Rat cerebral cortical slice respiration in media of various osmolarities

Brain Research, 249 (1982) 387-389

387

Elsevier Biomedical Press

Rat cerebral cortical slice respiration in media of various osmolarities DAVID HOLTZMAN, JAMES OLSON and HUNG NGUYEN Departments of Neurology and Pediatrics, Stanford University School of Medicine, Stanford, CA 94305 ( U.S.A)

(Accepted June 22nd, 1982) Key words: cell respiration - - cerebral cortex - - osmolarity - - maturation.

Freshly separated immature (5-day-old)and mature (60-day-old) rat cerebral cortical tissue demonstrates a Na+-dependent respiratory component similar to that recently described in primary cultured astrocytes. This respiratory component, which is not coupled to oxidative phosphorylation, increases as the osmolarity is increased above 354 mOsm in media with NaCI as the osmotic agent. With decreasing osmolarity from 354 to 80 mOsm, this respiratory component decreases more in cortical tissue than in cultured astrocytes. We propose that the respiratory response of neurons to decreasing extracellular osmolarity may differ from that of astrocytes in situ. We recently described the effects of altered ambient o3molarity on cell respiration in the primary cultured cerebral astrocyte and 3T3 fibroblast cell line4, 5. When NaCI is the predominant extracellular osmolar agent, a component of cell respiration, which apparently is not coupled to oxidative phosphorylation or to a Na+,K+-ATPase, increases with increasing osmolarity. At less than physiologic osmolarities, this respiratory component decreases more in the fibroblasts than in the astrocytes. In the primary cultured astrocytes, the Na÷-dependent respiratory response to increased osmolarity may be important in maintaining the osmotic properties of the astrocyte by preventing intracellular accumulation of permeant ions 4. Because of the potential importance of this respiratory response of the astrocyte in conditions of altered brain water or electrolyte concentrations (e.g., edema, hypernatremia), we have attempted to demonstrate its presence in cerebral cortical tissue freshly separated from immature (5-day-old) and adult (60-day-old) rats. Sprague-Dawley albino rats were used for all experiments. For studies with 5-day-old pups, pregnant females were obtained prior to delivery. Litters were reduced to 8 pups each on the day of birth. Adult animals were 60-day-old males. Animals were sacrificed by decapitation and the brains were remo-

ved immediately. The cerebral hemispheres were cut away from the brainstem. Ten slices, each approximately 300/~m tbick, were taken from each frontal lobe using a tissue slicer (DuPont Instruments, Newtown, CT). After teasing away the white matter, the slices were cut into thin strips. These strips were placed in 3 ml of a phosphate-buffered (pH 7.3) medium containing KCI (2.8 mM), CaClz (0.9 mM), MgC12 (0.5 mM), Na2HPO4 (8.0 mM), and KH2PO4 (1.5 mM). NaC1 was present in various concentrations from 0-548 mM. In some experiments with the mature cortical tissue, equi-osmolar sucrose was substituted for NaC1 in the media. Approximately 0.3 ml of this suspension ( l - 2 mg protein) was added to a 2 ml polarographic chamber (Yellow Springs Instrument Co., Yellow Springs, OH). The chamber was filled with art air-saturated medium. The temperature was maintained at 37 °C. Tissue strips were kept in suspension by means of a rotating magnet placed in a well in the chamber floor. Glucose was added to a final concentration of 50 mM. The concentration of Oz in the chamber was measured polarographically with an oxygraph (Gilson Co., Madison, WI) coupled to a Clark platinum cathode assembly polarized to --43.8 V. In most experiments, a constant respiratory rate was reached in 3-5 min. If the rate was not linear

388 within 10 rain, the results were excluded. The initial stable respiratory rate is termed the basal rate. Oligomycin, an inhibitor of oxidative phosphorylation 6, was then added in small quantities (25 nmol in 1.0 #1 EtOH) until a maximal inhibition of respiration was reached. In an additional set of experiments in the NaCI media, dinitrophenol ( D N P L an uncoupler o f oxidative phosphorylation~L was added to the basal respiring tissue suspension in small quantities (50 nmol in 1.0 /~1 EtOH) until a maximal respiratory rate was reached. After each experiment, the chamber contents were removed and frozen for later protein determination a. Respiratory rates, measured in cerebral cortical tissue f r o m 60-day-old rats, are shown in Fig. I A. As the ambient osmolarity decreased from 354 to 80 m O s m in the NaC1 media, b o t h the basal and oligomycin-insensitive respirations were inhibited by about 20-30 °,~i. As the ambient osmolarity increased from 354 to 902 mOsm, both basal respiration and oligomycin-insensitive respiration increased more than 50 o/o~ while the D N P-stimulated respiration was i

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Fig. 1. Respiratory rates of cerebral cortical tissue f r o m adult

rats (A) and from 5-day-old rat pups (B) measured in NaCI media of various osmolarities. Respiration was measured in a basal state and after adding oligomycin or dinitrophenol (see text). Each value is the mean ± S.E.M. of at least 6 experiments. P values <0.05 are shown for comparisons of respiratory rates in each medium with that measured in the 354 mOsm medium.

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Respiratory rates ( m e a n S.E. ~1.) ol" cerehral c . r t h ' a l lt~.sm, f i ' o m 60-tkO'-o/d rats measltred in sttcro,ve mcdio at two ,,,

mo/arilie.s The complete respiration medium is described in the text. Respiratory rates are defined in the text. P values, calculated by the Student*s t-test, are shown comparing respiratory rates measured in the media of 902 m O s m to those measured at 354 mOsm. Respiratory rates ( n~, at O/m K proteitl mitt)

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inhibited by about 15 i~i. In contrast, when sucrose replaced NaCI as the osmotic agent, basal respiration and oligomycin-insensitive respiration were markedly inhibited at 902 mOsm compared to 354 mOsm (Table I). In sucrose media, the basal respiration at 354 m O s m and both basal and oligomycininsensitive respirations at 902 m O s m were significantly less than the respiratory rates in NaCI media of the same osmolarities (p <.0.001 for the difference of each rate in sucrose c o m p a r e d to NaCI media). Respiratory rates, measured in cerebral cortical tissue from 5-day-old pups, are shown in Fig. lB. The immature tissue was studied only in NaC[ media. At 354 mOsm, respiratory rates were much lower than those measttred in mature cortical tissue, as observed in previous studies1, '. When the ambient osmolarity increased f r o m 354 to 902 mOsm. the basal respiration showed a small increase. As in the mature cortical tissue, oligomycin-insensitive respiration increased almost 50°, o in the immature cortical tissue with the increase in osmolarity. With decreasing osmolarity, basal respiration was stable from 354 to 166 m O s m and then dropped markedly from 166 to 80 mOsm, similar to changes in mature cortical tissue. The immature tissue also showed a similar decrease o f about 15--20 o / i n oligomycin-insensitive respiration as the osmolarity decreased from 354 to 80 mOsm. The respiratory changes in media of increased

389 osmolarity are qualitatively similar in freshly isolated cerebral cortical tissue and in primary cultured astrocytes 4. As in the astrocytes, respiration that is not coupled to oxidative phosphorylation (oligomycin-insensitive) and basal respiration increase with increasing NaCI concentration. These increases in respiratory rates are in contrast to the inhibition of cellular respiratory capacity (DNP-stimulated respiration) under the same condition. As in the astrocyte, the increases in respiratory rates do not occur when NaCI is replaced by the impermeant molecule, sucrose. There are some quantitative differences in the effects of altered N a + concentration on oligomycin-insensitive respiration in the freshly isolated cerebral cortical tissue compared to the effects in the primary cultured astrocyte. In hyper-osmolar media, this respiratory component increases only about 50% in mature cortical tissue and over 150 % in the cultured astrocytes 4. In cortical tissue, this respiratory component is progressively inhibited with decreasing osmolarity while, in the astrocytes, there is no further inhibition of th.e oligomycin-insensitive respiration below 270 m O s m 4. Assuming the astrocytes in freshly isolated cerebral cortical tissue show the same respiratory responses to altered osmolarity as the astrocytes from primary culture, the effects of altered osmolarity on neuronal respiration may differ markedly from the effects seen in the astrocyte.

The presence of a Na+-dependent, oxidative phosphorylation-independent respiratory component in freshly isolated cerebral cortical tissue strongly suggests that this is a property of cerebral cortical tissue in situ. We have proposed that this respiratory component is important in determining the osmotic properties of the primary cultured astrocyte 4. In hypo-osmolar NaCI media (less than 270 mOsm), the oligomycin-insensitive respiration in the astrocyte is independent of osmolarity (i.e., is not further inhibited) and cell volumes are greater than in equi-osmolar sucrose media. The Na + concentration-dependence of this respiratory component in the neuron in media of more extreme hypo-osmolarity would be consistent with the greater swelling of astrocytes than of neurons seen when the brain water content is increasedL The potential importance of this energy-coupled, cell-specific response to conditions of altered osmolarity in cerebral cortical tissue makes further study of the respiratory properties of bulk-separated and primary cultured brain cells and cell processes under these conditions necessary for an understanding of the in situ cellular responses to critical clinical states such as brain edema, dehydration and altered osmolarity.

1 Fazekas, J. F., Alexander, F. A. D. and Himwich, H. E., Tolerance of the newborn to anoxia, Amer. J. Physiol., 134 (1941) 281-287. 2 Holtzman, D., Nguyen, H., Zamvil, S. and Olson, J., In vitro cellular respiration at elevated temperature3 in developing rat cerebral cortex, Dev. Brain Res., in press. 3 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., Protein measurements with. the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. 40lson, J. and Holtzm~n, D., Respiration and cell volume of primary cultured cerebral astrocytes in media

of various osmolarities, Brain Research, in press. 50lson, J. and Holtzman, D., Sodium ion effects on the re3piratory properties of astrocytes from primary culture, Neuroscience Abstr., 7 (1931) 786. 6 Slater, E. C., Application of inhibitors and uncouplers for a study of oxidative phosphorylation. In R. W. Estabrook and M. E. Pullman (Eds.), Methods" in Enzymology, Vol. X, Academic Press, New York, pp. 4857. 7 Wasterlain, C. G. and Torack, R. M., Cerebral edema in water intoxication. I1. An ultrastructural study, Arch. Neurol., 19 (1968) 79-87.

This research was supported by grants to D.H. from the National Institutes of Health (NS 16256 and ES 02571).