CENTRAL NERVOUS SYSTEM METABOLISM IN VITRO 



I 83 I 



nervous system, but a method permitting study during 

 periods of a second or so have been described (74, 77, 

 •47)- 



Analytical Notes 



Fixing and extracting central nervous tissues for 

 analysis have employed many of the commonly used 

 reagents. Trichloroacetic acid has been used exten- 

 sively to obtain 'acid-soluble phosphates' (76, 112, 

 138, 194). Analysis for these compounds from cerebral 

 tissues meets special problems which require careful 

 consideration (76, 194). Trichloroacetic acid has been 

 employed in preparation for nucleic acid analyses 

 (116, 196). "Phosphoprotein' from cerebral tissues 

 also requires special care in interpretation of analyti- 

 cal values (48, 76, 196). 



Perchloric acid has been employed to yield acid- 

 soluble phosphates and has the virtue of yielding a 

 clear solution from which the excess perchloratc can 

 be largely removed as the potassium salt at o°C; this 

 is important in subsequent assays using enzymic 

 methods (100) or ion exchange (186). As a protein 

 precipitant and agent for extracting nucleotides, 

 perchloric acid has the advantage over trichloroacetic 

 acid of not absorbing light at wavelengths about jf>o 

 m/u, although when hot it may extract other substances 

 which absorb strongly at about 275 ium (1 16). 



Tungstic acid (20 mmole) is an effective extractant 

 of cerebral tissues for chloride analysis (208) as is 

 sulphosalicylic acid for glutathione. Hot ethanolic 

 potassium hydroxide, as commonlv used in determin- 

 ing glycogen in other tissues, yields from cerebral 

 tissues a glycogen precipitate contaminated with 

 carbohydrate-containing lipids which may be re- 

 moved by washing with chloroform-methanol (91, 

 109). 



NORMAL METABOLIC CHARACTERISTICS 



Water and Electrolytes, 



When cerebral slices are cut and incubated aerobi- 



cally in salines, there is an increase in weight of up to 

 50 per cent of that of the original slice within 1 hr. 

 (40, 162, 177, 191). Although this increase has been 

 ascribed variously to an increase in intracellular 

 water (4) and in extracellular water (191 ), it has re- 

 cently been shown (108, 162) that the fluid entering 

 the tissues is not water alone but a solution approxi- 

 mately isotonic with the medium. The problem has 

 been reinvestigated by Pappius & Elliott (162) using 



thiocyanate, sucrose and inulin to determine the 

 volume of the extracellular space. These agents are 

 assumed not to enter the cells and consequently the 

 amount taken up from solution by a slice under differ- 

 ing conditions affords a measure of the extracellular 

 volume. It was found that aerobic swelling in a glu- 

 cose bicarbonate medium was due to an increase in 

 the extracellular space, the "nonthiocyanate non- 

 sucrose' space remaining reasonably constant. The 

 inulin space was much less than the thiocyanate 

 space (41). The suggestion that this is owing to the 

 inulin molecule being too large to enter the whole of 

 the extracellular space is diflicult to reconcile with the 

 finding of Stern and co-workers (191) that glutamo- 

 aspartic transaminase, an enzyme of molecular 

 weight (io.ooo, can readily diffuse out of tissue sli< es 

 It was concluded ( [62, 163) that swelling involves an 

 uptake of fluid which need not be continuous with the 

 medium. 



Suppression of swelling by including high concen- 

 trations of sucrose or of polyvinylpyrollidone in the 

 medium reduces the intracellular space but not the 

 extracellular space. Swelling increases if slices are 

 incubated anaerobieally (162, 191 1, the increase being 

 intracellular, suggesting that energ) is essential for 

 the maintenance of the size of the intracellular space. 



Studies of the distribution of s.ilts, such as potassium 

 salts, between the fluid and the tissue slice show a 

 similar dependence upon energy-yielding processes 

 and other factors. I bus under anaerobic conditions 

 or immediately after cutting and placing in saline, 

 cerebral slices lose from v> to 70 per cent cil their 

 total potassium (162, 204). Under aerobic conditions, 

 the addition of glucose to the medium assists the re- 

 accumulation of some of the lost potassium. Under 

 anaerobic conditions glucose alone cannot support 

 such a recovery and glycolysis must be stimulated b\ 

 the addition of pyruvate to promote even a small re- 

 covery of lost potassium (163). 



The greatest reaccumulation of potassium occurred 

 if glutamate, but not glutaminc, was added to the 

 medium containing glucose (204). Glutamate alone 

 was not more effective than glucose alone. Of all 

 other amino acids tested only aspartic acid could 

 replace glutamate presumably because of its conver- 

 sion to glutamic acid in the tissue slice. The role of 

 glutamate is not clear. Since an equivalence exists 

 between the amounts of glutamate and potassium 

 accumulated by the slice, it is possible that the 

 7-carboxvl group is essential to potassium transport. 

 Glutamate causes swelling owing to an increase in 

 the intracellular space (163) and it has been calcu- 



