Retinal [125I]iodomelatonin binding sites in the tree shrew (Tupaiidae).

The characteristics of melatonin-binding sites labelled by [¹²⁵I]iodomelatonin in membrane preparations from the tree shrew retina were determined. Specific binding of [¹²⁵I]iodomelatonin to the membrane preparations of tree shrew retina was rapid, stable, saturable, and reversible. Among the indoles tested only 6-chloromelatonin, melatonin and N-acetylserotonin had significant affinities to the [¹²⁵I]iodomelatonin binding site. Scatchard analysis of the membrane preparations revealed a dissociation constant (K_d) of 51.0 ± 16 pM and total number of binding sites (B_max) of 1.97 ± 0.6 fmol/mg protein.     

Segmental, coronal and subcellular distribution of 2-[I]iodomelatonin binding sites in the chicken spinal cord

Radioreceptor and autoradiography studies using chicken spinal cords demonstrated that the binding density of 2-[¹²⁵I]iodomelationin ([¹²⁵I]MEL) was significantly higher in the lumbar segment and the specific binding of [¹²⁵I]MEL was localized in the gray matter. Subcellularly, different densities of binding sites were localized in the following order: nuclear > microsomal > mitochondrial > cytosolic. Localization of [¹²⁵I]MEL binding sites in the dorsal gray matter of the chicken spinal cord suggests that melatonin plays a role in regulating the spinal cord functions which may associate with the modulation of temperature and pain transmission and/or visceral and autonomic functions.     

Retinal [I]iodomelatonin binding sites in the tree shrew (Tupaiidae)

The characteristics of melatonin-binding sites labelled by [¹²⁵I]iodomelatonin in membrane preparations from the tree shrew retina were determined. Specific binding of [¹²⁵I]iodomelatonin to the membrane preparations of tree shrew retina was rapid, stable, saturable, and reversible. Among the indoles tested only 6-chloromelatonin, melatonin and N-acetylserotonin had significant affinities to the [¹²⁵I]iodomelatonin binding site. Scatchard analysis of the membrane preparations revealed a dissociation constant (K_d) of 51.0 ± 16 pM and total number of binding sites (B_max) of 1.97 ± 0.6 fmol/mg protein.     

The ontogeny of 2-[125I]iodomelatonin binding sites in chicken brain.

The characteristics of the binding sites for 2-[125I]iodomelatonin were studied in chicken brain membranes during development. Specific binding, defined using cold melatonin (1 microM), was detected as early as 8-day-old embryos. Scatchard analysis of saturation experiments showed that 2-[125I]iodomelatonin binds to a single class of site at all ages tested (8-day-old embryos to 3-month-old chicks). Binding affinity (Kd) did not change during development (18-31 pM), but the maximal number of binding sites (Bmax) increased until embryonic day 18, and then remained relatively constant until 30 days of age. A further increase in Bmax was seen at 3 months of age. Guanosine 5'-triphosphate (GTP, 1 mM) inhibited 2-[125I]iodomelatonin binding at all ages suggesting that the melatonin binding site is coupled to a guanine nucleotide binding protein at a very early stage of development. Competition experiments with a number of melatonin analogues indicated that the binding site detected in the brain at embryonic day 8 was pharmacologically identical to that observed 15 days after hatching.     

125I]iodomelatonin binding sites in spleens of birds and mammals.

The specific binding of [125I]iodomelatonin to duck spleen membrane preparations was studied in detail. These binding sites were stable, saturable, reversible and of high affinity. Scatchard analysis of the binding revealed a equilibrium binding constant (Kd) of 73.1 +/- 5.4 pM and a total number of binding sites (Bmax) of 3.64 +/- 1.38 fmol/mg protein. Studies on the relative binding capacities of [125I]iodomelatonin to the spleen in different species showed the following order: duck greater than chicken greater than guinea pig greater than pigeon greater than mouse. No binding site was detected in the rat spleen. The presence of [125I]iodomelatonin binding sites in the spleen of birds and mammals suggested a direct action of pineal melatonin on the immune system.     

Autoradiographic visualization of the binding sites for [125I]endothelin in rat and human brain

Putative receptors for endothelin were localised autoradiographically in frozen sections of rat and human brain using [125I]endothelin as a ligand. In the rat brain the highest densities were in the granular layer of the cerebellum, choroid plexus, hippocampus, and habenular nucleus. Similar brain high densities were found in the human cerebellum and hippocampus. The non-vascular pattern of distribution suggests that endothelin may have a function as a modulator of neuronal function in addition to its possible involvement in the regulation of vascular tone.     

Nicotinic receptor distribution in the human thalamus: autoradiographical localization of [H]nicotine and [I]α-bungarotoxin binding

The thalamus plays a major role in relaying and transforming information that is relayed to the cortex and in turn modulates cortical outputs. The reticular nucleus projects to the other thalamic nuclei, modulating and integrating their activity. The distribution of high affinity nicotine and α-bungarotoxin (αBTX) receptors in the human thalamus has been investigated by radioligand autoradiography in post mortem human tissue. [³H]nicotine binding in the human thalamus was high in most thalamic nuclei, especially in the lateral dorsal, the medial geniculate, lateral geniculate and anterior nuclei. The distribution of [¹²⁵I]αBTX binding was quite distinct from [³H]nicotine binding. [¹²⁵I]αBTX binding was generally lower (<0.26–11.62 fmol/mg protein compared with 6.68–36.17 fmol/mg protein for nicotine binding) and concentrated in the reticular nucleus, with discrete groups of cells displaying higher binding in the latter. These results indicate differences between the distribution of nicotinic receptors in humans and those previously reported in mice and monkeys. Changes in high affinity nicotine and αBTX receptors in the thalamus may contribute to symptoms observed in neuropathological conditions associated with disorders of perception and movement such as Dementia with Lewy Bodies, Alzheimer's Disease and Schizophrenia.     

The regional distribution of sulphonylurea binding sites in rat brain

Sulphonylureas such as glibenclamide, which are used in the treatment of Type-2 diabetes, are inhibitors of ATP-sensitive potassium channels. These channels link cellular metabolism to membrane electrical activity and it is likely that they are closely associated with glibenclamide binding sites. Quantitative autoradiography was used to localize high-affinity [3H]glibenclamide binding sites in coronal sections of rat brain. The relative density of binding sites was found to correlate well with the relative capacity of sites determined in homogenate assays. There was no evidence of any variation of affinity between brain regions. The highest levels of binding were found in the substantia nigra with high levels in the globus pallidus, cerebral cortex, hippocampus and caudate-putamen, intermediate levels in the cerebellum, and low levels in the hypothalamus and pons. The density of [3H]glibenclamide binding sites was low in glucose-responsive brain regions, known to contain ATP-sensitive potassium channels that are inhibited by sulphonylureas. However, higher densities were associated with brain regions (often limbic structures) active during temporal lobe epilepsy. In at least two of these structures, the CA3 region of the hippocampus and the substantia nigra, it is probable that these sites are coupled to ATP-sensitive potassium channels. These results are discussed with reference to the reported actions of ATP-sensitive potassium channels on CNS function.     

Characterization and regional distribution of strychnine-insensitive [3H]glycine binding sites in rat brain by quantitative receptor autoradiography

Recent evidence suggests that a strychnine-insensitive glycine modulatory site is associated with the N-methyl-D-aspartate receptor-channel complex. A quantitative autoradiographic method was used to characterize the pharmacological specificity and anatomical distribution of strychnine-insensitive [3H]glycine binding sites in rat brain. [3H]Glycine binding was specific, saturable, reversible, pH and temperature-sensitive and of high affinity. [3H]Glycine interacted with a single population of sites having a KD of approximately 200 nM and a maximum density of 6.2 pmol/mg protein (stratum radiatum, CA1). Binding exhibited a pharmacological profile similar to the physiologically defined strychnine-insensitive glycine modulatory site. Binding was stereoselective; the rank order of potency of simple amino acids as displacers of binding was: glycine greater than D-serine greater than D-alanine greater than L-serine greater than L-alanine greater than L-valine greater than D-valine. Binding was not altered by the inhibitory glycine receptor ligand, strychnine, by the glutamate agonists, quisqualate and kainate, or by GABA receptor selective ligands. Most competitive agonists or antagonists of the N-methyl-D-aspartate recognition site were ineffective displacers of glycine binding. The exceptions were the aminophosphono series of antagonists, D-alpha-aminoadipate, gamma-D-glutamyglycine and beta-D-aspartylaminomethylphosphonic acid. However, the inhibition of [3H]glycine binding produced by the aminophosphono compounds could be accounted for by the level of glycine contamination present in these compounds. The non-competitive NMDA receptor-channel blockers, phencyclidine, its thienyl derivative, and MK-801 did not alter glycine binding. Kynurenate, glycine methylester, L-serine-O-sulfate, L-homocysteic acid, and several glycine-containing dipeptides were effective displacers of glycine binding. Structure-activity relations of agonists and antagonists of the strychinine-insensitive glycine binding site are discussed. The distribution of strychnine-insensitive [3H]glycine binding was heterogeneous with the following rank order of binding densities: hippocampus greater than cerebral cortex greater than caudate-putamen greater than or equal to thalamus greater than cerebellum greater than brain stem. This distribution of binding was correlated with N-methyl-D-aspartate-sensitive [3H]glutamate binding (r2 = 0.77; P less than 0.001; Pearson product-moment) and [3H]thienylcyclohexylpiperidine binding (r2 = 0.72; P less than 0.001). These observations are consistent with the hypothesis that the strychnine-insensitive glycine binding site is closely associated with the N-methyl-D-aspartate receptor-channel complex.(ABSTRACT TRUNCATED AT 400 WORDS)     

Distribution and levels of [I]IGF-I, [I]IGF-II and [I]insulin receptor binding sites in the hippocampus of aged memory-unimpaired and -impaired rats

The insulin-like growth factors (IGF-I and IGF-II) and insulin are localized within distinct brain regions and their respective functions are mediated by specific membrane receptors. High densities of binding sites for these growth factors are discretely and differentially distributed throughout the brain, with prominent levels localized to the hippocampal formation. IGFs and insulin, in addition to their growth promoting actions, are considered to play important roles in the development and maintenance of normal cell functions throughout life. We compared the anatomical distribution and levels of IGF and insulin receptors in young (five month) and aged (25 month) memory-impaired and memory-unimpaired male Long–Evans rats as determined in the Morris water maze task in order to determine if alterations in IGF and insulin activity may be related to the emergence of cognitive deficits in the aged memory-impaired rat. In the hippocampus, [¹²⁵I]IGF-I receptors are concentrated primarily in the dentate gyrus (DG) and the CA3 sub-field while high amounts of [¹²⁵I]IGF-II binding sites are localized to the pyramidal cell layer, and the granular cell layer of the DG. [¹²⁵I]insulin binding sites are mostly found in the molecular layer of the DG and the CA1 sub-field. No significant differences were found in [¹²⁵I]IGF-I, [¹²⁵I]IGF-II or [¹²⁵I]insulin binding levels in any regions or laminae of the hippocampus of young vs aged rats, and deficits in cognitive performance did not relate to altered levels of these receptors in aged memory-impaired vs aged memory-unimpaired rats. Other regions, including various cortical areas, were also examined and failed to reveal any significant differences between the three groups studied.

It thus appears that IGF-I, IGF-II and insulin receptor sites are not markedly altered during the normal ageing process in the Long–Evans rat, in spite of significant learning deficits in a sub-group (memory-impaired) of aged animals. Hence, recently reported changes in IGF-I receptor messenger RNA levels in aged memory-impaired rats[42] are apparently not reflected at the level of the translated protein.     

Early developmental expression of leptin receptor gene and [125I]leptin binding in the rat forebrain

Leptin, via leptin receptors (Ob-R), regulates appetite and energy balance. Of the six isoforms of the receptor identified, so far, only the long form (Ob-Rb) can fully activate downstream signal transduction pathways. Although the expression and function of leptin receptors is well described in the adult brain, little is known about the ontogeny of leptin receptor system around the time of birth. In this study, the mRNA expression patterns of total leptin receptor, Ob-R, and the long signalling form of the receptor, Ob-Rb, were investigated in the brain of embryonic and newborn rats using in situ hybridisation and [125I]leptin binding. On embryonic day 18 (E18), Ob-R mRNA was detected in the choroid plexus and the ependymal layer of the third ventricle by in situ hybridisation. At E21, Ob-Rb mRNA was first observed in the arcuate and the ventral premammillary hypothalamic nuclei while at P3, receptor expression was also found in the dorsomedial nucleus. Other leptin target areas identified were the trigeminal ganglion, the thalamus and the hippocampus. Using quantitative receptor autoradiography specific [125I]leptin binding sites on the choroid plexus were found to increase with age in contrast to the ependymal layer of the third ventricle where levels decreased with age. Together these findings demonstrate that the leptin receptor system is differentially regulated during late gestation and early postnatal life in the rat.     

Autoradiographic localization of [125I]charybdotoxin binding sites in rat brain.

Charybdotoxin, a 37 amino acid peptide isolated from scorpion venom, is a potent inhibitor of potassium channel function. [125I]charybdotoxin was originally believed to be a selective ligand for the Ca(2+)-sensitive channel in many tissues, but it appears to bind only to a voltage-sensitive potassium channel in brain. We found high densities of [125I]charybdotoxin binding in lateral olfactory tract, interpeduncular nucleus and a variety of mesencephalic nuclei. Moderate levels were found in the cerebral cortex, medial thalamus, hypothalamus and selected thalamic nuclei. These results indicate that [125I]charybdotoxin identifies a potassium channel or channels with a unique distribution in the brain.     

Autoradiographic localization of [125I]-endothelin-1 binding sites in rat brain

Autoradiograms of [125I]-endothelin (ET) binding in the rat brain demonstrated that the receptors for endothelin are localized mainly in the brainstem, basal ganglia, and cerebellum. Among the many other nuclei in these regions, there also appeared nuclei which are considered to play important roles in the central nervous regulation of the cardiovascular system: they include the nuclei of the anteroventral third ventricle area, the supraoptic nucleus, and the subfornical organ, for example. From these findings, we suggest that ET-1 or its analogous peptide(s) may act as a neuropeptide regulating central nervous functions, including cardiovascular functions.     

Expanding the horizons of melatonin use: An immunohistochemical neuroanatomic distribution of MT1 and MT2 receptors in human brain and retina

Introduction

There is increasing evidence implicating the active role of melatonin beyond regulation of the human biological clock and reproduction. Its therapeutic use has been extended to neurodegenerative disorders, psychiatric disturbances, memory as well as a host of other neurological manifestations. This study was thus designed to identify regions of brain and retina for the expression of different types of melatonin receptors.

Autoradiographic distribution of non-dopaminergic binding sites labeled by [3H]haloperidol in rat brain

The regional distribution of binding sites labeled by [3H]haloperidol, in the presence of excess spiroperidol, was compared to the regional distribution of receptors labeled by [3H]SCH 23390 and [3H]sulpiride, [3H]SCH 23390 and [3H]sulpiride labeled distinct nuclei, such as the olfactory tubercle, caudate, globus pallidus, substantia nigra, and inferior and superior colliculi. In contrast, the distribution of binding sites labeled by [3H]haloperidol, in the presence of excess spiroperidol, were much more extensive. Some areas containing the highest density of sites labeled by [3H]haloperidol were the external plexiform layer of the olfactory bulb, the cerebral cortex, the paraventricular nuclei, the interpeduncular nucleus and the superior colliculus. The distribution of non-dopaminergic binding sites labeled by haloperidol was clearly quite different from that labeled by dopaminergic ligands.     

Specific [125I]Bolton-Hunter substance P binding sites in human and rat skin

[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.     

[I]Vasoactive intestinal polypeptide binding sites: Quantitative autoradiographic distribution in the rat spinal cord

The quantitative autoradiographic distribution of [¹²⁵I]vasoactive intestinal polypeptide (VIP) receptor binding sites was in vestigated in the rat spinal cord. [¹²⁵I]VIP binding sites are discretely distributed, with a rostro-caudal gradient, along the longitudinal length of the cord; highest densities of sites being observed in its lumbar and sacral segments. In transverse sections, highest levels of [¹²⁵I]VIP sites are present in laminae I and II, around the central canal, and in the parasympathetic lateral horn of the sacral segment. Moderate densities are seen along the medial border of the dorsal horn and the sympathetic lateral horn of the thoracic cord. Low amounts of labeling are observed in most structures of the ventral horn while white matter areas are apparently devoid of specific [¹²⁵I]VIP binding. Thus, the distribution of spinal [¹²⁵I]VIP receptor sites correlates well with that of VIP-like immunoreactive materials and support possible roles for this peptide in sensory neurotransmission and in the control of autonomic functions.     

Autoradiography of antidepressant binding sites in the human brain: localization using [3H]imipramine and [3H]paroxetine

[3H]Imipramine and [3H]paroxetine were used to label sites associated with serotonin uptake mechanisms in post-mortem brain tissue from control subjects. The anatomical localization of these sites was examined by autoradiography and densities measured by microdensitometry. We found [3H]imipramine binding to increase with age in the cortex and amygdala, but to be independent of gender and post-mortem delay. Preliminary results indicate that the binding of both [3H]imipramine and [3H]paroxetine is diminished in the brain of patients treated with imipramine. The distribution of [3H]imipramine and [3H]paroxetine high-affinity binding sites was very similar, and correlated well with the distribution of serotonergic presynaptic markers in the brain. The highest densities of binding sites were found in the raphé nuclei and the midline thalamic nuclei. Other structures presenting high levels of binding were the substantia nigra, nucleus interpeduncularis, locus coeruleus, nucleus nervi hypoglossi, nucleus nervi facialis, mammillary bodies and other parts of the hypothalamus. In contrast, regions such as the neocortex, hippocampus, amygdala and cerebellum showed low densities of [3H]imipramine and [3H]paroxetine binding sites. This distribution seems to indicate that the ascending serotonergic pathways are the main site of action of antidepressants.     

Systemic administration of kainic acid induces selective time dependent decrease in [I]insulin-like growth factor I, [I]insulin-like growth factor II and [I]insulin receptor binding sites in adult rat hippocampal formation

Administration of kainic acid evokes acute seizure in hippocampal pathways that results in a complex sequence of functional and structural alterations resembling human temporal lobe epilepsy. The structural alterations induced by kainic acid include selective loss of neurones in CA1–CA3 subfields and the hilar region of the dentate gyrus followed by sprouting and permanent reorganization of the synaptic connections of the mossy fibre pathways. Although the neuronal degeneration and process of reactive synaptogenesis have been extensively studied, at present little is known about means to prevent pathological conditions leading to kainate-induced cell death. In the present study, to address the role of insulin-like growth factors I and II, and insulin in neuronal survival as well as synaptic reorganization following kainate-induced seizure, the time course alterations of the corresponding receptors were evaluated. Additionally, using histological preparations, the temporal profile of neuronal degeneration and hypertrophy of resident astroglial cells were also studied. [¹²⁵I]Insulin-like growth factor I binding was found to be decreased transiently in almost all regions of the hippocampal formation at 12 h following treatment with kainic acid. The dentate hilar region however, exhibited protracted decreases in [¹²⁵I]insulin-like growth factor I receptor sites throughout (i.e. 30 days) the study. [¹²⁵I]Insulin-like growth factor II receptor binding sites in the hippocampal formation were found to be differentially altered following systemic administration of kainic acid. A significant decrease in [¹²⁵I]insulin-like growth factor II receptor sites was observed in CA1 subfield and the pyramidal cell layer of the Ammon's horn at all time points studied whereas the hilar region and the stratum radiatum did not exhibit alteration at any time. A kainate-induced decrease in [¹²⁵I]insulin receptor binding was noted at all time points in the molecular layer of the dentate gyrus whereas binding in CA1–CA3 subfields and discrete layers of the Ammon's horn was found to be affected only after 12 h of treatment.

These results, when analysed with reference to the observed histological changes and established neurotrophic/protective roles of insulin-like growth factors and insulin, suggest possible involvement of these growth factors in the cascade of neurotrophic events that is associated with the reorganization of the hippocampal formation observed following kainate-induced seizures.