OPTIMIZING LOW DENSITY PRIMARY CULTURES IN ORDER TO FIND A NEUROCHEMICAL MARKER FOR PACEMAKING CELLS IN THE SUPRACHIASMATIC NUCLEUS. Nikhil Angelo1, Alexis B. Webb1 and Erik D. Herzog1, Biology Department, Washington University, St. Louis, MO1.
The mammalian suprachiasmatic nucleus (SCN) is considered to be the central circadian pacemaker. We aimed to test the hypothesis that isolated SCN neurons are competent circadian pacemakers and that they comprise a unique cell class. We sought to develop methods to culture SCN neurons so that they could not communicate with each other and so that we could record their intrinsic Period2 (Per2) gene and neuropeptide expression. After recording bioluminescence of Per2::luc cultures or recording the firing rate of cultures on multi-electrode arrays (MEA), we performed dual-immunolabeling for the neuropeptides, arginine vasopressin (AVP) and vasoactive intestinal polypeptide (VIP). We have established methods for locating recorded cells after immunolabeling. Our preliminary findings confirm that AVP and VIP are expressed in low-density SCN cultures, but at lower proportions than in vivo 1. We found that only 30% of neurons plated at low density remain viable over the first seven days in vitro. To improve culture viability, we hypothesized that glial-conditioned medium (GCM) would enhance the health of the SCN cultures2. We raised low density primary cultures from the SCN in GCM and in CO2-buffered medium as a control. We counted the total number of cells, neurons, glia and cells with processes during their first week in vitro. Preliminary findings show that about 60% of the cells remained viable in GCM cultures. GCM’s primary effect seems to accelerate the development and persistence of neuronal processes. We are pursuing the optimal conditions to perform a low density experiment and beginning to immunolabel cells which have been scored as rhythmic by their Per2::luc expression.
1. Abrahamson, E.E. & Moore, R.Y. Suprachiasmatic nucleus in the mouse: retinal innervation, intrinsic organization and efferent projections. Brain Res. 916, 172-191 (2001).
2. Banker, G.A. Trophic Interactions Between Astroglial Cells and Hippocampal Neurons in Culture. Science. 209, 809-810 (1980).