Characterization of central melatonin receptors using 125I-melatonin

The binding of 125I-melatonin, a potent analog of melatonin, to rat brain synaptosomal preparations was investigated. 125I-melatonin bound with high affinity (Kd = 38 nM) to a single class of sites (Bmax = 81 fmol/mg protein). Kinetic studies indicated that binding was time-dependent and reversible. Specific 125I-melatonin binding was inhibited by melatonin, and was unaffected by other structurally related compounds including serotonin. Binding of 125I-melatonin was greatly reduced if the synaptosomal preparations were pretreated by heat or trypsin but was unaffected by freeze-thawing. These results suggest that 125I-melatonin may serve as a valuable probe for studying melatonin receptors.     

Asynchrony in the Diurnal Rhythms of Dopaminergic D2 Receptors in Different Brain Regions of the Rat

Diurnal variations of dopaminergic D₂ receptors have been described in the striatum of rats, while other dopaminergic regions remain unstudied. Diurnal variations of dopamine D₂ receptors in the striatum, frontal cortex, and amygdala of the rat, were characterized by the stereospecific binding of [³H]-spiperone. Clear rhythms were found in all these areas, but asynchronous to each other. Striatal receptors had diurnal variations with a single peak at 00:00 hours. Frontal cortex receptors showed two peaks at 00:00 and 12:00 hours. Amygdaline complex receptors had two peaks at 18:00 and 06:00 hours. Saturation binding curves and their Scatchard analysis indicated that the diurnal variations in [³H]-spiperone binding are related to changes in receptor density rather than its affinity. The diurnal variations asynchrony in [³H]-spiperone binding to dopaminergic D₂ receptors from different neural regions, suggest different regulation in each area. Other functional implications of these rhythms remains to be established.     

In vitro TRH release from hypothalamus slices varies during the diurnal cycle

We have previously described a daily rhythm in thyrotropin releasing hormone (TRH) and TRH mRNA in the rat hypothalamus. To determine whether TRH release fluctuates in a diurnal manner, we have measured basal and potassium stimulated release from hypothalamic slices, and compared it to release from olfactory bulb slices, during the diurnal cycle. Basal TRH release was higher at 7:00 h than at any other time (1:00, 13:00 or 19:00 h) in either hypothalamus or olfactory bulb. The ratio of stimulated over basal release was higher in the hypothalamus at 19:00 h, when TRH content was highest. Potassium stimulated TRH release from olfactory bulb was not different from basal release at any time. TRH release fluctuations were not due to a rhythm of extracellular inactivation: the activity of pyroglutamyl aminopeptidase II, an ectoenzyme responsible for TRH inactivation, was constant throughout the cycle. Our data demonstrate that diurnal variations of TRH release occur in vitro and that the enhanced responsiveness to potassium stimulation in hypothalamus is correlated with increased levels of peptide.     

Asynchrony in the Diurnal Rhythms of Dopaminergic D2 Receptors in Different Brain Regions of the Rat

Diurnal variations of dopaminergic D₂ receptors have been described in the striatum of rats, while other dopaminergic regions remain unstudied. Diurnal variations of dopamine D₂ receptors in the striatum, frontal cortex, and amygdala of the rat, were characterized by the stereospecific binding of [³H]-spiperone. Clear rhythms were found in all these areas, but asynchronous to each other. Striatal receptors had diurnal variations with a single peak at 00:00 hours. Frontal cortex receptors showed two peaks at 00:00 and 12:00 hours. Amygdaline complex receptors had two peaks at 18:00 and 06:00 hours. Saturation binding curves and their Scatchard analysis indicated that the diurnal variations in [³H]-spiperone binding are related to changes in receptor density rather than its affinity. The diurnal variations asynchrony in [³H]-spiperone binding to dopaminergic D₂ receptors from different neural regions, suggest different regulation in each area. Other functional implications of these rhythms remains to be established.     

Melatonin Binding Sites

The distribution and characterization of specific melatonin binding sites were studied using 125I-melatonin. Autoradiography revealed only three sites of specific melatonin binding in brain: the suprachiasmatic nuclei, the median eminence, and the small part of choroid plexus at the caudal end of the fourth ventricle. Two other sites were detected outside the CNS: the anterior pituitary and the retina. The specific binding of 125I-melatonin was saturable and reversible. The dissociation constant (KD) of the binding sites was 60 pM. The concentration of the binding sites (Bmax) in the median eminence was 26 fmol/mg protein, and in the pituitary 3 fmol/mg protein. Specificity of the binding sites was tested by displacement of 125I-melatonin. The order of potency--melatonin much less than N-acetyl-5-hydroxytryptamine less than 5-methoxytryptamine much less than 5-hydroxytryptamine = 3,4-dihydroxyphenylethylamine = noradrenaline--shows high specificity of the binding sites for melatonin.     

Further evidence for the presence of specific binding sites for prolactin in the rabbit brain. Preferential distribution in the hypothalamus and substantia nigra

In the present research we have extended our work on the presence of binding sites for prolactin in the rabbit brain focusing our attention on the brain areas with high dopamine cell bodies density. Among these areas the hypothalamus showed the highest specific binding of labeled ovine prolactin (oPRL). Clearly detectible specific binding was observed also in substantia nigra, whereas in other brain regions the specific binding was very small, except for the striatum where a low but not negligible binding was found in female rabbits. The binding of 125I-oPRL showed a hormonal specificity and Scatchard analysis of the binding showed no clear difference in dissociation constant (Kd) between hypothalamus, nigra and striatum.     

Marked Regional Heterogeniety of 125I-Bolton Hunter Substance P Binding and Substance P-Induced Activation of Phospholipase C in Astrocyte Cultures from the Embryonic or Newborn Rat

The specific binding of 125I-Bolton Hunter substance P (125I-BHSP) was estimated on 4- to 5-week-old primary cultures of astrocytes from several brain structures and the spinal cord of 16-day-old embryonic or newborn rats. In both cases, high levels of binding of 125I-BHSP were found on intact astrocytes from the brainstem, but this binding was low or negligible on cells from the cerebral cortex, striatum, hypothalamus, and mesencephalon. In addition, hippocampal astrocytes from newborn rats were also devoid of 125I-BHSP binding sites, while a binding of 125I-BHSP (half that of brainstem cells) was observed on astrocytes from the cerebellum and spinal cord. It was also shown that this regional heterogeneity in 125I-BHSP binding was not linked to differences in the inactivation of the ligand, cell plating density. or eventual cell contaminants. Five-day-old cultures from 16-day-old embryos were used to estimate 125I-BHSP binding on neuron-enriched cultures. Specific 125I-BHSP binding was found on cells from the brainstem, mesencephalon, and hypothalamus, but neurons from the cerebral cortex or the striatum contained low or negligible amounts of 125I-BHSP binding sites. Competition studies using tachykinins and SP analogues indicated that 125I-BHSP binding sites on brainstem astrocytes (16-day-old embryos) have the pharmacological profile expected for NK1 binding sites. SP (1 microM) stimulated phosphoinositide breakdown in cells rich in 125I-BHSP binding sites (brainstem) but not in those devoid of 125I-BHSP binding (striatum).(ABSTRACT TRUNCATED AT 250 WORDS)     

In vivo binding of 125I-LSD to serotonin 5-HT2 receptors in mouse brain

The binding of 125I-LSD (2-[125I]-lysergic acid diethylamide) was studied in various mouse brain regions following intravenous injection of the radioligand. The high specific activity of 125I-LSD enabled the injection of low mass doses (14 ng/kg), which are well below the threshold for induction of any known physiological effect of the probe. The highest levels of 125I-LSD binding were found in the frontal cortex, olfactory tubercles, extra-frontal cortex and striatum while the lowest level was found in the cerebellum. Binding was saturable in the frontal cortex but increased linearly in the cerebellum with increasing doses of 125I-LSD. Serotonergic compounds potently inhibited 125I-LSD binding in cortical regions, olfactory tubercles, and hypothalamus but had no effect in the cerebellum. Dopaminergic compounds caused partial inhibition of binding in the striatum while adrenergic compounds were inactive. From these studies we conclude that 125I-LSD labels serotonin 5-HT2 receptor sites in cortical regions with no indication that other receptor sites are labeled. In the olfactory tubercles and hypothalamus, 125I-LSD labeling occurs predominantly or entirely at serotonin 5-HT2 sites. In the striatum, 125I-LSD labels approximately equal proportions of serotonergic and dopaminergic sites. This data indicates that 125I-LSD labels serotonin receptors in vivo and suggests that appropriate derivatives of 2I-LSD may prove useful for tomographic imaging of serotonin 5-HT2 receptors in the mammalian cortex.     

Proliferation of thyrotropin releasing hormone receptors in specific brain regions during the development of hypertension in spontaneously hypertensive rats

The binding of [3H] [3-MeHis2] thyrotropin releasing hormone [( 3H]MeTRH) to brain membranes prepared from 8 week old spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats was determined. [3H]MeTRH bound specifically to rat brain membranes at a single high affinity site. The density (Bmax value) of [3H]MeTRH binding sites was significantly greater (28%) in SHR rats compared to WKY rats. The apparent dissociation constants (Kd values) for the binding of [3H]MeTRH in SHR and WKY rats did not differ. Binding in the various brain regions revealed that the density of [3H]MeTRH was highest in the hypothalamus followed in decreasing order by pons + medulla, midbrain, cortex and striatum. The binding of [3H]MeTRH was approximately 25% greater in cortex, hypothalamus and striatum of SHR rats in comparison to WKY rats. The binding in pons + medulla, midbrain and pituitary of SHR and WKY rats did not differ. To assess the significance of increased binding sites for [3H]MeTRH in some brain regions of SHR rats, the binding studies were carried out during normotensive and hypertensive stages of postnatal age in the two strains. In 3 and 4 week old SHR rats there was neither an increase in blood pressure nor any increase in [3H]MeTRH binding in the hypothalamus and striatum as compared to age matched WKY rats. With the development of elevated blood pressure at 6 weeks, an increase in [3H]MeTRH binding in the hypothalamus and striatum of SHR rats in comparison to the tissues from WKY rats was observed. The results provide, for the first time, evidence for a parallel increase in the density of brain TRH receptors with elevation of blood pressure, and suggest that brain TRH receptors may play an important role in the pathophysiology of hypertension.     

Opiate antagonist binding sites in discrete brain regions of spontaneously hypertensive and normotensive Wistar-Kyoto rats

The binding of 3H-naltrexone, an opiate receptor antagonist, to membranes of discrete brain regions and spinal cord of 10 week old spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats was determined. The brain regions examined were hypothalamus, amygdala, hippocampus, corpus striatum, pons and medulla, midbrain and cortex. 3H-Naltrexone bound to membranes of brain regions and spinal cord at a single high affinity site with an apparent dissociation constant value of 3 nM. The highest density of 3H-naltrexone binding sites were in hippocampus and lowest in the cerebral cortex. The receptor density (Bmax value) and apparent dissociation constant (Kd value) values of 3H-naltrexone to bind to opiate receptors on the membranes of amygdala, hippocampus, corpus striatum, pons and medulla, midbrain, cortex and spinal cord of WKY and SHR rats did not differ. The Bmax value of 3H-naltrexone binding to membranes of hypothalamus of SHR rats was 518% higher than WKY rats but the Kd values in the two strains did not differ. It is concluded that SHR rats have higher density of opiate receptors labeled with 3H-naltrexone in the hypothalamus only, in comparison with WKY rats, and that such a difference in the density of opiate receptors may be related to the elevated blood pressure in SHR rats.     

Diurnal Variations of Striatal D2 Dopaminergic Receptors and its Relation with Haloperidol-induced Catalepsy

In spite of the clear evidences for the blockade of dopaminergic D2 receptors as the mechanism of action for haloperidol-induced catalepsy, the contribution of pharmacokinetic and pharmacodynamic aspects on the diurnal modulation of haloperidol-induced catalepsy is controversial. We studied the diurnal variations of striatal dopamine receptors and its relation with catalepsy diurnal variations. The [ 3 H]-spiperone binding to dopamine receptors had a clear rhythm with a peak at 00:00 to 03:00 h, and a trough at 12:00 to 18:00 h. Haloperidol-produced catalepsy measured with the four-cork test, also showed a clear rhythm, with a peak at 00:00 h and trough at 9:00 h. The dose-response curves at peak and trough of catalepsy had the same ED 50 (0.12 mg), with time-related changes in the maximal effect. Similar diurnal variations between catalepsy and dopamine receptor binding, indicate a relevant role of temporal pharmacodynamics of haloperidol on the modulation of its behavioral effects.     

Diurnal Variations of Striatal D2 Dopaminergic Receptors and its Relation with Haloperidol-induced Catalepsy

In spite of the clear evidences for the blockade of dopaminergic D2 receptors as the mechanism of action for haloperidol-induced catalepsy, the contribution of pharmacokinetic and pharmacodynamic aspects on the diurnal modulation of haloperidol-induced catalepsy is controversial. We studied the diurnal variations of striatal dopamine receptors and its relation with catalepsy diurnal variations. The [3 H]-spiperone binding to dopamine receptors had a clear rhythm with a peak at 00:00 to 03:00 h, and a trough at 12:00 to 18:00 h. Haloperidol-produced catalepsy measured with the four-cork test, also showed a clear rhythm, with a peak at 00:00 h and trough at 9:00 h. The dose-response curves at peak and trough of catalepsy had the same ED 50 (0.12 mg), with time-related changes in the maximal effect. Similar diurnal variations between catalepsy and dopamine receptor binding, indicate a relevant role of temporal pharmacodynamics of haloperidol on the modulation of its behavioral effects.     

Daily changes of GABA and taurine concentrations in various hypothalamic areas are affected by chronic hyperprolactinemia

This study was designed to characterize, in anterior, mediobasal, and posterior hypothalamic and median eminence, the 24h changes of gamma aminobutyric acid (GABA) and taurine (TAU) contents in adult male rats and to analyze whether chronic hyperprolactinemia may affect these patterns. Rats were turned hyperprolactinemic by a pituitary graft. Plasma prolactin (PRL) levels increased after pituitary grafting at all time points examined. A disruption of the circadian rhythm was observed in pituitary-grafted rats, whereas GABA and TAU content followed daily rhythms in all areas studied in controls. In the mediobasal hypothalamus, two peaks for each amino acid were found at midnight and midday. In the anterior hypothalamus, GABA and TAU showed only one peak of concentration at midnight. In the posterior hypothalamus, the values of both GABA and TAU were higher during the light as compared to the dark phase of the photoperiod. In the median eminence GABA content peaked at 20:00h, the time when TAU exhibited the lowest values. Hyperprolactinemia abolished the 24h changes of GABA in the mediobasal hypothalamus and reduced its content as compared to controls. Hyperprolactinemia advanced the diurnal peak of TAU to 12:00h in the mediobasal hypothalamus and did not modify the 24:00h peak. In the anterior hypothalamus, hyperprolactinemia increased GABA and TAU contents during the light phase while it decreased them during the dark phase of the photoperiod. In the posterior hypothalamus hyperprolactinemia did not modify GABA or TAU patterns as compared to controls. In the median eminence hyperprolactinemia increased the 20:00h peak of GABA and shift advanced the decrease in TAU content at 20:00h and its maximum at 24:00h as compared to controls. These data show that GABA and TAU content exhibit specific daily patterns in each hypothalamic region studied. PRL differentially affects the daily pattern of these amino acids in each hypothalamic region analyzed.     

Daily Changes of GABA and Taurine Concentrations in Various Hypothalamic Areas Are Affected by Chronic Hyperprolactinemia

This study was designed to characterize, in anterior, mediobasal, and posterior hypothalamic and median eminence, the 24h changes of gamma aminobutyric acid (GABA) and taurine (TAU) contents in adult male rats and to analyze whether chronic hyperprolactinemia may affect these patterns. Rats were turned hyperprolactinemic by a pituitary graft. Plasma prolactin (PRL) levels increased after pituitary grafting at all time points examined. A disruption of the circadian rhythm was observed in pituitary-grafted rats, whereas GABA and TAU content followed daily rhythms in all areas studied in controls. In the mediobasal hypothalamus, two peaks for each amino acid were found at midnight and midday. In the anterior hypothalamus, GABA and TAU showed only one peak of concentration at midnight. In the posterior hypothalamus, the values of both GABA and TAU were higher during the light as compared to the dark phase of the photoperiod. In the median eminence GABA content peaked at 20:00h, the time when TAU exhibited the lowest values. Hyperprolactinemia abolished the 24h changes of GABA in the mediobasal hypothalamus and reduced its content as compared to controls. Hyperprolactinemia advanced the diurnal peak of TAU to 12:00h in the mediobasal hypothalamus and did not modify the 24:00h peak. In the anterior hypothalamus, hyperprolactinemia increased GABA and TAU contents during the light phase while it decreased them during the dark phase of the photoperiod. In the posterior hypothalamus hyperprolactinemia did not modify GABA or TAU patterns as compared to controls. In the median eminence hyperprolactinemia increased the 20:00h peak of GABA and shift advanced the decrease in TAU content at 20:00h and its maximum at 24:00h as compared to controls. These data show that GABA and TAU content exhibit specific daily patterns in each hypothalamic region studied. PRL differentially affects the daily pattern of these amino acids in each hypothalamic region analyzed.     

Day/night variations in membrane bound leucyl-2-naphthylamide hydrolysing activity in rat anterior hypothalamus, pituitary and retina

It is well known that the suprachiasmatic nucleus of the anterior hypothalamus acts as pacemaker regulating circadian rhythms in mammals. The daily variations of neuropeptides as well as their receptors depend on this system (Moore 1983). Aminopeptidases play an important role in regulating the activity of brain peptides (Bauer 1982). In this study we investigated membrane bound leucyl-2-naphthylamide hydrolysing activity in the anterior hypothalamus, pituitary and retina of adult male rats at six time points of a 12:12h light:dark schedule (light from 7:00 to 19:00 h), in order to analyse its day/night variation. The fluorometric assay evidenced significant differences between the three regions: in the anterior hypothalamus being higher during the dark period compared with the light period and in the pituitary higher during the light period compared with the dark period. In the retina the levels of this activity showed a higher heterogeneity during the day. Day - night differences in membrane bound leucyl-2-naphthylamide hydrolysing activity may reflect differences in its susceptible endogenous substrates.     

Apparent binding activity of [H]glutathione in rat central and peripheral tissues

An apparent binding activity of [³H]glutathione (GSH) was detected in the synaptic membranous preparations obtained from the rat brain. Both methionine- and leucine-enkephalins exhibited a profound diminution of the apparent binding at 100 μM in a naloxone-insensitive fashion. The retina was found to have the highest binding activity amongst various central structures examined, followed by the hypothalamus, striatum, spinal cord, midbrain, hippocampus, medulla-pons, cerebellum and cerebral cortex. In peripheral organs employed, the pituitary possessed an apparent binding activity higher than that in the retina, with progressively lower activities in the adrenal, liver, spleen, skeletal muscle and heart. No significant activity was detected in the kidney. These results suggest that specific binding sites of GSH may be located in the central and peripheral excitable tissues.     

Distribution of kappa opioid receptors in the brain of young and old male rats

The experiments to be described have been designed in order to: (a) provide new information on the concentrations of opioid kappa receptors in different regions of the brain of the male rats; and (b) to analyze whether the density of brain kappa receptors might be modified by the process of aging. The concentration of kappa receptors was investigated in the hypothalamus, amygdala, mesencephalon, corpus striatum, hippocampus, thalamus, frontal poles, anterior and posterior cortex collected from male rats of 2 and 19 months of age. 3H-bremazocine (BRZ) was used as the ligand of kappa receptors, after protection of mu and delta receptors respectively with dihydromorphine and d-ala-d-leu-enkephalin. The results obtained show that: (1) in young male rats, the number of kappa opioid receptors is different in the various brain areas examined: the hypothalamus and the striatum have a concentration of kappa binding sites which is significantly higher than that found in the mesencephalon and in the amygdala; much lower concentrations of kappa binding sites have been found in the thalamus, the frontal poles, the hippocampus, the anterior and posterior cerebral cortex. (2) Aging exerts little influence on the number of kappa receptors in the majority of the brain structures considered. However in the amygdala and in the thalamus the number of kappa receptors was increased in old animals. To the authors' knowledge, the data here presented are the first ones which suggest that age may increase rather than decrease the number of neurotransmitter receptors in the brain.     

Comparison of melatonin-binding sites in the brain of two amphibians: an autoradiographic study

Neuroanatomic comparison of the binding capability of 2-[¹²⁵I] iodomelatonin in the crested newt Triturus carnifex Laur. and the green frog Rana esculenta, using quantitative autoradiographic techniques, revealed a heterogeneous distribution pattern. The highest and relatively high binding activities were shown to occur in the optic tracts and in the suprachiasmatic area of the hypothalamus and the optic tectum, respectively, of both species. Low or no 2-[¹²⁵I] iodomelatonin binding values were obtained in the preoptic nucleus, the tuberal hypothalamus, the medulla oblongata, the septum and the dorsal pallium. A differential binding pattern was observed in the amygdaloid nucleus pars lateralis, the striatum and the hindbrain of these amphibians. Indeed, notably high binding levels were shown to occur in the former two brain areas of the crested newt, whereas high levels were displayed in the latter brain region of the green frog. On the basis of elevated quantities of melatonin receptors in mesencephalic, hypothalamic and telencephalic sites, it seems plausible to ascribe some important sensory functions to this receptor system in both species. The remarkably different binding activities in the brain of the two amphibians could be correlated with the simpler cytoarchitectonic brain structure of urodeles and with species-specific variations.     

Characterization and Regulation of Insulin Receptors in Rat Brain

An in vitro receptor binding assay, using filtration to separate bound from free [125I]insulin, was developed and used to characterize insulin receptors on membranes isolated from specific areas of rat brain. The kinetic and equilibrium binding properties of central receptors were similar to those of hepatic receptors. The binding profiles in all tissues were complex and were consistent with binding in multiple steps or to multiple sites. Similar binding properties were found among receptors in olfactory tubercle/bulb, cerebral cortex, hippocampus, striatum, hypothalamus, and cerebellum. High affinity [125I]insulin binding sites (KD = 3-11 nM) were distributed evenly between membranes isolated from P1 and P2 fractions of these brain areas, with the exception of the olfactory tubercle in which binding to P2 membranes was four-fold greater (Bmax = 150 fmol/mg protein). One difference between insulin receptors in brain and peripheral target tissues, however, was observed. Following exposure to 0.17 microM insulin for 3 h at 37 degrees C, the number of specific [125I]insulin binding sites on adipocytes decreased by 40%, while the number of binding sites on minces of cerebral cortex/olfactory tubercle remained constant. The results suggest that although the binding characteristics of central and peripheral insulin receptors are similar, these receptors do not appear to be regulated in the same manner.     

An attempt to correlate brain areas containing melatonin-binding sites with rhythmic functions: a study in five hibernator species

High affinity melatonin-binding sites have been described, by means of autoradiography with 2-¹²⁵I-melatonin as the ligand, in more than 60 brain areas of about 20 mammalian species, with dramatic variations in the nature and number of labelled structures among the different species studied. As melatonin is involved in the synchronization of biological rhythms, we have tried to correlate the brain areas containing melatonin-binding sites with some rhythmic functions typical of give species. Therefore, we have studied the location of melatonin-binding sites in the complete brain of five long-day breeders with hibernation cycles, viz. one insectivore and four rodents. With the exception of the suprachiasmatic nuclei and the pars tuberalis of the pituitary, both of which contain binding sites in all five species, few reactive structures are common, even among species from the same family, e.g. the edible dormouse and the garden dormouse.