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. 2019 May 21;13:37.
doi: 10.3389/fncir.2019.00037. eCollection 2019.

Cannabinoids Modulate Light Signaling in ON-Sustained Retinal Ganglion Cells of the Mouse

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Free PMC article

Cannabinoids Modulate Light Signaling in ON-Sustained Retinal Ganglion Cells of the Mouse

Terence Peter Middleton et al. Front Neural Circuits. .
Free PMC article

Abstract

The sole output of the retina to the brain is a signal that results from the integration of excitatory and inhibitory synaptic inputs at the level of retinal ganglion cells (RGCs). Endogenous cannabinoids (eCBs) are found throughout the central nervous system where they modulate synaptic excitability. Cannabinoid receptors and their ligands have been localized to most retinal neurons in mammals, yet their impact on retinal processing is not well known. Here, we set out to investigate the role of the cannabinoid system in retinal signaling using electrophysiological recordings from ON-sustained (ON-S) RGCs that displayed morphological and physiological signatures of ON alpha RGCs in dark adapted mouse retina. We studied the effect of the cannabinoid agonist WIN55212-2 and the inverse agonist AM251 on the spatial tuning of ON-S RGCs. WIN55212-2 significantly reduced their spontaneous spiking activity and responses to optimal spot size as well as altered their spatial tuning by reducing light driven excitatory and inhibitory inputs to RGCs. AM251 produced the opposite effect, increasing spontaneous spiking activity and peak response as well as increasing inhibitory and excitatory inputs. In addition, AM251 sharpened the spatial tuning of ON-S RGCs by increasing the inhibitory effect of the surround. These results demonstrate the presence of a functional cannabinergic system in the retina as well as sensitivity of ON-RGCs to cannabinoids. These results reveal a neuromodulatory system that can regulate the sensitivity and excitability of retinal synapses in a dynamic, activity dependent manner and that endocannabinoids may play a significant role in retinal processing.

Keywords: area response function; cannabinoids; receptive field; surround inhibition; synaptic modulation.

Figures

FIGURE 1
FIGURE 1
Effects of a cannabinoid agonist on spontaneous activity and receptive field properties of ON-S RGCs. (A) Representative traces showing the reducing effect of WIN55212-2 (10 μM) on spontaneous spike rate. (B) Bar plot showing that the spontaneous frequency of action potentials was significantly reduced in the presence of WIN55212-2 from 7.6 ± 2.38 to 2.0 ± 0.68 spikes/s (p < 0.05, n = 8). (C) Representative traces showing responses of an ON-S RGC to different sized spot stimuli. Stimulation of the cell’s receptive field with a small (154 μm) bright spot caused a response consisting of an increase in firing rate (first column). As the spot increases in size more of the receptive field is stimulated increasing the response. Stimulation of a larger area of the receptive field center causes a larger response (second column, spot size 308 μm). When the spot size increases further the antagonistic surround receptive field is activated and reduces the excitatory response (third column, spot size 1240 μm). After addition of a cannabinoid agonist WIN55212-2 (10 μM) the response to the light spot is decreased. (D) Number of spikes in response to spot stimuli of different sizes for a representative cell. The data is fitted to a Difference of Gaussians (DoG) function. Addition of a cannabinoid agonist lowered the peak response but also lowered the degree of surround inhibition that dampens the peak response observed at larger spot sizes. Symbols represent the average response to two stimulus presentations for each size, bars represent standard deviation. (E) Lucifer Yellow filled ON-S RGC that was treated with WIN55212-2. Scale bar = 50 μm. (F) Average curve fits from all cells tested with WIN55212-2 (n = 9) with the SEM shown in gray. Curves were normalized to the peak response as well as the stimulus size that elicited the peak response.
FIGURE 2
FIGURE 2
Effects of the CB1R inverse agonist on spontaneous activity and receptive field properties of ON-S RGCs. (A) Representative traces showing the increasing effect of AM251 (5 μM) on spontaneous firing rate. (B) Bar plot showing the significant increase in spontaneous firing rate induced by AM251 from 4.7 ± 1.5 to 15.8 ± 4.7 spikes/s (p < 0.05, n = 12). (C) A response from a representative cell to different sized spot stimuli. Small, medium, and large spot sizes were 154, 220, and 1240 μm, respectively. After addition of a CB1R inverse agonist AM251 (5 μM) the response to the light spot is increased. (D) Number of spikes in response to spot stimuli of different sizes for a representative cell. The data is fitted to a DoG function. Addition of AM251 increased the peak response but also increased the degree of surround inhibition that dampens the peak response observed at larger spot sizes. Symbols represent the average response to two stimulus presentations for each size, bars represent standard deviation. (E) Lucifer Yellow filled ON-S RGC that was treated with AM251. Scale bar = 50 μm. (F) Average curve fits from all cells tested with AM251 (n = 6) with the SEM shown in gray. Curves were normalized to the peak response to the stimulus size that elicited peak response.
FIGURE 3
FIGURE 3
WIN55212-2 reduces the strength of synaptic conductances in ON-S RGCs. (A) Representative ON-S cell showing the excitatory (Gexc) and inhibitory (Ginh) conductance in response to small (266 μm) bright (top left) and dark (top right) spot stimuli. Addition of WIN55212-2 (5 μM) reduced both the Gexc (57%) and the Ginh (17%) response to the light stimulus. Bottom traces show responses of the same cell to large (1275 μm) bright (left) and dark (right) stimuli. (B) Top plot illustrates the receptive field profile of Gexc represented by the mean DoG curve fits for all cells. Addition of WIN55212-2 (10 μM) reduced the peak response by 39 ± 15% and the surround inhibition by 9% (SI: 61 ± 6 to 52 ± 7%, p < 0.05, n = 5). Bottom plot shows that addition of WIN55212-2 (5 μM) reduced the peak Ginh response by 23 ± 17% (p < 0.05, n = 5) and the surround inhibition by 2% (SI: 52 ± 5 to 50 ± 9, n = 5).
FIGURE 4
FIGURE 4
AM251 increases the strength of synaptic conductances in ON-S RGCs. (A) Representative ON-S cell showing the excitatory (Gexc) and inhibitory (Ginh) conductance in response to small (266 μm) bright and dark stimuli. Addition of AM251 (5 μM) increased both the Gexc (75%) and the Ginh (14%) response to the bright stimulus. AM251 decreased the reduction in tonic excitation at the onset of a dark spot and increased the change in Gexc at the offset of a dark spot. (B) Receptive field profiles of Gexc and Ginh represented by the mean DoG curve fits for all cells. AM251 (5 μM) increased the peak response of Gexc by 69 ± 21% (p < 0.05, n = 4) and reduced surround inhibition by 4% (SI: 61 ± 16 to 57 ± 12%, n = 4). Addition of AM251 (5 μM) increased the peak Ginh response by 18 ± 21% (p < 0.05, n = 4) and the surround inhibition by 14% (SI: 80 ± 5 to 66 ± 13%, n = 4).

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