Cannabinoid Signaling Selectively Modulates GABAergic Inhibitory Input to OFF Bipolar Cells in Rat Retina

Abstract

Purpose: In the mammalian retina, cannabinoid type 1 receptors (CB1Rs) are well-positioned to alter inhibitory synaptic function from amacrine cells and, thus, might influence visual signal processing in the inner retina. However, it is not known if CB1R modulates amacrine cells feedback inhibition at retinal bipolar cell (BC) terminals.

Methods: Using whole-cell voltage-clamp recordings, we examined the pharmacological effect of CB1R activation and inhibition on spontaneous inhibitory postsynaptic currents (sIPSCs) and glutamate-evoked IPSCs (gIPSCs) from identified OFF BCs in light-adapted rat retinal slices.

Results: Activation of CB1R with WIN55212-2 selectively increased the frequency of GABAergic, but not glycinergic sIPSC in types 2, 3a, and 3b OFF BCs, and had no effect on inhibitory activity in type 4 OFF BCs. The increase in GABAergic activity was eliminated in axotomized BCs and can be suppressed by blocking CB1R with AM251 or GABAA and GABAρ receptors with SR-95531 and TPMPA, respectively. In all OFF BC types tested, a brief application of glutamate to the outer plexiform layer elicited gIPSCs comprising GABAergic and glycinergic components that were unaffected by CB1R activation. However, blocking CB1R selectively increased GABAergic gIPSCs, supporting a role for endocannabinoid signaling in the regulation of glutamate-evoked GABAergic inhibitory feedback to OFF BCs.

Conclusions: CB1R activation shape types 2, 3a, and 3b OFF BC responses by selectively regulate GABAergic feedback inhibition at their axon terminals, thus cannabinoid signaling might play an important role in the fine-tuning of visual signal processing in the mammalian inner retina.

Figure 1.

Rat OFF BCs display different backgrounds of spontaneous inhibitory activity under light-adapted conditions. (A) Fluorescence images (left) and sample traces (right) showing that type 2 and 3a/b OFF BCs display low background spontaneous inhibitory activity compared to type 4 OFF BCs. Scale bars: 10 µm. (B) Summary plot showing the difference in the amplitude and frequency of sIPSCs in the different OFF BC types recorded. Note that type 3a and 3b OFF BCs differ in their sIPSC amplitude, but not in frequency, whereas type 4 displays a comparatively higher amplitude and frequency compared to the other cell types. In all subsequent figures, types 2 and 3 OFF BCs are grouped as LFCs and type 4 OFF BC is referred to as HFCs. Summary data display mean ± SEM and open circles represent a single cell. **P < 0.01, ***P < 0.001; ns, not significant.

Figure 2.

Spontaneous activity in LFC comprises GABAergic and glycinergic inputs, whereas spontaneous activity in HFC is mainly mediated by glycinergic inputs. (A) sIPSC sample traces (left) and summary plot of frequency (middle) and amplitude (right) from the LFC group showing that sIPSC are partially reduced by GABA receptors antagonists (TPMPA, 50 µM/SR 10 µM) and the remaining component is eliminated by blocking glycine receptors with strychnine (5 µM). (B) The sIPSCs in HFC are mainly mediated by glycine receptors, as GABA receptors antagonists (TPMPA, 50 µM/SR 10 µM) had no significant effect on the frequency and amplitude of sIPSCs. Blocking glycine receptors with strychnine completely eliminated sIPSC in the HFC group. Summary data show mean ± SEM and open circles represent a single cell. **P < 0.01, ***P < 0.001; ns, not significant.

Figure 3.

Activation of CB1R increases GABAergic sIPSCs in OFF BCs. (A) Sample traces and cumulative probability plots, including summary bar graphs (inset), showing that blockage of CB1R with AM251 (5 µM) had no effect on spontaneous activity in LFC OFF BCs. (B), Bath application of 1 µM WIN produced a leftward shift consistent with an increase in sIPSC frequency, but no changes in the amplitude of spontaneous activity in types 2 and 3 OFF BCs were observed. (C) The CB1R inverse agonist (AM251, 5 µM) eliminated the effect of WIN on sIPSC frequency in LFC OFF BCs. Summary data display mean ± SEM and open circles in summary bar graphs represent a single cell. ***P < 0.001; ns, not significant.

Figure 4.

Activation of CB1R does not alter glycinergic sIPSCs recorded from type 4 OFF BCs. (A) Sample traces and cumulative probability plots, including summary bar graphs (inset) showing that blockage of CB1R with AM251 (5 µM) had no effect on spontaneous activity in type 4 OFF BCs. (B) Bath application of 1 µM WIN also had no effect on the spontaneous activity in type 4 OFF BCs. Summary data display mean ± SEM and open circles in bar graphs represent a single cell. ns, not significant.

Figure 5.

Inhibition of GABA receptors eliminates the effect of CB1R activation on sIPSCs in LFC OFF BCs. (A) Sample traces and cumulative probability plots, including summary bar graphs (inset), showing that activation of CB1R with WIN (1 µM) had no effect on spontaneous activity when both GABA_ARs and GABA_CRs were blocked with SR-95531 and TPMPA, respectively. (B), Blockage of GABA_ARs alone also eliminated the WIN-mediated increase in the frequency of sIPSC in LFC BCs. Summary data display mean ± SEM and open circles represent a single cell. ns, not significant.

Figure 6.

CB1R activation reduces spontaneous inhibitory activity in ON-OFF amacrine cell subtypes. (A) Activation of CB1R with WIN significantly reduces the frequency, but not the amplitude of spontaneous inhibitory activity recorded from morphologically identified ON-OFF subtypes of amacrine cells. (B), Inhibitory activity from morphological identified AII amacrine cells is unaffected by bath application of 1 µM WIN. (C,D) Bath application of WIN has no effect on the inhibitory activity in both morphological identified ON C and OFF D subtypes of amacrine cell recorded. All panels display representative fluorescence images of recorded amacrine cells (left), samples traces (middle), and cumulative probability plots (right), including summary bar graphs (inset). Images scale bars: 10 µm. Bars indicate mean ± SEM and open circles represent a single cell. *P < 0.05. ns, not significant.

Figure 7.

Brief application of glutamate to the OPL depolarizes OFF BCs and elicits IPSCs comprising GABAergic and glycinergic activity. (A) Sample traces (left panel) and summary plot (middle panel) showing that glutamate(Glu)-evoked inhibitory activity in the LFC group was partially reduced by GABA receptor antagonists (TPMPA, 50 µM/SR 10 µM) and the remaining component was eliminated by the glycine receptor antagonist strychnine (5 µM). Note that during glutamate-induced IPSCs, a significant increase in the frequency and amplitude of inhibitory activity was observed (right panels). (B) Glutamate-elicited IPSCs in HFC cells (left panel) were reduced by strychnine application and eliminated by GABA receptor antagonists (middle panel). Unlike LFC A, glycinergic spontaneous activity in the HFC decreases during the glutamate-evoked response (right panel). Summary data consists of mean ± SEM and open circles represent a single cell. The charge transfer is indicated as percentage of control; *P < 0.05, **P < 0.01, ***P < 0.001. ns, not significant.

Figure 8.

Activation of CB1Rs does not alter exogenous glutamate-evoked IPSCs, but increases the frequency of spontaneous GABAergic IPSCs. (A) Sample traces (left panel) and summary plot (middle panel) showing that bath application of 1 µM WIN did not exert any effect on the glutamate-evoked IPSC charge transfer, but in the same cell WIN significantly increased the frequency, but not the amplitude of GABAergic sIPSCs even after the exogenous application of glutamate (right panel). (B) Glutamate-elicited IPSCs in HFC cells (left panel) were unaffected by bath application of 1 µM WIN (middle panel). Summary data display mean ± SEM and open circles represent a single cell. *P < 0.05. ns, not significant.

Figure 9.

Inhibition of CB1R enhances exogenous glutamate-evoked IPSCs, but not spontaneous IPSCs. (A) Sample traces (left panel) and summary plot (right panel) showing glutamate-evoked IPSCs in LFC OFF BCs before and after bath application of the CB1R inverse agonist AM251. Although an increase in the charge transfer of evoked IPSCs was observed (middle panel) in the same cell sIPSC frequency and amplitude remained unchanged (right panel). (B) Charge transfer of glutamate-elicited IPSCs in type 4 OFF BCs (HFC; left panel) was also significantly increased by bath application of 5 µM AM251 (middle panel). No further effect of AM251 on sIPSC frequency was observed (right panel). (C) In the continuous presence of GABA receptor antagonists (SR/TPMPA), the charge transfer of glutamate-elicited IPSCs in types 2 and 3 OFF BCs (HFC; left panel) remained unchanged (middle panel), indicating that AM251 affected GABAergic but not glycinergic evoked transmission. Summary data display mean ± SEM and open circles represent a single cell. *P < 0.05, **P<0.01. ns, not significant.

Figure 10.

Potential mechanisms underlying CB1R-mediated effects in the OFF BCs. (A) Under light-adapted conditions, CB1R activation selectively increases GABAergic sIPSCs by a direct regulation of GABA release from a subset of amacrine cells (question mark) or indirectly by regulating serial lateral inhibition between GABAergic amacrine cells that mediates inhibitory inputs to types 2 and 3 OFF BCs, rather than by regulating glutamate release from OFF BCs. By shutting down inhibitory inputs between GABAergic amacrine cells (disinhibition), CB1Rs could influence the intrinsic excitability and, thus, produce an increase in the GABA release probability onto OFF BCs. (B), Depolarization of OFF BCs by brief application of glutamate in the OPL boosts amacrine cell activity, thus engaging the release of eCBs, which likely activates CB1Rs localized in a subtype of GABAergic amacrine cells that regulate evoked feedback IPSCs. Alternatively, a strong activation of GABAergic amacrine cells might produce a CB1R-dependent self-inhibition that inhibits neuronal firing, thereby reducing GABA release onto OFF BCs. AC, amacrine cell; Gly, glycine; ON and OFF BC, ON and OFF bipolar cell; (+), glutamate; (-), GABA/glycine.