Abstract
Abstract: :
Purpose: Recently, using a Per1::GFP transgenic mouse model we found that the circadian clock gene, Period 1 is expressed in dopaminergic amacrine cells with a circadian rhythm suggesting the possibility that dopamine neurons themselves may have endogenous circadian mechanisms (Witkovsky et al, 2003). To target clock gene expressing dopaminergic amacrine cells in vivo for functional and molecular analysis, we have now established a tyrosine hydroxylase (TH)–driven red fluorescent protein (RFP) transgenic mouse model. Methods: Transgenic mice were generated by injecting a construct carrying a 5.4 kb fragment of the rat TH gene linked to the dsRed2–1 reporter (Clontech). Retinas from transgenic mice harboring the TH::RFP reporter were screened for dsRed expression by using a dsRed filter set in conventional microscopy or by confocal microscopy. Rabbit polyclonal anti–dsRed and mouse monoclonal anti–TH were used as primary antibodies for ICC. 1% Lucifer Yellow was injected into RFP positive cells using a sharp electrode. Results: Six independent lines showing germ–line transmission to the transgene were generated while two of them (THRFP 5 and THRFP 81) were analyzed in detail. Two distinct types of TH–driven RFP expressing amacrine cells were visualized with RFP fluorescence. Type 1 cells were only seen in the inner nuclear layer and had relatively large cell bodies (13.7 ± 0.9 µm, SD, n=18) with 2–3 primary dendrites that further ramified with a number of swellings and varicosities in the outermost stratum of the IPL. A total of approximately 500 type 1 cells were found in each retina. Type 2 cells were located in both the INL and GCL with relatively small cell bodies (9.5 ± 1.1µm, SD, n=23). They had a single primary process that radially entered into the IPL and terminated in the middle of the IPL. Double label immunocytochemistry for dsRed and TH confirmed that type 1 dsRed+ cells were also immunoreactive for TH indicating that type 1 cells are dopaminergic neurons. In addition, TH–driven RFP expressing neurons were found in the substantia nigra of brain slices as well as in other areas of the brain. To examine the dendritic field of type 1 DA cells, Lucifer Yellow was injected into TH–RFP+ type 1 cells in retinal wholemount preparations. The diameter of average cell’s dendritic field was 589±113 µm (SD, n=6). Conclusions: Dopaminergic neurons of the living retina can be visualized with TH–driven RFP fluorescence for cell labeling and electrophysiological and optical imaging recordings. TH–RFP targeting of DA neurons of the retina will be a valuable approach in investigating the influence of clock genes on retinal dopamine.
Keywords: retina: proximal (bipolar, amacrine, and ganglion cells) • amacrine cells • neurotransmitters/neurotransmitter systems