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.     

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 evidence of [3H]neurotensin binding changes in discrete regions of brain in the rat model of persistent spasmodic dyskinesia induced by iminodipropionitrile

Chronic injection of iminodipropionitrile (IDPN) to rats causes a persistent set of abnormalities which includes hyperlocomotion, hyperexcitability, and dyskinetic movements of the neck. These behavioral changes are very similar to those observed after the acute administration of the dopamine (DA) agonist, amphetamine, in rodents. Because of the anatomical and functional evidence that neurotensin (NT) can modulate DA neurotransmission, the present receptor autoradiographic study investigated the binding of [3H]NT in the brains of IDPN-treated rats. There were significant decreases in binding in the frontal and cingulate cortices, the rhinal sulcus, the dorsolateral aspect of the caudate-putamen, and in the ventral tegmental area. These results provide the first evidence for the possible participation of the NT system in the manifestations of the IDPN-induced syndrome.     

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.     

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

A remarkable structural similarity exists between the parkinsonian neurotoxin, N-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP), and 2-[N]-methyl-tetrahydro-beta-carboline (2M-THBC), a tryptamine-derived alkaloid which can be biosynthesized in brain. To explore whether the beta-carboline also has neurotoxic effects, owl monkeys were treated daily with MPTP or 2M-THBC. Acute behavioral similarities were seen, but 2M-THBC did not induce persistent parkinsonism, nor did it cause apparent loss of nigral cells. However, 2M-THBC resembled MPTP in reducing 3,4-dihydroxyphenylacetic acid levels in caudate and in altering levels of serotonin and 5-hydroxyindoleacetic acid in substantia nigra. These limited similarities should be considered in the light of relationships between neurotoxicity, MPTP versus 2M-THBC oxidation, and the chronicity of human Parkinson's disease. We suggest that N-methylated beta-carboline species, possibly accumulating during stress and aging, could well be causative factors in parkinsonism.     

Quantitative autoradiography of [3H]norharman [( 3H]beta-carboline) binding sites in the rat brain

The anatomical distribution of [3H]norharman binding sites was determined by quantitative autoradiography in rat brain slices. They are enriched in hypothalamic, thalamic, accumbens and amygdaloid nuclei as well as in hippocampal, neocortical and olfactory-related structures. The distribution pattern differs from that of other previously described receptors or binding sites (e.g. monoamine oxidase, benzodiazepine, tryptamine, 5-hydroxytryptamine receptors (5-HT1A, 5-HT1B, 5-HT1C, 5HT2], which suggests that a unique class of [3H]norharman binding sites exists in the rat brain. The findings are consistent with previous experiments which showed high affinity binding sites for [3H]norharman in rat brain membranes (KD 1.552 nM; autoradiography KD 5.5 nM). A correspondence in the displacing activity of drugs was found for both methods (crude membrane fraction: harman much greater than tryptamine much greater than 5-hydroxytryptamine greater than N-methyl-beta-carboline-3-carboxamide (FG 7142) = diazepam; autoradiography: harman much greater than tryptamine much greater than FG 7142 greater than 5-hydroxytryptamine greater than diazepam). Provided that the binding sites represent functional receptors, the present anatomical findings may explain the biological effects of norharman, e. g. pro-conflict behaviour (limbic-hypothalamic structures), tonic-clonic convulsions (limbic-cortical structures) and alterations of locomotor activity (accumbens nucleus).     

Reduced number of [3H]nicotine and [3H]acetylcholine binding sites in the frontal cortex of Alzheimer brains

Nicotinic cholinergic receptors were measured in human frontal cortex using [3H]nicotine and [3H]acetylcholine (in the presence of atropine) as receptor ligands. A parallel marked reduction in number of [3H]nicotine (52%; P less than 0.01) and [3H]acetylcholine (-55%; P less than 0.05) binding was found in the frontal cortex of Alzheimer brains (AD/SDAT) when compared to age-matched control brains. As a comparison the number of muscarinic receptors was quantified using [3H]quinuclidinyl benzilate and found to be significantly increased (+23%; less than 0.01) in AD/SDAT compared to controls.     

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.     

Autoradiographic localization and pharmacology of unique [3H]tryptamine binding sites in rat brain

The distribution and pharmacological specificity of [3H]tryptamine binding to coronal and horizontal sections of the rat brain were investigated with computer-assisted autoradiography. [3H]Tryptamine bound to brain regions with up to 58% specificity, as determined with 10 microM tryptamine as a displacer. The capacity (Bmax) of saturable [3H]tryptamine binding sites was greatest in the nucleus accumbens and claustrum (660-760 fmol mg protein-1), with intermediate binding site concentrations in hippocampus, septum, olfactory tubercle, frontal cortex, cingulate cortex and caudate-putamen. The phenylalkylamine, p-methoxyphenylpropylamine and the beta-carboline, harmaline, as well as 5-methyl-tryptamine, displaced [3H]tryptamine from each of these brain regions with a potency that approximated the 5-9 nM affinity (Kd) of [3H]tryptamine binding to each site. Only micromolar concentrations of serotonin displaced [3H]tryptamine, which did not bind to S1, S2, D1, D2 or alpha- or beta-adrenergic sites. The unique pharmacology and the regional overlap of [3H]tryptamine binding sites with dopaminergic nerve terminals in the nucleus accumbens and caudate-putamen suggest that tryptamine-containing neurons in the mammalian brain may modulate behavioral functions such as locomotion.     

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.     

Septotemporal distribution of [3H]MK-801, [3H]AMPA and [3H]Kainate binding sites in the rat hippocampus

The distribution of glutamate receptors in transverse hippocampal sections has been well investigated. However, in spite of the known septotemporal gradients of hippocampal connectivity no systematic studies exist about the distribution of glutamate receptors along the septotemporal (longitudinal) hippocampal axis. Therefore, in the present study this issue was investigated using receptor autoradiography for the [³H]MK-801, [³H]AMPA and [³H]Kainate binding sites. Hippocampi from 30-day-old rats were sectioned perpendicularly to their longitudinal axis, yielding a total of 25–30 equidistantly spaced autoradiographs for each hippocampus. For each section layer-specific concentrations of binding sites were calculated by the aid of a computerized image analysing system. The dependency of concentrations of binding sites on the septotemporal position was evaluated by regression analysis. Gradients of binding were confined to distinct hippocampal layers. Significant septotemporal gradients of [³H]MK-801 binding were observed in selected layers of CA1 and the dentate gyrus, a septal to temporal decrease of binding in the oriens and radiatum layers of CA1 being most prominent. For [³H]AMPA, significant septotemporal gradients of binding were restricted to layers of CA3, CA4 and the dentate gyrus, with values generally increasing from septal to temporal levels. The observed septotemporal gradients possibly reflect functional segregations along the longitudinal hippocampal axis and could be important for the comparability of ligand binding studies using transverse hippocampal sections or hippocampal slice cultures.     

Autoradiographic localization in rat brain of kappa opiate binding sites labelled by [3H]bremazocine

[3H]Bremazocine, in the presence of saturating concentrations of mu and delta receptor blocking agents, was used to label putative kappa opiate binding sites in rat brain. The binding of [3H]bremazocine under these conditions was completely displaced with high affinity by U-50488H and dynorphin1-17, and the potency of a series of opiate ligands was consistent with an action at kappa receptors. Therefore, [3H]bremazocine, in the presence of mu and delta blockers, was used to localize U-50488H-displaceable kappa binding sites by autoradiography. A distribution different from that of mu and delta receptors was seen, with levels highest in the claustrum, striatum, medial preoptic area, suprachiasmatic nucleus, medial amygdala and superior layer of the superior colliculus. The results show that the U-50488H-displaceable kappa sites have a distinct distribution which is discussed in terms of the possible functional roles of kappa receptors.     

High- and low-affinity binding of [3H]acetylcholine at nicotinic cholinergic receptors in rat brain

There is both high-affinity and low-affinity nicotinic cholinergic binding of [3H]acetylcholine [( 3H]ACh) in rat brain membrane preparations. As determined by a filtration binding assay, [3H]ACh bound with Kd = 36.0 +/- 8.4 nM and Bmax = 19.4 +/- 4.5 fmol/mg protein or 3.3 +/- 0.7 fmol/mg tissue for high-affinity binding and Kd about 10(-7) to 10(-6) M and Bmax about 6-10 fmol/mg tissue or 40-60 fmol/mg protein for low-affinity binding. d-Tubocurarine (1 mM) inhibits high- as well as low-affinity binding, whereas 10 microM alpha-bungarotoxin does not compete at both binding sites. Substance P had no effect on the binding parameters of high-affinity nicotinic cholinergic binding.     

Status epilepticus alters zolpidem sensitivity of [3H]flunitrazepam binding in the developing rat brain

GABA, the main inhibitory neurotransmitter in the adult brain, exerts its effects through multiple GABA(A) receptor subtypes with different pharmacological profiles, the alpha subunit variant mainly determining the binding properties of benzodiazepine site on the receptor protein. In adult experimental epileptic animals and in humans with epilepsy, increased excitation, i.e. seizures, alters GABA(A) receptor subunit expression leading to changes in the receptor structure, function, and pharmacology. Whether this also occurs in the developing brain, in which GABA has a trophic, excitatory effect, is not known. We have now applied autoradiography to study properties of GABA(A)/benzodiazepine receptors in 9-day-old rats acutely (6 h) and sub-acutely (7 days) after kainic acid-induced status epilepticus by analyzing displacement of [(3)H]flunitrazepam binding by zolpidem, a ligand selective for the alpha1beta2gamma2 receptor subtype. Regional changes in the binding properties were further corroborated at the cellular level by immunocytochemistry. The results revealed that status epilepticus significantly decreased displacement of [(3)H]flunitrazepam binding by zolpidem 6 h after the kainic acid-treatment in the dentate gyrus of the hippocampus, parietal cortex, and thalamus, and in the hippocampal CA3 and CA1 cell layers 1 week after the treatment. Our results suggest that status epilepticus modifies region-specifically the pharmacological properties of GABA(A) receptors, and may thus disturb the normal, strictly developmentally-regulated maturation of zolpidem-sensitive GABA(A) receptors in the immature rat brain. A part of these changes could be due to alterations in the cell surface expression of receptor subtypes.     

Characterization of binding sites for spider toxin, [3H]NSTX-3, in the rat brain

A group of spider toxins (JSTX, NSTX, argiopin, argiotoxin etc.) share a basic common structure and have been reported to block strongly quisqualate- and kainate-sensitive glutamate responses in vertebrate and invertebrate nervous systems. They are presumed to be potent antagonists of both quisqualate and kainate receptors and may serve as useful tools for characterizing these receptors. We report here the synthesis of tritium-labeled NSTX-3 and the characterization of its binding sites in the rat brain. We found that high- and low-affinity binding sites exist in the cerebellum (Kd = 7.75 and 202 nM, Bmax = 0.37 and 5.54 pmol/mg protein, respectively). Synthetic NSTX analogs strongly inhibited [3H]NSTX-3 binding in the cerebellum (IC50 = 10(-7)-10(-6) M), whereas competitive agonists of glutamate receptors (AMPA, quisqualate, NMDA, kainate, glutamate and aspartate) exhibited weak or no inhibitory effects.     

Lanthanides stimulate [3H]tyramine binding in the rat striatum

Low (up to 100 nM) and high (approximately 100 microM) concentrations of lanthanides and Ca2+-ions, respectively, stimulated [3H]tyramine binding ([3H]TY) to rat striatal membranes, a putative marker for the vesicular transporter of dopamine. On the other hand, lanthanides (approximately 100 microM) inhibited or stimulated TY binding in striatal and extrastriatal (cortex, cerebellum) tissues, respectively. The binding increases by lanthanum (La3+) appeared to depend on endogenous Ca2+, whereas, those induced in EDTA-pretreated membranes were Ca2+-independent. The La3+-induced, apparent increase in the Bmax for [3H]TY binding seemed to reflect a retarded rate of dissociation of the ligand from its targets, rather than a larger availability of functionally-relevant, vesicular transport-related TY sites. This indicates uncertain mechanisms of present La3+ effects.     

Learned helplessness decreases [3H]imipramine binding in rat cortex

Specific binding of [3H]imipramine decreased in frontal neocortex from rats demonstrating learned helplessness, an animal model of depression. The decrease was in maximal binding but not in affinity for the receptor site. No change in [3H]imipramine binding was found in septum or hippocampus. The receptor changes found in frontal neocortex parallel behavioral and neurochemical changes produced by learned helplessness in this region. These changes are also similar to those found in the frontal neocortex from suicides and in platelets of patients with depression.     

In vivo binding of [3H]d-N-allylnormetazocine and [3H]haloperidol to sigma receptors in the mouse brain

Specific binding to sigma sites has been demonstrated and characterized in vitro using [3H]d-N-allylnormetazocine ([3H]d-NANM) and [3H]haloperidol ([3H]HAL) as ligands. As an extension of these experiments, we examined the regional in vivo specific binding of [3H]d-NANM and [3H]HAL in the mouse brain. Specific in vivo sigma binding was seen with both ligands; average estimates of specific binding across brain regions were 54 per cent and 56 per cent of total brain radioactivity, using [3H]d-NANM and [3H]HAL, respectively. Both ligands showed high levels of specific binding in the cerebellum, medulla-pons and midbrain, and lowest levels in the hippocampus. Estimated average [3H]d-NANM binding to phencyclidine (PCP) receptors across seven brain regions was only 13 per cent of total brain radioactivity, and showed a more uniform regional distribution than sigma binding. While the distributions of in vivo specific binding of [3H]d-NANM and [3H]HAL to sigma sites were comparable to findings obtained in vitro, the present estimates of in vivo [3H]d-NANM binding to PCP sites did not resemble the distribution of PCP receptors found in vitro. The results suggest that radiolabelled d-NANM and HAL may be useful for imaging sigma binding sites in vivo.