Membrane potentials in mammalian cerebral tissues in vitro: dependence on ionic environment

Abstract

Slices of cerebral tissues from guinea-pigs or cats can be maintained in moist oxygen at the surface of salines in a condition which permits the measurement of resting membrane potentials (Li & McIlwain, 1957). This technique gives an opportunity of comparing the effects of substances on polarization and metabolic phenomena in the slices. The present investigation examines the effect of the following cations on the membrane potential: potassium, sodium, calcium, magnesium and ammonium. L-and D-glutamate were also examined in view of their effect on the tissue content of potassium salts. METHODSIn most experiments the cerebral cortex of the guinea-pig was used; occasionally experiments were done with rat, human, calf or sheep slices. Human specimens were obtained at operation for non-malignant conditions by Mr V. Logue and Mr P. Schurr, to whom we are grateful. Calf and sheep specimens were excised after the animals had been anaesthetized and used for other experiments by Dr F. Bell, to whom also we are grateful. Guineapigs and rats were exsanguinated and the brain excised as quickly as possible. Slices were cut tangentially to the surface of the parietal cortex, with a strip of razor blading, using a glass guide which gave slices 0 3-035 mm thick; these were transferred immediately from the blade into the incubating medium in the slice chamber (see below). The time elapsing between exsanguination and the beginning of incubation was 2j-3 min. Usually the superficial slice only was used, and it was pre-incubated for 40 min before recording.The normal bicarbonate glucose saline contained (mM): NaCl 124, KCI 5, KH2PO4 1-24, MgSO4 1-3, CaCl2 2-8, NaHCO3 26 and glucose 10, and was equilibrated with 5 % C02 in 02. Its pH was 7-2-7-4 at the beginning of experiments, and never exceeded 7-8 by the end. Other salines are described in terms of their difference from this normal saline. Apparatu8sThe apparatus was basically that of Li & McIlwain (1957), but differed in the following ways. The slices rested on a nylon grid mounted on a Perspex ring. The slice chamber and the water jacket which maintained the temperature at 37-38°C were made of a single piece of glass. In the assembled apparatus, a Tempunit pump supplied water to the outer jacket in a closed circuit from the thermostatically controlled bath. The 95 % 02 + 5 % CO2 gas mixture was passed through a flowmeter and a sintered-glass bubbler immersed in the

The Effects of Sodium, Potassium and Calcium on Metabolism of Rat’s Cerebral Cortical Slice With or Without Electrical Stimulation

“…(21--23). A certain small amount of external sodium was necessary in the establishment of resting membrane potential in mammalian cerebral cortical slices, and when the medium was deprived of sodium, the resting potential was not observed (14), but the external sodium over a certain critical concentration scarcely affected the resting potential (14,21). In the same way, the production of action potential required the existence of external sodium and its concentra tion difference across the membrane, and the changes in the action potential were dependent on the external sodium concentration (21,(23)(24)(25).…”

“…was said that after the preparation of separated mammalian cerebral cortical tissue, the tissue reached approximate stability in its Na, K and Cl ions about 15 to 30 minutes after it was incubated by oxygenated glucose-containing medium (12) ; Pappius et al (13) have also reported on the same tendency concerning K ion and stated that such an equi librium state continued for about 60 minutes; the reappearance of the resting membrane potential in cerebral tissue in vitro also required some 20 to 30 minute incubation (14). Thus, it seems that the tissue in vitro requires incubation for a certain interval of time before it reaches astate of equilibrium.…”

“…Considering these experimental facts, the present results of the rise in inorganic phosphate and lactic acid levels in the unstimulated tissue with little or no potassium are supposed to have been induced by the increase in sodium adsorption in the tissue ; on the contrary, the respiratory and metabolic responses to electrical stimula tion were remarkably suppressed; this increase in sodium adsorption in the unstimulated tissue would have inversely reduced the responses to electrical stimulation. Hillman et al (14) observed that the resting membrane potential of guinea-pig cerebral slice incubated in normal medium was of about 50-60 mV and when the potassium concentration of the bathing fluid was increased above its normal value of 6.2 mm, the tissue potential diminished and fell to zero with about 20-50 mm-K+ in the outer medium ; below 6.2 mm-K' tissue potential also fell and could not be recorded in media of 1 mm-K+, when the tissue contained 44 aequiv K/g. According to this observation, the membrane poten tial seems to decrease in any case, whether the external potassium is more or less than its normal concentration.…”

“…For the uptake of K (41), or for the membrane depolarization due to potassium (52), or for the production of potassium stimulation, or to maintain the rate of oxygen uptake of cerebral cortical slices, or for the maintenance of resting membrane potential, a minimum concentration of sodium in the medium was necessary. And this sodium seems unable to be replaced by any other ion (14,26,53). In the present experiment, when the ex ternal K' ion concentration was changed to 7 mm, 21 mm or 50 mm, there was almost no change in the respiration and metabolism of the tissue in the absence of external sodium, either in the presence or in the absence of electrical stimulation, as compared with those of the tissue in NaCl-free medium ; that is, potassium alone seemed metabolically indifferent, when the external sodium was absent.…”

“…The absence of calcium caused refractory condition in squid axons, and in medium low in calcium, the squid axons were said to cause a change similar to depolarization of the system controlling Na and K permeability (62). It has been reported that the absence of calcium caused delay in repolarization on incubating the cerebral cortical slices (14). Then, in the present experiment, the responses to electrical stimulation were slightly depressed in addition of external calcium ; judging from the results of above observations, this fact would probably be due to the simple competition of sodium with calcium for sodium site, although the present data did not indicate it distinctly in the unstimulated tissue probably due to experimental error.…”