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.

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We have previously shown that chelated copper stimulates the release of luteinizing hormone-releasing hormone (LHRH) from isolated hypothalamic granules. In this study, we wished to ascertain if chelated copper acts on hypothalamic neurons to stimulate LHRH release and, if so, what is the ligand specificity of this interaction. An in vitro system of explants of the median eminence area (MEA) was established and characterized. MEA explants were exposed for 15 muin to 50 /uM copper, and then they were incubated for 75 min in copper-free medium. Copper led to a transient increase in the rate of LHRH release; the maximal rate was attained 15 min after transfer of the MEA to copper-free medium. In addition, we found that copper complexed to histidine (Cu-His), but not ionic copper, stimulated LHRH release, the magnitude of which was dependent on the dose of Cu-His. The chelator specificity for Cu complex action was such that Cu-His stimulated LHRH release 4.9-fold and Cu-Cys stimulated release 2.5-fold, whereas neither Cu-Thr, Cu-Gly-His-Lys, Cu-bovine serum albumin, nor ceruloplasmin stimulated LHRH release. Based on these results and those of others indicating that the concentration of copper in hypothalamic axonal terminals is 1-2 orders of magnitude greater than plasma, we propose that copper released in the vicinity of the LHRH neurons interacts with specific sites on the LHRH axonal terminals, which leads to release of the peptide.A small amount of cupric sulfate (50 ,g) leads to ovulation if it is administered into the posterior median eminence of the rabbit but not into other regions of the brain (1). Tsou et al.(2) observed increased levels of luteinizing hormone-releasing hormone (LHRH) in the pituitary stalk of the rabbit after the systemic administration of cupric acetate. We showed (3-5) that copper, at a concentration within the range required for the activity of several copper-dependent enzymes (6-9), markedly stimulates the release of LHRH from isolated hypothalamic granules. Based on these findings, we proposed (4) that copper plays a role in regulating LHRH release from LHRH neurons in the following manner. Newly taken up copper interacts with LHRH granules that are in close proximity to or in the process of fusing with the plasma membrane. Intraneuronal copper does not interact with LHRH granules, because it would be inactivated by reduced glutathione and copper-binding proteins (10,11).It is well established that tissues obtain their copper supply from the circulation, where copper is chelated by proteins (ceruloplasmin, albumin), peptides, and amino acids. The current view is that the readily exchangeable pool of copper is that associated with albumin, small peptides, and amino acids (12, 13). The following observations support the view that an active uptake mechanism for copper is operative in the hypothalamus. Copper is present in the hypothalamus in concentrations much greater than in blood (14, 15), and in hypothalamic homogenates, copper is highly concentrated in synaptosomes (pinche...

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A series of studies from our laboratory has established an aggregate culture system of fetal rat brain cells that can serve as a model for studying regulatory processes of the developing neuropeptide Y (NPY)-producing neurons. Using aggregate cultures derived from 17-day-old fetal rat cortex, we addressed these questions: 1) Does brain-derived neurotrophic factor (BDNF) stimulate NPY production, and if so, is stimulation a function of the developmental state of the cultured NPY neuron? 2) Does BDNF induce phenotypic differentiation of NPY neurons? BDNF led to an increase in NPY production and the accumulation of NPY-mRNA in a dose dependent manner. BDNF did not alter the stability of NPY-mRNA, judged by the disappearance rate of NPY-mRNA after blockade of RNA synthesis (estimated t1/2 was 6-8 hr). BDNF stimulation of NPY production was dependent on length of exposure to BDNF and on culture-age. A continuous 8-day exposure to BDNF resulted in a significantly higher level of NPY production than a pulse of 2 days (comparing BDNF exposure on days 0-8 vs. 6-8, or days 8-17 vs. 15-17). Moreover, older neurons (age 17 days) produced twice as much NPY as younger (age 8 days) neurons in response to a 2-day pulse of BDNF (50 ng/ml). BDNF was significantly more effective than NT-3 in inducing NPY production, and NGF was ineffective. Immunocytochemical analysis of 8-day NPY neurons revealed that a 2-day pulse of BDNF induced the appearance of an abundance of morphologically well-defined neurons bearing an elaborate network of neurites. This was in contrast to the control-treated NPY neurons, which were morphologically undefined. In summary, the age-dependent effect of BDNF on NPY production is consistent with induction of functional expression, rather than promotion of survival, of cultured NPY neurons. The neurotrophin specificity for stimulation of NPY production, and the lack of effect of BDNF on the stability of NPY-mRNA, implicate the TrkB receptor in mediating transcriptional activation of the NPY gene. Thus, BDNF exerts a dual effect on developing cultured NPY neurons: induction of functional expression, and phenotypic differentiation of immature neurons into mature neurite-bearing neurons.