Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2014 Jul 24;8:84.
doi: 10.3389/fncir.2014.00084. eCollection 2014.

GABAergic and Glycinergic Inhibitory Synaptic Transmission in the Ventral Cochlear Nucleus Studied in VGAT channelrhodopsin-2 Mice

Affiliations
Free PMC article

GABAergic and Glycinergic Inhibitory Synaptic Transmission in the Ventral Cochlear Nucleus Studied in VGAT channelrhodopsin-2 Mice

Ruili Xie et al. Front Neural Circuits. .
Free PMC article

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.

Keywords: IPSC; bushy; multipolar; stellate; target-specific inhibition.

Figures

Figure 1
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.
Figure 2
Figure 2
GABAergic inhibition is weak in bushy but strong in multipolar neurons. (A) Discharge pattern of a bushy neuron to direct current injection. The bushy neuron fires only one or a few spikes with depolarizing current injections. (B) Light pulses at different durations evoke brief IPSP responses in bushy neurons. Each trace is an average of six trials. Blue bars on top mark the timing of the light pulses with duration of 1, 5 and 20 ms. (C) Strychnine (stry) blocks the majority of the IPSP evoked by 20 ms light pulses in bushy neurons. Addition of SR95531 (stry+SR) fully blocks light evoked IPSPs. Traces are averages of 10 trials. Data in (A–C) are from the same bushy neuron. (D) Discharge pattern of an example multipolar neuron to direct current injections. Multipolar neurons fire a regular train of spikes throughout the current injection. (E) Light pulses at durations of 1, 5 and 20 ms evoke IPSPs in multipolar neurons. Note that the IPSPs have a wider half-width than those of bushy neurons in (B). (F) Strychnine only blocks about half of light evoked IPSP. The remainder of the current is fully blocked by the further addition of SR95531. Data in (D–F) are from the same multipolar neuron. (G–I) Summary data of the eIPSP half-width (G), eIPSP amplitude (H) and percentage of GABAergic IPSP (I). * p < 0.05; ** p < 0.01. Data is plotted as mean ± S.D.
Figure 3
Figure 3
Kinetics of GABAergic and glycinergic IPSCs in bushy and multipolar neurons. (A) A 2 ms light pulse (blue bar on top) evokes IPSCs in a bushy neuron. The IPSC is mostly blocked by strychnine (stry) and is fully blocked by a combination of strychnine and SR95531 (Stry + SR). Inset: magnified IPSC trace after strychnine block. The weighted decay time constants of the IPSCs are obtained by fitting the IPSC decay with double exponential functions (black curves) under both control and stry conditions. (B) 1 ms light pulse evokes IPSCs in a multipolar neuron. Plots are organized the same as in (A). Decay time constants of IPSCs are obtained by fitting the IPSC decay with single exponential functions (black curves). Traces in both (A) and (B) are averages of 10 trials. (C) Comparison of the light evoked IPSC amplitudes between bushy and multipolar neurons including the control IPSC amplitude (ctrl), glycinergic IPSC component (Gly), and GABAergic IPSC component (GABA). Abcissa: B: bushy neurons; M: multipolar neurons. (D) Comparison of the eIPSC decay time constants. * p < 0.05; ** p < 0.01. Data is plotted as mean ± S.D.
Figure 4
Figure 4
Glycinergic IPSCs are phasic compared to GABAergic IPSCs in response to sustained illumination. (A) IPSC responses evoked by different durations of light (2, 50, and 100 ms) in a bushy neuron. Notice that the glycinergic IPSCs peak shortly after the onset of the light pulse and decay rapidly even though the illumination is still on, whereas GABAergic IPSCs show little decay in amplitude. (B) Light evoked IPSC responses in a multipolar neuron. Traces in both (A) and (B) are averages of 10 trials. (C) Summary of the IPSC peak amplitudes (peak) and steady state amplitudes (SS) in bushy neurons. In (C-F): Gly: glycinergic IPSCs; GABA: GABAergic IPSCs. (D) Summary of the sustained current ratio in bushy neurons. (E) Summary of the IPSC peak amplitudes and steady state amplitudes in multipolar neurons. (F) Summary of the sustained current ratio in multipolar neurons. In (D) and (F): each connected pair represents a single neuron; black bar marks the average of the group. * p < 0.05; ** p < 0.01.
Figure 5
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).
Figure 6
Figure 6
GABAergic IPSCs are blocked by tetrodotoxin. Cells were tested in control conditions (red traces), in the presence of 2 µM strychnine to isolate the GABAergic IPSC (green trace), and in the presence of both strychnine and 1 µM tetrodotoxin to block action potential evoked release. No IPSC was evident in the presence of tetrodotoxin in any of the three cells tested, indicating that voltage-gated sodium channel activation is required for GABA release in response to ChR2 activation.
Figure 7
Figure 7
Light evoked inhibiton only entrains for low frequencies. (A) Example IPSCs to light trains consisting of ten 2-ms pulses. Notice that IPSCs are only effectively evoked throughout the trains at 20 Hz in this neuron. At 50 and 100 Hz, light failed to evoke IPSCs in the later phase of the trains. (B) Example IPSCs to light trains in a multipolar neuron. (C) Normalized IPSC amplitudes through the light stimulus trains at different frequencies in bushy neurons. (D) Normalized IPSC amplitudes through the light stimulus trains at different frequencies in multipolar neurons. In (C, D), thin lines show individual neurons; thick lines are group averages for each frequency.

Similar articles

See all similar articles

Cited by 9 articles

See all "Cited by" articles

References

    1. Adams J. C., Mugnaini E. (1987). Patterns of glutamate decarboxylase immunostaining in the feline cochlear nuclear complex studied with silver enhancement and electron microscopy. J. Comp. Neurol. 262, 375–401 10.1002/cne.902620305 - DOI - PubMed
    1. Altschuler R. A., Betz H., Parakkal M. H., Reeks K. A., Wenthold R. J. (1986). Identification of glycinergic synapses in the cochlear nucleus through immunocytochemical localization of the postsynaptic receptor. Brain Res. 369, 316–320 10.1016/0006-8993(86)90542-1 - DOI - PubMed
    1. Arnott R. H., Wallace M. N., Shackleton T. M., Palmer A. R. (2004). Onset neurones in the anteroventral cochlear nucleus project to the dorsal cochlear nucleus. J. Assoc. Res. Otolaryngol. 5, 153–170 10.1007/s10162-003-4036-8 - DOI - PMC - PubMed
    1. Backoff P. M., Shadduck Palombi P., Caspary D. M. (1999). Gamma-aminobutyric acidergic and glycinergic inputs shape coding of amplitude modulation in the chinchilla cochlear nucleus. Hear. Res. 134, 77–88 10.1016/s0378-5955(99)00071-4 - DOI - PubMed
    1. Bormann J., Hamill O. P., Sakmann B. (1987). Mechanism of anion permeation through channels gated by glycine and gamma-aminobutyric acid in mouse cultured spinal neurones. J. Physiol. 385, 243–286 - PMC - PubMed

Publication types

MeSH terms

Feedback