Autoradiographic localization of peripheral benzodiazepine binding sites in the cat brain with [3H]PK 11195

"Peripheral type" benzodiazepine binding sites were labelled in cat brain membranes by using [3H]PK 11195. This ligand binds to the "peripheral type" binding sites in a reversible, specific and saturable manner. Cat brain binding sites density (congruent to 6 pmol/mg prot.) was higher than in the rat. Pharmacological specificity was demonstrated by the potency order of displacing agents: PK 11195 greater than RO5-4864 greater than dipyridamole greater than diazepam greater than clonazepam. A similar characterization was performed in slide mounted brain sections where [3H]PK 11195 also labelled the "peripheral type" benzodiazepine binding sites. The high percentage of specific binding (80%) at 1 nM of [3H]PK 11195 made possible the autoradiographic studies on binding sites distribution. These sites were heterogeneously distributed in the grey matter and absent in white matter. Visual, auditory and other specific sensory relay stations were highly labelled. The blood pressure regulating nuclei, the vestibulo-cerebellar and the extrapyramidal motor system also presented a very high binding density. As previously described in the rat brain, choroid plexus was also strongly labelled by [3H]PK 11195 in the cat.     

Labelling of peripheral-type benzodiazepine binding sites in human brain with [3H]PK 11195: anatomical and subcellular distribution

The peripheral-type benzodiazepine binding site, erstwhile characterized in the rodent and feline brain, has now been characterized in post-mortem human brain using [3H]PK 11195. The kinetics and pharmacological properties of the binding of this ligand are similar to peripheral-type benzodiazepine binding sites elsewhere. The potency of RO5-4864 for this site in human brain is close to that seen in ruminant and carnivore tissues but considerably lower than in rodent tissues. The regional distribution of these binding sites would suggest a neuronal rather than a glial localization. [3H]PK 11195 bound in a similar fashion to slide-mounted sections of human brain, thus allowing quantitative studies of the regional distribution of peripheral-type benzodiazepine binding sites to be made. The binding sites were distributed heterogeneously, but were restricted to the grey matter. Highest densities of binding sites were found in forebrain structures. The localization was not limited to any functional system, nor did it resemble any previously described transmitter system. The similarities between peripheral-type benzodiazepine binding sites in human and in feline brain in terms of their pharmacological characteristics and their regional and subcellular distribution suggest that the cat, rather than the rat, may be the better model for studying a possible role for this site in human cerebral function.     

Autoradiographic localization of peripheral benzodiazepine binding sites in the cat brain with [H]PK 11195

“Peripheral type” benzodiazepine binding sites were labelled in cat brain membranes by using [³H]PK 11195. This ligand binds to the “peripheral type” binding sites in a reversible, specific and saturable manner. Cat brain binding sites density (6̃ pmol/mg prot.) was higher than in the rat. Pharmacological specificity was demonstrated by the potency order of displacing agents: PK 11195 > RO5-4864 > dipyridamole > diazepam > clonazepam. A similar characterization was performed in slide mounted brain sections where [³H]PK 11195 also labelled the “peripheral type” benzodiazepine binding sites. The high percentage of specific binding (80%) at 1 nM of [³H[PK 11195 made possible the autoradiographic studies on binding sites distribution. These sites were heterogeneously distributed in the grey matter and absent in white matter. Visual, auditory and other specific sensory relay stations were highly labelled. The blood pressure regulating nuclei, the vestibulo-cerebellar and the extrapyramidal motor system also presented a very high binding density. As previously described in the rat brain, choroid plexus was also strongly labelled by [³H]PK 11195 in the cat.     

Glutamic acid-insensitive [3H]kainic acid binding in goldfish brain.

Kainic acid is supposed to be a specific agonist for a subclass of excitatory glutamate receptors in the vertebrate CNS. An investigation of (2 nM) [3H]kainic acid binding sites in goldfish brain, using quantitative autoradiography, has revealed evidence for two types of kainic acid receptors which differ in sensitivity to glutamic acid. L-Glutamic acid (0.1-1 mM) displaced over 95% of specific [3H]kainic acid binding elsewhere in the brain but only 10-50% in the cerebellum and cerebellar crest. These structures apparently contain [3H]kainic acid binding sites that are extremely insensitive to glutamic acid. The glutamic acid-insensitive [3H]kainic acid binding was not displaced by quisqualic acid, kynurenic acid, alpha-amino-3-hydroxy-5-methylisoxazolepropionic acid (AMPA), or N-methyl-D-aspartatic acid, but was completely displaced by the kainic acid analogue domoic acid. The data indicate that two types of high affinity binding sites for [3H]kainic acid exist in the goldfish brain: glutamic acid-sensitive and glutamic acid-insensitive. High affinity [3H]kainic acid binding may therefore not always represent binding to subsets of glutamic acid receptors.     

The distribution of [3H]kainic acid binding sites in rat CNS as determined by autoradiography

The distribution of [3H]kainic acid (KA) binding sites in the rat CNS was determined by in vitro autoradiography. KA sites are distributed throughout the CNS gray matter in an anatomically specific pattern with telencephalic structures and the cerebellum accounting for the majority of the binding. These results, together with our previous finding that KA sites are greatly enriched at the synapse, suggest that KA binding sites are associated with select terminal fields, and hence may be involved in neurotransmission in certain CNS pathways.     

The binding properties and regional ontogeny for [3H]glutamic acid Na+-independent and [3H]kainic acid binding sites in chick brain

The binding kinetics, pharmacological properties and regional ontogeny of L-[3H]glutamic acid Na+-independent and [3H]kainic acid binding sites were studied in preparations of chick brain. One binding component was found for L-[3H]glutamic acid with a Kd value of 176 x 10(9) M. For [3H]kainic acid two binding components were found in the hemispheres, optic lobes and brain stem, one with high affinity and a Kd value of 12.5 x 10(9) M and one with low affinity and a Kd value of 260 x 10(9) M. In cerebellum only one binding site was detected for [3H]kainic acid with a Kd value of 144 x 10(9) M. The ontogeny of L-[3H]glutamic acid and [3H]kainic acid binding sites was studied using membrane preparations (48,000 g pellet) of hemispheres, optic lobes, brain stem and cerebellum. Binding of L-[3H]glutamic acid was already significant in all brain regions by embryonic day 11 but major increases in total receptor number per brain region or per mg of protein were apparent by embryonic day 19 and especially after hatching. Cerebral hemispheres, optic lobes and brain stem showed few [3H]kainic acid binding sites by day 13 in ovo. An increase follows which, in hemispheres and optic lobes, continues at the same rate during the first two weeks after hatching. In cerebellum, by contrast, the kainic acid binding site is almost undetectable until embryonic day 15. The appearance of these binding sites in cerebellum takes place during the restricted period between days 15 in ovo and 5 post-hatching. This pattern of development of [3H]kainic acid binding sites almost parallels the developmental patterns of the molecular layer of chick cerebellum and it is consistent with the results of our autoradiographic study showing that the great majority of kainic acid binding sites are localized in the molecular layer.     

The distribution of [H]kainic acid binding sites in rat CNS as determined by autoradiography

The distribution of [³H]kainic acid (KA) binding sites in the rat CNS was determined by in vitro autoradiography. KA sites are distributed throughout the CNS gray matter in an anatomically specific pattern with telencephalic structures and the cerebellum accounting for the majority of the binding. These results, together with our previous finding that KA sites are greatly enriched at the synapse, suggest that KA binding sites are associated with select terminal fields, and hence may be involved in neurotransmission in certain CNS pathways.     

Quantitative autoradiography of [3H]nitroimipramine binding sites in rat brain

We have used the recent tritium-sensitive film method of quantitative autoradiography to localize in rat brain high-affinity binding sites for nitroimipramine (NI), a long-lasting inhibitor of serotonin (5-HT) transport in platelets. The distribution of NI binding sites in rat brain closely parallels the location of 5-HT terminals. There are high concentrations of binding sites in the dorsal and medial raphe nuclei, the basal portion of the mammilary nuclei, the medial forebrain bundle, the olfactory tubercule and the posterior basal nucleus of the amygdala. The association of [3H]NI binding sites with regions having a high content of 5-HT supports the notion that the high-affinity binding site for [3H]NI corresponds to some aspect of the presynaptic uptake site for 5-HT.     

Autoradiography with [3H]PK11195 of spinal tract degeneration in amyotrophic lateral sclerosis

The diagnostic hallmarks of amyotrophic lateral sclerosis (ALS) are degeneration of upper and lower motor neurons and of corticospinal tracts. Here, we demonstrate the suitability of the gliosis marker [3H]PK11195 for quantitative evaluation of tract degeneration in ALS in vitro. Binding of [3H]PK11195 was increased in lateral and ventral white matter of ALS spinal cords but not in the anterior horn, in spite of a dramatic loss in muscarinic binding sites and a high level of oxidatively modified protein. Labeling of activated microglia with [11C]PK11195 may also allow tract degeneration in ALS to be visualized in vivo.     

Characterization and quantitative autoradiography of [3H]tryptamine binding sites in rat brain

[3H]Tryptamine binds with high affinity (Kd = 9.1 nM, Bmax = 54 fmol/mg wet wt.) to tissue sections of rat brain. The binding occurs rapidly and is reversible. Low concentrations of the beta-carbolines harmaline (IC50 = 25 nM) and tetrahydronorharman (tetrahydro-beta-carboline, IC50 = 50 nM) inhibit [3H]tryptamine binding. Serotonin (5-HT, IC50 = 2600 nM) as well as the 5-HT receptor antagonists methysergide and metergoline displace [3H]tryptamine at much higher concentrations from brain slices. The distribution of [3H]tryptamine binding sites in sections of rat brain has been analyzed by quantitative autoradiography. The highest density of binding sites is found in the nucleus (n.) interpeduncularis, a slightly lower one in the locus coeruleus. Moderately labelled are the n. accumbens septi, n. septi lateralis, n. medialis habenulae, n. tractus olfactorii lateralis, the central region of the amygdala, n. caudatus/putamen, n. reuniens and the hippocampal formation. A low density of binding sites is detected in the cerebral cortex and the subiculum. Even less binding sites are found in the n. dorsalis raphe and the substantia nigra. The pattern of distribution of [3H]tryptamine binding sites differs from that of [3H]5-HT (5-HT1), [3H]ketanserin (5-HT2) as well as [3H]imipramine binding sites. These data suggest unique tryptamine binding sites.     

Increased densities of binding sites for the 'peripheral-type' benzodiazepine receptor ligand [3H]PK 11195 in rat brain following portacaval anastomosis.

Using quantitative receptor radioautography, binding sites for the 'peripheral-type' benzodiazepine receptor ligand [3H]PK 11195 were studied in rats 4 week after end-to-side portacaval anastomosis and in sham-operated controls. Portacaval anastomosis resulted in region-selective increases in density of [3H]PK 11195 binding sites in cerebellum, pons greater than thalamus, cerebral cortex greater than hippocampus greater than striatum. Possible mechanisms implicated in these changes include (i) the action of endogenous ligands for the mitochondrial benzodiazepine receptor such as octadecaneuropeptide and (ii) neurotoxic actions of ammonia. In view of the proposed role of these receptors as modulators of intermediary metabolism and neurosteroid biosynthesis, such changes could contribute to the neurochemical mechanisms responsible for portal-systemic encephalopathy.     

Quantitative autoradiography of [H]nitroimipramine binding sites in rat brain

We have used the recent tritium-sensitive film method of quantitative autoradiography to localize in rat brain high-affinity binding sites for nitroimipramine (NI), a long-lasting inhibitor of serotonin (5-HT) transport in platlets. The distribution of NI binding sites in rat brain closely parallels the location of 5-HT terminals. There are high concentrations of binding sites in the dorsal and medial raphe nuclei, the basal portion of the mammilary nuclei, the medial forebrain bundle, the olfactory tubercule and the posterior basal nucleus of the amygdala. The association of [³H]NI binding sites with regions having a high content of 5-HT supports the notion that the high-affinity binding site for [³H]NI corresponding to some aspect of the presynaptic uptake site for 5-HT.     

Quantitative autoradiography of [H]ouabain binding sites in rat brain

In vitro quantitative autoradiography was used to localize in rat brain binding sites for [³H]ouabain, an inhibitor of the Na⁺,K⁺-ATPase. High levels of [³H]ouabain binding sites were found in the superior and inferior colliculi, the mammillary nucleus, the interpeduncular nucleus, and in various divisions of the olfactory, auditory and somatomotor systems. The heterogeneous distribution of [³H]ouabain binding closely parallels the regional brain glucose consumption as determined by the [¹⁴C]deoxyglucose method. Lesion studies of the rat hippocampus using the excitotoxin, ibotenic acid, showed both a marked decrease of neuronal cell types on the injected side and a corresponding decrease in [³H]ouabain binding, indicating that some of the [³H]ouabain binding sites are localized to neurons. The close correlation between [³H]-ouabain binding and regional glucose utilization provides further evidence for a linkage between glucose utilization and the neuronal Na⁺,K⁺-ATPase.     

Phylogenetic distribution of [3H]kainic acid receptor binding sites in neuronal tissue

The phylogenetic distribution of specific binding sites for kainic acid was determined in 14 species including invertebrates and vertebrates. The highest level of binding was observed in brains of the frog (Xenopus laevis), followed by the spiny dogfish (Heterodontus francisci), the goldfish (Carasius auratus) and the chick (Gallus domesticus). Although significant specific binding was noted in some of the lowest forms tested (e.g. Hydra littoralis), this was not a consistent observation in the invertebrates. In most cases, specific binding to both high and low affinity sites was detected; notable exceptions were the cockroach brain (Periplaneta americana), which had negligible high affinity binding, and the crayfish brain (Procambarus) which had negligible low affinity binding. In the spiny dogfish, the smooth dogfish and the chick, the highest level of binding occurred in cerebellum with less in the forebrain and the least in the medulla; in the mammalian species, the highest level of binding occurred in the forebrain structures with less in the cerebellum and least in the medulla. Eadie plots of the saturation isotherms for [3H]kainic acid revealed similar kinetics of binding for frog whole brain, rat forebrain and human parietal cortex with two apparent populations of binding sites: KD1 = 25--50 nM and KD2 = 3--14 nM. While binding in the spiny dogfish forebrain and human caudate nucleus occurred exclusively at a high affinity component, the cerebella of chick, rat and man exhibited only a low affinity binding site. In the 3 species studied most extensively, frog, rat and man, unlabeled kainic acid was the most potent inhibitor of the specific binding of [3H]-kainic acid. L-Glutamic acid was 20--20-fold less potent than kainic acid, and D-glutamic acid was 4--2500-fold less potent than its L-isomer. Reduction of the isopropylene side chain of kainic acid to form dihydrokainic acid decreased the affinity of the derivative 115--30,000-fold. Hill coefficients derived from these displacement curves were 1.0 for unlabeled kainic acid but approximately 0.5 for L- and D-glutamic acids and dihydrokainic acid, which is compatible with negative cooperativity. In summary, these studies demonstrated a widespread distribution throughout the animal kingdom of specific binding sites for kainic acid in neural tissue; the characteristics of these receptor sites are remarkably similar from primitive vertebrates to man.     

Distribution of PK11195 binding sites in porcine brain studied by autoradiography in vitro and by positron emission tomography

The cerebral distribution of peripheral-type benzodiazepine binding sites (PBBS) in human brain has been investigated by positron emission tomography (PET) with the specific radioligand [11C]PK11195 in diverse neuropathological conditions. However, little is known about the pattern of PK11195 binding sites in healthy brain. Therefore, we used quantitative autoradiography to measure the saturation binding parameters for [3H]PK11195 in cryostat sections from young Landrace pigs. Specific binding was lowest in the cerebellar white matter (85 fmol mg(-1)) and highest in the caudate nucleus (370 fmol mg(-1)), superior colliculus (400 fmol mg(-1)), and anterior thalamic nucleus (588 fmol mg(-1)). The apparent affinity was in the range of 2-6 nM in vitro, predicting high specific binding in PET studies of living brain. However, the distribution volume (V(d), ml g(-1)) of high specific activity [11C]PK11195 was nearly homogeneous (3 ml g(-1)) throughout brain of healthy Landrace pigs, and was nearly identical in studies with lower specific activity, suggesting that factors in vivo disfavor the detection of PBBS in Landrace pigs with this radioligand. In young, adult G?ttingen minipig brain, the magnitude of V(d) for [11C]PK11195 was in the range 5-10 ml g(-1), and had a heterogeneous distribution resembling the in vitro findings in Landrace pigs. There was a trend toward globally increased V(d) in a group of minipigs with acute MPTP-induced parkinsonism, but no increase in V(d) was evident in the same pigs rescanned at 2 weeks after grafting of fetal mesencephalon to the partially denervated striatum. Thus, [11C]PK11195 binding was not highly sensitive to constituitively expressed PBBS in brain of young Landrace pigs, and did not clearly demonstrate the expected microglial activation in the MPTP/xenograft model of minipigs.     

Quantitative brain autoradiography of [9,10-3H]palmitic acid incorporation into brain lipids

The distribution of radioactivity within brain metabolic compartments was examined following the intravenous injection of [9,10-3H]palmitate into awake rats. Brain radioactivity reached a maximum value by 15 min after [9,10-3H]palmitate injection and remained unchanged for at least 4 hr. Regional differences in radioactivity could be determined with high resolution by quantitative autoradiography, at the level of cell layers within the hippocampus and cerebral cortex, and between striosomes of the caudate nucleus. Regional brain radioactivities were converted to normalized regional radioactivities (k) by dividing them by the integrated plasma fatty acid radioactivity (integrated over the time course of the experiment). These values reflected incorporation mainly into brain phospholipids; radioactivity due to nonlipid components was minimal. Indeed, about 85% of brain radioactivity was within lipids between 5 min and 4 hr postinjection, the remainder being equally divided between protein-associated pellet and aqueous-soluble metabolites. The major lipids labeled were phospholipids, particularly phosphatidylcholine, which contained about 75% of phospholipid radioactivity. The results show that [9,10-3H]palmitate can be used to examine incorporation of plasma palmitate into individual brain regions via quantitative autoradiography. Furthermore, the tracer is a rather selective marker for phosphatidylcholine and can be used to examine turnover and synthesis of this phospholipid. [9,10-3H]palmitate has advantages over [U-14C]palmitate for autoradiographic studies of incorporation; following the 14C-tracer, significant label even at 4 hr after injection is in nonlipid compartments (glutamate and aspartate), and the long path length of 14C limits resolution at the cell layer level.     

Quantitative localization of [3H]TCP binding in rat brain by light microscopy autoradiography

The anatomical localization of phencyclidine (PCP)/sigma-opiate receptors in rat brain was determined by quantitative light microscopy autoradiography using the new ligand N-(1-[2-thienyl]cyclohexyl) [3H]piperidine ([3H]TCP). TCP is a potent analog of PCP which possesses a higher affinity for PCP/sigma-opiate receptor than does PCP itself. The highest level of [3H]TCP binding was detected in the hippocampus. Intermediate levels were found in frontal cortex, striatum, amygdala and cerebellum. Specific [3H]TCP binding was undetectable in anterior commissure and corpus callosum. The distribution pattern of [3H]TCP binding sites is similar to the pattern obtained with [3H]PCP but more sharply defined. On the basis of its greater potency and specificity, [3H]TCP may prove superior to [3H]PCP as a molecular probe for the study of brain sigma opiate/phencyclidine receptors.     

Analogue interactions with the brain receptor labeled by [H]kainic acid

The kinetics of interaction of kainic acid analogues and reputed antagonists of acidic excitatory amino acids with a specific binding site for [³H]α-kainic acid were examined in washed cerebellar membranes incubated at 4°C. The avidity of both l-glutamate (1.5 μM) and quisqualate (0.6 μM) suggests that proper orientation of the two car☐yls in the amino group is essential for binding to one component of the receptor. The high affinity of domoate, α-kainate and α-keto-kainate as compared to the low affinity of dihydrokainate and α-allo-kainate indicate that a pi electron group with appropriate cis-planar orientation versus the C2-car☐yl group is essential for high affinity binding to the receptor. These studies provide additional evidence that the recognition site labeled by [³H]kainic acid represents the receptor mediating its neurophysiologic and neurotoxic effects.     

Quantitative autoradiography of [H]forskolin binding sites in the rat brain

The binding sites for a radiolabeled form of the potent activator of adenylate cyclase, forskolin, have been localized in the rat brain, pituitary and spinal cord. Using the quantitative technique of in vitro autoradiography, a high density of [3H]forskolin binding was detected in brain structures such as the caudate-putamen, nucleus accumbens, olfactory tubercle, globus pallidus, substantia nigra and the hilus of the area dentata. A comparison of the distribution of [3H] forskolin binding sites with those reported for several neurotransmitter receptor types indicated that forskolin identified adenylate cyclase was probably not linked to any single type of neurotransmitter receptor. These results also presented several new brain areas in which to investigate the neuronal role of adenylate cyclase.     

Localization and characterization of [3H]desmethylimipramine binding sites in rat brain by quantitative autoradiography

The high affinity binding sites for the antidepressant desmethlyimipramine (DMI) have been localized in rat brain by quantitative autoradiography. There are high concentrations of binding sites in the locus ceruleus, the anterior ventral thalamus, the ventral portion of the bed nucleus of the stria terminalis, the paraventricular and the dorsomedial nuclei of the hypothalamus. The distribution of DMI binding sites is in striking accord with the distribution of norepinephrine terminals. Pretreatment of rats with the neurotoxin 6-hydroxydopamine, which causes a selective degeneration of catecholamine terminals, results in 60 to 90% decrease in DMI binding. These data support the idea that high affinity binding sites for DMI are located on presynaptic noradrenergic terminals.