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Insulin receptors are present in the hypothalamus, but the cell types bearing them are unknown. In order to test the hypothesis that some insulin receptors in the hypothalamus are associated with catecholamine terminals, rats were injected with 50 micrograms or 75 micrograms doses (intracerebroventricular) of 6-hydroxydopamine (6-OHDA). Control rats received vehicle only. The animals were sacrificed 7 days after injection, and catecholamine and indolamine levels in the hypothalamus were measured by high performance liquid chromatography with electrochemical detection. Localization of specific binding sites for [125I]-insulin in the arcuate (ARC), dorsomedial (DMN) and ventromedial (VMN) nuclei were determined by quantitative film autoradiography. Treatment with 6-OHDA resulted in a 70% reduction in hypothalamic norepinephrine content as compared to vehicle-treated controls (P less than 0.01). A slight depletion of epinephrine, dopamine and indolamines was also detected. Computerized image analysis of the autoradiograms was used to determine radioactivity bound (DPM/mm2) in each nucleus. Highest binding was in the ARC and DMN, with much lower binding in the VMN. Insulin binding in the ARC of the 6-OHDA-treated group was decreased by 25% compared to controls (P less than 0.01). No significant change in insulin binding was observed in the DMN or VMN. The 6-OHDA treatment had no significant effect on weight gain or on plasma insulin levels. The reduction of insulin binding in the ARC after 6-OHDA treatment supports the hypothesis that some insulin binding sites are located on catecholamine terminals in the arcuate nucleus.

In p-chlorophenylalanine (PCPA) pretreated insomniac cats, the intraventricular (i.v.t.) injection of artificial cerebrospinal fluid (CSF) did not impair the insomnia. CSF transfer from normal cats was followed in 4 out of 9 cats by the restoration of paradoxical sleep (PS). However, CSF transfer from paradoxical sleep deprived cats did result in 12 out of 13 experiments in a significant increase in slow wave sleep (SWS) and the induction of PS. Biochemical analysis of the CSF from normal or PS deprived cat has shown that the highest quantity of indolamines was at least 1000 times smaller than the threshold dose of 5-HTP (200 microgram) which has been shown to be able to restore sleep by i.v.t. injection in PCPA pretreated insomniac cats. These experiments provide evidence that the transfer of a small quantity of CSF (250 microliter) from a previously paradoxical sleep deprived cat can restore paradoxical sleep in an insomniac PCPA pretreated cat in bypassing the biosynthesis of serotonin (5-HT). These results suggest that a 'paradoxical sleep inducing factor' may be stored in the central nervous system during sleep deprivation.

Carbon fiber microelectrodes were used with the differential pulse voltammetry method for in vivo determination of indolamines within the extracellular space of the dorsal horn of the spinal cord or chloral hydrate-anesthetized rats. Under these conditions a peak of oxidation current which is characteristic of 5-hydroxyindoles is recorded at 280-300 mV. Stimulation of the nucleus raphe magnus (NRM) with stimulation parameters comparable to those used to elicit analgesia in freely moving animals produced marked alterations in the voltammograms: (1) stimulation of the NRM for 10 min induced an immediate and sustained increase in the peak amplitude; (2) post-effects of variable duration were observed; (3) the increase in the 5-hydroxyindolaminergic signal was significantly reduced during a second series of NRM stimulations indicating some degree of tolerance to central stimulation. The accuracy of these observations is strengthened by the fact that the basal 5 hydroxyindolaminergic signal is strongly depressed after pretreatment of the animal with p-chlorophenylalanine; in addition, under these conditions, NRM stimulation is totally inefficient. We suggest that these results reflect the in vivo modification of 5-HT metabolism. This represents the first evidence for an in vivo release of 5-HT during stimulation of brain stem areas which induces powerful analgesia in freely moving animals.