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Notes: Recent evidence suggests that the thyroid regulation of thyrotropin-releasing hormone (TRH)-containing neurons in the paraventricular nucleus of the hypothalamus involves the activation of other hypothalamic neural circuits. For example, the arcuate nucleus and not the paraventricular nucleus contains the highest enzyme activity of 5′-deiodinase type II, an enzyme that is pivotal for the local synthesis of T3. This experiment was undertaken to demonstrate whether a monosynaptic pathway exists between the arcuate nucleus and those TRH cells of the paraventricular nucleus that are neuroendocrine, i.e. project to the external layer of the median eminence. A specific cRNA probe derived from the coding region of deiodinase type II was used for the in situ hybridization histochemistry which was combined with immunocytochemistry for a specific marker of glial cells, glial fibrillary acidic protein (GFAP). The hybridization signals were present within the hypothalamus in the arcuate nucleus–median eminence region and in the periventricular area. The periventricular labeling was localized to the ependymal layer of the third ventricle and no hybridization product was detected in the paraventricular nucleus and other hypothalamic nuclei adjacent to the third ventricle. Within the median eminence, numerous cells containing the hybridization product were located in the internal layer adjacent to the floor of the third ventricle and in the external layer adjacent to the surface of the brain. In the dorso- and ventromedial regions of the arcuate nucleus, deiodinase type II mRNA-containing cells were also detected. Numerous type II deiodinase mRNA-containing cells in the median eminence and arcuate nucleus were also found to be immunopositive for GFAP. The abundance of arcuate cells expressing the hybridization product was lower than those in the periventricular region or in the median eminence. The anterograde tracer, Phaseolus vulgaris leucoagglutinin, was injected into the medial parts of the arcuate nucleus where the in situ hybridization experiment detected deiodinase type II mRNA. Simultaneously with the anterograde tracing, the retrograde tracer, Fluoro-Gold, was injected into either the median eminence or the general circulation. Light and electron microscopic double and triple immunolabeling experiments on vibratome sections of colchicine-pretreated animals revealed that arcuate fibers innervate TRH cells within the parvicellular region of the paraventricular nucleus. Populations of these TRH cells receiving afferents from the arcuate nucleus were also retrogradely labelled from either the median eminence or the general circulation indicating their direct role in the regulation of thyrotropin secretion from the anterior pituitary. The majority of arcuate nucleus efferents on TRH cells were found to establish symmetrical synaptic connections. The present results provided direct evidence of a monosynaptic pathway between the hypothalamic site of local thyroid hormone production, the arcuate nucleus, and neuroendocrine TRH cells in the paraventricular nucleus. This signalling modality may play an important role in thyroid feedback on TRH cells. Since the arcuate nucleus is involved in the regulation of central mechanisms controlling diverse homeostatic functions, including reproduction and feeding, the pathway described in this study may also carry integrated signals related to reproduction and ingestion to TRH-producing cells.

Notes: Peschke E, Peschke D, Hammer T, Csernus V. Influence of melatonin and serotonin on glucose-stimulated insulin release from perifused rat pancreatic islets in vitro. J. Pineal Res. 1997; 23:156–163. © Munksgaard, Copenhagen〈section xml:id="abs1-1"〉〈title type="main"〉AbstractInsulin plays a key role in the control of glucose homeostasis in mammals. Insulin secretion is regulated by a coordinated interplay of several factors. The role of the indoleamines in the control of insulin secretion has not been fully elucidated yet. The present study was addressed to investigate the function of melatonin and serotonin in the direct control of insulin secretion from the pancreatic islets. Explanted rat Langerhans' islets were treated with melatonin or serotonin while also being exposed to specific (glucose) or non-specific (KC1) stimulus either in a pulsatile or long-term manner in a perifusion system. Insulin content from the effluent tissue culture media was analyzed with RIA. Pulsatile administration of melatonin and serotonin alone did not alter the basal insulin secretion from the explanted islets even at pharmacological (5 μM) level. However, insulin response to specific (glucose) or non-specific (KC1) stimulus was significantly reduced while the islets were treated with melatonin (3 to 12 hr, 10 nM to 5 μM). This effect was reversible and repeatable. Both the start and end of the effect was rapid, evolving and disappearing within 10 min. On the other hand, under similar experimental protocol, serotonin (at 5 μM concentration) significantly enhanced both glucose and KC1 stimulated insulin release. Since the effect of the nonspecific stimulation (with KC1) was also altered, melatonin and serotonin seem to alter not only the release but also the synthesis of the insulin. Our data show that melatonin and serotonin have a direct effect on the insulin secretion from the pancreatic islets.

Notes: Abstract: Recent functional, autoradiographic, and molecular investigations have shown that the pineal secretory product melatonin reduces the forskolin-stimulated insulin secretion from isolated pancreatic islets of neonate rats. Autoradiographic and binding studies as well as reverse transcriptase-polymerase chain reaction (RT-PCR) experiments proved that these effects are mediated through specific, high-affinity pertussis-toxin-sensitive Gi-protein-coupled MT1 receptors and subsequent inhibition of the adenylyl cyclase/cyclic adenosine monophosphate (cAMP) system. This hypothesis was proved by blocking the intracellular signal transduction pathway using the non-hydrolyzable guanosine triphosphate analog guanosine 5′-O-(3-thiotriphosphate) (GTPγS) or the competitive melatonin receptor antagonist luzindole. Both GTPγS and luzindole diminished the melatonin effect. We have published these prior results elsewhere. So far, however, no information is available on both whether the MT1 receptors are located on the β-cells and whether the consecutive functional reactions are based on a direct influence of melatonin on the insulin producing β-cells. In order to examine this question, we used a glucose responsive insulin producing insulinoma cell line INS-1 isolated from rats. Comparable with the results of islets the competitive receptor antagonist luzindole diminished the insulin-decreasing effect of melatonin. In addition, our RT-PCR experiments, using specific primers for the rat melatonin receptor MT1 showed that this melatonin receptor mRNA is also expressed in the INS-1 cells. Furthermore we radioimmunologically analyzed the forskolin-stimulated cAMP concentration in the superfusate. Similar to insulin secretion, the cAMP concentration was significantly reduced by melatonin. Following the hypothesis that cAMP is actively secreted from INS-1 cells by an energy-dependent mechanism based on either a OAT1/ROAT1 like anion exchanger or MDR-like transport systems, we used probenecid (p-[dipropylsulfamoyl] benzoic acid), a known inhibitor of cAMP extrusion. Probenecid blocks the export of cAMP by acting on transport mechanisms which are as yet not completely understood. Consistently, insulin secretion was increased and cAMP concentration diminished. The application of the phosphodiesterase inhibitor IBMX (3-isobutyl-1-methylxanthine) caused a marked rise of insulin secretion as well as cAMP concentration in the perifusate. From these data we conclude that the MT1 receptor is located on the INS-1 cell and therefore in general on pancreatic β-cells.

Notes: Abstract We describe the synthesis of the first aviangalanin (GAL), chicken GAL, and its N-terminal and C-terminal segments by solid-phasesynthesis, using Boc/Bzl amino acidprotection groups and MBHA resin. The three peptides were prepared with purities of over97%, as determined by RP-HPLC, HPCE, FAB-MS or ESI-MSand amino acid analysis.Antibodies against these synthetic peptides wereraised in rabbits and used forimmunohistochemical localization of GAL-immunoreactiveneurons in chicken brain.

Notes: Summary We describe the synthesis of the first avian galanin (GAL), chicken GAL, and its N-terminal and C-terminal segments by solid-phase synthesis, using Boc/Bzl amino acid protection groups and MBHA resin. The three peptides were prepared with purities of over 97%, as determined by RP-HPLC, HPCE, FAB-MS or ESI-MS and amino acid analysis. Antibodies against these synthetic peptides were raised in rabbits and used for immunohistochemical localization of GAL-immunoreactive neurons in chicken brain.

Notes: An antiserum raised against the synthetic tripeptide pyroglutamyl-histidyl-proline (free acid) was used to localize thyrotropin-releasing hormone (TRH) in the rat central nervous system (CNS) by immunocytochemistry. The distribution of TRH-immunoreactive structures was similar to that reported earlier; i.e., most of the TRH-containing perikarya were located in the parvicellular part of the hypothalamic paraventricular nucleus, the suprachiasmatic portion of the preoptic nucleus, the dorsomedial nucleus, the lateral basal hypothalamus, and the raphe nuclei. Several new locations for TRH-immunoreactive neurons were also observed, including the glomerular layer of the olfactory bulb, the anterior olfactory nuclei, the diagonal band of Broca, the septal nuclei, the sexually dimorphic nucleus of the preoptic area, the reticular thalamic nucleus, the lateral reticular nucleus of the medulla oblongata, and the central gray matter of the mesencephalon. Immunoreactive fibers were seen in the median eminence, the organum vasculosum of the lamina terminalis, the lateral septal nucleus, the medial habenula, the dorsal and ventral parabrachial nuclei, the nucleus of the solitary tract, around the motor nuclei of the cranial nerves, the dorsal vagal complex, and in the reticular formation of the brainstem. In the spinal cord, no immunoreactive perikarya were observed. Immunoreactive processes were present in the lateral funiculus of the white matter and in laminae V-X in the gray matter. Dense terminal-like structures were seen around spinal motor neurons. The distribution of TRH-immunoreactive structures in the CNS suggests that TRH functions both as a neuroendocrine regulator in the hypothalamus and as a neurotransmitter or neuromodulator throughout the CNS.