Hz and 2 Hz average scaled and superimposed (lower traces; scale bar 8 pA for the 0.3 Hz). The similar time course of average and SDTC, and of averages obtained at different firing rates, indicates that all events included had a similar shape. (C) Average IPSCs elicited by two spike pairs. Average spontaneous IPSCs (sIPSCS, blue) with SDTC (gray) scaled and superimposed (5 pA scale bar; lower traces). (D) A near tonically firing MSN responding to sequential depolarizing current pulses, recorded at 14 DIV. Single sweeps of the responses of the simultaneously recorded postsynaptic MSN are shown below. (E) A MSN recorded at 16 DIV, displays more mature burst-firing behavior than the cell recorded at 14 DIV. Longer depolarizing current pulses elicit repetitive bursts (lower two records). (F) Response to 150 pA current injection in 7 DIV MSN.

Mentions: MSNs were recorded in whole-cell mode (using IR-DIC optics, Olympus BX51). The extracellular medium contained (in mM) 130 NaCl, 4 KCl, 10 HEPES, 20 NaHCO3, 10 glucose, 1 MgCl2 and 2 CaCl2, equilibrated with 5% CO2/95% O2 (pH 7.4, 330 mosmol.l−1) at 32°C (flow rate 1.8 ml.min−1). Whole cell pipettes had a final resistance of 3–8 MΩ when filled with intracellular solution, in mM: 130 KCl, 3 NaCl, 4.5 phosphocreatine, 10 HEPES, 1 EGTA, 3.5 Na-ATP, 0.45 Na-GTP and 2 MgCl2, adjusted to pH 7.2 with KOH, 290–300 mosmol.l−1. Recordings were discarded if access resistance exceeded 15 MΩ. The selection of cells for the analysis was made based on the quality and duration of recordings obtained. In many recordings the frequencies were too low and recordings too brief to allow confident analysis of enough events. The recordings selected for the analysis were the longer recordings of highest quality. Action potential (AP) amplitudes, widths at half amplitude, and AP afterhyperpolarization (AHP) amplitudes, were measured in current clamp mode, from the first AP triggered by a just suprathreshold depolarizing current pulse. Membrane time constants (τm) were measured from the decay of the response to a subthreshold depolarizing current pulse from a membrane potential of −70 mV. Continuous recordings of spontaneous synaptic events, recorded in voltage clamp mode, were filtered at 5 kHz, digitized at 10 kHz and collected with Spike2 (CED 1401, Cambridge Electronic Design), from a holding potential of −70 mV. Putative inhibitory postsynaptic currents (IPSCs) were detected off-line according to a current threshold and selected (manually, by shape) for further analysis (MSpike, D.C. West). Computed averages of miniature IPSCs (mIPSCs), spontaneous IPSCs (sIPSCs) and AP-IPSCs used the fast rising phase of the IPSC, or the fast rising phase of the presynaptic AP (dual recordings) as trigger. The shape of the average IPSC and timing of the peak, relative to the presynaptic spike, informed individual manual IPSC amplitude measurements. Wherever possible all spontaneous events were measured for the amplitude distribution plots, but the decay phases of some were contaminated by other spontaneous events. For averaged records (Figures 4B,C,F,G) therefore, those events that were uncontaminated for their entire time course were selected. The histograms and bar graphs give the more complete picture of IPSC amplitudes, but the shape and time course are better represented by averages of “clean” events. Standard deviation time course (SDTC) was computed in parallel with each average, to ensure that the events included in averages were of similar shape, i.e., were largely devoid of confounding spontaneous events and artifacts, and that triggers were accurately aligned for all contributory sweeps, before further analysis. Following collection of sIPSCs under control conditions in the presence of D-AP5 (50 μM) and DNQX (20 μM), mIPSCs were recorded in the presence of TTX (1 μM). In some experiments, bicuculline methochloride (10 μM) was then added. IPSC 10–90% rise time (RT) and width at half amplitude (HW) were measured from averages and IPSC amplitude distributions were constructed from single event measurements. Paired whole cell recordings were made from MSNs. APs were elicited with injected depolarizing current pulses in one neuron (current clamp recordings) and postsynaptic responses recorded in the other (voltage clamp recordings, from a membrane potential of -70 mV). For figures, some electrophysiological traces were smoothed (3 point running average) to reduce high frequency noise and enhance clarity. PSI-plot (Poly Software International), GraphPad Prism (GraphPad Software, Inc.), Excel (Microsoft) and OriginPro 9.1 were used for data plotting and statistical analysis. Unless otherwise stated, data are given as means and standard error of the mean (s.e.m.). Differences between means were tested with Student’s t test.


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)

Dual whole-cell recordings and firing characteristics of synaptically connected MSNs. The MSNs recordings were carried out at 14 DIV. (A) Single sweep inhibitory postsynaptic currents (IPSCs; lower traces; Vm = −70 mV) elicited by single spikes in the presynaptic MSN (upper trace). (B) Average IPSCs elicited by single spikes at 0.3 Hz, or 2 Hz (red), with standard deviation time course (SDTC, gray). The 0.3 Hz and 2 Hz average scaled and superimposed (lower traces; scale bar 8 pA for the 0.3 Hz). The similar time course of average and SDTC, and of averages obtained at different firing rates, indicates that all events included had a similar shape. (C) Average IPSCs elicited by two spike pairs. Average spontaneous IPSCs (sIPSCS, blue) with SDTC (gray) scaled and superimposed (5 pA scale bar; lower traces). (D) A near tonically firing MSN responding to sequential depolarizing current pulses, recorded at 14 DIV. Single sweeps of the responses of the simultaneously recorded postsynaptic MSN are shown below. (E) A MSN recorded at 16 DIV, displays more mature burst-firing behavior than the cell recorded at 14 DIV. Longer depolarizing current pulses elicit repetitive bursts (lower two records). (F) Response to 150 pA current injection in 7 DIV MSN.
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Related In: Results  -  Collection

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

Figure 3: Dual whole-cell recordings and firing characteristics of synaptically connected MSNs. The MSNs recordings were carried out at 14 DIV. (A) Single sweep inhibitory postsynaptic currents (IPSCs; lower traces; Vm = −70 mV) elicited by single spikes in the presynaptic MSN (upper trace). (B) Average IPSCs elicited by single spikes at 0.3 Hz, or 2 Hz (red), with standard deviation time course (SDTC, gray). The 0.3 Hz and 2 Hz average scaled and superimposed (lower traces; scale bar 8 pA for the 0.3 Hz). The similar time course of average and SDTC, and of averages obtained at different firing rates, indicates that all events included had a similar shape. (C) Average IPSCs elicited by two spike pairs. Average spontaneous IPSCs (sIPSCS, blue) with SDTC (gray) scaled and superimposed (5 pA scale bar; lower traces). (D) A near tonically firing MSN responding to sequential depolarizing current pulses, recorded at 14 DIV. Single sweeps of the responses of the simultaneously recorded postsynaptic MSN are shown below. (E) A MSN recorded at 16 DIV, displays more mature burst-firing behavior than the cell recorded at 14 DIV. Longer depolarizing current pulses elicit repetitive bursts (lower two records). (F) Response to 150 pA current injection in 7 DIV MSN.
Mentions: MSNs were recorded in whole-cell mode (using IR-DIC optics, Olympus BX51). The extracellular medium contained (in mM) 130 NaCl, 4 KCl, 10 HEPES, 20 NaHCO3, 10 glucose, 1 MgCl2 and 2 CaCl2, equilibrated with 5% CO2/95% O2 (pH 7.4, 330 mosmol.l−1) at 32°C (flow rate 1.8 ml.min−1). Whole cell pipettes had a final resistance of 3–8 MΩ when filled with intracellular solution, in mM: 130 KCl, 3 NaCl, 4.5 phosphocreatine, 10 HEPES, 1 EGTA, 3.5 Na-ATP, 0.45 Na-GTP and 2 MgCl2, adjusted to pH 7.2 with KOH, 290–300 mosmol.l−1. Recordings were discarded if access resistance exceeded 15 MΩ. The selection of cells for the analysis was made based on the quality and duration of recordings obtained. In many recordings the frequencies were too low and recordings too brief to allow confident analysis of enough events. The recordings selected for the analysis were the longer recordings of highest quality. Action potential (AP) amplitudes, widths at half amplitude, and AP afterhyperpolarization (AHP) amplitudes, were measured in current clamp mode, from the first AP triggered by a just suprathreshold depolarizing current pulse. Membrane time constants (τm) were measured from the decay of the response to a subthreshold depolarizing current pulse from a membrane potential of −70 mV. Continuous recordings of spontaneous synaptic events, recorded in voltage clamp mode, were filtered at 5 kHz, digitized at 10 kHz and collected with Spike2 (CED 1401, Cambridge Electronic Design), from a holding potential of −70 mV. Putative inhibitory postsynaptic currents (IPSCs) were detected off-line according to a current threshold and selected (manually, by shape) for further analysis (MSpike, D.C. West). Computed averages of miniature IPSCs (mIPSCs), spontaneous IPSCs (sIPSCs) and AP-IPSCs used the fast rising phase of the IPSC, or the fast rising phase of the presynaptic AP (dual recordings) as trigger. The shape of the average IPSC and timing of the peak, relative to the presynaptic spike, informed individual manual IPSC amplitude measurements. Wherever possible all spontaneous events were measured for the amplitude distribution plots, but the decay phases of some were contaminated by other spontaneous events. For averaged records (Figures 4B,C,F,G) therefore, those events that were uncontaminated for their entire time course were selected. The histograms and bar graphs give the more complete picture of IPSC amplitudes, but the shape and time course are better represented by averages of “clean” events. Standard deviation time course (SDTC) was computed in parallel with each average, to ensure that the events included in averages were of similar shape, i.e., were largely devoid of confounding spontaneous events and artifacts, and that triggers were accurately aligned for all contributory sweeps, before further analysis. Following collection of sIPSCs under control conditions in the presence of D-AP5 (50 μM) and DNQX (20 μM), mIPSCs were recorded in the presence of TTX (1 μM). In some experiments, bicuculline methochloride (10 μM) was then added. IPSC 10–90% rise time (RT) and width at half amplitude (HW) were measured from averages and IPSC amplitude distributions were constructed from single event measurements. Paired whole cell recordings were made from MSNs. APs were elicited with injected depolarizing current pulses in one neuron (current clamp recordings) and postsynaptic responses recorded in the other (voltage clamp recordings, from a membrane potential of -70 mV). For figures, some electrophysiological traces were smoothed (3 point running average) to reduce high frequency noise and enhance clarity. PSI-plot (Poly Software International), GraphPad Prism (GraphPad Software, Inc.), Excel (Microsoft) and OriginPro 9.1 were used for data plotting and statistical analysis. Unless otherwise stated, data are given as means and standard error of the mean (s.e.m.). Differences between means were tested with Student’s t test.

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