Distribution of neurotensin binding sites in rat brain: a light microscopic radioautographic study using monoiodo [125I]Tyr3-neurotensin

The topographic distribution of specifically labeled neurotensin binding sites was examined by light microscopic radioautography in rat brain sections incubated with monoiodo [125I]Tyr3-neurotensin. Preliminary experiments indicated that under the present experimental conditions [125I]neurotensin specifically binds to a single apparent population of sites with a dissociation constant of 7.7 +/- 0.3 nM, and that fixation of the labeled sections with glutaraldehyde ensures regionally proportional retention of more than 70% of bound [125I]neurotensin molecules. High concentrations of [125I]neurotensin binding sites were detected in the olfactory bulb and tubercle, parts of the neocortex, the lateral septum, the diagonal band of Broca, the caudate putamen, the amygdala, the dentate gyrus, the anterior dorsal nucleus of the thalamus, the suprachiasmatic nucleus of the hypothalamus, the medial habenula, the zona incerta, the substantia nigra and the ventral tegmental area. In certain areas, such as in the diagonal band of Broca, the substantia innominata, the nucleus basalis and the pars compacta of the substantia nigra, discrete accumulations of silver grains were apparent over neuronal perikarya and their proximal dendrites. In most areas, however, the label appeared more or less uniformly distributed over nerve cell bodies and surrounding neuropil. In several instances, the labeling conformed with the distribution of cell bodies of origin and terminal aborizations of specific projection systems, suggesting that neurotensin receptors might be distributed both proximally and distally on the plasma membrane of certain neurons. Such putative "neurotensinoceptive" projection systems might involve part of the mesostriatal, mesocortical and mesolimbic dopamine systems, as well as the raphe-prosencephalic serotonin system and the habenulo-interpeduncular and basal forebrain-cortical cholinergic systems. Finally, areas of dense [125I]neurotensin labeling often corresponded to zones previously shown to exhibit intense acetylcholinesterase staining, suggesting the existence of a possible link between the expression of neurotensin binding sites and that of acetylcholinesterase in certain neuronal populations.     

Binding of [3H]cimetidine to rat brain tissue

Binding of [³H]cimetidine to rat brain tissue was investigated, and a saturable binding with dissociation constant 0.22±0.05 M found. This binding is inhibited by a range of imidazole-derived histamine H₂-receptor antagonists, but not by a number of non-imidazole H₂-receptor antagonists. It is concluded that the [³H]cimetidine binding site in rat brain tissue that is labelled in these experiments is not the histamine H₂-receptor.     

Characterization of [3H]phenobarbital binding to rat brain membranes

The binding of [3H]phenobarbital to rat brain membranes was studied in order to determine its characteristics and specificity. The binding reaction was rapid and occurred at sites of low affinity. (Kd = 700 microM) and very high density (Bmax = 2.7 nmol/mg protein). It was unaffected by temperature changes from 0 degrees C to 95 degrees C and was maximal at pH 5. Detergents in low concentrations markedly decreased the binding, apparently without solubilizing the binding sites. It is concluded that the binding of [3H]phenobarbital is a rather non-specific interaction with the plasma membrane.     

Quantitative autoradiography of nicotinic [H]acetylcholine binding sites in rat brain

Quantitative autoradiography was used to localize nicotinic [³H]acetylcholine (ACh) binding sites in rat brain. High concentrations of nicotinic [³H]ACh binding sites were observed in the anterior and medial nuclei of the thalamus, the medial habenula and the superficial layer of the superior colliculus. Moderate levels of binding sites were observed in a variety of brain regions such as the frontoparietal cortex and the hippocampus. Low levels of nicotinic ACh sites occurred throughout the hypothalamus and the primary olfactory cortex.     

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.     

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.     

Autoradiographic localization of [H]-cyclic GMP binding sites in the rat brain

Both the atriopeptides and nitric oxide act in the nervous system by activating guanylyl cyclases to stimulate the production of cyclic GMP. Thus a key to understanding the roles of these messengers is to understand the functions of cyclic GMP in the nervous system. Three potential targets for cyclic GMP have been identified, phosphodiesterases, protein kinases and ion channels. In this study we describe a method using autoradiography to localize specific [³H]-cGMP binding sites in the brain. The specific binding of [³H]-cGMP to rat brain sections was saturable (B_max = 1.5 pmol/mg protein) and of high affinity (K_D = 164 nM). The pharmacological characteristics were consistent with binding to the cGMP-dependent protein kinase. Highest densities of binding were seen in the medial habenula, basal ganglia, locus ceruleus and nucleus of the solitary tract. The CA1 pyramidal cells of the hippocampus, the neocortex, thalamus and cerebellum were also labelled. This method should prove useful in studies of potential targets for cyclic GMP in the brain.     

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.     

Quantitative distribution of rat brain monoamine oxidase A by [14C]clorgyline autoradiography

The distribution of functionally active monoamine oxidase type A (MAO-A) was investigated by in vivo quantitative autoradiography using [¹⁴C]clorgyline in normal, conscious rat brain. [¹⁴C]clorgyline was synthesized by the methylation reaction of N-desmethylclorgyline using [¹⁴C]methyliodide. Sixty minutes after [¹⁴C]clorgyline administration (1.58 MBq/animal i.v.), the brains were removed and prepared for autoradiography by washing the brain sections with 5% trichloroacetic acid solution to remove the nonbinding free tracer. The amount of MAO-A was calculated from the regional acid-insoluble tissue radioactivity and the specific activity of the tracer. The highest amount of MAO-A (5.84 nmol/g tissue) was found in the locus coeruleus. The interpeduncular nucleus, habenular nucleus, fasciculus retroflexus, and solitary tract nucleus possessed over 1.6 nmol/g tissue of MAO-A. Among 23 regions of interest, the lowest amount of MAO-A (0.37 nmol/g tissue) was found in the globus pallidus. The findings of this study suggest that the pattern of MAO-A parallels both in neuroanatomical distribution and in density that of norepinephrine and serotonin innervation. The MAO-A concentration was, however, relatively low in the dopamine-related areas. This corresponded to the previous results obtained by histochemical analysis. In addition, among the white matter structures, a high amount of MAO-A was found specifically in the fasciculus retroflexus.     

Interaction of 4-aminopyridine with [3H]phencyclidine receptors in rat brain homogenates

The effect of 4-aminopyridine and its analogs on the specific binding of [3H]phencyclidine was investigated in rat brain homogenates. 4-Aminopyridine (4-AP) and 3,4-diaminopyridine displaced [3H]phencyclidine binding, while 3-aminopyridine was without effect. The concentrations of 4-AP required for inhibition of binding increased with increasing the ligand concentration, and the resultant Dixon plots indicated a competitive type of interaction. However, 4-AP also accelerated the dissociation rate of the ligand-receptor complex, suggesting that the effect of 4-AP on phencyclidine receptors in the brain might not be purely competitive.     

High-affinity binding of [3H]muscimol to subcellular particles of a neurone-enriched culture of embryonic rat brain.

High-affinity, Na+-independent binding of [3H]muscimol (KB approximately equal to 1.6 x 10(-8) M; Bmax approximately equal to 0.14 nmol/g pellet) occurred to a frozen-thawed particulate fraction of 74-h-old neurone-enriched cultures prepared from the cerebra of 12-13-day-old rat embryos. This finding provides evidence that GABA-receptors exist on cultured neurones which contain only a few synaptic connections.     

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.     

Proteolysis of endomorphin-1 by brain synaptic membrane-associated peptidases

The biotransformation of endomorphin-1 (EM-1) by brain synaptic membranes has been studied. Peptide fragments of EM-1 that were formed during incubation with membrane preparations in vitro were isolated by High-performance liquid chromatography (HPLC) and characterized using determination of amino acid composition. At pH 7.4, two degradation products, EM-1(1-3) and EM-1(1-2), were identified. EM-1 was degraded 77.5% at 30 min incubation with synaptic membranes. The time course of the experiments and the effect of carboxypeptidase inhibitor (CPI) demonstrated that the proteolysis reaction involves the participation of carboxypeptidase activity.     

Failure of omega-conotoxin to block L-channels associated with [3H]5-HT release in rat brain slices.

The effects of the mixed N- and L-type voltage-sensitive calcium channel (VSCC) antagonist, omega-conotoxin GVIA and the L-type VSCC agonist Bay K-8644 on calcium-dependent, potassium evoked release of [3H]5-hydroxtryptamine ([3H]5-HT) were investigated in slices of rat hippocampus. Bay K-8644 (1 microM) enhanced, whilst omega-conotoxin (10-30 nM) attenuated, but did not abolish, evoked release of [3H]5-HT. The facilitatory actions of Bay K-8644 on evoked release were unaffected by concentrations of omega conotoxin that significantly inhibited [3H]5-HT release. The experiments indicate that concentrations of omega-conotoxin which inhibit neurotransmitter release by blockade of N-type VSCC, may leave L-type calcium channel activity unaffected.     

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.