Responsiveness of Immature versus Adult Male Rat Hypothalami to Dibutyryl Cyclic AMP- and Forskolin-Induced LHRH Release in vitro

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Abstract
In the present study, we have investigated the effects of intermittent dibutyryl cyclic AMP (dbcAMP, 5 × 10–8 M), butyrate (5 × 10–8 M) and forskolin (10–4 M) on immunoreactive luteinizing hormone-releasing hormone (LHRH) release from superfused hypothalamic fragments from intact male rats of age 25, 30, 45, or 60–75 day (adult). The results indicate that at 25 days of age, male rat hypothalami were most responsive to cyclic AMP (162% of preinfusion basal LHRH release); by 30 days of age, dbcAMP also elicited increased LHRH release (120% of basal). By 45 days of age, the dbcAMP effect on in vitro LHRH release was slightly inhibitory (78% of basal); however, by adulthood, the effect of this cyclic nucleotide on LHRH release was minimal (92% of basal). Butyrate also induced age-dependent modifications in in vitro LHRH release from male rat hypothalami, with slight increases following butyrate delivery at 25 days of age (115%), slight decreases at 30 (82%) and 45 days of age (68%), and little change in adulthood (94%). This latter finding emphasizes the importance of using butyrate as a control for butyryl derivatives of cyclic AMP, which are known to liberate butyric acid as a product of hydrolysis of parent compounds. To address the possibility that the lack of effectiveness of dbcAMP in the older animal preparations was solely due to an increasing sensitivity of male rat medio-basal hypothalamus fragments to butyrate, we examined the effect of forskolin (10–4 M), an adenylate cyclase stimulator, on LHRH release. In the 30-day-old male rat preparation, continuous infusion of forskolin evoked marked increases in LHRH release, characterized by episodic surges; in contrast, this compound was relatively ineffective in inducing LHRH surges in the adult male rat condition. This study also shows that while hypothalamic LHRH concentration reached maximal levels by 45 days of age, spontaneous in vitro release of LHRH increased in a near-linear fashion as a function of age of male rat donor (from 25 days of age to adult), thus indicating a dissociation between spontaneous LHRH neuronal activity and dbcAMP-stimulated LHRH release. In sum, it can be concluded that the responsivity of the hypothalamic neural LHRH apparatus to cyclic AMP (either exogenously applied or generated through stimulation of adenylate cyclase) decreases as the rats mature, with no response close to puberty and during adulthood. In addition, maturational changes in LHRH release and LHRH concentration in superfused medio-basal hypothalamus fragments from intact male rats can be inferred using this in vitro superfusion system.