Biomedical subjects
Publications and source records attributed to J T Coyle.
The synthesis and the interaction with brain receptors of some 2-carboxy-5-isopropenylcyclopentaneacetic acids, C-analogues of kainic acids, are reported. They do not displace kainic acid at cortical or striatal receptors.
Huntington's Disease (HD) is a progressive neurologic disorder transmitted as autosomal dominant. The symptoms of HD, which typically appear in midlife, include disturbances in movement, psychiatric symptoms, and a progressive dementia. Neuropathologic studies indicate a distinct pattern of neuronal degeneration in HD that affects many areas of the brain but consistently and severely involves the basal ganglia including the caudate, putamen, and globus pallidus. The basal ganglia undergo a progressive atrophy due to degeneration of intrinsic neurons that results in ventricular enlargement. Over the last decade, detailed neurochemical analyses have been carried out on the brains of patients who have died with HD. These studies have demonstrated the selective degeneration of chemically defined neuronal systems including the striatal cholinergic intrinsic neurons, the striatal nigral GABAergic pathway, and striatal peptidergic neurons with the relative sparing of other systems such as the nigrostriatal pathway. These findings have resulted in a better understanding of the pathophysiologic basis for the movement disorder of HD and have led to the development of pharmacologic strategies to correct the synaptic neurochemical imbalances. Recent studies have begun to focus on mechanisms responsible for the selective neuronal degeneration in HD. One promising hypothesis evolved from the finding that intrastriatal injections of excitatory amino acid analogues reproduces the neurochemical and histologic pathology of HD in experimental animals; as a consequence, it has been hypothesized that dysfunction of excitatory amino acid neurotransmission may cause the selective neuronal degeneration of HD. Another hypothesis involves an abnormality of the plasma membrane based upon observations of differences in membrane characteristics in fibroblasts and red blood cells from HD patients as compared to suitable controls. The ultimate goals of these studies are to develop methods for identifying presymptomatic carriers of the HD gene as well as strategies for preventing the neuronal degeneration associated with expression of the gene.
Quinolinic acid evoked the release of [3H]acetylcholine from striatal slices with an EC50 of approximately 2 mM and an efficacy similar to that of N-methyl-D,L-aspartic acid. (+/-)-2-Amino-5-phosphonopentanoic acid and (-)-2-amino-7-phosphonoheptanoic acid antagonized quinolinic acid-evoked release, while glutamate diethylester and 2-amino-4-phosphonobutyric acid were ineffective as antagonists. Release of [3H]acetylcholine evoked by quinolinic acid was strongly attenuated by the presence of magnesium (1.2 mM) in the medium. The results are consistent with the interaction of quinolinic acid with NMDA-type excitatory amino acid receptors on the striatal cholinergic interneurons.
Slices prepared from hippocampus were dual prelabeled with [14C]L-glutamate and [3H]D-aspartate and superfused in the presence or absence of kainic acid (KA; 1 mM) or KCl (40 mM). Drug-evoked release of radioactivity as well as the endogenous amino acids, glutamate and aspartate were determined. Whereas KCl-induced a Ca2+-dependent release of all compounds, only the release of the endogenous amino acids was stimulated by KA. The results of the present study demonstrate that disparities exist between the Ca2+-dependent drug-evoked release of exogenously supplied and endogenous acidic amino acid neurotransmitters.
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The specific binding of [3H]+/- 2-amino-7-phosphono heptanoic acid (3H-APH), a potent N-methyl-D-aspartate (NMDA) antagonist, to extensively washed, previously frozen crude mitochondrial fractions of rat brain is described. Binding was optimal at physiological pH and temperature and, in Triscitrate buffer, attained equilibrium within 60 minutes. Scatchard analysis of the equilibrium data for forebrain revealed a single, non-interacting population of binding sites (BMapp = 15 picomoles/mg protein; KDapp = 3.6 uM; Hill coefficient = 0.92, r = 0.99; N = 5). Specific binding of the ligand was readily reversible by unlabeled APH and was absent in peripheral tissues including heart, lung, kidney, liver, spleen and striate muscle and in heat treated brain sonicates. An 8-fold variation in the amount of ligand bound to brain membranes prepared from different regions was observed with binding being greatest in the hippocampal formation and least in the midbrain. Kainic acid, NMDA and aspartic acid exhibited negligible affinity for the [3H]-APH site; in contrast, quisqualic acid, ibotenic acid, glutamatic acid, homocysteic acid and 2-amino-4-phosphono butyric acid were moderately potent displacers. The results indicate that [3H]-APH labels a quisqualate preferring site in vitro. Unlike the receptor labeled by [3H]-glutamate however, [3H]-APH binding was attenuated in the presence of chloride ions suggesting that this ligand may label a subpopulation of excitatory amino acid receptors.
The basal forebrain-cortex connections of the rat were topographically mapped by retrograde tracer methods; and their contribution to the cholinergic innervation of the cortex was assessed by excitotoxin lesions placed in the rostral and caudal aspects of the complex. Discrete injections of tracer into frontal cortex labeled the prominent multipolar acetylcholinesterase (AchE)-positive cells of the ventromedial globus pallidus. Injections of tracer into the parietal cortex labelled cells in the ventral globus pallidus, the underlying substantia innominata, and the lateral hypothalamus. Separate injections of Fast Blue and Nuclear Yellow in the frontal and in the parietal cortex resulted in double-labeled cells in the ventral globus pallidus, which indicates that at least some of these cells may possess collateralizing axons. The cingulate cortex is innervated predominantly by neurons in the nucleus of the horizontal limb of the diagonal band. The occipital cortex was also shown to receive a projection primarily from the nucleus of the horizontal limb of the diagonal band. The hippocampal formation is innervated primarily by cells located in the vertical limb of the diagonal band and in the medial septum. Consistent with the results of the retrograde tracing studies, excitotoxin lesions affecting the diagonal band and medial septum decreased choline acetyltransferase (CAT) activity up to 40% on the occipital cortex and by 64% in the hippocampus, but did not affect CAT activity in the rostral neocortex. In contrast, ibotenate lesions of the caudal ventral globus pallidus and substantia innominata caused decreases in CAT activity in the frontal cortex of up to 65% without affecting enzyme activity in the hippocampal formation. The results of the present study provide details on the topographic organization of the cortical projections originating in the basal forebrain complex and indicate that these neurons are the predominant source of cortical cholinergic innervation.
The kinetics of interaction of kainic acid analogues and reputed antagonists of acidic excitatory amino acids with a specific binding site for [3H]alpha-kainic acid were examined in washed cerebellar membranes incubated at 4 degrees C. The avidity of both L-glutamate (1.5 microM) and quisqualate (0.6 microM) suggests that proper orientation of the two carboxyls in the amino group is essential for binding to one component of the receptor. The high affinity of domoate, alpha-kainate and alpha-keto-kainate as compared to the low affinity of dihydrokainate and alpha-allo-kainate indicate that a pi electron group with appropriate cis-planar orientation versus the C2-carboxyl 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.
Great emphasis is being placed on identification of neurotransmitter systems involved in the symptomatic manifestations of neurological and psychiatric disorders. In the case of Alzheimer's disease, which now seems to be one of the most common causes of mental deterioration in the elderly, compelling evidence has been developed that acetylcholine-releasing neurons, whose cell bodies lie in the basal forebrain, selectively degenerate. These cholinergic neurons provide widespread innervation of the cerebral cortex and related structures and appear to play an important role in cognitive functions, especially memory. These advances reflect a close interaction between experimental and clinical neuroscientists in which information derived from basic neurobiology is rapidly utilized to analyze disorders of the human brain.
The effects of lesions induced by fetal treatment with methylazoxymethanol acetate (MAM) on the developmental synaptic chemistry of the frontal cortex of rats were examined. Treatment at 14 days' gestation (DG) resulted in a 50% decrease in frontal cortical mass whereas treatment at 15 DG caused a 40% decrement. No difference in concentration between MAM and control rats was observed in the postnatal development of the GABA-ergic markers, glutamate decarboxylase, and GABA. In contrast, the concentrations of the presynaptic markers for the dopaminergic, noradrenergic, and serotonergic terminals were generally elevated in the MAM lesioned frontal cortex throughout postnatal development. The effects of MAM lesion on cholinergic markers were mixed, with minimal alterations in concentration during the first 2 postnatal weeks followed by an increasing disparity thereafter. The results of this study provide additional evidence in support of the differential impact of the cortical hypoplasia on intrinsic neurons as contrasted to aminergic afferents.
A brain peptide with high affinity (420 nM) and marked specificity for brain receptor sites labeled with L-[3H]glutamate has been identified. Amino acid analysis and mass spectroscopy indicate that the peptide is N-acetylaspartylglutamate. The peptide exhibits potent convulsant properties when injected into the rat hippocampus, similar to those produced by the glutamate receptor agonist, quisqualic acid. These findings raise the question whether endogenous brain peptides enriched in acidic amino acids may serve as excitatory neurotransmitters.
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Kainic acid (KA) exhibits both neuroexcitatory and neurotoxic actions when applied to the brain. Whereas the neuroexcitatory actions are direct, the neurotoxic effects require the integrity of excitatory afferents. In addition, specific receptors for KA have been demonstrated on both pre- and postsynaptic neuronal elements. In the current study, both KA and KCl are shown to stimulate selectively and in a Ca++-dependent manner the efflux of endogenous Glu and Asp from hippocampal, striatal and cerebellar slices incubated in vitro. Unlike the release of other amino acids, the efflux induced by KCl, but not by KA, is attenuated by tetrodotoxin, suggesting that the action of KA is direct. Furthermore, the action of KA is not mimicked by GABAergic, cholinergic, dopaminergic or serotonergic agonists or antagonists, nor by the excitotoxins N-methyl-DL-aspartic acid or ibotenic acid. Both dihydrokainic acid and allokainic acid, which have low affinity for the KA receptor, also fail to stimulate Glu and Asp efflux. In the case of cerebella from 10-day-old rats, which lack a mature parallel fiber system, KA stimulated the efflux of several amino acids including Asp, but not Glu. Similarly, in slices prepared from adult granulo-prival mice, KA caused an efflux of Asp comparable with that observed in intact cerebellum, whereas the release of Glu was reduced by 75%. On the basis of these studies, the primary source of KA-stimulated Glu efflux in the cerebellar slices appears to be the granule cell-parallel fiber system. A hypothesis is proposed that KA stimulates the release of excitatory amino acids, independent of impulse flow, by activating presynaptic receptors located on excitatory nerve terminals. This additional site of action of KA at excitatory afferents may contribute to the potent but uneven neurotoxic effects of this compound.
The kinetics of interaction of excitatory amino acid analogues and reputed antagonists with a specific binding site for [3H]L-glutamate, having a Kd of 600 nM, were examined in washed cerebellar membranes incubated at 37 degrees C. Displacement curves were analyzed by an iterative computer program for a non-cooperative two-site competitive inhibition model. L-Glutamate, D-glutamate and D-aspartate exhibited simple, mass action kinetics with Hill coefficients near unity and Kis of 1.1, 9.3 and 23.3 microM, respectively. Quisqualate, ibotenate and cyclopentylglutamate had Hill coefficients less than 0.85 and bound to an high affinity component with KHS of 0.4, 0.8 and 1.7 microM, respectively. Neither N-methyl-D-aspartate nor derivatives of kainate, with the exception of a-keto kainate, had KiS less than 0.1 mM. Linear analogues of glutamate with reputed antagonistic properties all exhibited shallow displacement curves with Hill coefficients less than 0.6 and KHS varying from 0.5 to 6.6 microM. Notably, 2-amino-6-phosphonocaproic acid had negligible affinity for the site in contrast to the valeric and pimelic phosphono analogues. The results indicate that [3H]L-glutamate labels a single class of sites that can be resolved into subpopulations by agonists and antagonists and provide additional evidence of excitatory amino acid receptor heterogeneity.
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The somatostatin content and choline acetyltransferase activity of the rat cerebral cortex and hippocampus were examined after lesions to the nucleus basalis, the fornix and the dorsal hippocampus. Lesions of the nucleus basalis caused reductions in cortical choline acetyltransferase activity but had no effect on the concentration of somatostatin. Fornix transection caused a reduction in choline acetyltransferase activity in the dorsal hippocampus, but again was without effect on the somatostatin content. Excitotoxin lesions of the dorsal hippocampus caused a 69% reduction in somatostatin concentration in this structure, with no reduction in choline acetyltransferase activity. The results indicate that somatostatin and choline acetyltransferase are in separate populations of neurons in the rat cerebral cortex and hippocampus.