Effects of antiepileptic drugs on the developing central nervous system.
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Biomedical subjects
B R Ransom
Publications and source records attributed to B R Ransom.
Effects of polyvalent cations and dihydropyridine calcium channel blockers on recovery of CNS white matter from anoxia.
The effects of anoxic injury on the functional integrity of mammalian central white matter were studied electrophysiologically using the rat optic nerve model. Previous studies on this model have shown that extracellular Ca2+ is critical to the production of irreversible anoxic injury, and suggest that during anoxia Ca2+ crosses the membrane to enter the intracellular compartment. We attempted to elucidate the mechanism by which this damaging Ca2+ influx occurs. The inorganic Ca2+ channel blockers Mn2+ (1 mM), Co2+ (1 mM) or La3+ (0.1 mM) had no effect on recovery of the area under the compound action potential after a standard 60 min period of anoxia; only Mg2+ (10 mM) significantly improved recovery (54.9 +/- 8.9% vs. 28.7 +/- 10.1%, P less than 0.005). Treatment with organic Ca2+ channel blockers of the dihydropyridine class, nifedipine (1-10 microM) or nimodipine (1-40 microM), also had no effect on recovery from anoxia. We conclude that Ca2+ influx during anoxia does not occur via conventional Ca2+ channels sensitive to polyvalent cations or dihydropyridines.
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Anoxic injury of mammalian central white matter: decreased susceptibility in myelin-deficient optic nerve.
The rat optic nerve, a typical central nervous system white matter tract, rapidly loses excitability when it is exposed to anoxia and is irreversibly damaged by prolonged anoxia. Neonatal optic nerve is extremely resistant to anoxia-induced dysfunction and injury; the adult pattern of response to anoxia appears between 10 and 20 days postnatal, that is, during the period of oligodendroglial proliferation and myelination. To test the hypothesis that myelination, or associated events, confer anoxic susceptibility on developing white matter, we analyzed the effects of anoxia on the myelin-deficient (md) strain of rat. Acutely isolated optic nerves from 19- to 21-day-old md rats and control optic nerves from unaffected male littermates were maintained in vitro at 37 degrees C, and exposed to a standard 60-minute period of anoxia. The supramaximal compound action potential was recorded and amplitude of the compound action potential, expressed as % of amplitude before anoxic exposure, was determined. The compound action potential was nearly abolished within 3 to 6 minutes after onset of anoxia in control optic nerves, while optic nerves from md rats displayed a slower decrease in compound action potential amplitude during anoxia, with a distinct action potential present even after 60 minutes of anoxia. Optic nerves from md rats showed significantly greater recovery of compound action potential (71 +/- 25%) than did control optic nerves (33 +/- 21%; p less than 0.02) after 60 minutes of anoxia. These findings support the hypothesis that myelination, or changes associated with it, may be important in the development of anoxic susceptibility in central white matter.
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Intracellular pH shifts capable of uncoupling cultured oligodendrocytes are seen only in low HCO3- solution.
Electrical coupling between cultured mouse oligodendrocytes was transiently blocked when pHi was decreased below about 6.5 using the NH4+ prepulse method. This uncoupling could, however, only be achieved if the dominant pHi regulating mechanism in these cells, the Na+/HCO3- cotransporter, was blocked by lowering bath [HCO3-]. Under this condition, an NH4+ prepulse caused pHi to decrease toward the passive distribution for H+ (i.e., about pH 6.2). In the presence of normal bath [HCO3-] an NH4+ prepulse did not decrease pHi below 6.5 even when the second pHi regulating mechanism, the Na+/H+ exchanger, was blocked by amiloride, and consequently oligodendrocytes could not be uncoupled. Increasing CO2, which uncouples glial cells in situ (Connors et al: J. Neurosci. 4:1324-1330, 1984), did not uncouple cultured oligodendrocytes in the presence of normal bath [HCO3-], but did cause uncoupling in low [HCO3-] solution. These results indicate that electrical coupling between cultured oligodendrocytes is sensitive to pHi; in normal bath [HCO3-], however, the pHi regulation of these cells is so effective that standard techniques for intracellular acidification are unable to lower pHi to levels which cause the closure of oligodendrocyte gap junctions.
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Electrical coupling, without dye coupling, between mammalian astrocytes and oligodendrocytes in cell culture.
Evidence of electrical and dye coupling between oligodendrocytes and astrocytes was sought in cultures of mouse spinal cord. Cell identity was verified using cell specific antigenic markers. In most experiments current was injected into oligodendrocytes while recording voltage in nearby astrocytes. Nine of 17 oligodendrocyte-astrocyte cell pairs showed weak electrical coupling; the average estimated coupling ratio was 0.03 +/- 0.06 (cf. 0.11 for oligodendrocyte-oligodendrocyte and 0.44 for astrocyte-astrocyte pairs; Kettenmann and Ransom: Glia, 1: 64-73, 1988). Application of 0.5 mM BaCl2 or 44.6 mM CsCl depolarized astrocytes and oligodendrocytes and was estimated to increase the coupling ratio between these cells 3-5-fold; these effects were rapid in onset and completely reversible. In 5 of 7 cases, oligodendrocyte-astrocyte pairs that appeared uncoupled in normal solution exhibited coupling during Ba++ or Cs+ exposure. The actions of these cations are believed to be mediated by blockade of glial K+ channels. Depolarization, per se, as induced by increasing [K+]o, did not increase coupling ratio. The fluorescent dye lucifer yellow (LY) was injected into 10 oligodendrocytes, 8 of which were electrically coupled to nearby astrocytes, and never passed into astrocytes in detectable quantities. Likewise, astrocytes injected with LY stained other astrocytes, but never oligodendrocytes. These findings document the presence of weak electrical coupling between astrocytes and oligodendrocytes, in the absence of dye coupling. Weak coupling of this sort could subserve metabolic interactions between these cells mediated by the passage of small but important molecules such as cyclic AMP, but would not allow strong electrical interactions. If such coupling among glial cells is widespread, it would constitute a "metabolic syncytium" that could serve to coordinate glial behavior.
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Macromolecular structure of axon membrane and action potential conduction in myelin deficient and myelin deficient heterozygote rat optic nerves.
The macromolecular structure of the axon membrane in optic nerves from 25-day-old male littermate control and myelin deficient (md) rats and 16-month-old md heterozygotic rats was examined with quantitative freeze-fracture electron microscopy. The axon membrane of control optic nerves displayed an asymmetrical partitioning of intramembranous particles (IMPs); P-fracture faces of myelinated internodal axon membrane were more particulate than those of pre-myelinated axons (approximately 1600 v 1100 microns-2, respectively), while relatively few IMPs (approximately 150 microns-2) were present on external faces (E-faces) of internodal or pre-myelinated axon membrane. Amyelinated axons of md optic nerves also exhibited an asymmetrical partitioning of IMPs; protoplasmic membrane face (P-face) IMP densities, taken as a group, exhibited a wide range (approximately 600-2300 microns-2) and, in most regions, E-faces displayed a relatively low IMP density (approximately 175 microns-2). Axons of greater than 0.4 microns diameter exhibited significantly greater mean P-face IMP density than axons less than 0.4 microns diameter. Aggregations of E-face IMPs (approximately 350 microns-2) were occasionally observed along amyelinated axon membrane from md optic nerves. Optic nerves from md heterozygote rats exhibit myelin mosaicism, permitting examination of myelinated and amyelinated axon membrane along the same tract. The axon membrane exhibits different ultrastructure in these two domains. Myelinated internodal axon membrane from md heterozygote optic nerves exhibits similar P- and E-face IMP densities to those of control internodal axolemma (approximately 1800 and 140 microns-2, respectively). Amyelinated axons in the heterozygote exhibit a membrane structure similar to amyelinated axons in md optic nerve. P-face IMP density of large diameter (greater than 0.4 microns) amyelinated axons from md heterozygote optic nerves is significantly greater than that of small calibre (less than 0.4 microns) axons. In most regions, amyelinated axon membrane exhibits a relatively low E-face IMP density (approximately 200 microns-2); however, focal aggregations (approximately 400 microns-2) of E-face particles are present. Electrophysiological recordings demonstrate that amyelinated axons in md optic nerves support the conduction of action potentials. Compound action potentials in md optic nerves exhibit a monophasic configuration, even at 20-days postnatal, similar to that of pre-myelinated optic nerve of 7-day-old normal rats. Moreover, conduction velocities in the amyelinated 20-day-old md optic nerve are similar to those displayed by pre-myelinated axons from 7-day-old optic nerves.(ABSTRACT TRUNCATED AT 400 WORDS)
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Role of extracellular calcium in anoxic injury of mammalian central white matter.
White matter (WM) of the mammalian brain is susceptible to anoxic injury, but little is known about the pathophysiology of this process. We studied the mechanisms of anoxic injury in WM using the isolated rat optic nerve, a typical central nervous system WM tract. Optic nerve function, measured as the area under the compound action potential, rapidly failed when exposed to anoxia and recovered to 28.5% of control after a standard 60-min period of anoxia. Irreversible anoxic injury was critically dependent on the molar concentration of extracellular calcium [( Ca2+]o); maintaining the tissue in Ca2(+)-free solution throughout the anoxic period resulted in 100% compound action potential recovery. Increasing perfusion [Ca2+] during anoxia from zero to 4 mM resulted in progressively less recovery. As the introduction of the Ca2(+)-free solution was progressively delayed with respect to the onset of anoxia, a graded decrease in recovery of function was observed, suggesting that in WM the deleterious effects of Ca2+ accumulate slowly during anoxia. At the time of reoxygenation, an additional stepwise increase in injury was observed that was also Ca2(+)-dependent. Mammalian WM, which is relatively resistant to anoxic injury compared with gray matter, is damaged by anoxia in a manner that appears to depend on the gradual accumulation of Ca2+ in a cytoplasmic compartment.
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Specificity of cell-cell coupling in rat optic nerve astrocytes in vitro.
Intercellular coupling was studied in cultured rat optic nerve astrocytes individually characterized by A2B5 antibody staining. The presence of cell coupling was assessed by injecting single cells with the low molecular weight fluorescent dye Lucifer yellow and noting dye passage into adjacent cells; cell coupling was also studied by analyzing the decay phase of current transients recorded in response to small voltage steps using whole-cell patch-clamp recording. Cell coupling was restricted to A2B5- astrocytes, the majority of which had a flat fibroblast-like appearance and was never observed in A2B5+ stellate-shaped astrocytes. Furthermore, A2B5- astrocytes showed coupling only to A2B5- and never to A2B5+ astrocytes. Analysis of current transients provided an additional indicator for cell coupling. Astrocytes that showed dye coupling to at least one neighboring cell required the sum of two exponential functions to fit current transients, whereas a single exponential function sufficed to fit transients in cells that were not dye coupled. The specificity of cell coupling in cultured rat optic nerve astrocytes suggests that predominantly A2B5- astrocytes comprise a coupled glial syncytium; this physiological feature of these cells may be a specialized adaptation for "spatial buffering," the transport of K+ away from areas of focal extracellular accumulation. On the other hand, A2B5+ astrocytes form an uncoupled subpopulation of rat optic nerve glial cells that may serve different functions.
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Levodopa-responsive parkinsonism following central herniation due to bilateral subdural hematomas.
A 66-year-old man suffered bilateral subdural hematomas progressing to central herniation, despite repeated surgical evacuations. This eventually resolved, leaving him with a severe parkinsonian syndrome that was responsive to levodopa. MRI and CT showed midbrain compression from central herniation, and a follow-up MRI revealed thinning of the pars compacta. The clinical and radiologic evidence suggested that midbrain compression from central herniation was the probable cause of parkinsonism in this patient.
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Anoxic injury of CNS white matter: protective effect of ketamine.
Gray and white matter of the mammalian CNS are both damaged by anoxia. Anoxic injury in gray matter is mediated in part by excessive accumulation of excitotoxins like glutamate. Drugs such as ketamine, a dissociative anesthetic known to block glutamate (NMDA) receptors, reduce hypoxic neuronal injury in gray matter. In this study we used the isolated rat optic nerve preparation to determine if ketamine influences recovery after anoxia in a nonsynaptic system, ie, CNS white matter. Optic nerves from adult rats were exposed to a standard 60-minute period of anoxia. Ketamine (1 mM) improved recovery of the compound action potential (CAP) after anoxia. Since glutamate and aspartate (up to 10 mM) had no effect on CAP amplitude in the optic nerve, the effect of ketamine is probably not mediated by NMDA receptor blockade. These observations indicate that ketamine is able to protect CNS white matter, as well as gray matter, from anoxic injury.
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The pathophysiology of anoxic injury in central nervous system white matter.
White matter of the mammalian brain is susceptible to anoxic injury, but little is known about the pathophysiology of this process. We studied the mechanisms of anoxic injury in white matter using the isolated rat optic nerve, a typical central nervous system white-matter tract. Optic nerve function, measured as the area under the compound action potential, rapidly failed when exposed to anoxia. Postanoxic recovery was variable, depending on duration of the anoxic insult; after a standard 60-minute period of anoxia, the compound action potential recovered to 28.5% of control. Irreversible anoxic injury was critically dependent on extracellular Ca2+; maintaining the tissue in zero [Ca2+] solution throughout the anoxic period resulted in 100% compound action potential recovery. Increasing perfusate [Ca2+] during anoxia from zero to 4 mM resulted in progressively less recovery. Anoxic damage to the optic nerve appears to depend on the gradual accumulation (over tens of minutes) of Ca2+ in a cytoplasmic compartment. The inorganic Ca2+ channel blockers Mn2+ (1 mM), Co2+ (1 mM), or La3+ (0.1 mM) had no effect on recovery of the compound action potential after anoxia; only Mg2+ (10 mM) significantly improved recovery. Treatment with the dihydropyridine Ca2+ channel blockers nifedipine (1-10 microM) or nimodipine (1-40 microM) also had no effect on recovery from anoxia. Thus, Ca2+ influx during anoxia does not occur via conventional Ca2+ channels. Preliminary evidence suggests that this Ca2+ influx may occur via other cation channels that are imperfectly selective for Ca2+ or via the Na(+)-Ca2+ exchanger.(ABSTRACT TRUNCATED AT 250 WORDS)
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Visualization of oligodendrocytes and astrocytes in the intact rat optic nerve by intracellular injection of lucifer yellow and horseradish peroxidase.
The morphology of glial cells in the intact rat optic nerve, a central nervous system (CNS) white matter tract, was analysed by filling over 500 macroglial cells intracellularly with horseradish peroxidase (HRP) or Lucifer yellow (LY). Two main cell types were distinguished: fibrous astrocytes and cells presumed to be oligodendrocytes. Intracellularly stained astrocytes were highly complex, with 50-60 long branching processes which passed radially from the cell body and terminated in end-feet at the pial surface or on blood vessels; some processes ended freely in the nerve parenchyma. Astrocytes filled with LY were usually dye-coupled to other astrocytes after the first week of life. Filled oligodendrocytes had a unique appearance that unmistakably distinguished them from astrocytes and were occassionally dye-coupled to nearby oligodendrocytes. These cells had 20-30 longitudinally oriented processes 150-200 microns long, which passed exclusively along the long axis of the nerve parallel to axons; the longitudinal processes were connected to the cell body by thin branches 15-30 microns long. The longitudinal processes probably represent the tongue processes of the internodal myelin sheaths, and thus each oligodendrocyte appears to myelinate 20-30 axons with sheaths that are 150-200 microns in length.
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Antigenic abnormalities in fiber tract astrocytes of myelin-deficient rats: an immunocytochemical study in the olfactory cortex.
Intrafascicular oligodendrocytes in myelin-deficient (md) rats typically fail to form myelin sheaths or survive to maturity. The present study of the md olfactory cortex compares astrocytes that develop either among populations of pathological oligodendrocytes in a fiber tract or that develop in neuropil, which normally contains few oligodendrocytes. Antigenic profiles for md and normal astrocytes were defined by the monoclonal antibody Rat 401, and by antibodies that recognize vimentin and glial fibrillary acidic protein. In the olfactory cortex, antigenic abnormalities were typically restricted to astrocytes located in the vicinity of pathological oligodendrocytes.
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Effects of chronic phenobarbital exposure on cultured mouse spinal cord neurons.
The anticonvulsant phenobarbital (PB), at concentrations of 20, 40, and 90 micrograms/ml, was chronically applied to cell cultures of mouse spinal cord from day 2 or day 14 after initial plating, and the effects of this exposure on neuronal density and morphological characteristics were determined. Neuronal morphological characteristics were analyzed quantitatively following intracellular injection of the fluorescent dye Lucifer yellow. Cultures exposed to PB for 6 weeks, from day 14 after plating, showed concentration-dependent reductions in neuronal density; both large and small neurons were equally affected. PB exposure also reduced dendritic branching frequency, and the length of dendrites, of remaining large neurons. A higher percentage of these neurons had a bipolar branching pattern than was normally the case. Neurons in cultures exposed to PB from day 2 after plating, compared with those exposed from day 14, showed significantly less alteration in terms of density and morphological characteristics. Effects on neuronal morphological characteristics increased with duration of drug exposure. Equimolar concentrations of barbituric acid produced effects similar to those produced by PB. Chronic exposure to PB adversely affects survival and morphological characteristics of mammalian central neurons grown in cell culture. Curiously, exposure from the time of initial plating appears to be less deleterious than exposure initiated 2 weeks later. To the extent that neuronal development in vitro can be compared to the situation in vivo, these results, and those of other investigators, raise concerns about long-term exposure of the developing human nervous system to PB.
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Electrical coupling between astrocytes and between oligodendrocytes studied in mammalian cell cultures.
The characteristics of electrical coupling between astrocytes and between oligodendrocytes were analyzed in cell cultures derived from rodent central nervous system. Experiments were carried out by impaling one member of a glial pair with separate voltage recording and current passing electrodes (cell 1) and the other cell, a measured distance from the first, with a voltage-recording electrode (cell 2). Astrocyte pairs within 300 microns of one another were always coupled. The coupling ratio was determined for 23 astrocytic pairs various distances apart, and decreased with distance in a roughly exponential manner. The average coupling ratio of astrocytes within 100 microns of each other was 0.44 +/- 0.32. Oligodendrocytes were less strongly coupled to each other than astrocytes. Even cells immediately adjacent to one another were often uncoupled. Among coupled oligodendrocytes within 100 microns of each other, the average coupling ratio was 0.11 +/- 0.1. Current passage between pairs of astrocytes and pairs of oligodendrocytes was nonrectifying. Application of 0.5 mM BaCl2 or 44.6 mM CsCl (substituted for NaCl) depolarized and increased the input resistance of astrocytes and oligodendrocytes. These ions also increased the coupling ratio in astrocyte pairs and oligodendrocyte pairs; this effect was rapid in onset and completely reversible. Ba++ and Cs+ appear to block resting K+ conductance in glia and probably increase the coupling ratio by increasing the effective length constant of the glial membrane without any direct effect on junctional resistance. In three cases, oligodendrocyte pairs that appear uncoupled in normal solution exhibited coupling in the presence of BaCl2 or CsCl. This suggests that oligodendrocytes may be widely coupled by junctions that provide only weak electrical interaction; such junctions might be important for the exchange of small metabolically active molecules. The strong electrical coupling among astrocytes, in concert with their K+-selective membrane conductance, would provide for an electrical syncytium well designed to transport K+ away from areas of focal extracellular accumulation (i.e., the spatial buffer mechanism), and these cells, more than oligodendrocytes, may provide this function.
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Astrocytes convert the parkinsonism inducing neurotoxin, MPTP, to its active metabolite, MPP+.
The ability of astrocytes to convert 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP) to its toxic metabolite 1-methyl-4-phenylpyridinium ion (MPP+) was directly tested. Cultured astrocytes rapidly converted MPTP (25 micrograms/ml) to MPP+; after 6 h MPP+ concentrations reached 1.5 micrograms/ml, within the toxic range for neurons. MPTP (above 10 micrograms/ml) reduced glial density after 5 days of exposure. This toxic effect was blocked by pargyline, a monoamine oxidase inhibitor; pargyline also reduced the conversion of MPTP to MPP+ by 85%. When neurons were added to astrocyte cultures, MPTP conversion to MPP+ was not enhanced. Astrocytes appear critical in converting MPTP to MPP+, and are damaged by chronic exposure to MPTP.
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Carbonic anhydrase activity develops postnatally in the rat optic nerve.
We examined the appearance of carbonic anhydrase (CA) activity in rat optic nerves (RONs) 5-77 postnatal days of age and correlated the appearance of enzyme activity with structural and physiological alterations. CA activity was nearly absent before 10 days of age and appeared in this CNS white matter tract with a developmental time-course similar to that of oligodendrogliogenesis and myelinogenesis. When oligodendrocytes and myelin were depleted in the RON by treatment with a mitotic inhibitor, CA activity was markedly reduced. These observations support the hypothesis that CA is contained primarily in oligodendrocytes and myelin. Neural activity in the RON caused changes in extracellular pH (pHo) and the character of these pHo responses was very age dependent; older nerves exhibited much larger acid shifts than neonatal nerves. The development of CA activity may be a factor contributing to this physiological alteration.
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Electrophysiological properties of ependymal cells (radial glia) in dorsal cortex of the turtle, Pseudemys scripta.
1. We have investigated the electrophysiological properties of ependymal cells in the isolated dorsal cortex of the turtle, Pseudemys scripta. The cell bodies of these radial glia form an epithelium at the ventricular surface, and each cell sends one or more branching processes through the cortex to the pial surface. Very few non-ependymal glia exist in the dorsal cortex. 2. Ependymal cells had high resting membrane potentials (-90 mV), very fast time constants and a lack of intrinsic excitability or synaptic potentials. 3. Changes in the K+ concentration ([K+]) of the bathing solution caused near-Nernstian changes of ependymal membrane potentials. When local neuronal pathways were activated, ependymal cells slowly depolarized while extracellular voltage shifted negatively. Simultaneous measurements of extracellular [K+] ([K+]o) near the impaled ependymal cell body showed that these slow depolarizations were fully accounted for by activity-dependent increases in [K+]o. Similar measurements during focal pressure applications of solutions with high [K+] suggested that intrasomatic recordings reflect predominantly the [K+]o adjacent to the cell body, and not the intracortical process. 4. Intracellular injections of the fluorescent dye Lucifer Yellow CH, and simultaneous recordings from neighbouring cells, indicated that ependymal cells are chemically and electrically coupled to one another. Increasing the ambient CO2 level from 5 to 40% depolarized cells, increased their input resistance, and abolished interglial dye coupling. 5. The physiological properties of ependymal cells are very similar to those of a variety of glial cell types in a range of vertebrate and invertebrate species. In the absence of other types of glia, radial glia may function as the sole cellular mediators of K+ redistribution (i.e. K+ spatial buffering) following neural activity, as well as the generators of slow extracellular potentials.
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