To determine the oscillatory pattern of LHRH neurons in culture. RESULTS Previously, we have shown that 1) cultured LHRH neurons derived from the olfactory placode of monkey embryos release LHRH in a pulsatile manner at approximately 50-min intervals, and 2) individual LHRH cells exhibit [Ca2+]i oscillations. In order to understand the mechanism of LHRH-pulse generation, we further characterized the pattern of [Ca2+]i oscillations in LHRH neurons. The olfactory placode and the migratory pathway of LHRH neurons from monkey embryos at E35-37 were dissected out and cultured on glass coverslips in a growth medium. Two to five weeks later cultured cells were labeled with the calcium dye, fura-2, and examined for [Ca2+]i oscillations. [Ca2+]i changes were recorded every 10 seconds for 30 to 152 min using a video imaging system. After recording, cells were fixed and immunostained for LHRH. In 16 cultures, 359 LHRH cells (4-51 cells/culture) exhibiting [Ca2+]i oscillations were recorded. In 13 of 16 cultures, [Ca2+]i oscillations occurred synchronously in 50-100% of the individual cells in a culture, whereas [Ca2+]i oscillations in cells in the remaining 3 cultures did not synchronize. Strikingly, in 8 of 13 cultures the synchronization of [Ca2+]i oscillations repeatedly occurred in complete unison at 47.3q3.4 min, which is similar to the period observed for LHRH release, whereas in 5 of 13 cultures the less tight synchronization of [Ca2+]i oscillations repeatedly occurred at 23.4q4.2 min. Interpulse intervals (IPI, 6.9q0.8 min, n=134) of [Ca2+]i oscillations in cells with tight synchronization and IPI (9.2q1.0 min, n=130) in cells with less tight synchronization did not differ significantly from IPI (8.5q0.4 min, n=95) in cells without synchronization. Neither age of cultures, nor type of chambers (with or without media perifusion) was related to synchronization of oscillations. Results from this study indicate that LHRH neurons derived from monkey olfactory placode possess an endogenous mechanism for synchronization of [Ca2+]i oscillations, which may be related to the periodicity of LHRH release. FUTURE DIRECTIONS We will examine the mechanism of the synchronization. KEY WORDS LHRH neurons, Ca2+ oscillation, LHRH-pulse generation, olfactory placode
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