L. Hertz

NEUROGLIAL LOCALIZATION OF POTASSIUM AND SODIUM EFFECTS ON RESPIRATION IN BRAIN

BRAIN slices react to an increased concentration of potassium or a decreased concentration of sodium in a manner, which is unique to this tissue (ASHFORD and DIXON, 1935; DICKENS and GREVILLE, 1935; ELLIOTT and LIBET, 1942; HERTZ and CLAUSEN, 1963). During incubation in media with a ‘balanced’ ion composition (e.g. 120 mM-sodium and 5 mM-potassium), their rate of oxygen consumption is quite well maintained at about 90 pmoles/g fresh wt./hr. Exposure to sodium-free media, however, causes a very rapid decrease of the respiration, until the rate of oxygen uptake reaches a level at about 10 per cent of its original value; thereafter the decline stops and the residual respiration-which seems to be unaffected by lack of sodiumcontinues for hours. This peculiar course of the respiration may indicate that the slice contains two different fractions, e.g. two cell types, which do not react identically to lack of sodium. The originally larger fraction shows a rapidly declining rate of oxygen uptake, whereas the respiration in the originally smaller fraction remains stable and accordingly is unmasked when the total respiration declines. Knowledge of the importance of sodium for the maintenance of respiration by various cell types in a brain slice may therefore yield information about their relative contributions to the total respiration. The rate of oxygen uptake by a brain slice incubated in the ‘balanced’ medium does not represent its maximum respiratory intensity. Addition to the medium of more than 50 mwpotassium leads to an initial increase of about 70 per cent in the respiratory rate; this ‘stimulation’ is, however, followed by a rapid exponential decline, and eventually the ‘stimulated’ respiration becomes lower than the rate of oxygen consumption in a ‘balanced’ medium. The resemblance between this secondary depressing effect on respiration by high concentrations of potassium and the rapid respiratory decline during incubation in sodium-deficient media may indicate that the same cell type reacts to lack of sodium and excess of potassium. This idea is supported by the fact that both the potassium and sodium effects are limited to grey matter from certain regions (e.g. cerebral and cerebellar cortex, basal ganglia) of the central nervous system (HERTZ and CLAUSEN, 1963). The macroscopic localization does not allow any conclusion whether the reactions to deviations in the ionic composition of the medium occur in the nerve cells or in the non-neuronal elements. In the present study the rate of oxygen uptake by single isolated nerve cell perikarya and by lumps of intercellular grey was therefore followed during incubation in ‘balanced’, sodium-deficient and potassium-rich media. Since the non-neuronal ‘intercellular grey’ (the neuropile) mainly consists of glia cells, the isolated samples of non-neuronal elements will be referred to as neuroglia.

POTASSIUM EFFECTS ON ION TRANSPORT IN BRAIN SLICES

—(1) Fluxes of sodium, potassium, chloride and glutamate ions were studied in brain slices by aid of radio‐isotopes. Desaturation curves showed the efflux to occur from at least two compartments with widely different kinetics.

CONTENT OF ATP IN CULTIVATED NEURONS AND ASTROCYTES EXPOSED TO BALANCED AND POTASSIUM‐RICH MEDIA

Abstract— The content of ATP was measured in neurons from dorsal root ganglia and in cortical and spinal astrocytes with the luciferase method. No ATP could be demonstrated in the cortical cells but in the dorsal root neurons and spinal astrocytes an average amount of 7·9 and 11·9 × 10−15 mol per cell respectively was found. An increase in the external potassium concentration had no effect on the content of ATP in neurons (7·1 × 10−15 mol/cell) but led to a significant (P < 0·005) decline to 6·4 × 10−15 mol per cell in the astrocytes.

RESPIRATION BY CULTIVATED ASTROCYTES AND NEURONS FROM THE CEREBRAL HEMISPHERES

‐Rates of oxygen uptake were measured in chick and/or rat astrocytes and neuronal cells cultivated for 2–4 weeks in Falcon flasks or Rose chambers. All the preparations were found to have respiratory rates between 0.4 and 0.8 × 10−5μl/h O2 per cell. Based upon measurements of cell diameters these values were recalculated to about 570 μmol/g wet wt. for the neuronal cells and 130 μmol/g wet wt. for the glial cells. The results are compared with previous data of oxygen uptake by neurons and glial cells separated by other procedures.

REGULATION OF OXYGEN CONSUMPTION IN NEUROBLASTOMA CELLS: EFFECTS OF DIFFERENTIATION AND OF POTASSIUM

Abstract— The rate of oxygen consumption has been measured micromanometrically in fresh mouse neuroblastoma cells and in corresponding cells cultivated with and without 20% calf serum known to suppress differentiation. Fresh cells and cells cultivated in the serum depleted medium showed a relatively intense respiration (0.5–1.0 × 10−5μl/h/cell), whereas the proliferating, cultivated cells had a low rate of oxygen uptake (0.2 × 10‐5μl/h/cell), provided they did not differentiate morphologically during the 4 h of measurement in the serum‐free diver medium. Both morphological and metabolic differentiation of such cells occurs rapidly but may be completely prevented by siliconization of the divers.

Energy metabolism of nerve cells during differentiation. O2 uptake, lactate production and ATP content of chick embryo brain cells before and after cultivation in the Rose chamber.

Abstract Mechanically dissociated brain cells from 7-day-old chick embryos were grown in Rose chambers. Cells in such cultures show a marked morphological differentiation into neuronal cells resting on a confluent monolayer. In the present study the rate of O 2 uptake and lactate production and the content of ATP per neuronal cell were measured at different times of cultivation and compared with the same parameters in cells obtained from the brain of the living embryo. In 2–4-day-old cultures, both O 2 uptake, lactate production, and ATP content were identical to the values observed in the ‘fresh’ cells from which the cultures had been prepared. The rate of O 2 uptake increased as a function of the age of the culture in parallel with the increase in the rate of O 2 uptake observed in the embryo. The rate of anaerobic lactate production increased during cultivation as a function of the age of the culture (as known from other in vitro systems), whereas the glycolytic rate in the embryo decreased with development. The ATP content remained approximately constant. The observations suggest that cultures of dissociated brain cells may undergo a considerable degree of metabolic differentiation and thus be well suited for studies of metabolic events in the nervous system.

UPTAKE OF GLUCOSE ANALOGUES BY RAT BRAIN CORTEX SLICES: Na+-INDEPENDENT MEMBRANE TRANSPORT

Abstract— The glucose analogues 3‐O‐methyl‐D‐glucose and α‐methyl‐D‐glucoside were not metabolized in brain tissue.

Rate of oxygen uptake by the N N cell line of hamster astroglia: lack of effect by excess potassium

GLIA cell function and metabolism have attracted considerable interest during recent years. Investigations are, however, hampered by difficulties in obtaining intact cells from living animals in sufficient amounts, and cultured cells have been resortcd to. Both primary cultures and established cell lines have been used. An easily replicable, commercially available preparation is the spontaneously transformed astroglia line, N N , which originally was developed from the brains of new-born hamsters (Smm et al., 1970; EICHBERG et a/. , 1971; SILBERSTEIN et al., 1972). These cells have been well characterized with respect to their content of Na’ and K + (LEES & SHEIN, 1970; GILL et al., 1974), of free amino acids (MOKRASCH, 1971), and of nuclcic acids and glycolipids (SHEIN et al., 1970; ROBERT et al., 1975) as well as the uptake kinetics for chloride (GILL e t al., 1974), and the activities of several enzymes including the Na+-K+-ATPase (EMBREE et al., 1971; MANDEL et al., 1974). No information seems, however, to be available about the energy metabolism, and the purpose of the present work has been to study the oxygen uptake of the N N cells. This was done using either whole cultures or microsamples of selected cells and after cultivation in both the absence and the presence of 0 1 mM-dibutyryl-CAMP (BcAMP), which enhanccs the biochemical maturation of primary cultures of glia cells (SCHOUSBOE et a/., 1975). The oxygen consumption was followed during incubation in both ‘physiological’ and potassium-rich media, because the respiration of mature brain-cortex tissue is increased by a substantial elevation of the K + concentration in the medium (ASHFORD & Drxo~, 1935; DICKENS & GREVILLE, 1935; HIMWICH et al., 1942). This response seems to occur in glia cells (HERTZ, 1966; ALEKSIDZE & BLOMSTRAND, 1969; HALJAM~E & HAMBERGER, 1971; HERTZ e t al., 1973).

Some Histochemical, Biochemical, and Pharmacological Aspects of Differentiation of Neuroblastoma Cells of Mouse

C1300 neuroblastoma cells in vitro retain the ability of neuronal differentiation (Augusti- Tocco and Sato, 1969; Schubert et al., 1969). This maturation of neoplastic immature neuroblasts can be induced by various molecular environments: suppression of fetal serum in the culture medium (Seeds et al., 1970) incubation with BrdU (Schubert and Jacob, 1970) or BcAMP (Furmanski et al., 1971), X-ray irradiation (Prasad, 1971), cultivation with NGF (Hermetet et al., 1972a). Different cellular events were used as proofs of neuronal differentiation: growth of expansions and the presence of enzymes involved in the biosynthesis of neurotransmitters (Augusti-Tocco and Sato, 1969); changes in oxidative metabolism (Ciesielski-Treska et al., 1972); neuronal type pharmacological reactivity (Hermetet et al., 1972b). The cultures of neuroblastoma cells may be useful for genetic mapping, in studying the maturation of neural morphology and function, as well as the effects of the molecular microenvironment.