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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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Photostimulation drives excitatory responses in EYFP expressing cells in VGAT-ChR2-EYFP mice. (A) Expression pattern of ChR2 in cochlear nucleus as visualized by EYFP fluorescence. Notice that expression is absent in the 8th nerve root region, moderate in anteroventral cochlear nucleus (AVCN) and posteroventral cochlear nucleus (PVCN), and high in the DCN. The image is a mosaic assembled from different areas of the cochlear nuclei. (B–C) Multiphoton images of EYFP expressing cells from areas as marked in (A). Expression of the EYFP-ChR2 construct is present in both membrane and cytoplasm. Arrows mark expressing neurons. Asterisks mark non-expressing cells whose soma is surrounded by expressing terminals. (D) Example responses from an EYFP-ChR2 expressing cell to different durations of 470 nm illumination from 0 (no light) to 1.0 ms. The threshold of the light duration was 0.8 ms in this cell, which evoked an action potential as shown in red. All sub-threshold traces are averages of 5–10 trials; traces with spikes are single trials. (E) Longer duration illumination reliably evoked a single spike or trains of spikes (same cell in (D)). Each plot shows the responses to 5–10 trials. (F) Ten 2-ms light pulses at 10, 20, 50, and 100 Hz evoke trains of spikes. Top: single trial; bottom: superimposed traces from four trials. Note that tonic firing is evoked at higher frequencies, although the cell no longer entrains to individual flashes.
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Figure 1: Photostimulation drives excitatory responses in EYFP expressing cells in VGAT-ChR2-EYFP mice. (A) Expression pattern of ChR2 in cochlear nucleus as visualized by EYFP fluorescence. Notice that expression is absent in the 8th nerve root region, moderate in anteroventral cochlear nucleus (AVCN) and posteroventral cochlear nucleus (PVCN), and high in the DCN. The image is a mosaic assembled from different areas of the cochlear nuclei. (B–C) Multiphoton images of EYFP expressing cells from areas as marked in (A). Expression of the EYFP-ChR2 construct is present in both membrane and cytoplasm. Arrows mark expressing neurons. Asterisks mark non-expressing cells whose soma is surrounded by expressing terminals. (D) Example responses from an EYFP-ChR2 expressing cell to different durations of 470 nm illumination from 0 (no light) to 1.0 ms. The threshold of the light duration was 0.8 ms in this cell, which evoked an action potential as shown in red. All sub-threshold traces are averages of 5–10 trials; traces with spikes are single trials. (E) Longer duration illumination reliably evoked a single spike or trains of spikes (same cell in (D)). Each plot shows the responses to 5–10 trials. (F) Ten 2-ms light pulses at 10, 20, 50, and 100 Hz evoke trains of spikes. Top: single trial; bottom: superimposed traces from four trials. Note that tonic firing is evoked at higher frequencies, although the cell no longer entrains to individual flashes.

Mentions: VGAT-ChR2-EYFP mice (B6.Cg-Tg(Slc32a1-COP4*H134R/EYFP)8Gfng/J; (Zhao et al., 2011)) were purchased from Jackson Laboratories (stock #014548) and maintained in our breeding colony. The mice incorporate a BAC transgene that expresses ChR2 and enhanced yellow fluorescent protein (EYFP) under the control of the VGAT promoter. Because ChR2 is fused to EYFP, EYFP fluorescence directly reports the cellular localization of ChR2 (see Figure 1). All animal procedures were approved by the University of North Carolina Internal Animal Concerns and Use Committee (IACUC).


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)

Photostimulation drives excitatory responses in EYFP expressing cells in VGAT-ChR2-EYFP mice. (A) Expression pattern of ChR2 in cochlear nucleus as visualized by EYFP fluorescence. Notice that expression is absent in the 8th nerve root region, moderate in anteroventral cochlear nucleus (AVCN) and posteroventral cochlear nucleus (PVCN), and high in the DCN. The image is a mosaic assembled from different areas of the cochlear nuclei. (B–C) Multiphoton images of EYFP expressing cells from areas as marked in (A). Expression of the EYFP-ChR2 construct is present in both membrane and cytoplasm. Arrows mark expressing neurons. Asterisks mark non-expressing cells whose soma is surrounded by expressing terminals. (D) Example responses from an EYFP-ChR2 expressing cell to different durations of 470 nm illumination from 0 (no light) to 1.0 ms. The threshold of the light duration was 0.8 ms in this cell, which evoked an action potential as shown in red. All sub-threshold traces are averages of 5–10 trials; traces with spikes are single trials. (E) Longer duration illumination reliably evoked a single spike or trains of spikes (same cell in (D)). Each plot shows the responses to 5–10 trials. (F) Ten 2-ms light pulses at 10, 20, 50, and 100 Hz evoke trains of spikes. Top: single trial; bottom: superimposed traces from four trials. Note that tonic firing is evoked at higher frequencies, although the cell no longer entrains to individual flashes.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4109614&req=5

Figure 1: Photostimulation drives excitatory responses in EYFP expressing cells in VGAT-ChR2-EYFP mice. (A) Expression pattern of ChR2 in cochlear nucleus as visualized by EYFP fluorescence. Notice that expression is absent in the 8th nerve root region, moderate in anteroventral cochlear nucleus (AVCN) and posteroventral cochlear nucleus (PVCN), and high in the DCN. The image is a mosaic assembled from different areas of the cochlear nuclei. (B–C) Multiphoton images of EYFP expressing cells from areas as marked in (A). Expression of the EYFP-ChR2 construct is present in both membrane and cytoplasm. Arrows mark expressing neurons. Asterisks mark non-expressing cells whose soma is surrounded by expressing terminals. (D) Example responses from an EYFP-ChR2 expressing cell to different durations of 470 nm illumination from 0 (no light) to 1.0 ms. The threshold of the light duration was 0.8 ms in this cell, which evoked an action potential as shown in red. All sub-threshold traces are averages of 5–10 trials; traces with spikes are single trials. (E) Longer duration illumination reliably evoked a single spike or trains of spikes (same cell in (D)). Each plot shows the responses to 5–10 trials. (F) Ten 2-ms light pulses at 10, 20, 50, and 100 Hz evoke trains of spikes. Top: single trial; bottom: superimposed traces from four trials. Note that tonic firing is evoked at higher frequencies, although the cell no longer entrains to individual flashes.
Mentions: VGAT-ChR2-EYFP mice (B6.Cg-Tg(Slc32a1-COP4*H134R/EYFP)8Gfng/J; (Zhao et al., 2011)) were purchased from Jackson Laboratories (stock #014548) and maintained in our breeding colony. The mice incorporate a BAC transgene that expresses ChR2 and enhanced yellow fluorescent protein (EYFP) under the control of the VGAT promoter. Because ChR2 is fused to EYFP, EYFP fluorescence directly reports the cellular localization of ChR2 (see Figure 1). All animal procedures were approved by the University of North Carolina Internal Animal Concerns and Use Committee (IACUC).

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