Distribution of neurotensin binding sites in the caudal brainstem of the rat: a light microscopic radioautographic study

Specific high-affinity neurotensin binding sites were labeled in sections of the rat caudal brainstem using a monoiodinated ligand, and their distribution was examined by light microscopic radioautography after fixation with glutaraldehyde. In the medulla, labeled binding sites were mainly concentrated within the dorsal motor nucleus of the vagus, the nucleus of the solitary tract, the external cuneate nucleus, the lateral reticular nucleus, the medial vestibular nucleus, the retrofacial nucleus, the linearis nucleus, the paragigantocellular nucleus and the nucleus raphe pallidus. Within the pons, neurotensin binding sites were detected in the reticulotegmental nucleus, the pontine nuclei, the dorsal tegmental nucleus, the laterodorsal and pedunculopontine tegmental nuclei and the nuclei raphe dorsalis and medianus. Most nuclei found here to contain high densities of neurotensin binding sites have been shown to stain intensely for acetylcholinesterase, suggesting a possible association between this enzyme and neurotensin receptors.     

Distribution of [125I]angiotensin II binding sites in the rat brain: a quantitative autoradiographic study

Angiotensin II receptors have been localized by quantitative autoradiography in the rat central nervous system after labeling with [125I]angiotensin II. A highly discrete distribution of these receptors was found throughout the rat brain. The highest density was seen in regions of the medulla, hypothalamus and circumventricular organs where angiotensin II could potentially produce cardiovascular, dipsogenic and neuroendocrine responses. The distribution of angiotensin II receptors correlates relatively well with the previously reported distribution of angiotensin immunoreactive nerve terminals as well as areas determined by various physiological techniques to be sensitive to angiotensin II. Finally, the anatomical localization of angiotensin II receptor populations has revealed several areas of the brain where the effects of this peptide have not been investigated. Many of these nuclei are involved in the transmission and processing of somatic and visceral sensory information. These results suggest a broader role for the central renin-angiotensin system in modulating several types of sensory input.     

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.     

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 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.     

Light microscopic autoradiographic localization of [3H]arginine-vasopressin binding sites in the rat brain and kidney

Specific binding sites for arginine-vasopressin (VP) were demonstrated in various major target areas of VP in the rat brain and kidney by light microscopic autoradiography. In the kidney moderate and intense labelling was found in the cortical and medullar areas, respectively. Within the brain intense labelling was shown in the lateral septum, which lends further support to the hypothesis that VP acts as a neurotransmitter. In the hypophysis moderate and heavy labelling was found in the anterior and neural lobes, respectively, which is in agreement with the idea that VP influences hypophyseal functioning.     

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.     

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.     

Autoradiographic mapping of [mono[125I]iodo-Tyr10, MetO17]vasoactive intestinal peptide binding sites in the rat brain

The distribution of vasoactive intestinal peptide binding sites in the rat brain was examined by in vitro autoradiography on slide-mounted sections. A fully characterized monoiodinated form of vasoactive intestinal peptide (M-[125I]VIP) previously shown to maintain in the central nervous system the full biological activity of native vasoactive intestinal peptide was used for this study. In initial kinetic and pharmacological experiments the binding of M-[125I]vasoactive intestinal peptide to slide-mounted sections was shown to be time-dependent, saturable and reversible. Association of M-[125I]VIP specific binding was maximal within 90-120 minutes. Specific binding, corresponding to approximately 50% of total binding was saturable, of high affinity (Kd of 76.6 pM) and low capacity (fmol/mg prot range). Dissociation of M-[125I]VIP was maximal at 10 minutes. Unlabeled vasoactive intestinal peptide and the two structurally related peptides "peptide-histidine-isoleucine" (PHI) and secretin competed in a concentration-dependent manner for sites labeled by M-[125I]vasoactive intestinal peptide with the following rank order of potencies: vasoactive intestinal peptide greater than PHI greater than secretin. Vasoactive intestinal peptide receptors, as revealed by quantitative autoradiography, are present at various levels of the neuraxis. High densities were observed in olfactory bulb, cerebral cortex (highest in layers I, II, IV and VI), dentate gyrus, subiculum, various thalamic and hypothalamic nuclei, superior colliculus, locus coeruleus, area postrema, subependymal layer and pineal gland. Intermediate densities were found in the amygdala, nucleus accumbens, caudate-putamen, septum, bed nucleus of the stria terminalis, CA1 to CA4 fields of the hippocampus and central gray. No specific binding of M-[125I]vasoactive intestinal peptide was observed in white matter tracts such as corpus callosum, anterior commissure, medial forebrain bundle and fornix. The mapping of M-[125I]vasoactive intestinal peptide binding sites as revealed by autoradiography on slide-mounted sections indicates an association, although not exclusive, of vasoactive intestinal peptide receptors with brain regions involved in the processing of specific sensory inputs.     

Autoradiographic localization of [3H]buspirone binding sites in rat brain

In the C-neurons of rabbit nodose ganglion there is a persistent slow outward current at Vm levels positive to -80 mV. This current was detectable in Na+-free Ringer and disappeared in Ca2+-free medium. Therefore it may be the Ca2+-activated K+ current. This K+ current shows a unique time and voltage dependency, suggesting that it may have a regulatory role on the excitability of C-neurons. Two other types of current also observed in C-neurons were IQ- and IA-like currents. In A-neurons, however, a Ca2+-activated K+ current was not observed at all.     

Stability of [D-Trp11]-neurotensin to rat brain peptidases

The stability of neurotensin (NT) and a potent, long lasting analogue, [D-Trp11]-NT, to rat brain peptidases was compared by incubating the peptides with subcellular fractions (synaptosomes, synaptic membranes) and a purified endopeptidase from rat brain. Degradation of the peptides with time was followed by high performance liquid chromatography (HPLC). The rates of degradation (pmol/min/mg prot.) in synaptosomes were 890 (NT) and 59 [D-Trp11]-NT), and in synaptic membranes were 1180 (NT) and 12 ([D-Trp11]-NT). The main products of the degradation of [D-Trp11]-NT by synaptic peptidases (isolated by HPLC and characterized by amino acid analysis) were the 1-3, 1-4 and 6-13 fragments implying cleavage of [D-Trp11]-NT at the Tyr3-Glu4, Glu4-Asn5 and Asn5-Lys6 bonds. The rates of degradation of NT and [D-Trp11]-NT by the purified endopeptidase from rat brain were 27.2 and 0.76 pmol/min/microliter of enzyme solution respectively. This endopeptidase, which hydrolyses NT at Arg8-Arg9, may be responsible along with other endopeptidases for NT degradation at nerve terminals.     

The localization and distribution of high affinity beta-nerve growth factor binding sites in the central nervous system of the adult rat. A light microscopic autoradiographic study using [125I]beta-nerve growth factor

Although beta-nerve growth factor is primarily known for its trophic role in the peripheral nervous system, recent reports have also revealed an inductive effect of beta-nerve growth factor on the cholinergic metabolism of the forebrain. To learn more about the significance and location of beta-nerve growth factor action in the central nervous system, the distribution of [125I]beta-nerve growth factor binding sites was studied by using the method of in situ receptor autoradiography and compared with the distribution of acetylcholinesterase, a sensitive enzyme marker of cholinergic neurons. The autoradiographic studies demonstrated strong, specific and saturable [125I]beta-nerve growth factor binding to several neuronal groupings in the forebrain and brainstem. beta-Nerve growth factor binding sites and strong acetylcholinesterase reactivity were jointly distributed in the forebrain on the medial septal nucleus, the diagonal band of Broca, the magnocellular basal nucleus and in the striatum. In the brainstem, beta-nerve growth factor binding sites were located on a number of neuronal groups in the reticular formation, the dorsolateral lemniscus and the cochlear nuclei. In contrast to the forebrain, less correlation was found with the distribution of acetylcholinesterase; no beta-nerve growth factor receptor expression was recorded on the cholinergic motor nuclei of the brainstem, while specific [125I]beta-nerve growth factor labeling could be located on the non-cholinergic cochlear nuclei. The present autoradiographic studies reveal a variety of tentatively beta-nerve growth factor receptor-positive neurons in the central nervous system. While strong correlation between the cholinergic metabolism and the presence of specific beta-nerve growth factor binding is demonstrated in the forebrain, this observation could not be extended to the brainstem, indicating the chemical diversity of central beta-nerve growth factor receptor-positive neurons.     

Quantitative autoradiography of sodium-dependent [3H]D-aspartate binding sites in rat brain

In vitro autoradiography was employed to localize and quantify Na-dependent [³H]d-aspartate binding sites in rat brain. LKB autoradiograms revealed a 15-fold variation in the concentration of [³H]d-aspartate binding sites among 40 brain regions. These results indicate that high-affinity uptakes sites for aspartic and/or glutamic acid are present ubiquitously, though not uniformlly, throughout the rat brain.     

Distribution of [125I]omega-conotoxin GVIA and [3H]isradipine binding sites in the central nervous system of rats of different ages

Potential age-related changes in L- and N-type voltage-sensitive calcium channels (L- and N-VSCCs) were assessed by the in vitro binding of [3H]isradipine ([3H]ISR, 150 pM) and [125]omega-conotoxin GVIA ([125I]omega-CT, 4 pM) to membranes prepared from discrete central nervous system regions of 0.5-, 2-, and 18-month-old rats. The rank orders of [3H]ISR and [125I]omega-CT binding, although differing, indicated that the highest binding was in neocortex, corpus striatum, and hippocampus; radioligand binding was generally not affected by the variable of age. These results suggest that the nonidentical [3H]ISR and [125I]omega-CT binding sites are concentrated in those regions characterized by high densities of synaptic connections, and that these sites, as presumed components of L- and N-VSCCs, are relatively stable during the aging process.     

Quantitative autoradiographic localization of alpha 2-antagonist binding sites in rat brain using [3H]idazoxan

The distribution of alpha 2-receptors was determined by quantitative autoradiography with the antagonist [3H]idazoxan. Apart from regions which are known to be enriched in alpha 2-receptors, the highest silver grain densities were located over the subfornical organ and the area postrema. However, binding in these regions was not displaced by yohimbine and therefore, according to the present understanding, cannot be considered to represent alpha 2-receptors. Within the cerebellum, alpha 2-receptors were found to be arranged in 3 sagitally oriented strips within the molecular layer of lobules 9 and 10, suggesting a co-incidence with dopamine and substance P receptors in this structure.     

Quantitative light microscopic autoradiographic localization of binding sites labelled with [3H]vasopressin antagonist d(CH2)5Tyr(Me)VP in the rat brain, pituitary and kidney

Binding sites for the vasopressin (VP) antagonist d(CH2)5Tyr(Me)VP, were located in various brain areas (e.g. the lateral septum, amygdala, choroid plexus and nucleus of the solitary tract) using light microscopic autoradiography. A number of areas (e.g. suprachiasmatic and arcuate nucleus, pineal gland) which previously showed no VP binding were labelled in the present study. The olfactory nucleus and ventromedial hypothalamic nucleus were not labelled. It therefore appears that d(CH2)5Tyr(Me)VP is capable of discriminating between VP and oxytocin binding sites and a more sensitive means of detecting VP binding sites than VP alone.     

Distribution of nicotinic cholinergic receptors in the rat tel- and diencephalon: a quantitative receptor autoradiographical study using [3H]-acetylcholine, [alpha-125I]bungarotoxin and [3H]nicotine

The topographical distribution of [alpha-125I]bungarotoxin [125I]BTX, [3H]nicotine ([3H]Nic), [3H]acetylcholine ([3H]ACh) (in the presence of atropine) binding in rat tel- and diencephalon was investigated using a quantitative receptor autoradiographical technique. With the [3H]ACh and [3H]Nic radioligands, a strong labelling was observed in various thalamic nuclei, including the medial habenula, a moderate labelling in different areas of the cortex cerebri, the nucleus caudatus putamen, the nucleus accumbens and tuberculum olfactorium and a uniform weak labelling in the hypothalamus. When the binding data for [3H]Nic were plotted against binding data for [3H]ACh in various brain nuclei, a significant correlation was obtained. Considering [125I]BTX, the strongest labelling was observed in the lateral mammillary nucleus and the hilus gyrus dentatus of the hippocampal formation. A weak labelling occurred in areas such as the nucleus causatus putamen, the thalamus and the cerebral cortex. No significant correlation was therefore obtained between the degree of [125I]BTX binding in various brain nuclei and the degree of binding observed with [3H]Nic or [3H]ACh. The present results underline the view that the high-affinity [3H]Nic and [3H]ACh binding sites label the same cholinergic nicotinic receptor binding site, while [125I]BTX labels another subpopulation of nicotinic cholinergic receptors, predominantly found in discrete areas of the hypothalamus and the limbic cortex.     

Transport of [125I] IgG and [125I] ovalbumin between lymphocyte-like and macrophage-like rat spleen cells: chromatin binding

Spleen cells from rats were incubated for 10 min in Eagle's medium containing [125I] IgG or [125I] ovalbumin. Lymphocyte-like and macrophage-like cells were separated by centrifugation in Ficoll-metrizoate solution (d = 1.095). Control spleen cells were incubated in Eagle's medium without [125I] IgG or [125I] ovalbumin and both subpopulations were separated analogically. Lymphocyte-like cells labelled with [125I] IgG or [125I] ovalbumin were incubated for 10 min with control macrophage-like cells and labelled macrophage-like cells with control lymphocyte-like cells. After the incubation time all subpopulations were separated again in Ficoll-metrizoate solution. Radioactivity of [125I] IgG or [125I] ovalbumin was found in cytoplasmic fraction, sap protein fraction (nucleoplasm) and chromatin of control lymphocyte-like and macrophage-like cells. It may be concluded that transport of these potential antigenic compounds occurs directly in lymphocyte-like and macrophage-like cells as well as transport between these two subpopulations.     

Quantitative autoradiography of the rat brain vesicular monoamine transporter using the binding of [3H]dihydrotetrabenazine and 7-amino-8-[125I]iodoketanserin

The binding of [3H]dihydrotetrabenazine, a specific ligand of the monoamine transporter present on serotonin and catecholamine synaptic vesicles, was studied on rat brain sections. The characteristics of binding (Kd = 5.0 nM, k1 = 0.13 x 10(6) M-1 s-1; k-1 = 0.66 x 10(-3) s-1) were similar to those previously observed on tissue homogenates. The rostrocaudal topographical distribution of dihydrotetrabenazine binding sites was analysed by quantitative autoradiography. High labelling was observed in regions richly innervated by monoaminergic systems: dopamine in the striatum and olfactory tubercles, noradrenaline in the striatal fissure and in the paraventricular and dorsomedial hypothalamus and serotonin in the lateral septum, islands of Calleja and suprachiasmatic nucleus. Cell bodies were also labelled in the substantia nigra and ventral tegmental area (dopamine), in locus coeruleus (noradrenaline) and in raphe nucleus (serotonin). The pituitary gland (particularly the neural lobe) and the pineal gland were also labelled. Low labelling was observed in various areas of the cerebral cortex and in the cerebellum. Unilateral 6-hydroxydopamine lesion of the substantia nigra dramatically reduced [3H]dihydrotetrabenazine labelling in the ipsilateral striatum. Moreover, ketanserin has recently been shown to possess a nanomolar affinity for the vesicular monoamine transporter, and autoradiographic localization of brain monoaminergic synaptic vesicles was also obtained by means of the derivative 7-amino-8-[125I]iodoketanserin in the presence of 5-hydroxytryptamine2 and alpha 1 antagonists, although the non-specific labelling was higher than with [3H]dihydrotetrabenazine. It is concluded that [3H]dihydrotetrabenazine may represent a valuable monoaminergic marker in in vitro autoradiographic studies.