Quercetin Enhances Inhibitory Synaptic Inputs and Reduces Excitatory Synaptic Inputs to OFF- and ON-Type Retinal Ganglion Cells in a Chronic Glaucoma Rat Model

Abstract

Background: Glaucoma is a neurodegenerative disease caused by excitotoxic injury of retinal ganglion cells (RGCs). In our previous model of high intraocular pressure, prepared by injecting magnetic beads into the anterior chamber, we demonstrated that an important natural dietary flavonoid compound (quercetin) can improve RGC function. However, it is unclear whether quercetin can improve the synaptic function of RGCs and how quercetin regulates synaptic transmission in rat models of chronic glaucoma.

Methods: A rat model of chronic glaucoma was prepared by electrocoagulation of the superior scleral vein. Electrophysiological electroretinography was used to detect the photopic negative response (PhNR). The whole-cell patch-clamp technique was used to clamp ON- and OFF- type RGCs in sections from normal retinas and from retinas that had been subjected to glaucoma for 4 weeks.

Results: Quercetin can reverse the decrease in PhNR amplitude caused by chronic glaucoma. The baseline frequency of miniature GABAergic inhibitory postsynaptic currents (mIPSCs) in the RGCs of glaucomatous retinal slices was lower than that of the control group. The frequencies of miniature excitatory postsynaptic currents (mEPSCs) were not significantly different between control and glaucomatous RGCs. The baseline frequencies of GABAergic mIPSCs and mEPSCs in OFF-type glaucomatous RGCs were greater than those in ON-type glaucomatous RGCs. Quercetin increased miniature GABAergic inhibitory neurotransmission to RGCs and decreased miniature glutamatergic excitatory neurotransmission, reducing the excitability of the RGCs themselves, thus alleviating the excitability of RGCs in glaucomatous slices.

Conclusion: Quercetin may be a promising therapeutic agent for improving RGC survival and function in glaucomatous neurodegeneration. Quercetin exerted direct protective effects on RGCs by increasing inhibitory neurotransmission and decreasing excitatory neurotransmission to RGCs, thus reducing excitotoxic damage to those cells in glaucoma.

Keywords: excitatory synaptic inputs; glaucoma; inhibitory synaptic inputs; patch-clamp; quercetin; retinal ganglion cells.

FIGURE 1

PhNR of normal and glaucomatous rat eyes. (A,B) Representative traces of the “a” wave, the “b” wave, and PhNR in a control eye (A) and a glaucomatous eye (B) in step 3 with the stimulus applied at 22.76 cd.s/m²–0.33 Hz. (C,D) Representative waves in a PBS-treated glaucomatous eye (C) and quercetin-treated glaucomatous eye (D) in the same step and with the same stimulus used in (A,B). (E) Quantitative analysis of PhNR amplitude (n = 12). The amplitude was normalized to the amplitude in control retinas. ^∗∗p < 0.01 (one-way analysis of variance). a, “a” wave; b, “b” wave; PhNR, photopic negative response.

FIGURE 2

Baseline sIPSCs and sEPSCs were more common in OFF-type RGCs than in ON-type RGCs. (A,B) The infrared interference phase microscope image in (A) shows all of the retinal layers. Representative Lucifer Yellow-filled OFF-type RGCs with dendritic arborizations in the distal (b) parts of the IPL are shown (A1, A2). Representative Lucifer Yellow-filled OFF-type RGCs with dendritic arborizations in the proximal (a) and distal (b) parts of the IPL are also shown (B1, B2). GCL, ganglion cell layer; IPL, inner plexiform layer. Scale bar, 5 μm. (C) Representative traces showing the frequency of GABAergic mIPSCs in both OFF-type RGCs (C1) and ON-type RGCs (C2). Vertical scale bar, 20 pA; horizontal scale bar, 1 s. (D) Representative traces showing the frequency of glutamatergic sEPSCs in both OFF-type RGCs (D1) and ON-type RGCs (D2). Vertical scale bar, 10 pA; horizontal scale bar, 1 s. (E) Summarized data from 14 RGCs showing that baseline mIPSCs were more common in OFF-type RGCs than in ON-type RGCs. (F) Summarized data from 12 RGCs showing that baseline mEPSCs were more common in OFF-type RGCs than in ON-type RGCs. ^∗p < 0.05 and ^∗∗p < 0.01, unpaired Student’s t-test.

FIGURE 3

Effects of chronic glaucoma on retinal mIPSCs and mEPSCs. (A) Representative traces of voltage clamp recordings of GABAergic mIPSCs in the presence of TTX (1 μM) in control and glaucomatous retinas. The baseline frequency of mIPSCs was markedly lower in glaucomatous retinas than in control retinas. Vertical scale bar, 20 pA; horizontal scale bar, 2 s. (B) Bar graphs showing the mean frequency of mIPSCs in control and glaucomatous retinas. (C) Representative traces of voltage-clamp recordings of glutamatergic mEPSCs in the presence of TTX (1 μM) in control and glaucomatous retinas. The baseline frequency of mEPSCs did not differ between glaucomatous retinas and control retinas. Vertical scale bar, 10 pA; horizontal scale bar, 0.5 s. (D) Bar graphs showing the mean frequency of mEPSCs in control and glaucomatous retinas. ^∗∗∗p < 0.001, unpaired Student’s t-test.

FIGURE 4

Quercetin increased the frequency and amplitude of GABAergic mIPSCs in OFF-type RGCs. (A) Representative traces showing the effect of 10 μM quercetin on mIPSCs. Vertical scale bar, 20 pA; horizontal scale bar, 1 min. (B) Recordings from a large-scale representative experiment under control conditions (B1), during quercetin treatment (B2), and during recovery (B3). (C,D) Cumulative frequency and amplitude distributions of GABAergic mIPSCs in a representative neuron under control conditions and during quercetin treatment showing that quercetin caused the cumulative frequency curve to shift significantly to the left and the cumulative amplitude curve to shift significantly to the right. (E,F) Summarized data on the frequency (E) and amplitude (F) of GABAergic mIPSCs (n = 9; from six slices). ^∗∗p < 0.01 and ^∗∗∗p < 0.001 compared with the control conditions; paired Student’s t-test.

FIGURE 5

Quercetin significantly increased the frequency, but not the amplitude, of mIPSCs in ON-type RGCs. (A) Representative recordings showing that quercetin increased the frequency but not amplitude of mIPSCs. (A, top) control condition; (A, middle) during quercetin treatment; (A, bottom) recovery period. Vertical bar, 10 pA; horizontal bar, 1 s. (B,C) Cumulative interevent interval and amplitude distributions of mIPSCs in a representative neuron during a control recording and after quercetin application. Quercetin significantly shifted the distribution of interevent intervals to the left (B) but did not shift the distribution of mIPSC amplitudes (C). The quercetin-induced changes in the distribution of interevent intervals were statistically significant (n = 11). ^∗∗p < 0.01, Kolmogorov–Smirnov test. (D,E) Summarized data for the frequency (D) and amplitude (E) of mIPSCs (frequency: n = 13; amplitude: n = 9). ^∗∗p < 0.01, paired Student’s t-test.

FIGURE 6

Quercetin significantly inhibited the frequency, but not the amplitude, of mEPSCs in OFF-type RGCs. (A) Representative traces showing the effect of 10 μM quercetin on glutamatergic mEPSCs in OFF-type RGCs. (A1) Control conditions; (A2) during quercetin treatment; (A3) recovery. Vertical bar, 5 pA; horizontal bar, 2 s. (B) Summarized data from 14 OFF-type RGCs showing that 10 μM quercetin significantly decreased the average frequency of glutamatergic mEPSCs. (C) Summarized data from 14 OFF-type RGCs showing that 10 μM quercetin did not change the average amplitude of glutamatergic mEPSCs. ^∗∗p < 0.01, paired Student’s t-test.

FIGURE 7

Quercetin significantly inhibited the frequency, but not the amplitude, of mEPSCs in ON-type RGCs. (A) Representative traces showing the effect of 10 μM quercetin on glutamatergic mEPSCs in ON-type RGCs. (A1) Control conditions; (A2) during quercetin treatment; (A3) recovery. Vertical bar, 10 pA; horizontal bar, 1 s. (B) Summarized data from 10 ON-type RGCs showing that 10 μM quercetin significantly decreased the average frequency of glutamatergic mEPSCs. (C) Summarized data from 10 ON-type RGCs showing that 10 μM quercetin did not change the average amplitude of glutamatergic mEPSCs. ^∗∗∗p < 0.001, paired Student’s t-test.

FIGURE 8

Quercetin hyperpolarized RGCs and decreased the firing rate. (A,B) Top trace: responses to a –200 pA current step in OFF-type RGCs (A) and ON-type RGCs (B). Negative current injection led to rebound burst firing in OFF-type RGCs but not in ON-type RGCs. Vertical bar, 20 mV; horizontal bar, 50 ms. (C) Current clamp recording of a representative RGC. Note that quercetin caused hyperpolarization and decreased the firing rate. Recordings under control conditions (C1), during application of quercetin (C2), and during washout (C3) are shown on an expanded time scale. (D) Bar graph summarizing the changes in MP. Quercetin can hyperpolarize MP. MP, membrane potential. ^∗p < 0.05, paired Student’s t-test.