3H]Ro5-4864 benzodiazepine binding in the kainate lesioned striatum and Huntington's diseased basal ganglia

[³H]Ro5-4864-labeled peripheral-type benzodiazepine binding sites in the brain were studied after kainic acid lesions of the rat striatum. Following intrastrial kainate injections [³H]Ro5-4864 binding increased to approximately 1000% of control over a period of 1 week and was maintained at this level for up to 6 weeks. Two weeks after lesioning the number of binding sites (B_max) was selectively increased while the dissociation constant (K_d) was only minimally affected. [³H]Ro5-4864 binding in the Huntington's diseased (HD) basal ganglia was not changed as compared to non-neurological control in the caudate nucleus and globus pallidus. A highly significant 51% increase was found in the HD putamen. It is concluded that the peripheral-type, Ro5-4864-sensitive benzodiazepine receptor in the brain may be predominantly localized on glial elements.     

Characteristics of an atypical benzodiazepine,Ro 5-4864

Ro 5-4864 is a 1,4 benzodiazepine which, atypically, does not bind to the classical CNS benzodiazepine receptors, but has high affinity for the peripheral type of binding site found both in the periphery and in the brain. Biochemical evidence for alternative sites of action for this compound is discussed. We review the behavioral profile of Ro 5-4864 (sedative, convulsant and anxiogenic in rodents) and also describe the behavioral effects of combining Ro 5-4864 treatment with benzodiazepines (e.g., diazepam, chlordiazepoxide) and with other drugs that modify the activity of benzodiazepines (Ro 15-1788, CGS 8216, picrotoxin, PK 11195, phenytoin). In the light of these interactions and electrophysiological evidence we conclude that the actions of Ro 5-4864 are most likely to be mediated at the GABA-benzodiazepine receptor complex in the CNS.     

Binding of [H]Ro 5–4864 in primary cultures of astrocytes

Intact primary cultures of astrocytes display benzodiazepine receptors, which can be labeled with [³H]Ro 5–4864. Binding of [³H]Ro 5–4864 is specific, saturable and temperature dependent, being maximal at 0 °C. Scatchard analyses show a single population of high affinity binding sites with aK_d value of 6.7 nM and a B_max value of 12,000 fmol/mg protein. The binding reaches equilibrium at 100 min, with k₊₁ of 0.0078 nM⁻¹ · min⁻¹ and is rapidly reversible with k₋₁ of 0.057 min⁻¹. [³H]Ro 5–4864 binding is not modulated by GABA. Certain benzodiazepines (flunitrazepam, diazepam, Ro 7–3351) and dipyridamole displace this binding with IC₅₀ values in the nanomolar range, whereas other benzodiazepines (alprazolam, clonazepam, chlordiazepoxide) as well as carbamazepine, phenytoin and phenobarbital have IC₅₀ values in the micromolar range. These characteristics resemble those of Ro 5–4864 binding to brain membrane preparations reported by other authors and thus indicate that the cultured astrocytes are good models of their in vivo counterparts.     

Diazepam and its Anomalousp-Chloro-derivative Ro 5-4864: Comparative effects on mouse neurons in cell culture

The actions of diazepam and its p-chloro-derivative Ro 5-4864 were compared on mouse spinal cord and dorsal root ganglion neurons in cell culture. Diazepam enhanced but Ro 5-4864 reduced iontophoretic GABA responses in a concentration-dependent manner. Both diazepam and Ro 5-4864 limited sustained, high frequency repetitive firing of spinal cord neurons but diazepam was more potent. Ro 5-4864 was, however, more potent than diazepam in inhibiting spontaneous neuronal activity of spinal cord neurons and reducing the duration of calcium-dependent action potentials of dorsal root ganglion neurons. The differing actions of diazepam and Ro 5-4864 may account for the contrasting pharmacological spectra of the two benzodiazepines.     

Binding of [H]flunitrazepam and [H]Ro5-4864 to crude homogenates of amygdala-kindled rat brain: two months post-seizure

The binding of [3H]flunitrazepam and [3H]Ro5-4864 to crude homogenates of amygdala-kindled and 'yoked' control rat brains was evaluated in subjects sacrificed 60 days after the sixth generalized convulsion. Decreases in the Bmax for [3H]flunitrazepam binding to 'central-type' benzodiazepine receptors were observed in the hypothalamus and ipsilateral cortex of kindled animals. The binding of [3H]Ro5-4864 to 'peripheral-type' benzodiazepine receptors was unchanged.     

Adenosine potentiation and antagonism may account for the diverse behavioral actions of Ro 5-4864

The actions of the p-chloroderivative of diazepam, Ro 5-4864 on the spontaneous discharges of rat cerebral cortical neurons and its interactions with depressions evoked by adenosine and adenosine 5'-N-ethylcarboxamide (NECA) were observed. Iontophoretically applied Ro 5-4864 had variable actions on cortical neuronal activity, exciting some neurons and depressing others. During the period of applications. Ro 5-4864 antagonized the depressant effects of NECA, whilst having less of an action on adenosine depressions. Following Ro 5-4864 application adenosine, but not NECA, depressions were potentiated in amplitude and duration for periods of up to 20 min. It is suggested that Ro 5-4864 has both antagonistic and potentiative interactions with adenosine.     

Development of kainate binding sites in chick cerebellum demonstrated with the photoaffinity ligand [H]ABCPA

The development of kainate binding proteins in membrane preparations of chick cerebellum was studied with the novel photoaffinity ligand [³H](2S,3S,4S)-4-[1-(4-azidobenzamidomethylethenyl)-2-carboxy-3-pyrrolidineacetic acid (ABCPA). Electrophoretic analysis revealed two major radioactive bands with apparent molecular masses of 45 and 33.5 kDa and pharmacological characteristics of high (45 kDa) and low (33.5 kDa) affinity kainate binding sites. The 33.5 kDa polypeptide is present already at embryonic day (E) 13, and the amount of incorporated radioactivity remains practically constant until post-hatching day (P) 48. In contrast, the 45 kDa polypeptide is undetectable at E13. Its appearance takes place at E17, and the amount of incorporated radioactivity dramatically increases until P6. Its developmental profile is identical to that observed for [³H] kainic acid reversible binding in the same tissue.It is suggested that the 33.5 kDa polypeptide which appears in the early stages of development may participate in receptor complexes which play an important role in developmental phenomena.     

Anticonvulsant actions of nefiracetam on epileptic EL mice and their relation to peripheral-type benzodiazepine receptors

Anticonvulsant actions of the nootropic drug nefiracetam were studied using EL mice, an animal model of epilepsy, in which peripheral-type benzodiazepine receptors (PBRs) might be involved in their epileptogenesis. Nefiracetam, when administered orally t o EL mice, inhibited convulsions induced by the PBR agonist, Ro 5-4864, with an ED(50) of 17.2 mg/kg, whereas it did not inhibit the drug-induced convulsions in control DDY mice. When administered intravenously (i.v.) to DDY mice, nefiracetam and other piracetam-like nootropics inhibited the Ro 5-4864-induced convulsions in the sequence of nefiracetam>aniracetam>oxiracetam, piracetam. Spontaneous EL mouse seizures were also inhibited by these nootropics with a similar rank order of potencies. Binding studies for PBRs, performed on crude membranes of brain tissues of these mice, revealed that [3H]Ro 5-4864 and [3H]PK 11195 bindings were both inhibited by micromolar concentrations of nootropic agents in the sequence of nefiracetam> aniracetam>oxiracetam, piracetam. The results suggest that nefiracetam may exert an anticonvulsant action through interacting with a low-affinity type of PBR in the brain, and could be developed as a promising therapeutic drug for neurological disorders including epilepsies.     

Enhancement of estradiol-induced DNA synthesis in the anterior pituitary gland by the peripheral-type benzodiazepine receptor ligand RO 5-4864

Summary The effects of peripheral (Ro 5-4864) and central-type (Ro 15-1788) benzodiazepine receptor ligands on estrogen-induced DNA synthesis in the rat anterior pituitary gland was investigated. As expected, a single injection of estradiol (250g per rat) significantly increased the uptake of³H-thymidine by anterior pituitary cells. Additional treatment with Ro 5-4864 potentiated the effects of estradiol on pituitary DNA synthesis. Under the same experimental conditions, no effect of Ro 15-1788 and sodium valproate, a GABA-transaminase inhibitor, was detected. These findings indicate the involvement of peripheral-type benzodiazepine receptors in the control of anterior pituitary cell proliferation.     

Effect of chronic ethanol consumption on central and peripheral type benzodiazepine binding sites in the mouse brain

Following chronic exposure of C57/BL6 mice to ethanol the binding of [3H]Ro5-4864 and [3H]propyl-beta-carboline-3-carboxylate to benzodiazepine binding sites in the brain was studied. Peripheral-type benzodiazepine binding sites were measured using the probe [3H]Ro5-4864. Chronic ethanol treatment resulted in a significant increase in [3H]Ro5-4864 binding due to a 43% increase in receptor density. The affinity of [3H]Ro5-4864 for the receptor was not significantly affected. The binding of [3H]propyl-beta-carboline-3-carboxylate to central-type benzodiazepine receptors was not affected by chronic ethanol treatment.     

Pharmacokinetic studies on Ro 15-1788, a benzodiazepine receptor ligand, in the brain of the rat

Methods for determining Ro 15-1788 in brain tissue were developed using gas chromatography with nitrogen-phosphorus detection, and using reverse-phase high performance liquid chromatography. Application of the methods to pharmacokinetic studies in the rat found the elimination half-life of Ro 15-1788 from rat brain to be 16 min. Ro 15-1788 was undetectable in rat plasma at the time points studied. Concentrations of Ro 15-1788 in the brain were reduced if chlordiazepoxide was coadministered.     

Distribution of histamine H3-receptor binding in the normal human basal ganglia: comparison with Huntington's and Parkinson's disease cases

It is now widely recognized that histamine acts as a neurotransmitter in the mammalian central nervous system. Three selective histamine receptors have been described, all of which are present in the basal ganglia. This study is a detailed, quantitative, autoradiographical examination of the densities of histamine H3-receptors in coronal sections of human basal ganglia, using the selective ligand [3H]-(R)-alpha-methylhistamine. [3H]-(R)-alpha-methylhistamine binding was highest within the external and internal segments of the globus pallidus together with the substantia nigra. High levels were also found in the striatum, where density was significantly higher (P < 0.05) at a pre-, as opposed to post-, anterior commissure coronal level. Within the striatum, binding was noticeably higher in both the nucleus accumbens and acetylcholinesterase-deficient striosomes, while being undetectable in the subthalamic nucleus and very low in both the ventroanterior and ventrolateral thalamic nuclei. An intermediate level of binding, often with a laminar distribution, was seen in the insular cortex. [3H]-(R)-alpha-methylhistamine binding was also examined in both Parkinson's disease and Huntington's disease. No difference from control receptor density was found in any area examined in Parkinson's disease, while values were significantly lower in caudate (P < 0.001), putamen (P < 0.001), external (P < 0.001) and internal (P < 0.05) globus pallidus, although not the insular cortex, in Huntington's disease cases. These data suggest that H3-receptors are present upon striatonigral projection neurons of the direct and indirect movement pathways thus providing histaminergic control over the activity of both these circuits.     

Localization of [H]N-propylnorapomorphine binding in mouse striatum

The anatomic localization of specific striatal [³H]N-propylnorapomorphine ([³H]PNA) binding was determined in male C57BL/6J mice. Striatal [³H]PNA binding was of high affinity and sensitive to guanine nucleotides. Frontal cortical ablation did not alter striatal [³H]PNA binding, but reduced [³H]spiperone binding by 36%. Kainic acid reduced and 6-hydroxydopamine elevated [³H]PNA binding. A combined frontal cortical ablation and striatal kainic acid lesion was similar to that of kainate alone. These data are consistent with a localization of [³H]PNA binding sites on neurons intrinsic to the mouse striatum.     

The effects of chemically and electrically-induced convulsions on [H]Nitrendipine binding in mouse brain

The effects of chemically and electrically-evoked seizures on [³H]nitrendipine binding to voltage-dependent calcium channels in mouse brain were determined 30 and 60 min following the initiation of convulsions. While maximal electroconvulsive shock, pentylenetetrazol and strychnine exhibited either no or marginal effects, Ro 5-4864 produced a decrease (14%) in the B_max of [³H]nitrendipine at 30 min but not 60 min. The convulsant dihydropyridine calcium channel activator, BAY K 8644, produced a significant increase in the K_d (31%) of [³H]nitrendipine at 30 min, and a significant increase in both the B_max (21%) and K_d (28%) of [³H]nitrendipine 60 min following the initiation of convulsions. While maximal electroconvulsive shock, pentylenetetrazol and strychnine exhibited either no or marginal effects, Ro 5-4864 produced a decrease (14%) in the B_max of [³H]nitrendipine at 30 min but not 60 min following the initiation of convulsions. These findings indicate that modulation of voltage-dependent calcium channels by certain convulsants may be important in the genesis of seizures or in post-ictal compensatory processes.     

Maximal electroshock increases the density of [3H]Ro 5-4864 binding to mouse cerebral cortex

The effects of chemically and electrically-induced convulsions on the binding of [3H]Ro 5-4864 to peripheral benzodiazepine receptors (PBR) was studied in both peripheral tissues and the central nervous system (CNS). Acute, maximal electroshock (MES) increased the density of PBR in mouse cerebral cortex as evidenced by a 30% increase in the Bmax of this archetypic ligand. These values returned to control levels by 60 minutes after MES treatment. In contrast, thirty and sixty minutes after convulsions induced by Ro 5-4864, strychnine, or pentylenetetrazol, neither the Bmax nor Kd of [3H]Ro 5-4864 binding to mouse cerebral cortical membranes was altered. The increase in [3H]Ro 5-4864 binding to cortex observed 30 minutes after MES was blocked by anticonvulsant doses of phenobarbital, phenytoin and clonazepam. No changes in the characteristics of [3H]Ro 5-4864 binding was observed in cerebellar or hippocampal membranes 30 minutes following acute MES. Further, after long-term MES administration (1 treatment/day, 5 days), no change in PBR density could be detected 30 minutes after the last MES. Finally, while no change in PBR density was noted in the kidneys 30 minutes after the MES, a significant increase in PBR density was seen in the cardiac ventricles. These results demonstrate a selective modulation of PBR density by MES, suggesting that the PBR could be involved in either the generation of seizures or in postictal compensatory processes.     

Changes in [H]zolpidem and [H]Ro 15-1788 binding in rat globus pallidus and substantia nigra pars reticulata following a nigrostriatal tract lesion

Changes in GABA_A receptor α₁ subunit gene expression occur in the globus pallidus and substantia nigra pars reticulata following lesions of the nigrostriatal tract. To determine whether these changes are translated at the protein level, we performed quantitative autoradiography with the α₁ selective ligand, [³H]zolpidem, and the non-selective benzodiazepine site ligand, [³H]Ro 15-1788. Binding of both [³H]zolpidem and [³H]Ro 15-1788 was significantly increased in the substantia nigra pars reticulata (13.5±4.1 and 26.3±2.9%, respectively) and significantly reduced in the globus pallidus (20.9±0.8 and 18.3±1.3%, respectively). These changes in α₁ subunit protein expression may help to compensate for the pathological changes in GABAergic activity that occur after striatal dopamine depletion.     

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.     

Visualization of alpha5 subunit of GABAA/benzodiazepine receptor by 11C Ro15-4513 using positron emission tomography

Although [(11)C]Ro15-4513 and [(11)C]flumazenil both bind to the central benzodiazepine (BZ) receptors, the distributions of the two ligands are not identical in vivo. Moreover, the in vivo pharmacological properties of [(11)C]Ro15-4513 have not been thoroughly examined. In the present study, we examined the pharmacological profile of [(11)C]Ro15-4513 binding in the monkey brain using positron emission tomography (PET). [(11)C]Ro15-4513 showed relatively high accumulation in the anterior cingulate cortex, hippocampus, and insular cortex, with the lowest uptake being observed in the pons. Accumulation in the cerebral cortex was significantly diminished by the BZ antagonist flumazenil (0.1 mg/kg, i.v.), but not that in the pons. Using the pons as a reference region, the specific binding of [(11)C]Ro15-4513 in most of the cerebral cortex including the limbic regions clearly revealed two different affinity sites. On the other hand, specific binding in the occipital cortex and cerebellum showed only a low affinity site. Zolpidem with affinity for alpha1, alpha2, and alpha3 subunits of GABA(A)/BZ receptor fully inhibited [(11)C]Ro15-4513 binding in the occipital cortex and cerebellum, while only about 23% of the binding was blocked in the anterior cingulate cortex. Diazepam with affinity for alpha1, alpha2, alpha3, and alpha5 subunits inhibited the binding in all brain regions. Since Ro15-4513 has relatively high affinity for the alpha5 subunit in vitro, these in vivo bindings of [(11)C]Ro15-4513 can be interpreted as the relatively high accumulation in the fronto-temporal limbic regions representing binding to the GABA(A)/BZ receptor alpha5 subunit.     

Localization of [H cyclohexyladenosine and [H nitrobenzylthionosine binding sites in rat striatum and superior colliculus

The localization of adenosine receptors labelled wit[³H cyclohexyladenosine ([³H CHA) and adenosine transport sites labelled wit[³H nitrobenzylthioinosine ([³H NBI) was examined in striatum and superior colliculus (SC) using radioligand binding and lesioning methods. Striatal kainic acid lesions significantly reduced the number (B_max) of a single class of higaffinity binding sites for [³H CHA by 50% and that for [³H NBI by 15% without alteringK_d values for either ligand. In SC, enucleations significantly reduced bothigand low affinity [³H CHA binding sites by about 60% while levels of [³H NBI binding were unaffected. Thus, adenosine receptors are present on striatal interneurons and retinal projections to the SC and some [³H NBI binding sites are located on striatal interneurons.