1828 



II Willi' i( ik < II- l'll\ s A 



NEUROPHYSIOLOGY III 



absence of an adequate supply of materials, cell 

 integrity fails and, with such failure, metabolic and 

 other characteristics which depend on cell structure 

 also change. The totality of substances which must be 

 supplied to these tissues to maintain them chemically 

 and otherwise similar to their in vivo condition is not 

 yet known; approximations which have proved ade- 

 quate for many studies are described in the following 

 section. The two substances which are most important 

 are oxygen and glucose; of these, oxygen is the sub- 

 stance which b) its diffusion brings defined physical 

 limits to the size of the preparation which can be 

 studied. Calculation and experiment indicate that 

 with a tissue of respiratory rate of some ioo pmole < >j 

 per gm fresh wt. per hr., such as cerebral cortex, 

 diffusion of oxygen from a solution in equilibrium 

 with an atmosphere of ioo per cent O s gives adequate 

 oxygenation to a depth of about 0.15 to 0.2 mm. 

 Accepting this limitation, the tissue can therefore be 

 prepared with minimal damage in the form of a sheet 

 0.3 to 0.4 mm in thickness. Corresponding thickness 

 for white matter of respiratory rate 25 or 50 pinole O a 

 per gm per hr. would be about 0.5 to 0.7 mm. It will 

 be noted that these arrangements, which are the ones 

 most frequently adopted, expose the outer parts of the 

 tissue to unusually high oxygen tensions while in the 

 center of the slice tension may be lower than in vivo. 

 Methods for slicing the tissue to this form have been 

 described 1 1 25, i<|o). 



From small or irregularly shaped pieces of tissue, 

 slices are best prepared bv chopping ( I ;-;()). A mechan- 

 ical chopper has been devised which enables blocks of 

 white matter, otherwise difficult to slice, to be uni- 

 formly chopped. 1 From most parts of the central 

 nervous system, cell-containing suspensions which 

 can be pipetted may be obtained by chopping twice 

 in two directions at right angles to each other. The 

 mechanical chopper enables the transition from cell- 

 containing to cell-free systems to be appraised. When 

 cuts are made at intervals of 0.02 mm in two directions 



.11 righl angles to each Other, svstems intermediate in 

 properties between the normal slice and homogenate 

 are obtained. Intermediate preparations may also be 

 obi. lined l>\ enzymic treatment ol the cell-containing 

 tissue, yielding from cerebral cortex an approxima- 

 tion to a cell-suspension 1 1 32 1. 



Systems in which cell structure has been destroyed 

 in normall) prepared b) grinding tissues in aqueous 

 fluids; other procedures are noted subsequently. 

 Grinding in water or in greatly hypotonic solutions 



11 \ln ki. \iiii Works, GomshaU, Surrey, England. 



disturbs not only cell structure but also subcellular 

 elements and is to be regarded as a possible pre- 

 liminary to systems containing individual enzymes 

 rather than to those now under consideration. These 

 typically employ dispersion in approximately isotonic 

 or hyptertonic solutions at o~4°C. Most use (1, 22, 

 ")9- 79> • 58, 183) has been made of 0.25 M sucrose 

 following procedures developed primarily in connec- 

 tion with tissues other than those of the central ner- 

 vous system. In first using a given homogenizer for a 

 given tissue, an investigator must examine micro- 

 scopically the resulting suspension or preparations 

 from it in order to choose grinding conditions which 

 do in fact give minimal residual cell structure. The 

 breakdown is of course gradual, and with brief grind- 

 ing or a loosely fitting pestle even large cells will re- 

 main intact, shorn of the greater part of their axons 

 and dendrites. 



Manx" studies have employed unfractionated 

 homogenates; in others, separation into three or four 

 fractions has been carried out by centrifuging, most 

 frequently in the sucrose in which the tissue was 

 ground (22, 23, [61 I. Chemical and enzymic analyses 

 of the fractions have been made (2, 22, ",<)). As in 

 other tissues, the fractions recognized have been pre- 

 dominantly unchanged cells, nuclei, mitochondria, 

 microsomes and soluble components. Probably more 

 consideration should be given to the fate of the myelin, 

 to subcellular entities other than those named and to 

 selection of discrete parts of the central nervous sys- 

 tem before homogenizing. Separation of secretory 

 granules from the pituitary gives a valuable example 

 (228). 



Preparation of tissues for histochemical stud} can 

 often be regarded from a metabolic point of view as 



yielding systems of the 1 t\ pe, for although relative 

 positions of subcellular entities in the cell mav be 

 preserved, selective permeability of the cell is usually 

 not. Freeze-drying and sectioning methods have been 



developed iiifi, tig, 1 v,, 156, 200) specifically for 

 displaying chemical and metabolic attributes of cen- 

 tral nervous tissues. In these methods the tissue may 

 be frozen, sectioned, dehydrated while frozen and 

 microdissected while dr) (118, tig) or while in oils 

 |8); or frozen, dried, embedded in paraffin, sec- 

 tioned and extracted with a fat solvent (1 |6) Cognate 

 procedures, on a preparative scale, have been to 

 lieeze-drv blocks oi tissue, grind these in nonaqueous 

 solvents, and separate therefrom nuclei and other sub- 

 cellulai pai deles. 



For stud) oi spec i he enzyme systems, a few illustra- 

 tive examples, specific to central nervous tissues, may 



