Neurotransmitter molecules are implicated in regulating cell proliferation and fate determination in the CNS. During development, the nuclei of retinal progenitor cells (PCs) undergo interkinetic nuclear migration (INM), moving from the ventricular zone (VZ) to the prospective ganglion cell layer in G1 of the cell cycle, before duplicating their DNA in S phase, and returning to the VZ in G2 and undergoing mitosis. Previous work (Pearson et al. 2002) has shown that purinergic and muscarinic receptors are involved in generating spontaneous [Ca2+]i transients amongst VZ cells, and that both stimulation and blockade of these receptors affect the rate of mitosis. However, muscarinic and purinergic receptors may further affect the rate of the cell cycle through regulating the time it takes for INM. To investigate this we have visualised cells within embryonic day five (E5) chick retina. Embryos were decapitated, retinas dissected out, and labelled using DiI coated particles fired from a gene gun. This technique produces sparse labelling of both differentiated cells and PCs on a dark background. The movements of the cell nuclei and cell bodies labelled in this way can be followed on a confocal microscope for more than 2 h. Using these methods we have examined the effects of UTP (10 µM), carbachol (50 µM), GABA, glutamate (both 20 µM), PPADS (30 µM), pirenzipine, bicuculline and NBQX (all 25 µM), on cell movement. The mean rate of these movements was significantly decreased by bath application of UTP (rate in µm h-1: 17.4 ± 1.0, mean ± S.E.M., N = 6 retinas, n = 59 cells, compared with paired controls (21.7 ± 1.4, n = 48, P = 0.01 (2-tailed Student’s unpaired t test)), and significantly increased in the presence of the purinergic antagonist PPADS (22.1 ± 1.3, N = 6, n = 55 in PPADS compared with 17.6 ± 0.5, n = 77 in control, P = 0.001). Carbachol caused an increase (18.3 ± 0.7, N = 4, n = 93 in carbachol, 15.2 ± 1.1, n = 33 in control, P = 0.03), and pirenzipine a decrease (17.0 ± 0.9, N = 4, n = 48 in pirenzipine, 18.3 ± 1.1, n = 40 in control, P = 0.37), in the rate of movement, although only for carbachol was this significant. GABA, glutamate, bicuculline and NBQX were without effect. Purinergic and muscarinic stimulation and blockade affected only movements away from the VZ. Movements in this direction arise from both INM within PCs and the migration of differentiated cells. Since these two populations cannot be distinguished on basis of their morphology alone it was not possible to determine whether or not movements in one or both populations were affected by these drugs.
This work was supported by The Wellcome Trust, MRC and Royal Society.