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A trp E fusion protein containing a C-terminal portion of the rat substance P receptor (SPR) was expressed in bacteria and used to produce an antibody. The antibody specifically reacted with SPR expressed in a mammalian cell line and rat striatum. Light and electron microscope analyses of the rat striatum revealed intense SPR-like immunoreactivity in neuronal somata and dendrites. These immunoreactive neurons constituted approximately 3% of the total population of striatal neurons; they were putative interneurons of large and medium-sized aspiny type.

The strict photoperiodic dependence of gonadotropic function observed in mink provides an excellent physiological model for studying the activity of GnRH hypothalamic neurons. In mink, exposure to more than 10 h light inhibits the activity of this neurohormonal system. Melatonin plays an essential role in this type of regulation of gonadotropic function. In mink, contrary to results in many other photosensitive species, melatonin was observed to mediate the gonadostimulating effect of short days. However, the binding sites and action of this substance have not yet been defined. We thus attempted to identify melatonin binding sites in the mink brain. The study was carried out at three times during the seasonal testicular cycle in male minks maintained under natural environmental conditions. Coronal sections were taken from the whole brain and pituitary gland. Using 2-[125I]iodomelatonin, we observed a strong concentration of binding sites in the pars tuberalis and not in the part of the median eminence surrounded by the pars tuberalis. This concentrated localization of melatonin binding sites brings up the problem of defining the action of this substance in photoregulating the activity of the GnRH neurohormonal system.

Changes in the sleep-waking cycle of freely moving cats were studied during application of excitatory amino acid antagonists in the ventro-posterolateral thalamic nuclei by microdialysis. DL-2-Amino-5-phosphono-pentanoic acid (APV), a selective N-methyl-D-aspartate (NMDA) receptor antagonist, produced an increase in the deep stages of slow wave sleep and in paradoxical sleep and a decrease in the light stages of slow wave sleep (SWS1), while 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), at a concentration selective for the non-NMDA receptors, produced a marked increase in SWS1. These results indicate a strong sleep-promoting action of excitatory amino acid antagonists and suggest that thalamic NMDA and non-NMDA receptors may play different roles in sleep regulation. Thus, changes in the sleep-waking cycle should be carefully evaluated when assessing the potential clinical use of excitatory amino acid antagonists.

Homogenates from guinea pig vas deferens were examined for possible existence of sigma receptor and phencyclidine (PCP) receptor using a radioligand binding assay. [3H](+)-SKF-10047 bound saturably in the homogenates to a single population of binding sites with a Kd of 311 and a Bmax of 1090 fmol/mg protein. IC50S of several representative sigma in displacing [3H](+)-SKF-10047 from this binding site were very much similar to the IC50S of those drugs in the brain sigma receptor binding assay. A correlation coefficient of 0.96 was obtained when IC50S from vas deferens were compared with those from the brain assay. Attempts to demonstrate PCP receptors in this tissue using [3H]TCP (N-[1-(2-thienyl)cyclohexyl]piperidine), however, yielded negative results. The results indicate that while guinea pig vas deferens contains sigma receptors, PCP receptors may be absent in this tissue.

We have previously shown that D-cycloserine displaces [3H]glycine binding to a recognition site with properties consistent with an N-methyl-D-aspartate (NMDA) receptor modulatory site. Additionally, D-cycloserine positively modulates the NMDA receptor as evidenced by its dose-dependent enhancement of [3H]1-[1-(2-thienyl)cyclohexyl]piperidine ([3H]TCP) binding to the NMDA receptor-coupled ionophore. Further evaluation of this compound indicates that the maximal stimulation of [3H]TCP binding induced by D-cycloserine is lower than that produced by other compounds acting at the NMDA receptor associated glycine modulatory site (glycine and D-serine). Moreover, the stimulation of [3H]TCP binding induced by D-cycloserine in the presence of various fixed concentrations of glycine results in a family of dose-response curves which asymptotically converge to 40-50% of the maximal stimulation induced by glycine alone. These results are consistent with D-cycloserine acting as a partial agonist of the NMDA receptor via its interaction with the coupled glycine modulatory site.

Interruption of the ascending serotonin axons of the medial forebrain bundle (MFB) in the rat brain produced a progressive time-dependent accumulation of imipramine receptors (labeled for autoradiography with [3H]imipramine). The largest accumulation of receptors occurred during the first 12 h at the caudal aspect of the lesion. An electrolytic lesion of the nucleus raphe dorsalis, administered 24 h prior to interruption of the medial forebrain bundle, markedly reduced the number of imipramine receptors on the caudal side of the lesion, while a significant accumulation was still evident on the rostral aspect. These results suggest that imipramine receptors are undergoing the process of orthograde axonal transport to terminals in the forebrain from the neuronal perikarya found in the nucleus raphe dorsalis. These receptors may also be undergoing retrograde transport back to their cell bodies of origin.

The density of the spinal cord thyrotropin-releasing hormone (TRH) receptor in male and female rats was measured during postnatal development. The younger rats of both sexes (11-16 days old) exhibited higher levels of TRH-binding than did adults. A comparison of the properties of receptor binding for young and adult spinal cords showed that the increased binding resulted solely from an increase in maximum binding capacity rather than a change in receptor affinity. Furthermore, no sex-dependent difference in the receptor property was found at any stage of development. In conclusion, these findings demonstrate that the differences in the maturation of TRH receptor itself may not be the cause of known gender-specific differences in the physiologic actions of TRH in the spinal cord of young rats.

Substance P (SP), a relatively non-selective tachykinin receptor agonist, and Septide and Senktide, highly selective NK-1 and NK-3 tachykinin receptor agonists, respectively, were injected intradermally in rats. The resulting cutaneous plasma extravasation (PE) was evaluated by measuring the amount of Evans blue that leached from the circulation into the skin. SP and Septide produced dose dependent PE, Septide being the more potent of the two. Senktide did not produce PE, even at doses 10,000 times higher. Neonatal capsaicin treatment significantly reduced SP- and Septide-induced PE. These data indicate that SP-induced PE is mediated by NK-1 tachykinin receptors.

The 6,7-dichloro derivative of 3-hydroxy-2-quinoxalinecarboxylic acid (diCl-HQC) is a relatively potent antagonist at the two excitatory amino acid receptor subtypes activated by N-methyl-D-aspartate (NMDA) and kainic acid. It antagonizes NMDA-induced excitation in the frog spinal cord (pA2 5.8), i.e. with a potency similar to D-2-amino-7-phosphonoheptanoate (D-AP-7). It also antagonizes NMDA-induced sodium efflux from rat brain slices (pA2 5.6). The compound inhibits kainic acid-induced sodium efflux from rat brain slices with a pA2 of 5.4 and it inhibits [3H]kainic acid binding to rat brain membranes with a pKi of 5.4. DiCl-HQC is only weakly active at the quisqualate receptor. This spectrum of activities may make this compound a useful tool to investigate the pharmacology of excitatory amino acid receptors.

Well washed membranes have been prepared from samples of cerebral cortex of control subjects and patients with Alzheimer's disease, obtained both at post mortem and by neurosurgical procedures earlier in the course of the disease. Binding to these membranes of two radioligands for the N-methyl-D-aspartate-phencyclidine receptor complex has been determined in the presence and absence of glycine. Glycine increased the binding in both control and Alzheimer tissue samples. At one concentration of radioligand, in the presence of glycine there was less binding to post-mortem samples, which Scatchard analysis showed was associated with a 36% loss of sites. In rare neurosurgical samples, there was also a loss of binding of radioligand which suggests that the effect is not due to post-mortem artefacts or epiphenomena. These new results may have implications for the symptomatic and preventative treatment of Alzheimer's disease.

N-Methyl-D-aspartate (NMDA)-stimulated [3H]noradrenaline release from rat hippocampal slices was blocked stereospecifically and non-competitively by MK-801 with the (+)-isomer achieving 50% blockade of 100 microM NMDA at 16 nM. The results indicate that MK-801 is the most potent NMDA antagonist yet described and that it blocks NMDA-stimulated neurotransmitter release by an action at the so-called 'phencyclidine (PCP) site'.

Agonist-induced accumulation of [3H]inositol-1-phosphate ([3H]IP1) was studied using human embryonic pituitary tumour cells (Flow 9000). Stimulation of Flow 9000 cells, prelabelled with [3H]inositol, with the nonapeptide bradykinin (BK), or its analogues and fragments produced a differential accumulation of [3H]IP1. BK-related peptides exhibited the following rank order of potency in this assay: BK = [Lys]BK greater than [Met-Lys]Bk much greater than [Des-Arg9]BK much greater than BK(1-6) = BK(2-7) = BK(2-9). BK and [Lys]BK produced half-maximal effects at 2-3 nM. [3H]BK receptor binding studies showed that BK and [Des-Arg9]BK produced a concentration-dependent inhibition of [3H]BK binding with Ki values of 4.8 +/- 1.9 nM (n = 3) and 6.8 +/- 0.7 microM (n = 3) respectively. These studies suggest the presence of B2-bradykinin receptors on the human embryonic pituitary tumour cell-line which appear to be coupled to the phosphatidyl inositol turnover signal transduction mechanism. Cholecystokinin, angiotensin II, vasopressin, thyrotropin-releasing hormone and bombesin also stimulated [3H]IP1 production but were generally much weaker than BK. In contrast, substance P, eledoisin, somatostatin, neurotensin, VIP, NPY, CGRP, U50488, DAGO and DADLE appeared inactive in this system at 10 microM.

The effect of guanine nucleotides and ions on (+)-[3H]3-(3-hydroxyphenyl)-N-(1-propyl)piperidine [+ )-[3H]3-PPP), (+)-N-[3H]allylnormetazocine ((+)-[3H]SKF 10047) and [3H]1-[1-(3-hydroxyphenyl)-cyclohexyl]piperidine ([3H]PCP-3-OH) specific binding to rat brain membranes was examined. These 3 compounds are proposed as prototypical ligands for the labeling of the sigma- and phencyclidine (PCP)-receptor subtypes. Competition binding experiments of (+)-SKF 10047 with (+)-[3H]3-PPP yielded a biphasic inhibition curve which transformed to a monophasic curve when membranes were incubated in the presence of Gpp(NH)p (0.1 mM). The common (+)-[3H]3-PPP/(+)-SKF 10047 binding component is more susceptible to Gpp(NH)p than the high affinity [3H]PCP-3-OH/(+)-SKF 10047 common binding component. Low affinity [3H]PCP-3-OH binding, which may represent a PCP-selective site, is not affected by GTP and Gpp(NH)p. Mono- and divalent cations markedly inhibit high affinity [3H]PCP-3-OH binding but they had a differential inhibitory effect on the binding of the other radioligands tested. These findings suggest differences in the regulation of multiple psychotomimetic (sigma- and PCP) binding sites by guanine nucleotides and ions.

The binding of the somatostatin analogue, 125I-iodo-Tyr-[Tyr0,D-Trp8]S14, to the foetal (18- and 24-week-old) and infant (newborn and 17-month-old) spinal cord was examined using in vitro autoradiography. Somatostatin binding sites were detected at cervical, thoracic and lumbosacral levels in foetal as well as in infant spinal cord. The radiolabelling was localized over the grey especially in the superficial layers of the dorsal horn including the substantia gelatinosa and the marginal zone. In foetal and newborn spinal cord, the direct and crossed pyramidal paths exhibited a substantial binding of the ligand. A similar labelling was not observed in the pyramidal paths of a 17-month-old child or in anencephalic newborn spinal cord or previously described in adult. These results emphasize the early presence of somatostatin binding sites during the ontogeny of the human spinal cord. Further, the transient appearance of somatostatin binding sites in the pyramidal paths, prior the myelination, raises the question whether somatostatin receptors could be involved in the maturation of certain normal paths.

[125I]Bolton-Hunter substance P [( 125I]BH-SP) binding sites in rat and human skin were investigated, using quantitative receptor autoradiographic and emulsion autoradiographic methods. [125I]BH-SP binding sites were discretely localized in skin areas anatomically corresponding to dermal papillae, sweat glands, and hair follicles. The highest density of the binding sites was in the dermal papilla of the finger, followed by the sweat gland. [125I]BH-SP binding to the dermal papillae of the human finger pad skin and rat paw pad skin was displaced by unlabeled SP, with a high affinity, and Kd values were calculated to be 744 pM and 297 pM, respectively. The existence of [125I]BH-SP binding sites supports the idea of the neurotransmitter role of substance P in skin dermal papilla.

Transplants of fetal neocortex (E15-19) within the minimally traumatized fourth ventricle were examined for the presence of receptors for the peptides bombesin (BN) and vasoactive intestinal peptide (VIP). These peptides are involved in cellular growth or regulation of cerebral blood flow. Although fetal and adult cortex in situ have low and moderate receptor densities for BN-like peptides respectively, neocortical transplants developed high densities between 4 and 16 weeks postoperative. VIP receptors, which have moderate density in adult cortex, were initially high but later showed a variable distribution in the transplants. The results suggest that neuropeptides, particularly BN and VIP may have a role in the growth and vascularization of central nervous system transplants.

Activity of presumed serotonin-containing neurons in the dorsal raphe nucleus of the cat was recorded across the sleep--waking cycle every 4 h, for 24 or 48 h. These neurons showed no diurnal variation in activity independent of behavioral state. This suggests that reports of diurnal fluctuations in serotonin metabolism are due either to differential patterns of sleep--wakefulness across the 24 h cycle and/or to a lack of tight coupling between neuronal activity and neurotransmitter metabolism.

The venom of the Joro spider, reported to block glutamate action selectively at some glutaminergic synapses, was applied focally to rod horizontal and on-bipolar cells in dark-adapted retinal slices. The venom hyperpolarized horizontal cells in the dark and blocked their responses to light. Depolarization of horizontal cells by ionophoretic pulses of L-glutamate, L-aspartate or kainate was antagonized by the toxin in a slowly reversible manner. These results are consistent with other evidence for glutamate as the rod neurotransmitter acting on a single postsynaptic receptor type on horizontal cells. In contrast, the venom, like glutamate, closed the same ion channels of on-bipolar cells as the transmitter, thereby blocking light responses of on-bipolars. Joro spider toxin distinguishes the glutamate receptor-channel complexes of rod horizontal and on-bipolar cells.

Polyvalent cations that block excitatory responses to N-methyl-D-aspartate inhibited the binding of [3H]MK-801 to putative N-methyl-D-aspartate receptor-gated channels in brain membranes. The order of potency was Zn2+ greater than La3+ = Cd2+ greater than Mn2+ greater than Co2+ greater than Ni2+ = Mg2+. These findings support the existence of interacting sites on the N-methyl-D-aspartate channel for ionic and organic antagonists, and provide a molecular mechanism for the modulation of excitatory neurotransmission and excitotoxicity by endogenous polyvalent cations.