Synaptic inputs to retinal ganglion cells that set the circadian clock

Abstract

Melanopsin-containing retinal ganglion cells (RGCs) project to the suprachiasmatic nuclei (SCN) and mediate photoentrainment of the circadian system. Melanopsin is a novel retinal-based photopigment that renders these cells intrinsically photosensitive (ip). Although genetic ablation of melanopsin abolishes the intrinsic light response, it has a surprisingly minor effect on circadian photoentrainment. This and other non-visual responses to light are lost only when the melanopsin deficiency is coupled with mutations that disable classical rod and cone photoreceptors, suggesting that melanopsin-containing RGCs also receive rod- and cone-driven synaptic inputs. Using whole-cell patch-clamp recording, we demonstrate that light triggers synaptic currents in ipRGCs via activation of ionotropic glutamate and gamma-aminobutyric acid (GABA) receptors. Miniature postsynaptic currents (mPSCs) were clearly observed in ipRGCs, although they were less robust and were seen less frequently than those seen in non-ip cells. Pharmacological treatments revealed that the majority of ipRGCs receive excitatory glutamatergic inputs that were blocked by DNQX and/or kynurenic acid, as well as inhibitory GABAergic inputs that were blocked by bicuculline. Other ipRGCs received either glutamatergic or GABAergic inputs nearly exclusively. Although strychnine (Strych)-sensitive mPSCs were evident on many non-ipRGCs, indicating the presence of glycinergic inputs, we saw no evidence of Strych-sensitive events in ipRGCs. Based on these results, it is clear that SCN-projecting RGCs can respond to light both via an intrinsic melanopsin-based signaling cascade and via a synaptic pathway driven by classical rod and/or cone photoreceptors. It remains to be determined how the ipRGCs integrate these temporally distinct inputs to generate the signals that mediate circadian photoentrainment and other non-visual responses to light.

FIG. 1

Synaptic inputs in intrinsically photosensitive (ip)- and non-ip retinal ganglion cells (RGCs). (A) Whole-cell currents were recorded from RGCs in a flat mount preparation. Retinas were bathed in control external solution at a holding potential of −60 mV (left) In an ipRGC, the light stimulus (bar) evoked a short-latency, transient inward current, followed by the long-lasting inward current of the intrinsic light response. The transient responses were reduced by application of bicuculline (Bicuc, 10 μM), and abolished by subsequent application of DNQX (20 μM). (right) In a non-ipRGC, a light stimulus evoked a burst of short-latency, transient inward currents. The later currents were blocked by application of Bicuc, and the initial spike was blocked by kynurenic acid (Kyn, 1.0 mM). (B) Comparison of spontaneous synaptic activity in non-ip and ipRGCs. Representative traces of robust (> 5 events/s), moderate (between 0.1 and 5 events/s) and silent (< 0.1 events/s) spontaneous synaptic activity in a non-ipRGC (left) and an ipRGC (right).

FIG. 2

Excitatory and inhibitory miniature synaptic currents in an ipRGC. (A) Plot of time vs. amplitude of mPSCs. Pharmacological blockers were applied as indicated. Concentrations were as noted in Fig. 1. (B) Selected recordings obtained in the presence of the pharmacological agents indicated. Arrow indicates a GABAergic event, and asterisks indicate glutamatergic events. (C) Comparison of averaged traces of small (< 12 pA) and large (> 12 pA) events. (D) Histogram of mPSCs before and after application of bicuculline (Bicuc), a blocker of ionotropic GABA receptors. (E) Cumulative probability plot comparing decay time constants of events before and after application of Bicuc.

FIG. 3

Some ipRGCs exhibit predominantly GABAergic (A–C) or glutamatergic (D–F) mPSCs. (A and D) Time vs. amplitude plots. (B and E) Representative current traces. (C and F) Amplitude histograms in the presence of synaptic blockers.