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The convulsant actions of methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM) and of methyl beta-carboline-3-carboxylate (beta-CCM) were evaluated in the baboon (Papio papio). DMCM, 0.6-4 mg/kg, induced epileptic seizures with short latency. DMCM convulsive seizures could be blocked by i.v. administration of the benzodiazepine agonist diazepam (10 mg). Similarly, beta-CCM, 0.3-3 mg/kg i.v., provoked generalized seizures in the baboons. These seizures were also reversed by the administration of propyl beta-carboline-3-carboxylate (3 mg/kg) or of diazepam (5 mg/kg). Combining the results from Positron Emission Tomography and the EEG assessments, benzodiazepine receptor occupancy by beta-CCM and DMCM was directly correlated with their convulsant actions in the living baboon. beta-CCM exerted its convulsant action in the living baboon at 76 and 74% benzodiazepine receptor occupancy in, respectively, occipital and temporal cortices whereas DMCM displayed a similar convulsive activity when only 58 and 65% of these receptors in the above regions were occupied.

It is reported that LY81067, a new diaryltriazine, possesses anticonvulsant properties against grand mal status epilepticus induced by intravenous administration of picrotoxin binding site ligands (Ro 5-4864 and pentylenetetrazole) in the baboon. Intravenous administration of LY81067 during the seizures blocked grand mal type electroencephalographic (EEG) paroxysmal discharges and led to a long electrical silence, progressively replaced by spike-and-wave discharges of low frequency (2 c/sec). A transient blocking effect was also observed when LY81067 was injected during grand mal status epilepticus induced by the benzodiazepine inverse agonist methyl beta-carboline-3-carboxylate; however, the long electrical silence observed after administration of LY81067 was rapidly followed by grand mal type paroxysmal discharges in the EEG, which could be stopped by a subsequent injection of Ro 15-1788. However, LY81067 also displayed intrinsic epileptogenic properties. Administration of this drug alone led to the appearance of rhythmic EEG (2-3 c/sec) associated with myoclonia. Concomitantly with the EEG studies, interactions of all these drugs with benzodiazepine receptors were observed in vivo using [11C]Ro 15-1788 as radioligand and positron emission tomography (PET) as a non-invasive technique to measure the binding of the [11C]benzodiazepine antagonist in brain, in vivo. The [11C]Ro 15-1788 bound in the brain could not be displaced by the administration of LY81067 but rather, the [11C]antagonist binding in the brain was somewhat enhanced. Administration of pentylenetetrazole or Ro 5-4864 decreased the rate of wash-out of the radioligand. This fast effect of these two convulsant drugs was partially inhibited by the subsequent administration of LY81067. The concomitant blocking of the grand mal status epilepticus was also observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Positron emission tomography (PET) was used to investigate, in the living baboon, the in vivo modulation of [11C]Ro 15-1788 binding to benzodiazepine receptors in brain and the changes with ligands acting at the supramolecular complex during status epilepticus induced by pentylenetetrazole. The central type benzodiazepine receptors were labelled in vivo by intravenous injection of [11C]Ro 15-1788. Simultaneous positron emission tomography and electroencephalographic activity recording evidenced a modulation of the brain binding of [11C]Ro 15-1788 during pentylenetetrazole-induced status epilepticus. We investigated the changes in the modulation of radioligand kinetics and in seizure activity after intravenous administration of a benzodiazepine agonist (diazepam, 1.5 mg/kg), a benzodiazepine antagonist (Ro 15-1788, 2 mg/kg), a GABA agonist (progabide, 50 mg/kg) and a ligand of the picrotoxin/barbiturate binding sites (LY81067, 3.5 mg/kg). The results showed that there is an in vivo competitive interaction of pentylenetetrazole with the benzodiazepine receptors, as reflected by the low displacement of [11C]Ro 15-1788 in the first 10 min of the status epilepticus. However, in contrast to diazepam, progabide and LY81067, a dose (2 mg/kg) of Ro 15-1788 that saturates the benzodiazepine receptors was unable to block the seizures induced by pentylenetetrazole. This indicates that the benzodiazepine receptors play only a minor role in the status epilepticus induced by pentylenetetrazole. The contribution of other binding sites within the supramolecular complex is assessed.

The brain regional distribution and kinetics of RO 15-1788, a benzodiazepine (BZD) antagonist labeled with 11C was studied by time-of-flight positron tomography after intravenous injection in four normal human volunteers. In two control studies, there was a high uptake of [11C]RO 15-1788 in gray matter structures initially (brain/blood ratio approximately 3), and subsequent retention that was highest in cerebral cortex, a structure known to have a high density of BZD receptors in vitro. Variation in tissue kinetics of [11C]RO among different gray matter structures may, however, suggest regional differences in binding characteristics or environment of BZD receptors. In two displacement studies, unlabeled RO 15-1788 was injected ten minutes after the radioligand: there was an immediate and marked washout of [11C]brain radioactivity that reached 70% in the occipital cortex with a 0.05 mg/kg dose (indicating a high specific to non-specific binding ratio) but was less prominent with a 0.01 mg/kg dose. These data suggest that [11C]RO 15-1788 may be useful for in vivo mapping of human brain BZD receptors using positron tomography.

A method of labelling ethyl 8-fluoro-5,6-dihydro-5-[11C]methyl-6-oxo-4H-imidazo[1,5-a][1,4] benzodiazepine-3-carboxylate (RO 15.1788 11C), a benzodiazepine antagonist with carbon-11 has been developed. RO 15.1788-11C was prepared by methylation of the nor derivative by I11CH3. About 100 mCi (maximum 153 mCi, 5.66 GBq) of the chemically and radiochemically pure labelled product were obtained within 25 min with a specific activity on average of 1100 mCi/mumol (maximum 1740 mCi/mu mol--64.4 GBq/mu mol). Preliminary results obtained after i.v. administration in the baboon have shown RO 15.1788-11C to be of interest as a benzodiazepine radioligand for the in vivo study of benzodiazepine receptors by positron emission tomography.

Bromolisuride, an ergoline derivative, was labeled with the positron emitter radionuclide, bromine 76. In vitro and in vivo binding and competition studies in rats demonstrated a high affinity (KD = 0.3 nM) and a high specificity of this new radioligand for D-2 dopamine receptors. PET kinetic studies in baboons showed an accumulation of [76Br]bromolisuride in the striatum which reached a maximum 30 min post-injection and which could be displaced by haloperidol. All these results indicated that this new ligand is certainly suitable for the non-invasive in vivo quantitative imaging of D-2 dopamine receptor sites in human brain.

The selective benzodiazepine antagonist RO 15-1788, labelled with carbon 11 [11C] RO 15-1788, as a specific marker, together with positron emission tomography, allows the in vivo study of benzodiazepine receptors in primates. In addition, when coupled with recordings of electroencephalographic activity, this method offers the feasibility of studying the correlation between occupancy of benzodiazepine receptors and the convulsant action of drugs acting at the benzodiazepine-GABA receptor complex in vivo. The present study showed that convulsant doses of pentylenetetrazol (PTZ) could affect the binding of [11C] RO 15-1788 in vivo in two ways, depending on the doses tested: at concentrations of 20 and 30 mg/kg, pentylenetetrazol increased the binding of [11C] RO 15-1788 whereas larger concentrations displaced the binding of [11C] RO 15-1788. The direct correlation between the occupancy of respective benzodiazepine receptors, afforded by increasing convulsant doses of pentylenetetrazol, revealed that competitive interaction with benzodiazepine receptors was not necessary for pentylenetetrazol to induce the appearance of seizures in vivo.

After labelling the brain benzodiazepine receptors of sub-human primates with [11C]RO15-1788, the interactions of different benzodiazepine receptor antagonist ligands were studied by positron emission tomography (PET). Various doses of either RO15-1788, RO15-3505 or propyl beta-carboline-3-carboxylate were injected intravenously 20 min after the radiotracer, and induced an immediate and specific dose-dependent displacement of the brain radioactivity. However, a comparison of the dose-receptor occupancy patterns of these three antagonists established from the displacement experiments revealed that only propyl beta-carboline-3-carboxylate displayed clear biphasic dose-receptor occupancy curves. This indicates that, in the living primate brain, there are two different benzodiazepine receptor subpopulations (which can be either different benzodiazepine receptor subtypes or distinct conformational states of a single receptor).

Using Positron Emission Tomography (PET) and specific radioligands, dopaminergic D2 (DA-D2 receptors) and benzodiazepine receptors (BZ-receptors) were studied in living animals during normal and pathological conditions. In vivo characterization of both receptors was performed using two highly specific antagonists namely: 11C-Ro 15 1788 for BZ-receptors and 76 Br-Bromospiperone for DA-D2 receptors. Changes in 11c-Ro 15 1788 specific binding to BZ-receptors were observed during convulsive seizures. After MPTP treatment, a decrease in the 76 Br-Bromospiperone striatal specific binding was observed, correlated with the establishment of a Parkinson-like syndrome.

In vivo binding of a benzodiazepine (flunitrazepam-C11) and a benzodiazepine antagonist (Ro 15-1788-C11) were studied with positron emission tomography. Advantages and disadvantages of each drug for studying specific in vivo binding sites are presented. The results obtained indicate that Ro 15-1788-C11 is a better in vivo radiocoordinat than flunitrazepam-C11.

An anatomical atlas has been constructed of the brain of the baboon (Papio papio) in the orbito-meatal plane (OM-plane) which is frequently used in experimental positron emission tomography (PET) investigations. The atlas comprises 12 photographic reproductions of histological brain sections separated by 2.5 mm intervals, and covers telencephalic to pontine brain stem levels. The anatomical atlas was used in analysis of some PET scan images obtained after administration of either a benzodiazepine (BZ) antagonist, (11C)-Ro 15-1788, or a dopamine D2 receptor antagonist, (76Br)-bromospiperone. Since PET camera detects radiation emitted from a slice of tissue of 15 mm thickness, each PET image corresponds to the tissue represented on six levels of the anatomical atlas. In optimal conditions, the PET image shows a pattern of receptor labelling reminiscent of anatomical structures in the atlas. Sometimes, however, the superimposition of different labelled structures yields a PET image which lacks any apparent resemblance with individual anatomical structures. In these cases, the analysis of the PET scan must rely on the anatomical atlas, as well as available data on the distribution of specific binding sites.

An in vivo characterization of specific central type benzodiazepine (BZD) binding sites, labelled with [11C]Ro 15-1788 was performed, using positron emission tomography. After i.v. injection of 10 mCi [11C]Ro 15-1788 (corresponding to 1 nmol/kg), sequential quantitative tomographic slices of the brain were obtained during 80 min. In some experiments various doses of different cold drugs (BZD agonist or antagonist) were injected i.v. subsequently in order to explore the specificity of the binding of the radioligand in brain structures. The main criteria usually utilized in vitro to demonstrate a specific binding to receptors, such as regional distribution, stereospecificity and saturability of the binding and pharmacological effect linked to the receptor's occupancy, were demonstrated in the brain of a living baboon.

The feasibility of visualizing the heterogeneity of benzodiazepine (BDZ) receptors in the brain of living baboons was investigated using Positron Emission Tomography. Ethyl 8-fluoro-5,6-dihydro-5-methyl 6-oxo-4H-imidazo (1,5-a) (1, 4) benzodiazepine-3-carboxylate (RO 15 1788) labelled by carbon 11 (11C-RO 15 1788) was I.V. injected for the "in vivo" labelling of the central type BDZ receptors. Displacement experiments were performed 20 minutes after the administration of the radioligand by two different cold drugs: RO 15 1788 which has an equal affinity for central type BDZ receptors, and propyl B-Carboline-3-carboxylate (B-CCP) which favours the sites located in the cerebellum. Different sensitivities to these two drugs displacement of 11C-RO 15 1788 binding "in vivo" were observed: on the one hand in the regional localization of the displacement, and on the other hand, in the amount of the radioactivity displaced. The apparent interregional heterogeneity of the displacement seen in the cerebellum and in the temporal cortex are discussed in terms of discrepancies observed "in vitro" at physiological temperature, between cerebellar and non-cerebellar BDZ central type binding sites.

Bromine-76 labeled bromospiperone has been prepared with a very high specific activity (greater than 1 Ci/mumole). In-vivo studies in rat corroborated by PET studies in baboon have shown that the regional concentration of this radioligand parallels the morphologic distribution of dopamine receptors and that its binding in the striatum is saturable and displaceable. Tomographic images show a clear delineation of the striatal region 2.5 hours after administration of the radioligand.

Ibotenic acid was injected unilaterally into the baboon caudate-putamen (CP) to achieve a neural degeneration model in the primate, with a neuropathology similar to Huntington's disease. Four to six weeks later injections of cell suspensions of striatal precursor cells, obtained by dissection of the fetal rat striatal region (13-15 days gestational age), were made into the excitotoxically lesioned CP of 3 baboons immunosuppressed by Cyclosporin A. Morphological analysis indicated that in one of the baboons, which had the largest lesion of the CP and the shortest survival time (6 weeks after implantation), there was a surviving striatal implant. The implanted neurons grew in high densities in cellular aggregates within the host gliotic CP. These neurons had a neuronal size phenotypical for rat striatum, i.e. on average about a 25% smaller neuronal cell diameter than a similar population in the baboon caudate-putamen. Glial-fibrillary-acid-protein immunoreactivity was present on large astrocytes within the striatal implant, with a distinct border towards the lesion-induced astrogliosis of the host. Neuronal markers for acetylcholinesterase and Leu-enkephalin were distributed in a typical patchy manner in the striatal implants along with fiber staining for tyrosine-hydroxylase-like immunoreactivity (TH) possibly derived from afferent host dopaminergic axons. Some of these fibers in the implants came from intrinsic TH-positive neuronal somata, probably of neocortical fetal origin and transiently expressing the enzyme. In conclusion, the results indicate that neuronal replacement can be achieved by cross-species implantation of fetal striatal precursor cells to the previously neuron depleted primate CP under immunosuppression but that the survival and growth of such implants may be variable and subject to unfavourable trophic conditions.