We investigated the postnatal development of the thyrotropin-releasing hormone (TRH)-containing raphe system in the brainstem of neonatal rats. Postnatal changes in TRH expression in nucleus (n.) raphe obscurus (ROb) and n. raphe pallidus (RPa) were evaluated by in situ hybridization using an 35S-labeled oligonucleotide probe complementary to TRH precursor mRNA. TRH mRNA expression was low at birth [postnatal day 0 (P0)], but was clearly evident by P7 and increased from that time to reach sustained high levels from P14 to P28. Consistent with this postnatal increase in TRH expression, we found increases in the density of TRH-immunoreactive (IR) fibers, which are derived from ROb and RPa, in the hypoglossal nucleus (nXII). TRH-IR fibers in nXII were very sparse at P0, but increased markedly over the first 2 postnatal weeks. The change in TRH innervation of nXII was closely matched by concomitant increases in 3H-methyl-TRH binding in nXII; specific TRH binding increased from very low levels at birth to high levels of P14. Finally, we recorded intracellularly the electrophysiological responses to TRH of hypoglossal motoneurons (HMs; n = 42) of neonatal rats (P0-P21) in a brainstem slice preparation. The response of neonatal HMs to TRH, in contrast to adult HMs, was highly variable. In some neonatal HMs, even at P0, TRH caused a depolarization with a decrease in input conductance (GN) that was characteristic of the response of all adult HMs. However, in other neonatal HMs, TRH was either without effect or caused a slight depolarization with no apparent change in GN, responses that were unlike those of adult HMs. A response was considered typical (i.e., "adult-like") if GN decreased to < 85% of control. The percentage of cells responding in a typical manner increased progressively from 25% at P0-P2 to 100% after P11. In addition, we found that the density of TRH-sensitive current (normalized to cell capacitance) increased with postnatal age in HMs that responded in a typical manner, suggesting that expression of the TRH-sensitive conductance is also developmentally regulated. Together, these data indicate that the TRH raphe neuronal system of the rat brainstem is not fully mature at the time of birth but develops over the first few postnatal weeks. This was true of levels of TRH mRNA in caudal raphe nuclei, density of TRH-IR fibers and 3H-methyl-TRH binding in nXII, and also the manner and magnitude of electrophysiological responses of HMs to exogenously applied TRH.

SUMMARYInsulin and glucagon release from monolayer pancreatic islet cell cultures were inhibited in a dose-response fashion by various enkephalins. Morphine, however, stimulated insulin and glucagon release. Both effects were blocked by naloxone, while naloxone alone had no effect. In isolated and denervated islet cells, opiates directly influence secretion from islet cells. DIABETES 29:84-86, January 1980. E ndogenous opiates (endorphins and enkephalins) are widely distributed in the nervous system and gastrointestinal tract of animals, including man. They have been shown to affect rates of neuronal transmission and the secretion of several pituitary hormones (ADH, prolactin, growth hormone, and gonadotrophins).1 " 3 By immunocytochemical techniques, endorphin-like substances have been demonstrated in the A-cells of the islets of Langerhans of the rat, 4 and morphine and B-endorphin have been reported to enhance insulin and glucagon release from the isolated perfused dog pancreas. 5Since islets are richly enervated by sympathetic, parasympathetic, and peptidergic systems, opiates might alter islet secretion indirectly by modulating these neuronal influences or directly by interacting with receptors on the islet secretory cells. We have used pancreatic islet cell monolayer cultures, an isolated and denervated islet cell system, to determine if opiates have a direct effect on islet cells and if all opiates act in a similar fashion. MATERIALS AND METHODSPancreatic monolayer cultures were established according to previously described techniques.6 " 8 These cultures have been shown by immunocytochemistry to contain cells that stain for insulin, glucagon, somatostatin (SRIF), gastric inhibitory polypeptide, secretin, gastrin, and pancreatic polypeptide.9 " 15 Experiments were performed on the fourth day of culture. Experimental incubation medium was Eagle's minimal essential medium supplemented with nonessential amino acids, Earle's salts, and 0.5% (v/v) heat-inactivated fetal bovine serum. All dishes were preincubated for 2 h in 5.5 mM glucose. This was followed by a 1-h incubation during which secretagogues plus varying concentrations of opiate were added to experimental cultures and compared with control cultures with segretagogues alone. Insulin and glucagon were measured in the incubation media by radioimmunoassay. 16 ' 17 Statistical analysis was performed using Newman-Keul's multiple comparison procedure. 18 The naturally occurring peptides leucine-and methionine-enkephalin, D-ala 2 -leucine-, and D-phe 4 -leucine-enkephalin analogues were generously provided by Dr. Richard J. Miller of the University of Chicago. RESULTSWith glucose 16.5 mM, the naturally occurring opiates leucine-enkephalin (LEK) and methionine-enkephalin (MEK) produced, over a concentration range of 0.02 to 2.0 /Jvi, a dose-related suppression of both insulin and glucagon release (Figure 1). D-ala 2 -leucine-enkephalin (DLE) is an enkephalin analogue that has been reported to resist metabolic degradation.1 Over the same concentration range, ...