Muscarinic cholinergic receptor subtypes expression by human placenta

The presence of a cholinergic system in the placenta is suggested by several data, but no information is available concerning cholinergic receptor expression by placenta. The present study was designed to investigate muscarinic cholinergic receptors in sections of human placenta using a radioligand binding techniques with [3H]N-methyl scopolamine ([3H]NMS) as a ligand. [3H]NMS was bound to sections of human placenta in a manner consistent with the labelling of muscarinic cholinergic receptors. The dissociation constant (Kd) value was 0.1 +/- 0.03 nM and the maximum density of binding site (Bmax) value was 10.82 +/- 0.09 fmol/mg of tissue. The binding was time-, temperature- and concentration-dependent, belonging to one class of high affinity sites. Analysis of [3H]NMS displacement curves by compounds acting on the different subtypes of muscarinic cholinergic receptor subtypes suggests that human placenta expresses the four subtypes (M1-M4) of muscarinic cholinergic receptor assayable with radioligand binding assay techniques. The demonstration of muscarinic cholinergic recognition sites in human placenta may contribute to define the possible significance of placental cholinergic system. Moreover, human placenta can be used as an easily obtainable human source of M1-M4 muscarinic cholinergic receptor subtypes.     

Sequestration of muscarinic acetylcholine receptor m2 subtypes. Facilitation by G protein-coupled receptor kinase (GRK2) and attenuation by a dominant-negative mutant of GRK2

Sequestration of m2 receptors (muscarinic acetylcholine receptor m2 subtypes), which was assessed as loss of N-[3H]methylscopolamine ([3H]NMS) binding activity from the cell surface, was examined in COS 7 and BHK-21 cells that had been transfected with expression vectors encoding the m2 receptor and, independently, vectors encoding a G protein-coupled receptor kinase (GRK2) (beta-adrenergic receptor kinase 1) or a GRK2 dominant-negative mutant (DN-GRK2). The sequestration of m2 receptors became apparent when the cells were treated with 10(-5) M or higher concentrations of carbamylcholine. In this case, approximately 40% or 20-25% of the [3H]NMS binding sites on COS 7 or BHK-21 cells, respectively, were sequestered with a half-life of 15-25 min. In cells in which GRK2 was also expressed, the sequestration became apparent in the presence of 10(-7) M carbamylcholine. Approximately 40% of the [3H]NMS binding sites on both COS 7 and BHK-21 cells were sequestered in the presence of 10(-6) M or higher concentrations of carbamylcholine. When DN-GRK2 was expressed in COS 7 cells, the proportion of [3H]NMS binding sites sequestered in the presence of 10(-5) M or higher concentrations of carbamylcholine was reduced to 20-30%. These results indicate that the phosphorylation of m2 receptors by GRK2 facilitates their sequestration. These results are in contrast with the absence of a correlation between sequestration and the phosphorylation of beta-adrenergic receptors by the GRK2 and suggests that the consequences of phosphorylation by GRK2 are different for different receptors.     

Neuropeptide Y phase shifts the circadian clock in vitro via a Y2 receptor

The suprachiasmatic nuclei (SCN) contain a circadian clock whose activity can be recorded in vitro for several days. This clock can be reset by the application of neuropeptide Y. In this study, we focused on determination of the receptor responsible for neuropeptide Y phase shifts of the hamster circadian clock in vitro. Coronal hypothalamic slices containing the SCN were prepared from Syrian hamsters housed under a 14 h:10 h light:dark cycle. Tissue was bathed in artificial cerebrospinal fluid (ACSF), and the firing rates of individual cells were sampled throughout a 12 h period. Control slices received either no application or application of 200 nl ACSF to the SCN at zeitgeber time 6 (ZT6; ZT12 was defined as the time of lights off). Application of 200 ng/200 nl of neuropeptide Y at ZT6 resulted in a phase advance of 3.4 h. Application of the Y2 receptor agonist, neuropeptide Y (3-36), induced a similar phase advance in the rhythm, while the Y1 receptor agonist, [Leu31, Pro34]-neuropeptide Y had no effect. Pancreatic polypeptide (rat or avian) also had no measurable phase-shifting effect. Neuropeptide Y applied at ZT20 or 22 had no detectable phase-shifting effect. These results suggest that the phase-shifting effects of neuropeptide Y are mediated through a Y2 receptor, similar to results found in vivo.     

Plutonium contamination in soils in open space and residential areas near Rocky Flats, Colorado

The present study investigated the interaction between the muscarinic acetylcholine receptor (mAChR) allosteric modulator heptane-1,7-bis-(dimethyl-3'-phthalimidopropyl) ammonium bromide (C(7)/3-phth) and the orthosteric antagonist [3H]N-methylscopolamine ([3H]NMS) at the five cloned human mAChRs expressed in Chinese hamster ovary cells. Equilibrium binding studies, using two different concentrations of radioligand, showed the interaction between C(7)/3-phth and [3H]NMS to be characterized by different degrees of negative cooperativity, depending on the receptor subtype. The modulator exhibited the highest affinity (85 nM) for the unoccupied M2 receptor and the lowest affinity for the unoccupied M5 receptor, the latter being approximately 100-fold lower. In contrast, the highest degree of negative cooperativity was observed at the M5 receptor, whereas lowest negative cooperativity was found at the M1 and M4 receptors. Non-equilibrium dissociation kinetic studies also confirmed the allosteric properties of C(7)/3-phth at all five mAChRs and yielded independent estimates of the modulator affinity for the occupied receptor. The latter estimates showed good agreement with those calculated using parameter values determined from the equilibrium experiments. The present results extend previous findings that C(7)/3-phth is a potent allosteric modulator at mAChRs, particularly the M2 subtype, and also highlight the effects of cooperativity on apparent drug-receptor subtype selectivity.     

Partial purification of an endogenous inhibitor of muscarinic ligand binding

An endogenous inhibitory factor (EIF alpha) to the binding of a muscarinic antagonist, [3H]N-methyl scopolamine ([3H]NMS), has been partially (12-fold) purified from the soluble fraction of the ileal longitudinal muscle of guinea-pigs using a heat-treatment, isoelectric fractionation, and DEAE-column chromatography. The EIF alpha inhibited the [3H]NMS binding to the longitudinal muscle membrane with an IC50 of 53.6 micrograms/ml. This was about 230-fold potent than the non-specific inhibition of [3H]NMS binding by bovine serum albumin (BSA). Zn2+ (0.1 mM) almost completely blocked the inhibitory activity of EIF alpha, whereas such the effect of Zn2+ was not observed in the inhibition by BSA. These results suggest that EIF alpha inhibits the [3H]NMS binding to the muscarinic acetylcholine receptor in a different manner from non-specific interaction with the receptor.     

Rapid resetting of the mammalian circadian clock

The suprachiasmatic nuclei (SCN) contain the principal circadian clock governing overt daily rhythms of physiology and behavior. The endogenous circadian cycle is entrained to the light/dark via direct glutamatergic retinal afferents to the SCN. To understand the molecular basis of entrainment, it is first necessary to define how rapidly the clock is reset by a light pulse. We used a two-pulse paradigm, in combination with cellular and behavioral analyses of SCN function, to explore the speed of resetting of the circadian oscillator in Syrian hamster and mouse. Analysis of c-fos induction and cAMP response element-binding protein phosphorylation in the retinorecipient SCN demonstrated that the SCN are able to resolve and respond to light pulses presented 1 or 2 hr apart. Analysis of the phase shifts of the circadian wheel-running activity rhythm of hamsters presented with single or double pulses demonstrated that resetting of the oscillator occurred within 2 hr. This was the case for both delaying and advancing phase shifts. Examination of delaying shifts in the mouse showed resetting within 2 hr and in addition showed that resetting is not completed within 1 hr of a light pulse. These results establish the temporal window within which to define the primary molecular mechanisms of circadian resetting in the mammal.     

Small-scale characterization of wet powder masses suitable for extrusion-spheronization

Phthalimide-containing heptane-bisammonium-type compounds retard the dissociation of the antagonist [3H]-N-methylscopolamine ([3H]NMS) from muscarinic M2-receptor allosterically with high potency. To study the contribution of the lateral substituents to this effect, a series of derivatives was synthesized in which the phthalimide moiety was truncated. The potency of the compounds to delay [3H]NMS dissociation was measured in porcine heart homogenates (50 mM Tris-HCl, 3 mM MgHPO4, pH 7.3, 37 degrees C). Potency declined with diminuition of the lateral substituents, e.g. loss of the aromatic ring of the phthalimide resulted in a 400fold reduction in potency. In the hexahydrophthalimide derivatives, the cis-stereoisomer was about fivefold more potent than the trans-isomer. In conclusion, almost flat hydrophobic lateral moieties appear to be pivotal for high allosteric potency, suggesting a hydrophobic interaction of these parts of the molecule with the [3H]NMS occupied receptor protein.     

Molecular rigidity and potency of bispyridinium type allosteric modulators at muscarinic M2-receptors

Several bispyridinium compounds have been shown to be potent allosteric modulators of ligand binding to muscarinic M2-receptors. ,,Uno compounds" are benzyl derivatives of the bispyridinium "TMB4" (trimethylene-bis-[4-hydroxy-iminomethyl-pyridinium]). To gain more insight into structure activity relationships, eleven derivatives with varying structure of the oxime-linked aromatic substituent were tested for their ability to inhibit the equilibrium-binding of [3H]N-methylscopolamine ([3H]NMS) in guinea pig cardiac membranes and to retard [3H]NMS-dissociation allosterically. At a concentration of 3 microM, all compounds reduced [3H]NMS-binding to about 40 % of the control level, indicating a similar potency to inhibit the association of [3H]NMS. Allosteric retardation of [3H]NMS-dissociation required higher concentrations. Comparing the effects of the compounds at 30 and 300 microM, respectively, revealed considerable differences in potency. Therefore, the concentration-dependency of the delay of [3H]NMS-dissociation was determined for selected compounds. The results indicate that introduction of a benzyl-moiety into TMB4 leads to a 20-fold increase in allosteric potency. A further increment by a factor of 10 is obtained with the 2,6-dichlorobenzyl-substitution and with the naphthyl-derivative. The other compounds were less potent. An inverse correlation was found between the rotational freedom of the aromatic substituent and the allosteric potency. In conclusion, the aromatic moiety of non-symmetric bispyridinium-type modulators does not seem to be part of the pharmacophore involved in the inhibitory effect on the association of [3H]NMS. In contrast, a rigid aromatic lateral moiety appears to be essential for the interaction with the recognition site mediating the allosteric delay of [3H]NMS dissociation from muscarinic M2-receptors.     

Cholinergic regulation of the suprachiasmatic nucleus circadian rhythm via a muscarinic mechanism at night

In mammals, the suprachiasmatic nucleus (SCN) is responsible for the generation of most circadian rhythms and for their entrainment to environmental cues. Carbachol, an agonist of acetylcholine (ACh), has been shown to shift the phase of circadian rhythms in rodents when injected intracerebroventricularly. However, the site and receptor type mediating this action have been unknown. In the present experiments, we used the hypothalamic brain-slice technique to study the regulation of the SCN circadian rhythm of neuronal firing rate by cholinergic agonists and to identify the receptor subtypes involved. We found that the phase of the oscillation in SCN neuronal activity was reset by a 5 min treatment with a carbachol microdrop (1 microliter, 100 microM), but only when applied during the subjective night, with the largest phase shift (+ 6 hr) elicited during the middle of the subjective night. This effect also was produced by ACh and two muscarinic receptor (mAChR) agonists, muscarine and McN-A-343 (M1-selective), but not by nicotine. Furthermore, the effect of carbachol was blocked by the mAChR antagonist atropine (0.1 microM), not by two nicotinic antagonists, dihydro-beta-erythroidine (10 microM) and d-tubocurarine (10 microM). The M1-selective mAChR antagonist pirenzepine completely blocked the carbachol effect at 1 microM, whereas an M3-selective antagonist, 4,2-(4,4'-diacetoxydiphenylmethyl)pyridine, partially blocked the effect at the same concentration. These results demonstrate that carbachol acts directly on the SCN to reset the phase of its firing rhythm during the subjective night via an M1-like mAChR.     

Diurnal changes of GABA turnover rate in brain and pineal gland of Syrian hamsters

Circadian rhythms of GABA turnover rate in cerebral cortex, preoptic area-medial basal hypothalamus (PMBH), cerebellum, and pineal gland were examined in Syrian hamsters kept for 3 months under either long (14 h of light/day) or short days (10 h of light/day). In vivo GABA turnover rate was measured by the increase of GABA levels following inhibition of GABA-transaminase by gamma-acetylenic GABA. Under long photoperiods, a significant rhythm of GABA turnover was detected in the four areas studied (cerebral cortex, PMBH, cerebellum, and pineal gland), with maxima at night. A Cosinor analysis indicated acrophases which varied from 2300 to 0400 h (3rd to 8th h of darkness). Under short photoperiods, there were no significant circadian variations in GABA turnover in the cerebral cortex, and the synchronization in turnover rate among the remaining regions was lost, with acrophases being detectable either during the light phase of daily photoperiod (PMBH) or at night (cerebellum, pineal gland). Steady state levels of GABA also changed periodically in the same brain regions under both lighting environments, although phase relationships of circadian rhythms in GABA content and turnover rate varied significantly among tissues, as well as on photoperiodic conditions.     

Determination of pindolol enantiomers in human plasma and urine by simple liquid-liquid extraction and high-performance liquid chromatography

Ethanol disrupts signal transduction mediated by a variety of G-protein coupled receptors. We examined the effects of ethanol on arachidonic acid release mediated by muscarinic acetylcholine receptors. Chinese hamster ovary (CHO) cells transfected with the different subtypes of human muscarinic receptors (M1 to M5) were incubated with [3H]arachidonic acid ([3H]AA) for 18 hr, washed, and exposed to the cholinergic agonist carbamylcholine for 15 min. Carbamylcholine induced [3H]AA release from CHO cells expressing M1, M3, or M5, but not M2 or M4, muscarinic receptors. Dose response curves revealed that carbamylcholine stimulated [3H]AA release by up to 12-fold with an ECo of approximately 0.4 microM; maximal responses were obtained with 10 microM carbamylcholine. Exposure of M1-, M3-, or M5-expressing cells to ethanol for 5 min before stimulating with carbamylcholine reduced [3H]AA release by 40 to 65%; 50% of the maximal inhibition was obtained with an ethanol concentration of 30 to 50 mM. Ethanol did not affect basal [3H]AA release measured in the absence of carbamylcholine. Dose response curves suggest that ethanol acts as a noncompetitive inhibitor of muscarinic receptor-induced [3H]AA release insofar as maximal [3H]AA release was depressed in the presence of ethanol with no apparent change in the EC50 for stimulation by carbamylcholine. Exposure of CHO cells to 38 mM ethanol for 48 hr increased [3H]AA release induced by carbamylcholine without affecting basal [3H]AA release or altering the EC50 for carbamylcholine. These results indicate that ethanol acutely inhibits muscarinic receptor signaling through the arachidonic acid pathway in a noncompetitive manner, but chronically enhances muscarinic signaling through the same pathway.     

M2, M3 and M4, but not M1, muscarinic receptor subtypes are present in rat spinal cord

Muscarinic receptors in the spinal cord have been shown to mediate antinociception and alter blood pressure. Currently, there is much interest in identifying which muscarinic receptor subtypes regulate these functions. Toward that end, this study aimed to identify and localize the muscarinic receptor subtypes present in spinal cord using in vitro receptor autoradiography with [3H]-pirenzepine and [3H]-N-methylscopolamine. The results showed that M2 binding sites were distributed throughout the dorsal and ventral horns, whereas M3 binding sites were localized to laminae I to III of the dorsal horn. Only background levels of M1 binding sites were detected. Saturation binding assays using [3H]-pirenzepine in spinal cord homogenates confirmed the absence of M1 receptors. Competition membrane receptor assays using [3H]-N-methylscopolamine and the unlabeled antagonists pirenzepine, 11-2[(-[(diethylamino)methyl]-1-piperidinyl)-acetyl]-5, 11-dihydro 6H-pyrido(2, 3-b)(1, 4) benzodiazepine-one, methoctramine, and methoctramine in combination with atropine corroborated the autoradiographic findings and also revealed the presence of M4 binding sites. The finding that M2 and M3 binding sites were localized to the superficial laminae of the dorsal horn where nociceptive A delta and C fibers terminate suggests the possibility that either or both of these muscarinic receptor subtypes modulate antinociception. The present demonstration of M4 binding sites in spinal cord is consistent with the possibility that M2 and/or M4 receptors are involved in the regulation of blood pressure at the spinal level.     

Phenylclindine-induced alteration in rat muscarinic cholinergic receptor regulation

A muscarinic cholinergic receptor binding profile was performed on 3 separate regions of the rat brain following administration of the drug phencyclidine (10 mg/kg, twice a day) for 10 days. Binding assays were performed on homogenates of the hippocampus, striatum and cortex using the ligand [3H]QNB. Scatchard analysis of the binding data revealed a statistically significant decrease in the number of [3H]QNB binding sites in all phencyclidine treated animals. Binding was 22% lower in the cortex and 16% lower in the hippocampus and striatum. Phencyclidine treatment did not significantly alter the affinity of the receptor for the ligands [3H]QNB, acetylcholine or phencyclidine. This decrease in CNS muscarinic receptors could be contributing to the underlying biochemical mechanism of tolerance to the drug.     

Selection and analysis of rare second-site suppressors of Drosophila RNA polymerase II mutations

The disruptive effects on the activity-rest cycle of the monoamine oxidase inhibitor (MAOI) clorgyline and of continuous light were examined in Syrian hamsters. When administered in dim and moderate light intensities, clorgyline delayed the daily onset of wheel-running. When administered in bright light, it dissociated the circadian rhythm of wheel-running. This dissociation was prevented by lesions of the intergeniculate leaflet of the ventral lateral geniculate nucleus. Constant darkness restored the circadian rhythm of wheel-running in hamsters with disrupted circadian rhythms. The phase of the restored rhythm of wheel-running was shifted 6-12 h later than the phase of wheel-running prior to dissociation. Our results suggest that MAOI treatment weakens the coupling between oscillators that comprise the circadian pacemaker, and augments the disruptive effects of continuous light acting via the intergeniculate leaflet region of the ventral lateral geniculate nucleus. These effects on the circadian pacemaker may be responsible for disruptions of the sleep-wake cycle that occur as side effects when MAOIs are used clinically to treat depression and might play a role in the induction of mania and rapid cycling by antidepressants.     

Comparative laminar distribution of various autoradiographic cholinergic markers in adult rat main olfactory bulb

To provide anatomical information on the complex effects of acetylcholine (ACh) in the olfactory bulb (OB), the distribution of different cholinergic muscarinic and nicotinic receptor sub-types was studied by quantitative in vitro autoradiography. The muscarinic M1-like and M2-like sub-types, as well as the nicotinic bungarotoxin-insensitive (alpha 4 beta 2-like) and bungarotoxin-sensitive (alpha 7-like) receptors were visualized using [3H]pirenzepine, [3H]AF-DX 384, [3H]cytisine and [125I] alpha-bungarotoxin (BTX), respectively. In parallel, labelling patterns of [3H]vesamicol (vesicular acetylcholine transport sites) and [3H]hemicholinium-3 (high-affinity choline uptake sites), two putative markers of cholinergic nerve terminals, were investigated. Specific labelling for each cholinergic radioligand is distributed according to a characteristic laminar and regional pattern within the OB revealing the lack of a clear overlap between cholinergic afferents and receptors. The presynaptic markers, [3H]vesamicol and [3H]hemicholinium-3, demonstrated similar laminar pattern of distribution with two strongly labelled bands corresponding to the glomerular layer and the area around the mitral cell layer. Muscarinic M1-like and M2-like receptor sub-types exhibited unique distribution with their highest levels seen in the external plexiform layer (EPL). Intermediate M1-like and M2-like binding densities were found throughout the deeper bulbar layers. In the glomerular layer, the levels of muscarinic receptor subtypes were low, the level of M2-like sites being higher than M1. Both types of nicotinic receptor sub-types displayed distinct distribution pattern. Whereas [125I] alpha-BTX binding sites were mostly concentrated in the superficial bulbar layers, [3H]cytisine binding was found in the glomerular layers, as well as the mitral cell layer and the underlying laminae. An interesting feature of the present study is the visualization of two distinct cholinoceptive glomerular subsets in the posterior OB. The first one exhibited high levels of both [3H]vesamicol and [3H]hemicholinium-3 sites. It corresponds to the previously identified atypical glomeruli and apparently failed to express any of the cholinergic receptors under study. In contrast, the second subset of glomeruli is not enriched with cholinergic nerve terminal markers but displayed high amounts of [3H]cytisine/nicotinic binding sites. Taken together, these results suggest that although muscarinic receptors have been hypothesized to be mostly involved in cholinergic olfactory processing and short-term memory in the OB, nicotinic receptors, especially of the cytisine/ alpha 4 beta 2 sub-type, may have important roles in mediating olfactory transmission of efferent neurons as well as in a subset of olfactory glomeruli.     

Reentrainment of motor activity and spontaneous neuronal activity in the suprachiasmatic nucleus of Djungarian hamsters

Neurons in the suprachiasmatic nucleus (SCN) of the hypothalamus exhibit a daily rhythm in spontaneous electrical activity. Essentially two methods have been employed to record this circadian rhythm: (1) an in vitro brain slice technique and (2) in vivo multiunit recordings. Reentrainment of a circadian output to a shifted light:dark cycle commonly takes several cycles (depending on the amount of shift) until completed. Such a resetting kinetic has also been shown to be valid for SCN electrical activity if recorded in vivo. In an in vitro slice preparation, however, pharmacologically induced resetting is much faster and lacks transients; that is, a shift is completed within one cycle. This study was designed to probe for the presence of transients in the neuronal activity of the SCN in a brain slice preparation. The authors exposed Djungarian hamsters to an 8-h advanced or delayed light:dark cycle and monitored wheel-running activity during reentrainment. Additional groups of identically treated hamsters were used to record the pattern of spontaneous neuronal activity within the SCN using the brain slice preparation. Neuronal activity exhibited the usual rhythm with high firing rates during the projected day and low firing rates during the projected night. However, following 1 day of exposure to the 8-h advanced light:dark cycle, this rhythm disappeared in 6 of 7 slices. Rhythmicity was still absent following 3 days of exposure to the advanced light:dark cycle (n = 4). By contrast, 3 of 7 slices prepared from hamsters exposed to a delayed light:dark cycle for 3 days exhibited a daily rhythm in electrical activity. Although pharmacological agents reset the in vitro SCN neuronal activity almost instantaneously and in in vivo studies a stable phase relationship to a shifted light:dark cycle occurs gradually over several cycles, the authors did not detect either of these patterns. Such differences in resetting kinetics (e.g., rapid resetting, gradual reentrainment, temporary lack of measurable rhythmicity) may be due to (a) application of a resetting stimulus in vivo versus in vitro, (b) duration of the resetting stimulus, (c) the nature of the resetting stimulus, or (d) the recording technique employed.     

Brain histamine H3 receptors in rats with portacaval anastomosis: in vitro and in vivo studies

The long-term effects of portacaval anastomosis (PCA) on histamine H3 receptors in rat brain were studied by in vitro and in vivo methods. The overflow of histamine from the anterior hypothalamus and from cortex after long-term PCA was determined by in vivo microdialysis. The binding properties of [3H]-R-alpha-methylhistamine in membranes from cortex, cerebellum, and rest of brain (ROB) were examined with saturation binding experiments. The regional distribution of [3H]-R-alpha-methylhistamine binding sites in the brain of sham- and PCA-operated rats was assessed also with autoradiography. The tissue levels of histamine were significantly elevated in cortex and ROB of PCA-operated rats. In addition, the spontaneous and K+-evoked overflow of histamine from anterior hypothalamus, and the thioperamide-induced overflow from both anterior hypothalamus and cortex were increased after chronic PCA. In spite of the significantly elevated tissue concentrations and the moderate increase in histamine release, the binding properties of [3HI-R-alpha-methylhistamine to cortical membranes were not significantly changed. However, the autoradiography study did reveal a decrease in [3H]-R-alpha-methylhistamine binding density, particularly in striatum and cortex, where H3 receptors are located mainly at non-histaminergic neurons. In conclusion, we suggest that there is a region-selective increase in the histaminergic activity in chronic PCA, which leads to the down-regulation of somadendritic and pre-synaptic H3 receptors located at non-histaminergic neurons. At the same time, the autoreceptor mediated control of histamine neuronal activity via pre-synaptic H3 receptors located at histaminergic neurons is preserved after long-term PCA.     

Melatonin binding sites in the harderian gland of the rat and Syrian hamster

Specific melatonin binding sites in the harderian gland of both rat and Syrian hamster were studied using [125I]melatonin. In both species, binding of [125I]melatonin by harderian gland membranes exhibited properties such as dependence on time, temperature, membrane concentration, saturability, and high specificity. Only one class of high-affinity binding sites was found with a Kd of 0.19 and 6.47 nM for the rat and Syrian hamster, respectively. The binding capacity in the rat harderian gland was 4.00 fmol/mg protein; in the Syrian hamster it was 7.58 fmol/mg protein. In the rat, no sex differences were found in the binding of the tracer to the membranes. However, in the Syrian hamster, binding of [125I]melatonin by the harderian gland was twice higher in the female than in the male. No changes were found in the Kd values (6.47 vs. 6.94 nM), while binding capacity was significantly increased in the female (13.50 fmol/mg protein) when compared to the male hamster (7.58 fmol/mg protein). Binding of [125I]melatonin by the harderian gland of male hamsters was modified by castration but not by melatonin treatment. Castration induced an increase of binding up to the level of females. However, chronic melatonin administration did not alter the [125I]melatonin binding in either intact or gonadectomized male hamsters. Binding studies also showed diurnal variations. There was a diurnal rhythm of [125I]melatonin binding by Syrian hamster harderian glands with the peak at the end of the light period and the trough late in the dark period. This rhythm in the binding is observed in both male and female hamsters, although binding in females was always higher than that in males.(ABSTRACT TRUNCATED AT 250 WORDS)     

Roles of suprachiasmatic nuclei and intergeniculate leaflets in mediating the phase-shifting effects of a serotonergic agonist and their photic modulation during subjective day

Serotonin (5-HT) has been implicated in the phase adjustment of the circadian system during the subjective day in response to nonphotic stimuli. Two components of the circadian system, the suprachiasmatic nucleus (SCN) (site of the circadian clock) and the intergeniculate leaflet (IGL), receive serotonergic projections from the median raphe nucleus and the dorsal raphe nucleus, respectively. Experiment 1, performed in golden hamsters housed in constant darkness, compared the effects of bilateral microinjections of the 5-HT1A/7 receptor agonist, 8-hydroxydipropylaminotetralin (8-OH-DPAT; 0.5 microgram in 0.2 microliter saline per side), into the IGL or the SCN during the mid-subjective day. Bilateral 8-OH-DPAT injections into either the SCN or the IGL led to significant phase advances of the circadian rhythm of wheel-running activity (p < .001). The phase advances following 8-OH-DPAT injections in the IGL were dose department (p < .001). Because a light pulse administered during the middle of the subjective day can attenuate the phase-resetting effect of a systemic injection of 8-OH-DPAT, Experiment 2 was designed to determine whether light could modulate 5-HT agonist activity at the level of the SCN and/or the IGL. Serotonergic receptor activation within the SCN, followed by a pulse of light (300 lux of white light lasting 30 min), still induced phase advances. In contrast, the effect of serotonergic stimulation within the IGL was blocked by a light pulse. These results indicate that the respective 5-HT projections to the SCN and IGL subserve different functions in the circadian responses to photic and nonphotic stimuli.