Faculty Bibliography

Ion-selective microelectrodes can be used to evaluate the characteristics and laminar distribution of excitatory amino acid agonist-induced K+ and Ca2+ shifts in the extracellular environment of brain cells. This report describes the pattern of K+ increases and Ca2+ decreases elicited by glutamate and aspartate at 100 microns intervals in the isolated turtle cerebellum. These responses were compared to ion shifts evoked by kainate, quisqualate and N-methyl-D-aspartate. Glutamate and aspartate produced indistinguishable laminar patterns of ion shifts, with the greatest [K+]o and [Ca2+]o shifts in the granular layer. The average maximum granular and molecular layer increases in [K+]o were, respectively, 130% and 24% larger than the increase in the Purkinje cell layer. Kainate-induced increases in [K+]o also followed this granular greater than molecular greater than Purkinje cell layer pattern; however, the corresponding [Ca2+]o decreases were smaller and more variable. Quisqualate-evoked ion shifts in the molecular layer closely mimicked the shape of glutamate- and aspartate-induced responses. In the granular layer, however, quisqualate caused little ion change during iontophoresis followed by large [K+]o and [Ca+]o shifts after the end of the pulse. The minimal ion shifts induced during quisqualate application in the granular layer gave this agonist the distinction of being the only agent tested to have its greatest direct effect in the molecular layer. N-Methyl-D-aspartate caused large, two-phase [K+]o and [Ca2+]o shifts in the granular layer, only small [K+]o rises in the Purkinje cell and ventral molecular layers, and no response in the dorsal molecular layer. The lack of similarity between glutamate- and aspartate-induced ion shifts in the granular layer and those of any one agonist demonstrate the mixed agonist action of glutamate and aspartate in the cerebellum. These studies provide new information about the dynamics of excitatory amino acid receptor activation that is complementary to autoradiographic receptor mapping data and to single cell electrophysiological studies

Several improvements in the fabrication and use of carbon fiber voltammetric microelectrodes (CFVMs) are described. These procedures did not involve oxidative treatment, but resulted in sensitivities and selectivities approaching those of treated CFVMs, without the inherent slow response times associated with the latter electrodes. To accomplish this we reduced CFVM noise by (1) improving the adhesive seal between the 8 microns o.d. carbon fiber and the glass insulation using vacuum, (2) snapping rather than cutting or beveling the fiber to be flush with the glass, and (3) using a concentrated electrolyte solution to make electrical contact with the fiber. System noise was reduced by digital smoothing and signal averaging. Selectivity of the CFVMs for dopamine over ascorbate was enhanced to better than 2000:1 by coating with Naflon, a perfluorinated cation exchange polymer, using a low (+0.5 V vs Ag/AgCl) electroplating potential. This low voltage also prevented electrode surface oxidation. To demonstrate the performance of our CFVMs, we used them in conjunction with high-speed cyclic voltammetry to accurately measure the diffusion coefficient of iontophoretically released dopamine at concentrations as low as 35 nM over distances of less than 200 microns in agarose gel

The isolated turtle cerebellum was used as a model system to study effects of depolarizing conditions on interstitial ascorbic acid concentration. The depolarizing stimulus was Leao's spreading depression, which is characterized by transient negative extracellular potentials, high potassium levels (20-60 mM), and local depression of neuronal activity. Interstitial concentrations of ascorbate (200-400 microM) and other electroactive species were monitored voltammetrically, using graphite fiber microelectrodes. Total tissue ascorbate (1,810 nmol/g tissue wet weight) was similar to mammalian levels and was several orders of magnitude higher than catecholamine and indoleamine content. During spreading depression, a large (up to 200 microM) increase in concentration of interstitial electroactive species was monitored. Use of Nafion- and ascorbate oxidase-coated electrodes and uricase confirmed that ascorbate was the only substance detected. Simultaneous monitoring of ascorbate, extracellular potential, and extracellular volume (using tetramethylammonium and ion-selective microelectrodes) indicated that (a) the ascorbate increase began with the decrease in extracellular volume during spreading depression, and (b) much of the increase was the result of extracellular volume decrease. In sucrose-substituted medium, in which volume changes are eliminated, a 50 microM increase in interstitial ascorbate, caused by release from intracellular stores, was also seen. The ascorbate concentration increase was prolonged in sucrose medium, suggesting that an uptake process involving sodium may further regulate interstitial ascorbate concentration

A review of some of the literature on Ca2+ diffusion in free media and a variety of nervous tissues is presented. In the majority of tissue studies the apparent diffusion coefficient of Ca2+ is three to nine times smaller than that in a free aqueous medium. The methodology of using pressure microejection and Ca2+ ion-selective microelectrodes to measure Ca2+ diffusion is discussed. Our ongoing studies of Ca2+ diffusion in the cerebral cortex of the rat, using these methods, also confirm that Ca2+ diffusion is mainly influenced by the tortuosity of the tissue rather than other factors such as binding to extracellular charge sites or uptake