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GABAergic and glycinergic inhibitory synaptic transmission in the ventral cochlear nucleus studied in VGAT channelrhodopsin-2 mice.

Xie R, Manis PB - Front Neural Circuits (2014)

Bottom Line: During prolonged stimulation, the ratio of steady state vs. peak IPSC amplitude was significantly lower for glycinergic IPSCs.In the absence of receptor blockers, repetitive light stimulation was only able to effectively evoke IPSCs up to 20 Hz in both bushy and multipolar neurons.We conclude that local GABAergic release within the VCN can differentially influence bushy and multipolar cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Otolaryngology/Head and Neck Surgery, University of North Carolina at Chapel Hill Chapel Hill, NC, USA.

ABSTRACT
Both glycine and GABA mediate inhibitory synaptic transmission in the ventral cochlear nucleus (VCN). In mice, the time course of glycinergic inhibition is slow in bushy cells and fast in multipolar (stellate) cells, and is proposed to contribute to the processing of temporal cues in both cell types. Much less is known about GABAergic synaptic transmission in this circuit. Electrical stimulation of the auditory nerve or the tuberculoventral pathway evokes little GABAergic synaptic current in brain slice preparations, and spontaneous GABAergic miniature synaptic currents occur infrequently. To investigate synaptic currents carried by GABA receptors in bushy and multipolar cells, we used transgenic mice in which channelrhodopsin-2 and EYFP is driven by the vesicular GABA transporter (VGAT-ChR2-EYFP) and is expressed in both GABAergic and glycinergic neurons. Light stimulation evoked action potentials in EYFP-expressing presynaptic cells, and evoked inhibitory postsynaptic potentials (IPSPs) in non-expressing bushy and planar multipolar cells. Less than 10% of the IPSP amplitude in bushy cells arose from GABAergic synapses, whereas 40% of the IPSP in multipolar neurons was GABAergic. In voltage clamp, glycinergic IPSCs were significantly slower in bushy neurons than in multipolar neurons, whereas there was little difference in the kinetics of the GABAergic IPSCs between two cell types. During prolonged stimulation, the ratio of steady state vs. peak IPSC amplitude was significantly lower for glycinergic IPSCs. Surprisingly, the reversal potentials of GABAergic IPSCs were negative to those of glycinergic IPSCs in both bushy and multipolar neurons. In the absence of receptor blockers, repetitive light stimulation was only able to effectively evoke IPSCs up to 20 Hz in both bushy and multipolar neurons. We conclude that local GABAergic release within the VCN can differentially influence bushy and multipolar cells.

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GABAergic and glycinergic IPSCs show different reversal potentials in multipolar neurons. (A) Multipolar neuron held at −57 mV using 38 mM Cl− electrode solution. IPSC traces show currents in control solution (red), in strychnine (stry, green), and in both strychnine and SR95531 (stry + SR, black). Inset: Isolated glycinergic IPSC (blue), GABAergic IPSC (green), and complete block in stry + SR (black). The glycinergic IPSC is inward, whereas the GABAergic IPSC is outward, suggesting different IPSC reversal potentials. (B) IPSCs recorded at holding potential of −47 mV from the same neuron as in (A). Traces in (A) and (B) are the averages of 20 trials. (C) Response to a 20 ms light flash (blue bar below traces) in a voltage-clamped multipolar cell with 38 mM [Cl−]i at different voltage steps. The light-evoked currents are superimposed on unblocked currents. Each trace is the average of four trials; peaks of capacitative transients at onset and offset of voltage steps have been clipped. (D) Same cell as in (C), in the presence of 2 µM strychnine to isolate the GABAergic component. Voltage steps are indicated below the traces. Current and voltage scales are the same in (C) and (D). (E) Current-voltage relationship of the light-evoked current (see Section Materials and Methods for analysis details). Large red circles: mean of currents across four trials in control conditions; small circles show responses for individual trials. Red line: cubic spline fit to the data. Large green triangles: responses in the presence of strychnine; small triangles show responses for individual trials. Green line: cubic spline fit to the data. (F) Reversal potentials measured as in (E) for, for total (glycinergic + GABAergic) currents, and isolated GABAergic currents. Measurements made sequentially in the same cell are connected. Asterisk indicates ANOVA post tests, p < 0.05. (G) Conductance at −60 mV in individual cells. (H) Ratio of GABAergic to glycinergic conductance at −60 mV for individual cells (asterisk, p < 0.05).
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Figure 5: GABAergic and glycinergic IPSCs show different reversal potentials in multipolar neurons. (A) Multipolar neuron held at −57 mV using 38 mM Cl− electrode solution. IPSC traces show currents in control solution (red), in strychnine (stry, green), and in both strychnine and SR95531 (stry + SR, black). Inset: Isolated glycinergic IPSC (blue), GABAergic IPSC (green), and complete block in stry + SR (black). The glycinergic IPSC is inward, whereas the GABAergic IPSC is outward, suggesting different IPSC reversal potentials. (B) IPSCs recorded at holding potential of −47 mV from the same neuron as in (A). Traces in (A) and (B) are the averages of 20 trials. (C) Response to a 20 ms light flash (blue bar below traces) in a voltage-clamped multipolar cell with 38 mM [Cl−]i at different voltage steps. The light-evoked currents are superimposed on unblocked currents. Each trace is the average of four trials; peaks of capacitative transients at onset and offset of voltage steps have been clipped. (D) Same cell as in (C), in the presence of 2 µM strychnine to isolate the GABAergic component. Voltage steps are indicated below the traces. Current and voltage scales are the same in (C) and (D). (E) Current-voltage relationship of the light-evoked current (see Section Materials and Methods for analysis details). Large red circles: mean of currents across four trials in control conditions; small circles show responses for individual trials. Red line: cubic spline fit to the data. Large green triangles: responses in the presence of strychnine; small triangles show responses for individual trials. Green line: cubic spline fit to the data. (F) Reversal potentials measured as in (E) for, for total (glycinergic + GABAergic) currents, and isolated GABAergic currents. Measurements made sequentially in the same cell are connected. Asterisk indicates ANOVA post tests, p < 0.05. (G) Conductance at −60 mV in individual cells. (H) Ratio of GABAergic to glycinergic conductance at −60 mV for individual cells (asterisk, p < 0.05).

Mentions: The difference in the reversal potential between glycinergic and GABAergic IPSCs was initially observed as different directions of currents in three different multipolar neurons when recorded using Cs-based electrode solution containing 38 mM Cl− (Figure 5). At a holding potential of −57 mV, the IPSCs of one multipolar neuron (Figure 5A) under control conditions showed an initial inward current followed by mixed inward and outward currents. When the glycinergic IPSCs were blocked with strychnine, an outward GABAergic IPSC was revealed. This IPSC in turn was completely blocked by the subsequent addition of SR95531, confirming that it was mediated by GABAA receptors. The isolated glycinergic IPSC was entirely inward at this holding potential. Similar features were also apparent in this cell when held at −47 mV (Figure 5B). These results suggest that the reversal potentials of the glycinergic and GABAergic IPSCs are different.


GABAergic and glycinergic inhibitory synaptic transmission in the ventral cochlear nucleus studied in VGAT channelrhodopsin-2 mice.

Xie R, Manis PB - Front Neural Circuits (2014)

GABAergic and glycinergic IPSCs show different reversal potentials in multipolar neurons. (A) Multipolar neuron held at −57 mV using 38 mM Cl− electrode solution. IPSC traces show currents in control solution (red), in strychnine (stry, green), and in both strychnine and SR95531 (stry + SR, black). Inset: Isolated glycinergic IPSC (blue), GABAergic IPSC (green), and complete block in stry + SR (black). The glycinergic IPSC is inward, whereas the GABAergic IPSC is outward, suggesting different IPSC reversal potentials. (B) IPSCs recorded at holding potential of −47 mV from the same neuron as in (A). Traces in (A) and (B) are the averages of 20 trials. (C) Response to a 20 ms light flash (blue bar below traces) in a voltage-clamped multipolar cell with 38 mM [Cl−]i at different voltage steps. The light-evoked currents are superimposed on unblocked currents. Each trace is the average of four trials; peaks of capacitative transients at onset and offset of voltage steps have been clipped. (D) Same cell as in (C), in the presence of 2 µM strychnine to isolate the GABAergic component. Voltage steps are indicated below the traces. Current and voltage scales are the same in (C) and (D). (E) Current-voltage relationship of the light-evoked current (see Section Materials and Methods for analysis details). Large red circles: mean of currents across four trials in control conditions; small circles show responses for individual trials. Red line: cubic spline fit to the data. Large green triangles: responses in the presence of strychnine; small triangles show responses for individual trials. Green line: cubic spline fit to the data. (F) Reversal potentials measured as in (E) for, for total (glycinergic + GABAergic) currents, and isolated GABAergic currents. Measurements made sequentially in the same cell are connected. Asterisk indicates ANOVA post tests, p < 0.05. (G) Conductance at −60 mV in individual cells. (H) Ratio of GABAergic to glycinergic conductance at −60 mV for individual cells (asterisk, p < 0.05).
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Related In: Results  -  Collection

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Figure 5: GABAergic and glycinergic IPSCs show different reversal potentials in multipolar neurons. (A) Multipolar neuron held at −57 mV using 38 mM Cl− electrode solution. IPSC traces show currents in control solution (red), in strychnine (stry, green), and in both strychnine and SR95531 (stry + SR, black). Inset: Isolated glycinergic IPSC (blue), GABAergic IPSC (green), and complete block in stry + SR (black). The glycinergic IPSC is inward, whereas the GABAergic IPSC is outward, suggesting different IPSC reversal potentials. (B) IPSCs recorded at holding potential of −47 mV from the same neuron as in (A). Traces in (A) and (B) are the averages of 20 trials. (C) Response to a 20 ms light flash (blue bar below traces) in a voltage-clamped multipolar cell with 38 mM [Cl−]i at different voltage steps. The light-evoked currents are superimposed on unblocked currents. Each trace is the average of four trials; peaks of capacitative transients at onset and offset of voltage steps have been clipped. (D) Same cell as in (C), in the presence of 2 µM strychnine to isolate the GABAergic component. Voltage steps are indicated below the traces. Current and voltage scales are the same in (C) and (D). (E) Current-voltage relationship of the light-evoked current (see Section Materials and Methods for analysis details). Large red circles: mean of currents across four trials in control conditions; small circles show responses for individual trials. Red line: cubic spline fit to the data. Large green triangles: responses in the presence of strychnine; small triangles show responses for individual trials. Green line: cubic spline fit to the data. (F) Reversal potentials measured as in (E) for, for total (glycinergic + GABAergic) currents, and isolated GABAergic currents. Measurements made sequentially in the same cell are connected. Asterisk indicates ANOVA post tests, p < 0.05. (G) Conductance at −60 mV in individual cells. (H) Ratio of GABAergic to glycinergic conductance at −60 mV for individual cells (asterisk, p < 0.05).
Mentions: The difference in the reversal potential between glycinergic and GABAergic IPSCs was initially observed as different directions of currents in three different multipolar neurons when recorded using Cs-based electrode solution containing 38 mM Cl− (Figure 5). At a holding potential of −57 mV, the IPSCs of one multipolar neuron (Figure 5A) under control conditions showed an initial inward current followed by mixed inward and outward currents. When the glycinergic IPSCs were blocked with strychnine, an outward GABAergic IPSC was revealed. This IPSC in turn was completely blocked by the subsequent addition of SR95531, confirming that it was mediated by GABAA receptors. The isolated glycinergic IPSC was entirely inward at this holding potential. Similar features were also apparent in this cell when held at −47 mV (Figure 5B). These results suggest that the reversal potentials of the glycinergic and GABAergic IPSCs are different.

Bottom Line: During prolonged stimulation, the ratio of steady state vs. peak IPSC amplitude was significantly lower for glycinergic IPSCs.In the absence of receptor blockers, repetitive light stimulation was only able to effectively evoke IPSCs up to 20 Hz in both bushy and multipolar neurons.We conclude that local GABAergic release within the VCN can differentially influence bushy and multipolar cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Otolaryngology/Head and Neck Surgery, University of North Carolina at Chapel Hill Chapel Hill, NC, USA.

ABSTRACT
Both glycine and GABA mediate inhibitory synaptic transmission in the ventral cochlear nucleus (VCN). In mice, the time course of glycinergic inhibition is slow in bushy cells and fast in multipolar (stellate) cells, and is proposed to contribute to the processing of temporal cues in both cell types. Much less is known about GABAergic synaptic transmission in this circuit. Electrical stimulation of the auditory nerve or the tuberculoventral pathway evokes little GABAergic synaptic current in brain slice preparations, and spontaneous GABAergic miniature synaptic currents occur infrequently. To investigate synaptic currents carried by GABA receptors in bushy and multipolar cells, we used transgenic mice in which channelrhodopsin-2 and EYFP is driven by the vesicular GABA transporter (VGAT-ChR2-EYFP) and is expressed in both GABAergic and glycinergic neurons. Light stimulation evoked action potentials in EYFP-expressing presynaptic cells, and evoked inhibitory postsynaptic potentials (IPSPs) in non-expressing bushy and planar multipolar cells. Less than 10% of the IPSP amplitude in bushy cells arose from GABAergic synapses, whereas 40% of the IPSP in multipolar neurons was GABAergic. In voltage clamp, glycinergic IPSCs were significantly slower in bushy neurons than in multipolar neurons, whereas there was little difference in the kinetics of the GABAergic IPSCs between two cell types. During prolonged stimulation, the ratio of steady state vs. peak IPSC amplitude was significantly lower for glycinergic IPSCs. Surprisingly, the reversal potentials of GABAergic IPSCs were negative to those of glycinergic IPSCs in both bushy and multipolar neurons. In the absence of receptor blockers, repetitive light stimulation was only able to effectively evoke IPSCs up to 20 Hz in both bushy and multipolar neurons. We conclude that local GABAergic release within the VCN can differentially influence bushy and multipolar cells.

Show MeSH
Related in: MedlinePlus