In mammals, the master circadian clock resides in the suprachiasmatic nuclei (SCN) of the hypothalamus. The period and phase of the circadian pacemaker are calibrated by direct photic input from retinal ganglion cells (RGCs). SCN-projecting RGCs respond to light in the absence of rod- and cone-driven synaptic input, a property for which they are termed intrinsically photosensitive. In SCN-projecting RGCs, light activates a nonselective cationic current that displays inward and outward rectification. The goal of the present study was to investigate the identity of the light-activated ion channel and the intracellular signaling pathway leading to its activation. We considered two candidate channels, cyclic nucleotide-gated (CNG) channels and transient receptor potential (TRP) channels, which mediate vertebrate and invertebrate phototransduction, respectively. We report that the intrinsic light response relies upon a G-protein-dependent process. Although our data indicate that cyclic nucleotides modulate the signaling pathway, CNG channels do not appear to conduct the light-activated current because (i) cyclic nucleotides in the pipette solution do not activate a conductance or completely block the light response, (ii) CNG channel blockers fail to inhibit the light response, (iii) the effects of internal and external divalent cations are inconsistent with their effects on CNG channels, and (iv) immunohistochemistry reveals no CNG channels in SCN-projecting RGCs. Finally, we show that the pharmacology of the light-activated channel resembles that of some TRPC channel family members; the response is blocked by lanthanides and ruthenium red and SK&F 96365, and is enhanced by flufenamic acid and 1-oleoyl-2-acetyl-sn-glycerol. Furthermore, immunohistochemical experiments reveal that TRPC6 is expressed in many RGCs, including those that express melanopsin.

In mammals, light entrainment of the circadian clock, located in the suprachiasmatic nuclei (SCN), requires retinal input. Traditional rod and cone photoreceptors, however, are not required. Instead, the SCN-projecting retinal ganglion cells (RGCs) function as autonomous photoreceptors and exhibit light responses independent of rod-and cone-driven input. Using whole-cell patch-clamp recording techniques, we have investigated the morphological and electrophysiological properties of this unique class of RGCs. Although SCN-projecting RGCs resemble Type III cells in form, they display strikingly different physiological properties from these neurons. First, in response to the injection of a sustained depolarizing current, SCN-projecting cells fired in a transient fashion, in contrast to most RGCs which fired robust trains of action potentials. Second, in response to light, SCN-projecting RGCs exhibited an intensity-dependent transient depolarization in the absence of rod and cone input. This depolarization reached a peak within 5 s and generated increased spiking activity before decaying to a plateau. Voltage-clamp recordings were used to characterize the light-activated conductance which generated this depolarization. In response to varying light intensities, SCNprojecting RGCs exhibited a graded transient inward current which peaked within 5 s and decayed to a plateau. The voltage dependence of the light-activated current was obtained by subtracting currents elicited by a voltage ramp before and during illumination. The light-activated current displayed both inward and outward rectification and was largely unaffected by substitution of extracellular Na + with choline. In both respects, the intrinsic light-activated current observed in SCNprojecting RGCs resembles currents carried by ion channels of the transient receptor potential (trp) family, which are known to mediate the light response of invertebrate photoreceptors.

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 wholecell patch-clamp recording, we demonstrate that light triggers synaptic currents in ipRGCs via activation of ionotropic glutamate and γ-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.

SUMMARY:The objective of the study was to determine the role of the angiopoietins in the regulation of gelatinase expression during angiogenesis, and whether inhibition of the angiopoietin/Tek interaction in vivo can suppress the extent of retinal neovascularization. Retinal microvascular endothelial cells were treated with angiopoietins and examined for the production of gelatinases. The effects of inhibiting angiopoietin binding to the Tie-2 receptor was studied in newborn mice with experimentally induced retinal neovascularization. Animals were treated with an ip injection of the Tie-2 antagonist, muTek delta Fc, while oxygen-exposed mice treated with similar concentrations of murine IgG were used as controls. The effect of muTek delta Fc on the gelatinase expression in the retina was examined by real-time RT-PCR analysis. The stimulation of cultured retinal endothelial cells with Ang-1 and -2 resulted in the increased expression of matrix metalloproteinase (MMP)-9. Ang-2 expression was up-regulated in experimental animals during the period of angiogenesis and was the greatest on Day 17 (the time of maximal angiogenic response). Histologic analysis of mice treated with the Tie-2 antagonist, muTek delta Fc, showed significant (87%; p ϭ 0.001) inhibition of retinal neovascularization, and the response was dose-dependent. In vitro binding data support the fact that both Ang-1 and Ang-2 bind with high avidity to muTek delta Fc. The RT-PCR analysis of the retinas of the Tek-treated animals showed a similar (80%; p ϭ 0.001) inhibition of the MMP-9 expression, which correlated with the decrease in angiogenesis. The up-regulation of gelatinases in microvascular endothelial cells by Ang-2 may be an important early response during the development of retinal neovascularization. Inhibition of the binding activity of the angiopoietins in vivo suppressed retinal neovascularization concomitant with a reduction in the expression of MMP-9. (Lab Invest 2003, 83:1637-1645.

Expression of the urokinase receptor uPAR is essential to the development of retinal neovascularization. Inhibition of the activity of uPAR suppresses retinal neovascularization, possibly through a reduction in cell-associated proteolytic activity, cell signaling, or cell-matrix adhesion necessary for cell migration during angiogenesis. The uPA/uPAR interaction may be an important therapeutic target in the management of proliferative retinopathies.