Autoradiographic analysis of [H]ryanodine binding sites in rat brain: Regional distribution and the effects of lesions on sites in the hippocampus

Quantitative and qualitative autoradiographic methods together with lesion approaches were used to determine the distribution of [3H]ryanodine binding sites in rat brain and the neuronal localization of these sites in the hippocampus. In normal animals, levels of [3H]ryanodine binding sites ranged from a low of about 1 fmol/mg tissue in subcortical structures to a high of 12-18 fmol/mg tissue in subregions of the hippocampus and the olfactory bulb. Relatively high densities of sites (5-9 fmol/mg tissue) were also seen in the olfactory tubercle, most areas of the cerebral cortex, accumbens nucleus, striatum, lateral septal nuclei, pontine nucleus, superior colliculus and granule cell layer of the cerebellum. Specific binding was undetectable in white matter. In experimental animals, intracerebral injections of kainic acid caused neuronal degeneration and a near total depletion of [3H]ryanodine binding sites in the dentate gyrus and in fields CA1, CA2 and CA3 of the hippocampus. Injections of kainic acid that left dentate granule cells largely intact while destroying all neurons in field CA3 had no effect on binding sites in the dentate gyrus. However, these lesions substantially reduced the density of binding in field CA3, leaving a narrow band of sites outlining the position of the degenerated CA3 pyramidal cells. Mechanical knife-cut lesions that severed the granule cell mossy fiber input to field CA3 reduced the density of binding sites in the CA3 region. The results indicate that [3H]ryanodine binding sites in brain are heterogeneously distributed and suggest that a proportion of these sites in the hippocampus may be contained in mossy fiber terminals where a presumptive calcium channel/ryanodine receptor complex may be involved in the regulation of calcium mobilization and/or neurotransmitter release.     

Galanin receptors in the post-mortem human brain. Regional distribution of I-galanin binding sites using the method of in vitro receptor autoradiography

The distribution of putative receptors for the peptide galanin was studied in the normal post-mortem human brain by using ¹²⁵I-galanin (0.5 nM) in combination with in vitro receptor autoradiography. Specific binding of ¹²⁵I-galanin was found in a large number of brain areas throughout the neuraxis. Highest binding densities occurred in the basal forebrain and hypothalamus, while the basal ganglia, major parts of the thalamus and the tectum were found to be poor in binding sites. All cortical areas harboured ¹²⁵I-galanin binding, and in the visual cortex a laminated pattern was present. In the hippocampus, ¹²⁵I-galanin binding occurred in layer 2 of the entorhinal cortex, in the uncus and in the hippocampal-amygdala area. In the brain-stem, ¹²⁵I-galanin binding was found in serotoninergic and noradrenergic cellgroups as well as in the reticular formation and in the parabrachial nuclei. Galanin receptors may, thus, mediate the response of galanin in numerous structures in the human brain.     

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.     

Distribution of [3H]cholecystokinin octapeptide binding sites in the hippocampal region of the rat brain as shown by in vitro receptor autoradiography

The distribution of binding sites for the neuropeptide cholecystokinin octapeptide in the rat hippocampal region was studied by using quantitative in vitro receptor autoradiography. Biochemical analysis of [3H]cholecystokinin octapeptide binding to tissue sections of the hippocampal region showed it to be of high affinity, to be saturable and approximately 50% specific at saturating concentrations. The binding of [3H]cholecystokinin octapeptide to hippocampal sections was dose-dependently blocked by cholecystokinin octapeptide, cholecystokinin and by pentagastrin. The autoradiographic analysis showed high densities of [3H]cholecystokinin octapeptide binding sites in the hilus of the area dentata, the outer three layers of the retrosplenial area and the presubiculum, layer 3 of the medial, but not the lateral, entorhinal area and the deep and superficial parts of layer 1 and 2, respectively of both the medial and the lateral entorhinal area. Medium binding densities were found in the parasubiculum and remaining layers of the entorhinal area and low densities occurred in the subiculum and in all subfields of Ammon's horn. The angular bundle and fornix-fimbria lacked specific [3H] cholecystokinin octapeptide binding sites. A very similar pattern of binding densities was found for [3H]pentagastrin. Comparisons of the cholecystokinin octapeptide receptor distribution with the cholecystokinin octapeptide innervation of the hippocampal region suggest that there exists a relatively good concordance in some hippocampal subfields such as the presubiculum and the entorhinal area between binding sites for [3H]cholecystokinin octapeptide and cholecystokinin-immunoreactive afferent input.     

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 cocaine recognition sites in rat brain: In vitro and ex vivo autoradiography with [I]RTI-55

The distribution of binding sites of [¹²⁵I]RTI-55 (3β-(4-iodophenyl)tropan-2β-carboxylic acid methyl ester), a phenyl tropane analog of cocaine, and the selective labelling of the dopamine transporter (DAT) were studied by in vitro and ex vivo autoradiography in the rat whole brain. Recent evidence has shown that RTI-55 binds to not only DAT but also serotonin transporter (5HTT). In the present study, in vitro autoradiography revealed that [¹²⁵I]RTI-55 bound to the olfactory tubercle, the caudate putamen, the accumbens nucleus, the midline and lateral geniculate nuclei of the thalamus, the hypothalamic nuclei, the substantia nigra compact part, the subthalamic nucleus, the ventral tegmental area, the superior colliculus, the dorsal raphe nucleus, and the facial nucleus. Further, in the presence of clomipramine, a selective ligand for 5HTT, [¹²⁵I]RTI-55 binding was remarkably inhibited in the midline and lateral geniculate nuclei of the thalamus, the hypothalamic nuclei, the superior colliculus, the dorsal raphe nucleus, and the facial nucleus, while [¹²⁵I]RTI-55 binding remained in the olfactory tubercle, the caudate putamen, the accumbens nucleus, the substantia nigra compact part, the subthalamic nucleus, and the ventral tegmental area. These findings suggest that [¹²⁵I]RTI-55 binds to 5HTT in the former areas and to DAT in the latter areas. It is therefore concluded that RTI-55 is a suitable ligand for studying the action of cocaine in whole brain regions, including the thalamus, the hypothalamus and the dorsal raphe nucleus, regions in which cocaine is thought to act evoking several neurological effects, e.g., analgesia and elevation of adrenocorticotropic hormone. DAT was also labelled selectively both in vitro and in vivo using [¹²⁵I]RTI-55 combined with clomipramine. Therefore, radiolabelled RTI-55, combined with unlabelled clomipramine, which displaces its binding to 5HTT, also appears to be suitable for the selective imaging of DAT in vivo.     

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.     

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)     

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).     

Intracellular electrophysiology of CA1 pyramidal neurones in slices of the kainic acid lesioned hippocampus of the rat

Summary Intracellular recordings were made from hippocampal CA1 pyramidal cells in slices where the CA3/CA4 region had been lesioned using intracerebroventricular kainic acid. In 55% of the cells studied orthodromic excitation evoked bursts of action potentials. This bursting activity was associated with a decrease in or loss of the early phase to the hyperpolarisation which normally follows orthodromically evoked action potentials. The recurrent inhibitory post-synaptic potential produced by antidromic activation of pyramidal cells was also reduced or absent. A late phase to the orthodromic hyperpolarisation was reduced in cells from lesioned slices. However, in normal slices treated with bicuculline this potential showed an apparent increase. The afterhyperpolarisation which follows a short current evoked burst of action potentials was reduced in bursting cells from lesioned slices. In addition, a silent period in the firing pattern produced by long depolarising current pulses was reduced or absent in these cells. These results together with observations made with bicuculline suggest that the bursting activity in lesioned slices is largely due to a loss of inhibition mediated by -aminobutyric acid. It is proposed that the kainic acid-lesioned in vitro hippocampus may be a suitable preparation for studying the electrophysiology of temporal lobe epilepsy.     

Propidium iodide in vivo: an early marker of neuronal damage in rat hippocampus

We have investigated the use of the fluorescent exclusion dye propidium iodide as a marker for acutely degenerating cells in vivo, and report here that combined injection of kainic acid and propidium iodide into the lateral cerebral ventricle results in labelling of CA3 pyramidal cells 1 and 6 h after injection. Alternate sections stained with thionin at these early times revealed little evidence of histologically detectable cell damage.     

In vitro autoradiographic localization of calcitonin and amylin binding sites in monkey brain

Calcitonin (CT) and amylin are related peptides with potent central actions, including suppression of appetite and gastric acid secretion. Little is known about the distribution and binding characteristics of amylin receptors in species other than rat; therefore, in this study, by using in vitro autoradiography, we have mapped the distribution of ¹²⁵I-rat amylin binding sites in the monkey brain and compared this distribution to that of binding sites for ¹²⁵I-salmon CT (¹²⁵I-sCT). Highest densities of ¹²⁵I-amylin binding were in the hypothalamus, including the arcuate nucleus and parts of the ventromedial hypothalamic nuclei, and the solitary nucleus. Rostrally, moderate to high density binding was present in parts of the preoptic area, bed nucleus of the stria terminalis, amygdala and accumbens nucleus (Acb). Caudally, binding of amylin was more restricted, with moderate to high density binding present only in dorsal raphe, and area postrema. The primary visual cortex displayed strong and periodic CT binding in layer 4. The subcortical pattern of distribution of amylin and CT receptors in the monkey was similar to that seen previously in the rat, although the relative densities of binding to different brain structures were not always conserved. As with rat, monkey amylin receptors were a subset of the sites labeled with ¹²⁵I-sCT. Analysis of receptor specificity indicated a greater relative potency of CT peptides in competing for ¹²⁵I-amylin binding in monkey, when compared to rat, while, there was a decrease in the relative potency of CT gene-related peptides, potentially due to differences the level of receptor activity modifying proteins (RAMPs) in monkey versus rat brain. Amylin receptors in primates are likely to perform a similar role to those in rats; however, the interaction of the receptors with related peptides may differ.     

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.     

Some behavioral effects of selective neuronal depletion by kainic acid in the dorsal hippocampus of rats

Iontophoretic injections of the neurotoxin kainic acid (KA) into the dorsal hippocampus resulted in an essentially complete attrition of neurons throughout the dorsal hippocampus but spared the fimbria/fornix system. The ventral hippocampus, subiculum, and associated aspects of the entorhinal area appeared completely intact. There was also no detectable damage to neocortex or thalamus adjacent to the dorsal hippocampus or to such KA-sensitive distant regions of the brain as the piriform cortex or striatum. Bilateral depletion of neurons from the dorsal hippocampus retarded the acquisition of a brightness discrimination (because the animals developed position habits during the early phases of acquisition) but had no deleterious effects on brightness discrimination reversal. The KA injections also sharply reduced spontaneous alternation in a T maze and increased locomotor activity in an open field. Food and water intake as well as saline and sucrose preferences were normal after the initial post-operative period but the animals continued to spill more of their food than controls.     

Increased [H]kainic acid binding in the prefrontal cortex in schizophrenia

[³H]Kainic acid binding sites were measured post mortem in the putamenand prefrontal cortex areas from 10 control subjects and 12 schizophrenic patients. A 25–50% increase in [³H]kainic acid binding was observed in the medial frontal (Brodmann areas 9, 10 and 46) and eye-movement areas (8), but not in the other regions of schizophrenic brains. No significant correlation between the binding and either age at death, storage of the brains, duration of illness or neuroleptics-free period was observed. These findings suggest that a dysfunction of cortical excitatory amino acidergic transmission may be involved in schizophrenia.     

In vivo autoradiography of [H]SCH 39166 in rat brain: selective displacement by D1/D5 antagonists

The purpose of this study was to examine the receptor occupancy of D1/D5 antagonists for D1-like dopamine receptors in rat brain using [³H]SCH 39166, a highly selective D1/D5 antagonist with low affinity for 5HT2 receptors. A single concentration of triated SCH 39166 was administered to rats, with or without competing doses of the D1/D5 antagonist SCH 23390 and unlabeled SCH 39166, the D2-like antagonists haloperidol or the 5-HT₂ antagonist ketanserin. The bound radioactivity in the cortex, striatum, nucleus accumbens and olfactory tubercle was then quantified using an in vivo autoradiographic procedure. The results indicated that [³H]SCH 39166 was dose dependently displaced by the D1/D5 antagonists in regions associated with both the nigro-striatal pathway and the mesolimbic dopamine pathway, particularly the nucleus accumbens. Neither haloperidol nor ketanserin displaced [³H]SCH 39166 in any of the regions examined. The data were compared with previously published data examining the in vivo binding of [³H]SCH 39166 in rat brain homogenates. The relative values obtained were comparable to values detected in rat brain homogenates after in vivo binding of [³H]SCH 39166.     

Autoradiographic localization of binding sites for vasotocin in the brain and pituitary of the sea bass (Dicentrarchus labrax)

Specific binding sites for vasotocin (VT) were detected in brain and pituitary of a teleost fish, the sea bass, after in vitro incubation of tissue sections with [³H]arginine-vasopressin (AVP) and light microscopic autoradiography. Conditions for the binding assay were optimized and as a result the binding was saturable and specific. In the brain [³H]AVP binding was found to occur in the pars lateralis and the pars ventralis of the ventral telencephalon, in the pars centralis of the dorsal telencephalon, in the hypothalamic region (especially in the nucleus preopticus, in the tuberal hypothalamus and around the posterior recess), in the tectum opticum and in the noncellular layer of the corpus cerebelli. In the pituitary a high density of [³H]AVP binding was observed in the areas of the pars distalis (PD) occupied by ACTH-, TSH- and GH-cells and also in the pars intermedia (PI). The present study presents the first anatomical evidence for the presence of VT specific binding sites in teleost brain and pituitary.     

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.