In vitro GnRH release from the isolated medial basal hypothalamus of the male guinea pig: evidence for the existence of two pools of releasable GnRH

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We studied in vitro GnRH output from the isolated medial basal hypothalamus (MBH) of the adult intact male guinea pig. Both basal release and the response to veratridine (V), which exerts a depolarizing action by increasing Na + permeability, were assessed using a static incubation system. Spontaneous GnRH release from single MBHs was pulsatile, with mean pulse frequency (‘Ultra’ program; 3 CV threshold) of 0.86 ± 0.17 pulse/h (range: 0.17–1.75) for 7.5 min fractions collected over a 240 to 360 min observation period ( N=10). Exposure to 20 μM V for a 15–60 min period resulted in increased GnRH output characterized by a consistently biphasic pattern of release, with an immediate response, and a secondary increase usually observed with a delay of 30 to 60 min relative to the introduction of V. This characteristic pattern of response was seen for tested V concentrations ranging between 0.1 and 50 μM. The Na + channel blocker tetrodotoxin (TTX, 3 μM), the calcium chelator EGTA (20 mM) and the calcium channel blocker verapamil (1 mM) significantly suppressed basal GnRH release and effectively prevented the GnRH response to V when added simultaneously with V. The secondary response to V could not be blocked selectively by tetrodotoxin introduced following V or from the last 7.5 min of V exposure onwards. In contrast, this secondary response could be selectively suppressed with either EGTA or verapamil. We conclude that the secondary component of the GnRH response to V is the result of delayed, calcium dependent secretory events triggered by the initial depolarizing action of V. These findings indicate the presence of two distinct pools of releasable GnRH in the MBH of the adult male guinea pig. The relevance of this finding to the pulsatile mode of GnRH release remains to be established. When, for purposes of comparison, isolated MBHs from 50-day-old male rats were studied, a clearly different, monophasic pattern of response to V was observed. Whether this observation is related to the known differences in distribution of hypothalamic GnRH between the rat and the guinea pig, still needs to be elucidated.