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.     

Increased densities of binding sites for the ‘peripheral-type’ benzodiazepine receptor ligand [H]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 > thalamus, cerebral cortex > hippocampus > 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.     

Ontogenetic development of peripheral benzodiazepine binding sites in rat brain, heart and lung

In rats, the brain exhibits negligible ontogenetic changes, while a steady marked increase in the density of peripheral benzodiazepine binding sites (PBS) has been demonstrated in the heart and lungs, reaching maximal levels at 31 days after birth. It may be that PBS play a role in the cellular proliferation of these peripheral organs. The present findings are consistent with current evidence of a relationship between these binding sites and mitochondrial function.     

Regulation of central and peripheral benzodiazepine receptors in progesterone-treated rats

The effects of progesterone treatment to male rats on cortical and hippocampal central benzodiazepine (BZ) receptors and peripheral BZ binding sites (PBS) in kidney, testis and heart were evaluated. Chronic progesterone treatment resulted in a 30% elevation of the density of central BZ receptors in cerebral cortex accompanied by a 24% augmentation of PBS in the kidney. No significant change in the density of central BZ receptors in the hippocampus or of PBS in the testis or heart was observed. No change in the affinity of [³H]flunitrazepam and [³H]PK 11195 binding to central BZ receptors and PBS, respectively, was observed. The increase in PBS in the kidney might be associated with the natriuretic effect of this hormone and the adaptatory increase in aldosterone secretion. The up-regulatory effect of progesterone on cortical central BZ receptors may be involved in the neurobehavioral activities of progesterone.     

Evidence for loss of brain [H]spiroperidol and [H]ADTN binding sites in rabbit brain with aging

[³H]Spiroperidol and [³H]2-amino-6,7-dihydroxyl-1,2,3,4,-tetrahydronaphthalene hydrochloride (ADTN) binding were measured in various central nervous system regions of 5 month and 5.5 year old rabbits. In striatum, young animals had a 38% higher number of [³H]spiroperidol binding sites and a 140% higher number of [³H]ADTN binding sites than did the older animals. In frontal cortex and anterior limbic cortex there were respectively 42% and 26% more [³H]spiroperidol binding sites in the young animals. There was no change in the binding site number or affinity for [³H]spiroperidol in retina with aging. Pharmacological characterization demonstrated that [³H]spiroperidol binds to a dopamine receptor in striatum and to a serotonin receptor in cortex.     

Two binding sites for [3H]PBR28 in human brain: implications for TSPO PET imaging of neuroinflammation

[¹¹C]PBR28, a radioligand targeting the translocator protein (TSPO), does not produce a specific binding signal in approximately 14% of healthy volunteers. This phenomenon has not been reported for [¹¹C]PK11195, another TSPO radioligand. We measured the specific binding signals with [³H]PK11195 and [³H]PBR28 in brain tissue from 22 donors. Overall, 23% of the samples did not generate a visually detectable specific autoradiographic signal with [³H]PBR28, although all samples showed [³H]PK11195 binding. There was a marked reduction in the affinity of [³H]PBR28 for TSPO in samples with no visible [³H]PBR28 autoradiographic signal (K_i=188±15.6 nmol/L), relative to those showing normal signal (K_i=3.4±0.5 nmol/L, P<0.001). Of this latter group, [³H]PBR28 bound with a two-site fit in 40% of cases, with affinities (K_i) of 4.0±2.4 nmol/L (high-affinity site) and 313±77 nmol/L (low-affinity site). There was no difference in K_d or B_max for [³H]PK11195 in samples showing no [³H]PBR28 autoradiographic signal relative to those showing normal [³H]PBR28 autoradiographic signal. [³H]PK11195 bound with a single site for all samples. The existence of three different binding patterns with PBR28 (high-affinity binding (46%), low-affinity binding (23%), and two-site binding (31%)) suggests that a reduction in [¹¹C]PBR28 binding may not be interpreted simply as a reduction in TSPO density. The functional significance of differences in binding characteristics warrants further investigation.     

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

[³H]tryptamine binds with high affinity (K_d = 9.1nM, B_max= 54fmol/mg wet wt.) to tissue sections of rat brain. The binding occurs rapidly and is reversible. Low concentrations of the β-carbolines harmaline (IC₅₀ = 25nM) and tetrahydronorharman (tetrahydro-β-carboline), IC₅₀ = 50nM) inhibit [³H]tryptamine binding. Serotonin (5-HT, IC₅₀ = 2600nM) 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 section of rat brain has been analyzed by quantitative autoradiography. The highest density of binding sites is found in the nucleus (n.) interpreduncularis, a slightly lower one in the locus coeruleus. Moderately labelled are the n. accumbens septi, n. septi lateralis, n. medalis habenulae, n. tractus olfactorii lateralis, the central region of the amydgala, n. caudatsu/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.     

Differences in the efficiency and pattern of incorporation of [H]leucine and [H]proline into proteins of adult cat brain

Previous EM autoradiographic studies have shown that injection of [3H]proline ([3H]Pro) into the cat dorsal column nuclei (DCN) results in heavy labeling of macroglia but negligible labeling of DCN neurons. [3H]Leucine ([3H]Leu), in contrast, was extensively incorporated into both neurons and glia. We now report preliminary assessment of differences in molecular labeling patterns produced by the two amino acid precursors in DCN injection sites (24 h following injection; equal amounts of [3H]Pro and [3H]Leu of equal specific radioactivity). [3H]Pro, despite its lack of incorporation into neurons, labeled DCN to a significantly greater extent than [3H]Leu (30.0 dpm/micrograms protein/microCi for [3H]Pro vs 11.7 dpm/micrograms protein/microCi for [3H]Leu). Greater than 90% of the radioactivity from both precursors was recovered in protein as opposed to TCA soluble or ethanol soluble molecules. Of the [3H]Pro- or [3H]Leu-derived radioactivity recovered in protein, greater than 94% was found to remain in the original precursor form. Fluorographic analysis of SDS acrylamide gels showed labeling of a wide variety of individual proteins with either amino acid. However, particular molecular weight classes were relatively more heavily labeled with either [3H]Leu (47, 63, 77 kDa) or [3H]Pro (22, 45, 50, 66, 80 kDa). Overall the results indicate that the difference in cellular distribution of incorporated [3H]Pro and [3H]Leu, as observed by EM autoradiography is a reflection of the extent of labeling and specific labeling pattern of proteins isolated from the tissue.     

Regional [H]acetylcholine and [H]nicotine binding in developing mouse brain

The postnatal development of nicotine-like binding sites in the cortex, hippocampus, midbrain and cerebellum of 3-, 7-, 12-, 17- and 30-day-old mice was studied. Two different nicotinic cholinergic ligands, namely [³H]acetylcholine ([³H]ACh) and [³H]nicotine ([³H]NIC) were used to detect the nicotine-like binding sites in in vitro binding assays. The postnatal development of the binding sites of [³H]NIC increased gradually with age in all brain regions studied. The [³H]ACh binding, on the other hand, showed a marked peak on day 12 in the cerebellum and midbrain but did not change notably with age in the hippocampus and cortex, except for a slight temporary increase in the cortex on day 7. The time-course for the appearance of nicotinic binding sites as observed with [³H]ACh was found to be rather similar to that earlier described for [³H]alpha-bungarotoxin binding sites, whereas that for [³H]NIC differed from that described for other nicotinic ligands.     

Autoradiographic localization of muscarinic agonist binding sites in the rat central nervous system with (+)-cis-[H]methyldioxolane

(+)-cis-[³H]Methyldioxolane ((+)-[³H]CD), a potent muscarinic agonist, was used to label high-affinity agonist states of muscarinic receptors in thin tissue sections of the rat central nervous system. Light microscopic autoradiography of atropine-sensitive (+)-[³H]CD binding sites revealed regions of dense labeling (superior colliculus, inferior colliculus, lateral geniculate body, hypoglossal (XII) nucleus, facial (VII) nucleus, tractus diagonalis) and regions of sparse labeling (hippocampus, dentate gyrus). The inverse regional correlation between high-affinity (+)-[³H]CD states and binding sites for the muscarinic antagonists [³H]pirenzepine (r = −0.79) and (-)-[³H]quinuclidinyl benzilate (r = −0.30) underscores potentially important differences between agonist and antagonist binding to CNS tissue slices.     

Effect of chronic haloperidol treatment on peripheral benzodiazepine binding sites in cerebral cortex of rats

Summary The effects of 21 days of haloperidol treatment on central benzodiazepine (BZ) receptors in the cerebral cortex of rats and on peripheral-type BZ binding sites (PBS) in the cerebral cortex and heart of rats were studied. Neuroleptic treatment did not affect the maximal binding capacity or the affinity of the central BZ receptor to³H-flunitrazepam. Chronic haloperidol treatment resulted in a significant increase of 38% in PBS density in the cerebral cortex, with no alteration in PBS density in the heart. No alteration in PBS affinity for its ligand³H-PK 11195 was observed, either in the cerebral cortex or in the heart. The modulatory effect of chronic haloperidol administration on PBS density in the brain may be related to some of the neurobehavioral or hormonal effects of the drug.     

“Peripheral” benzodiazepine binding sites in the maudsley reactive rat: Selective decrease confined to peripheral tissues

"Peripheral" benzodiazepine binding sites (PBS) were studied in both the CNS and peripheral tissues of Maudsley reactive (MR) and Maudsley non-reactive (MNR) rats using the PBS specific ligand [3H]Ro 5-4864. A statistically significant reduction in the density of PBS was found in heart and kidney of the MR compared to the MNR. Similar reductions in the density of PBS were not observed in a number of areas of the central nervous system (including cortex, hippocampus, and hypothalamus) or other peripheral tissues, such as lung and adrenal. This selective decrease in PBS in a strain selectively bred for a high degree of "fearfulness" may be related to previous findings of a reduction in the density of PBS in the same tissues in rats subjected to uncontrollable shock. These observations suggest that PBS in heart and kidney may be altered in response to fear or anxiety.     

[H]WIN 35,428 binding in the human brain

The binding of [³H]WIN 35,428 was studied in post-mortem human brain, including extrastriatal regions. In the putamen, dopamine almost completely inhibited the [³H]WIN 35,428 binding. Paroxetine inhibited the binding with similar affinity as cocaine, in the range 200–300 nM. In the frontal cortex, [³H]WIN 35,428 labelled cocaine- and alaproclate sensitive binding sites, of which a major fraction was of protein nature. The elucidation of the cocaine sensitive sites in the frontal cortex should be the subject of further research.     

Muscarinic cholinergic receptors in the human spinal cord: differential localization of [H]pirenzepine and [H]quinuclidinylbenzilate binding sites

The localization of muscarinic cholinergic receptor subtypes was studied in the human spinal cord using in vitro labelling of cryostat sections with [³H]quinuclidinylbenzilate (QNB) and [³H]pirenzepine (PZ) followed by autoradiography. The highest densities of [³H]QNB binding were localized in laminae II (substantia gelatinosa) and IX (motor neurons); in contrast, the highest density of [³H]PZ binding was localized to lamina II where the binding density was 22—32% higher than in lamina IX. These results suggest that the M₁ and M₂ muscarinic cholinergic receptor subtypes may be differentially localized in sensory and motor regions of the human spinal cord.     

Up-regulation of the peripheral-type benzodiazepine receptor expression and [(3)H]PK11195 binding in gerbil hippocampus after transient forebrain ischemia

In mammalian CNS, the peripheral-type benzodiazepine receptor (PTBR) is localized on the outer mitochondrial membrane within the astrocytes and microglia. The main function of PTBR is to transport cholesterol across the mitochondrial membrane to the site of neurosteroid biosynthesis. The present study evaluated the changes in the PTBR density, gene expression and immunoreactivity in gerbil hippocampus as a function of reperfusion time after transient forebrain ischemia. Between 3 to 7 days of reperfusion, there was a significant increase in the maximal binding site density (B(max)) of the PTBR antagonist [(3)H]PK11195 (by 94-156%; P < 0.01) and PTBR mRNA levels (by 1.8- to 2.9-fold; P < 0.01). At 7 days of reperfusion, in the hippocampal CA1 (the brain region manifesting selective neuronal death), PTBR immunoreactivity increased significantly. Increased PTBR expression after transient forebrain ischemia may lead to increased neurosteroid biosynthesis, and thus may play a role in the ischemic pathophysiology.     

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.     

Autoradiographic localization of binding sites for the delta sleep-inducing peptide ([H]DSIP) on neurons of cultured rat brainstem

Binding of the delta sleep-inducing peptide (DSIP) was studied in cultures of rat brainstem by means of autoradiography. Binding sites for [³H]DSIP were observed on small, medium-sized and large brainstem neurons but not on glial cells. Addition of unlabeled DSIP inhibited or markedly reduced binding of [³H]DSIP. It is suggested that brainstem neurons might possess receptors for this sleep-inducing peptide.     

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.     

Rapid regulation of [H]hemicholinium-3 binding sites in the rat brain

The effects of various drug known to affect the sodium-dependent high-affinity choline-uptake system (SDHACU) in the brain were examined for their action upon the [³H]hemicholinium-3 ([³H]HCh-3) binding site, which is associated with the choline carrier. The [³H]HCh-3 binding sites are affected in a similar way to the SDHACU system. Thus, alterations in the velocity of choline-uptake are mediated through changes in the apparent number of available transport sites at cholinergic terminals.     

Effect of GABA and benzodiazepine antagonists on [H]flunitrazepam binding to cerebral cortical membrane

The effects of GABA on the binding of [3H]flunitrazepam to bovine cerebral cortical membranes was investigated in presence and in absence of benzodiazepine antagonists. The percentage stimulation of [3H]flunitrazepam binding by GABA is higher when approximately 50% of the binding is displaced by benzodiazepine antagonists. The observed increase in percent stimulation of [3H]flunitrazepam binding by GABA might reflect the preferential displacement of the ligand by benzodiazepine antagonists from a GABA-insensitive conformational state or site of the receptor.