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.     

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.     

Quantitative autoradiographic characterization of l-[3H] glutamate binding sites in rat vestibular nuclei

Summary Quantitative autoradiography has been used to characterize l-[³H] glutamate binding sites and to describe their distribution in frozen sections of rat vestibular nuclei. Scatchard plots and Hill coefficients of glutamate binding suggest that glutamate interacts with a single population of sites having a K_D of about 126 nM and a capacity of 2.5 pmol/mg of protein. Although the level of glutamate binding was not very high compared to the highest levels described for some other brain regions, it was nonetheless substantial. The sites were distributed unevenly in the four vestibular nuclei and their distribution correlated well with the projection areas of the vestibular nerve, which has been described as a glutamate-mediated pathway. The highest numbers of glutamate binding sites were observed in the medial vestibular nuclei. This technique provides a very sensitive assay for characterizing the pharmacological subtypes of glutamate binding in the vestibular nuclei and for analyzing changes in these sites during development or after deafferentation of the vestibular nuclei.     

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.     

Imaging of the super high affinity binding sites for [H]Ro15–4513 in rat hippocampus: comparison between in vitro and in vivo binding

The super high affinity binding sites for [³H]Ro15–4513, a partial inverse agonist of central benzodiazepine receptors, were analyzed in rat hippocampus both in vivo and in vitro. An ultra high sensitive method of autoradiography with an imaging plate system was employed for quantitative analysis of [³H]Ro15–4513 binding. In in vitro binding, the super high affinity binding sites in the hippocampus were observed when the [³H]Ro15–4513 concentration was below 0.5 nM. In vivo, the super high affinity binding sites were only found when the injected dose of Ro15–4513 was below 3.6 μg/kg and almost disappeared when the dose was increased to 10 μg/kg. These results both in vivo and in vitro indicate that there is a significant discrepancy between actual free ligand concentration in vivo and in vitro, and that concentrations in intact brain may be much lower than previously thought.     

The subcellular distribution of DL-[3H]2-amino-4-phosphonobutyrate binding sites in the rat brain

The subcellular distribution of DL-[3H]2-amino-4-phosphonobutyrate binding sites in rat brain was examined. Binding sites for this radioligand, which selectively labels a chloride/calcium-dependent subpopulation of glutamate binding sites, were highly concentrated in synaptic membrane fractions. This is wholly consistent with the proposed involvement of this site in physiological responses.     

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.     

[3H]Nicotine binding sites in developing fetal brains in rats

[³H]Nicotine binding sites were examined in developing fetal brains in rats. The fetal brain membranes bound [³H]nicotine with a similar affinity to that of adult brain membranes. This binding was displaced by unlabelled nicotine or carbamylcholine, the inhibition concentrations being approximately the same for fetal and adult brain preparations. α-Bungarotoxin had no effect on [³H]nicotine binding to fetal brain membranes as well as to adult brain preparations. The specific [³H]nicotine binding was first detectable on day 16 of gestation and it increased several folds until birth.     

Possible presence of [3H]glutathione (GSH) binding sites in synaptic membranes from rat brain

Reduced as well as oxidized forms of glutathione exhibited a significant displacement of the specific binding of [3H]L-glutamic acid (Glu), a potential candidate for the central excitatory neurotransmitter, to the rat brain synaptic membranes. In order to elucidate these findings, an attempt was made to determine whether or not the synaptic membranes contained the binding sites for this peptide using [3H]glutathione (GSH) as a ligand. The specific binding activity was detected in the synaptic membranous preparations and found to be dependent on the incubation temperature and incubation time. The binding reached a plateau within 60 min of incubation at 2 degrees C and 30 degrees C. [3H]GSH binding increased linearly with increasing concentrations of membranous proteins employed. Scatchard analysis revealed that the binding sites consisted of two separate independent components rather than being comprised of a single constituent. A significant and concentration-dependent displacement of the binding was induced not only by the addition of GSH, but also by the inclusion of some GSH derivatives without SH-moiety, such as the oxidized form of glutathione, S-methyl-glutathione and S-hexyl-glutathione. The binding was also significantly inhibited by various alpha- and gamma-peptides containing L-Glu, but not by those containing D-Glu. Amongst 4 different agonists and antagonists used for the subclassification of the central Glu receptors, an agonist, quisqualic acid, and an antagonist, 2-amino-4-phosphonobutyric acid, exhibited a significant inhibition of the binding at the highest concentration employed. These results suggest that the rat brain synaptic membranes may contain structure-selective, temperature-dependent, high affinity and saturable binding sites for glutathione.     

~3H—酪氨酸与大鼠几种组织的细胞膜制备物的结合

将大鼠不同组织的细胞膜制备物与~3H-酪氨酸在24℃下孵育5 min,结果在肝脏、肾脏、脾脏、睾丸、黄体、垂体和肾上腺均发现了酪氨酸的高亲合力和低亲合力两类结合位点;在子宫、肌肉和下丘脑仅有低亲合力结合位点。这提示酪氨酸可能在多种组织发挥调节作用。     

Quantitative autoradiography of [H]prazosin binding sites in rat forebrain

We have used the LKB Ultrofilm method of autoradiography to localize and quantify in rat forebrain the binding sites for [³H]prazosin, a highly-selective antagonist for the ₁ adrenoreceptor subtype. Frozen 32 μm thick brain sections were labeled in vitro with 1 nM [³H]prazosin and applied against LKB Ultrofilm for 60 days to generate autoradiograms. Non-specific binding was defined as the labeling in the presence of 10 μM phentolamine. The highest levels of prazosin binding were found in layer V of the motor portion of the frontoparietal cortex and in all nuclei of the thalamus. Moderate levels of ₁ receptors were observed in the remaining layers of the cerebral cortex and in most regions of the limbic system. Low levels of prazosin binding occurred in the caudate-putamen and the accumbens nucleus. Our results indicate that ₁ adrenoreceptors are distributed heterogenously throughout the rat forebrain.     

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).     

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.     

The association of [3H]d-aspartate binding and high-affinity glutamate uptake in the human brain

The binding of [³H]d-aspartate ([³H]d-Asp) to human cerebellum homogenate was compared with the uptake of [³H]glutamate ([³H]Glu) by homogenates prepared from rapidly frozen human cerebral cortex. There was a close correlation between the potencies of a range of drugs for inhibiting the binding of [³H]d-Asp and the uptake of [³H]l-Glu. Compounds selective for postsynaptic Glu receptors were inactive. The findings are consistent with the labelling of high-affinity Glu uptake sites by [³H]d-Asp, which may be a valuable ligand for studying excitatory amino acid terminals in human brain.     

Muscarinic receptors on intact, cultured neurons. Characterization by [3H]quinuclidinylbenzilate binding

High affinity [3H]diazepam binding sites were identified on neurons prepared from the hemispheres of 8-day-old chick embryos and grown in serum-containing or serum-free medium. Clonazepam (IC50 = 3 nM) was more potent than Ro 5-4864 (IC50 greater than 1000 nM) in displacing [3H]diazepam binding. GABA and pentobarbital, in the presence of chloride ions were able to stimulate [3H]diazepam binding synergistically. These interactions were found to be comparable to those observed in mammalian brain.     

Circadian variation of [3H]N6-(L-phenylisopropyl)adenosine binding in rat brain

Since considerable recent experimental evidence suggests a role for the purine nucleoside adenosine in the regulation of mammalian sleep, circadian variations of adenosine receptor binding were examined in whole rat brain using [3H]N6-(L-phenylisopropyl)adenosine ([3H]L-PIA). These results demonstrate a significant circadian variation in the number of [3H]L-PIA binding sites (Bmax) with a maximum 3 h after the beginning of the dark phase of a 12 h light/12 h dark cycle, and a minimum 8 h later (P less than 0.025). The dissociation constant (Kd) of [3H]L-PIA at adenosine receptors did not exhibit any statistically significant circadian variation. These data indicate a daily rhythm in the number of adenosine receptors without a change in Kd and may support the hypothesized involvement of adenosine in the regulation of sleep in rats.     

Evidence for interactions between [3H]glutamate and [3H]kainic acid binding sites in rat striatal membranes. Possible relevance for kainic acid neurotoxicity

The projection of the substantia nigra-ventral tegmental area (SN-VTA) to the ipsilateral and contralateral caudate-putamen was studied with double-labeling fluorescence techniques in the albino rat. By combining glyoxylic acid histofluorescence with retrograde fluorescence tracers, we were able to determine that 93% of the minor (1–2%) contralateral mesostriatal pathwayis catecholaminergic. In addition, combined immunofluorescence and retrograde fluorescence tracer studies showed that 50% of the crossed mesostriatal pathway also arises from cholecystokinin-containing neurons. These results provide evidence for crossed catecholamine- and peptide-containing pathways in the mesostriatal system which is part of the ‘prefrontal system’ that links the prefrontal cortex through the caudate-putamen, with the thalamus and tectum of each hemisphere.     

Prolactin binding sites in rat brain and liver: effects of long-term ovariectomy and ovarian steroids

The effects of long-term ovariectomy on the levels of brain and liver lactogenic binding sites as well as plasma and liver prolactin (PRL) have been investigated in sham-operated and ovariectomized rats receiving either 17β estradiol (OVX-E), progesterone (OVX-P), or vehicle (OVX-V). The levels of lactogenic binding sites in the parietal and piriform cortices, amygdala, thalamus, hypothalamus, as well as in the liver were significantly decreased after long-term ovariectomy. Moreover, the levels of plasma and liver PRL were also significantly decreased. Exogenous estradiol and progesterone replacement restored the levels of lactogenic binding sites in the parietal cortex and hypothalamus as well as in the liver. However, plasma and liver PRL levels were significantly increased by estradiol but only restored by progesterone. These results suggest that ovarian steroids influence the levels of lactogenic binding sites and prolactin.