The potential importance of copper (Cu) in neurosecretion can be inferred from the demonstration that extracellular Cu modulates the secretory function of peptidergic neurons (in vitro studies) and from the presence of high Cu concentrations in nerve terminals and secretory vesicles, primarily within the soluble matrix of the latter. We have previously hypothesized that vesicular Cu is released from neurons undergoing exocytosis and that such extracellular Cu plays an important modulatory role in the central nervous system. To test this Cu release hypothesis, rat hypothalami were incubated under in vitro conditions for 1 or 2 hr with 20 nM radiolabeled Cu (67Cu), and then 67Cu release was stimulated by a depolarizing concentration (60 mM) of K+. K+ markedly (P less than 0.001) stimulated 67Cu release in a Ca2+-dependent manner (stimulated release was 95 fmol/10 min/mg protein after 1 hr 67Cu loading and 160 after 2 hr). These amounts of released 67Cu account for about 10% of the total 67Cu taken up by the tissue. These results indicate that part of the 67Cu taken up by hypothalamic explants is directed into an intracellular compartment from where it can be released by a Ca2+-dependent mechanism, thus providing strong support to our hypothesis that release of copper is operative in situ in the brain.

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.

This paper deals with the effect of temperature (t degrees), ions, prostaglandin (PGE2) and testosterone on the rate of release of LHRH from the superfused medial basal hypothalamus (MBH) of normal or castrated male rats in an effort to further validate the biological properties of this preparation. LHRH in the perfusate is nondetectable when the MBH is at 0 C, warming to 37 C produces a sharp rise in LHRH and then stabilization at a level at least threefold the one calculated for the 0 C experiment. Basal release rate of LHRH is t degrees and calcium dependent, as is the stimulatory action of potassium. Infusion of high K+ induces approximately a fivefold increase in the release rate of LHRH and stays at this high level for as long as the infusion lasts (50 min). LHRH stimulation by PGE2 (pulse) proceeds in the absence of external calcium, and the effect lasts longer than the potassium (pulse) action. Surprisingly, MBHs from long-ter castrated males (60-90 days) release less LHRH per minute than intact male MBHs during the steady state phase of LHRH secretion; testosterone propionate (TP, 200 microgram/100 g BW x 3 days) increases significantly the rate of release in castrate rats and is not related to the amount of LHRH assayed in the MBH. MBHs of untreated castrated rats or castrated rats treated with TP, show a reduced LHRH secretion in response to PGE2. It is concluded that this preparation can be used at advantage to study the rate of LHRH release in a variety of experimental paradigms.

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