G.N. Woodruff

MK-801 is neuroprotective in gerbils when administered during the post-ischaemic period

The neuroprotective effects of the non-competitive N-methyl-D-aspartate receptor antagonist (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801) have been evaluated in the gerbil hippocampus when the drug was administered i.p. at various times during and after a 5 min period of transient forebrain ischaemia, induced by bilateral common carotid artery occlusion. A single dose of 1, 3 or 10 mg/kg of MK-801 gave significant protection of hippocampal CA1 and CA2 pyramidal neurons when administered during the occlusion and up to 24 h following the period of ischaemia. A dose of 0.3 mg/kg was effective when administered during the occlusion period but gave no protection at 30 min or 2 h post-ischaemia. Experiments in which MK-801 was administered in repeated doses indicated that significant protection was achieved with 1 mg/kg of MK-801 repeated post-ischaemically and with 1 mg/kg MK-801 supplemented with repeated doses of 0.3 mg/kg of MK-801. However 0.3 mg/kg of MK-801 followed by repeated doses of 0.03 mg/kg administered post-ischaemically was not neuroprotective. These results indicate that MK-801 can protect hippocampal neurons from ischaemia-induced neuronal degeneration when it is administered up to 24 h after the insult. These data provide further evidence that therapeutic intervention in the post-ischaemic period can successfully prevent neurodegenerative events, and that the delayed degeneration of hippocampal neurons following an ischaemic insult occurs by an N-methyl-D-aspartate receptor-mediated process.

Differentiation of central cholecystokinin receptor binding sites using the non-peptide antagonists MK-329 and L-365,260

Cholecystokinin (CCK) receptor binding was measured in rodent and primate brain and spinal cord using 125I-Bolton Hunter CCK-8 (125I-BH-CCK) and the selective non-peptide CCK antagonists MK-329 and L-365,260. In homogenate binding studies, L-365,260 displayed nanomolar affinity for CCK-B receptors in the cerebral cortex of several species including man (pIC50 congruent to 8.2) but showed low affinity for CCK-A receptors in the rat pancreas (pIC50 congruent to 6.3). By contrast, the CCK-A antagonist MK-329 showed the reverse selectivity (cortex: pIC50 congruent to 6.9, pancreas: pIC50 = 9.6). In autoradiographs of rat and monkey brain. 125I-BH-CCK binding was localized regionally with high levels being detected in the cerebral cortex, basal ganglia and some mid- and hindbrain nuclei. Specific 125I-BH-CCK binding was also localized to the substantia gelatinosa of the rat, monkey and human spinal cord. L-365,260 inhibited binding to most areas of the brain, but in the rat medial nucleus tractus solitarii and the monkey nucleus tractus solitarii. dorsomedial nucleus and infundibular hypothalamic nuclei together with the dorsomedial aspects of the caudate nucleus, where CCK-A sites are present, L-365,260 failed to displace all 125I-BH-CCK binding. In the primate spinal cord, L-365,260 was a relatively weak inhibitor of 125I-BH-CCK binding (pIC50 congruent to 6.0) whereas MK-329 showed high affinity for the CCK-A sites present there (pIC50 congruent to 9.6).(ABSTRACT TRUNCATED AT 250 WORDS)

The interaction between MK-801 and receptors for N-methyl-D-aspartate: functional consequences.

Electrophysiological studies using a cortical slice preparation revealed that MK-801 is a potent, non-competitive, antagonist of N-methyl-D-aspartate (NMDA)-induced depolarisations. Also, MK-801 was a highly selective antagonist, exhibiting a high degree of use-dependency. It completely blocked responses to NMDA and quinolinic acid but had no effect on responses produced by kainic acid or quisqualic acid. Using [3H]MK-801, high affinity binding sites for MK-801 were detected in membranes of the rat brain. The pharmacological specificity and regional distribution of binding sites for MK-801 were consistent with an action of the compound at the level of the NMDA receptor-associated ion channel. Administered parenterally, MK-801 had a remarkable neuroprotective role. It caused essentially complete protection against loss of neurones produced by injection of neurotoxic doses of NMDA or quinolinic acid into the striatum or hippocampus. Furthermore, MK-801 was highly effective in preventing loss of hippocampal neurones following bilateral occlusion of the common carotid arteries in the gerbil.

Systemic administration of MK-801 prevents N-methyl-d-aspartate-induced neuronal degeneration in rat brain

MK-801 is a novel, potent and selective non-competitive antagonist of the N-methyl-D-aspartate (NMDA) subtype of excitatory amino acid receptors. Pretreatment of rats with MK-801 (1-10 mg/kg, i.p.) prevented neuronal degeneration in the hippocampus and striatum caused by direct intracranial injections of NMDA (20-120 nmol), but did not protect against the loss of neurones induced by kainate (2.5 nmol) injected into the striatum. Thus, MK-801 is a selective antagonist of neuronal degeneration caused by excessive stimulation of NMDA receptors in vivo.

Autoradiographic studies in animal models of hemi-parkinsonism reveal dopamine D2 but not D1 receptor supersensitivity. I. 6-OHDA lesions of ascending mesencephalic dopaminergic pathways in the rat

The selective dopaminergic antagonist ligands [3H]SCH 23390 and [3H]sulpiride were used to reveal autoradiographically dopamine D1 and D2 receptors, respectively, in brain sections from rats which had received unilateral 6-hydroxydopamine (6-OHDA) injections destroying ascending nigrostriatal neurones. The binding of both ligands to striatal sections was first shown to be saturable, reversible and of high affinity and specificity [( 3H]SCH 23390: Bmax 2.16 pmol/mg protein, Kd 1.4 nM; [3H]sulpiride; Bmax 0.67 pmol/mg protein, Kd 10.7 nM). After unilateral stereotaxic 6-OHDA injections, rats rotated contralaterally when challenged with apomorphine (0.5 mg/kg), or specific D1 or D2 agonists, SKF 38393 (1.0-5.0 mg/kg) and LY 171555 (0.05-0.5 mg/kg), respectively. Loss of forebrain dopaminergic terminals was assessed autoradiographically using [3H]mazindol to label dopamine uptake sites. A loss of approximately 90-95% of uptake sites was reproducibly accompanied by an enhanced density of binding ipsilaterally for the D2 ligand, [3H]sulpiride, in all areas of the striatum, but most markedly in the lateral areas. An increase in the D2 binding site density was also seen in the ipsilateral nucleus accumbens and the olfactory tubercle. In contrast, in the same animals, the striatal D1 receptors were far less affected by dopaminergic denervation, with no consistent changes seen in the binding of [3H]SCH 23390. These results suggest that dopamine D2 receptors are more susceptible than D1 receptors to changes after dopaminergic denervation, which is expressed as an increase in the density of binding sites revealed here with [3H]sulpiride.

Autoradiographic studies in animal models of hemi-parkinsonism reveal dopamine D2 but not D1 receptor supersensitivity. II. Unilateral intra-carotid infusion of MPTP in the monkey (Macaca fascicularis)

The selective dopaminergic antagonist ligands [3H]SCH 23390 and [3H]sulpiride were used to reveal autoradiographically dopamine D1 and D2 receptors, respectively, in brain sections from monkeys which had received unilateral intracarotid infusions of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), causing loss of dopamine-containing neurones of the substantia nigra pars compacta. The monkeys developed hemi-parkinsonian symptoms (tremor, bradykinesia) in limbs contralateral to the side of the toxin infusion. Administration of apomorphine (0.05-0.25 mg/kg) caused contralateral rotational behaviour, and reversal of the parkinsonian symptoms. Loss of forebrain dopaminergic terminals was assessed autoradiographically using [3H]mazindol to label dopamine uptake sites. A reduction in these sites of 97% (mean brain value) in the caudate nucleus, and 91% in the putamen, as compared with binding values from untreated control monkeys, was accompanied by a significant increase in the binding of [3H]sulpiride (D2) in these structures. In contrast, in the same animals there was no similar increase in [3H]SCH 23390 binding to D1 receptors in the denervated areas. These results suggest that in the parkinsonian brain, where the dopaminergic innervation of the caudate nucleus and putamen has been lost, D2 receptors may be more susceptible than D1 receptors to changes, revealed here as an increase in [3H]sulpiride binding sites.

Species differences in the localization of ‘peripheral’ type cholecystokinin receptors in rodent brain

A comparison of cholecystokinin (CCK) receptor binding to sections of rat, mouse and guinea pig brain has been performed using 125I-Bolton Hunter CCK and the selective peripheral CCK receptor antagonist L-365,031. In both rat and mouse, 125I-Bolton Hunter CCK binding in the region of the interpeduncular nucleus (IPN) was inhibited by L-365,031 indicating that these receptors resemble CCK receptors found in peripheral tissues. In the mouse especially, dense regions of peripheral CCK receptors were detected either side of the IPN. By contrast, in the guinea pig IPN no evidence of L-365,031-sensitive binding could be found. The present reports shows that in different species, regional variations in brain CCK receptor binding occur not only in the case of classical brain receptors, but also for the more discretely localised peripheral type CCK receptors.

Binding sites for 125I-cholecystokinin in primate spinal cord are of the CCK-A subclass

Cholecystokinin (CCK) receptor binding was measured in sections of human, monkey and rat spinal cord using autoradiographical techniques. In each species, high levels of specific 125I-Bolton-Hunter CCK binding were detected in the superficial layers of the dorsal horn (the substantia gelatinosa). In monkey and human but not rat spinal cord, 125I-CCK binding was dose-dependently inhibited by low concentrations of the selective CCK-A antagonist L-364,718. Binding of [3H]L-364,718, which was saturable (Bmax = 29.0 +/- 0.95 pmol/g wet wt.) and of high affinity (pKd) = 9.92 +/- 0.16) was also detected in sections of monkey spinal cord and had a similar localization to that of specific 125I-CCK binding. These data indicate that in striking contrast to CCK receptors in rat spinal cord, those in the primate cord are of the CCK-A receptor subclass.

CCK-A receptors in the rat interpeduncular nucleus: evidence for a presynaptic location.

Using autoradiography, peripheral type or cholecystokinin-A (CCK-A) receptor binding was measured in the interpeduncular nucleus (IPN) of rats which had received electrolytic lesions of the habenular nucleus. Presynaptic GABAB receptor binding was also determined in the IPN in order to verify the loss of input to the nucleus. In animals which showed almost total loss of GABAB receptors. 125I-Bolton Hunter CCK binding in the IPN was reduced from 1.83 +/- 0.28 pmol/g wet weight to 0.87 +/- 0.16 pmol/g wet weight. This was not significantly different from non-specific levels, determined in the same region using 10(-7) M L-365,031 (0.97 +/- 0.14 pmol/g wet weight). These data suggest that CCK-A receptors in rat IPN are localized on presynaptic terminals within the nucleus.

Reduction of [125I]Bolton Hunter CCK8 and [3H]MK-329 (devazepide) binding to CCK receptors in the substantia nigra/VTA complex and its forebrain projection areas following MPTP-induced hemi-parkinsonism in the monkey

Cholecystokinin (CCK) receptors were visualized autoradiographically using [125I]Bolton Hunter CCK8 ([125I]BHCCK8) in the fore- and midbrain of 3 monkeys rendered hemi-parkinsonian by unilateral intra-carotid infusion of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). More specifically, CCK-A receptors were detected using [3H]MK-329 (devazepide), a peripheral-type (CCK-A) receptor antagonist. In the substantia nigra pars compacta, ipsilateral to the toxin infusion, where dopamine D2 receptors (labelled with [3H]sulpiride) were lost, there was a decrease in the binding of both [125I]BHCCK8 and [3H]MK-329. Binding of the two CCK ligands was also reduced in the ipsilateral nucleus accumbens and most medial part of the caudate nucleus, whereas 3H-sulpiride binding was increased in the lateral caudate nucleus and putamen. These results indicate that CCK-A receptors may be located on dopaminergic cells within the substantia nigra, which are lost in the parkinsonian brain, and may also be present on dopaminergic terminals within restricted regions of nigral/ventral tegmental area projection sites.