Period1-Driven GFP Expression in Retinal Ganglion Cells of Transgenic Mice

Abstract

Abstract: : Purpose: We have produced transgenic mice in which neurons expressing the Period1 circadian clock gene are labeled by a Per1-driven GFP reporter and reported that GFP expressing cells were detected in both the inner nuclear and ganglion cell layers (Kuhlman et al., 2000). In this study, we sought to determine if the Per1::GFP expressing neurons on the GCL are ganglion cells or displaced amacrine cells. Methods: Transgenic mice hemizygous for the Per1::GFP transgene were used for all experiments. The retrograde tracer dye Di-I was injected into each side of either the superior colliculus or lateral geniculate. After 3-5 days, retinas were removed and retinal neurons double labeled with Di-I and GFP were visualized by fluorescence microscopy in living whole mounts. Visualized neurons were recorded using a standard whole-cell patch clamp techniques. GFP and thy1,2 immunocytochemistry were carried out by using a rabbit polyclonal anti-GFP and a purified rat anti-mouse monoclonal antibody against thy1,2, respectively. Secondary antibodies included Alexa 488 and Rhodamine TRITC. Results: A minority of Per1::GFP expressing cells in the GCL were labeled with retrograde transport of the dye Di-I from the superior colliculus (4 mice) or LGN (3 mice). To further examine the population of ganglion cells among the Per1::GFP expressing cells in the GCL, anti-thy1,2 antibody, a specific mouse ganglion cell marker, was introduced along with anti-GFP antibody. In three retinas, 14% of the GFP positive cells were co-stained with anti-thy1.2 antibody (GFP positive cells: 456 ± 50 cells/mm²; GFP⁺ + Thy1,2 ⁺ cells: 65 ± 24 cells/mm²). Using whole-cell current patch clamp recording the resting membrane potential of GFP⁺/Di-I⁺ cells was -62.5 ± 2.5 mV (n=4) and repetitive firing was evoked by a depolarization current pulse in all cells tested. When the holding potential was stepped from –70 mV to -45 mV, a 1997 ± 308 pA inward current with kinetic characteristics consistent with a sodium current, was produced in whole-cell voltage clamp mode (n=4). Conclusions: Whereas the majority of Per1::GFP expressing neurons in the GCL are displaced amacrine cells, some ganglion cells also express the Per1 clock gene. Since the Per1 gene plays a critical role in the molecular feedback loop that generates circadian rhythms, this raises the possibility of circadian clocks in ganglion cells. Future studies will focus on identifying the specific subtypes of ganglion cells that express Per1 and their potential role in retinal circadian rhythms. Supported by NIH EY09256 to DGM.

Keywords: ganglion cells • circadian rhythms • electrophysiology: non-clinical