a study of extracellular space in central...

A STUDY OF EXTRACELLULAR

SPACE IN CENTRAL NERVOUS TISSUE

BY FREEZE-SUBSTITUTION

A. VAN HARREVELD, M.D., JANE CROWELL, Ph.D., and

S. K. MALHOTRA, D.Phil.

From the Kerckhoff Laboratories of the Biological Sciences, California Institute of Technology,Pasadena, California

ABSTRACT

It was attempted to preserve the water distribution in central nervous tissue by rapid freezingfollowed by substitution fixation at low temperature. The vermis of the cerebellum of whitemice was frozen by bringing it into contact with a polished silver mirror maintained at atemperature of about -207C. The tissue was subjected to substitution fixation in acetonecontaining 2 per cent Os0 4 at -85°C for 2 days, and then prepared for electron micros-copy by embedding in Maraglas, sectioning, and staining with lead citrate or uranylacetate and lead. Cerebellum frozen within 30 seconds of circulatory arrest was comparedwith cerebellum frozen after 8 minutes' asphyxiation. From impedance measurementsunder these conditions, it could be expected that in the former tissue the electrolyte andwater distribution is similar to that in the normal, oxygenated cerebellum, whereas in theasphyxiated tissue a transport of water and electrolytes into the intracellular compartmenthas taken place. Electron micrographs of tissue frozen shortly after circulatory arrest re-vealed the presence of an appreciable extracellular space between the axons of granularlayer cells. Between glia, dendrites, and presynaptic endings the usual narrow clefts andeven tight junctions were found. Also the synaptic cleft was of the usual width (250 to300 A). In asphyxiated tissue, the extracellular space between the axons is either completelyobliterated (tight junctions) or reduced to narrow clefts between apposing cell surfaces.

An apparent paucity of extracellular space in thecentral nervous system has been demonstratedwith the electron microscope by many authors.Horstmann and Meves (15) determined theextracellular space from the mean distancesbetween the cell membranes (about 200 A) andthe mean diameters of the cellular elements. Avalue not exceeding 5 per cent was in this waycomputed for the extracellular volume in theshark's optic tectum. An extracellular space of 3to 5 per cent of the tissue volume has been esti-mated for mammalian central nervous tissue (17).Recently, a tight apposition of cell membranes incentral nervous tissue was described in materialfixed by perfusion with glutaraldehyde or formal-

dehyde, reducing the extracellular spaces evenmore (16).

These findings apparently conflict with therelatively high concentration of the traditionallyextracellular ions, sodium and chloride, in thecentral nervous system. On the assumption thatall the sodium and chloride is extracellular andthat the extracellular material has the same ioniccomposition as cerebrospinal fluid, Manery (19)and Manery and Hastings (20) estimated achloride space of 25 to 35 per cent and a sodiumspace of 38 per cent of the tissue volume. Wood-bury et al. (45) found after 8 hours' equilibrationwith Cl36 a chloride space of 25 per cent. Aprisonet al. (2) reported estimates of extracellular water

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FIGURE 1 The apparatus for rapid freezing consistedof a Dewar vessel (a) closed with a bakelite plate (b).A brass tube (c) was fastened to the bakelite under ahole in the plate. The bottom of the tube was formedby a silver plug (d) the upper surface of which had beenpolished mirror smooth. The pressure in the Dewarvessel which was filled with liquid nitrogen could bereduced by evacuating N2 through a tube passingthrough the bakelite plate. Helium, after flowingthrough a spiral (e) immersed in the liquid nitrogen,was released over the surface of the silver plug. A glassplate placed over the hole in the bakelite retarded theescape of helium from tube e. The tissue was attached

varying from 36 per cent for the cerebral cortex to27 per cent for the medulla. Since some of thesodium and chloride is present in the cellular ele-ments, for instance in the neurons (7), the sodiumand chloride spaces will be larger than the actualextracellular space. Methods for determiningextracellular space based on the comparison inplasma and tissue of the concentrations of com-pounds which do not readily pass cell membranesare less suitable for the central nervous system dueto the presence of the blood-brain barrier. Fromsuch efforts, estimates between 4 per cent and 22per cent have resulted, depending on the com-pounds used and the measures taken to circumventthe barrier (1, 6, 25, 34, 44).

The extracellular space can also be estimatedfrom the electrical impedance of a tissue. Thecurrents used for impedance measurements arecarried mainly by extracellular electrolytes, sincethe high resistance of the limiting cell membranesimpedes movements of intracellular ions. It hasbeen possible to compute accurately the volumepercentage of the particles in cell suspensions,which can be considered as models for tissues, fromthe specific resistances of the suspension and of thesubstrate (5, 12). A mean specific impedance of208 to 256 2 cm has been determined for thecerebral cortex (11, 24, 40). From this low specificimpedance, which is not more than 4 to 5 timesthat of a tyrode solution, an extracellular space inexcess of 25 per cent was estimated (43).

Although there are uncertainties in the indirectestimates of extracellular space in central nervoustissue, the values obtained are so high that theystand in striking contrast with the electron micro-scope observations. These discrepancies can bereconciled by assuming that the sodium andchloride present in the tissue is contained incellular elements, especially in the glia (17, 32,37). Furthermore, to account for the low specificimpedance of the tissue, the assumption has to bemade that the membranes of glia cells are ionpermeable and do not markedly impede currentflow (17, 37, 42, 43). Alternately, it has been sug-gested that an appreciable space exists in theliving nervous tissue, but that the preparationnecessary for electron microscopy results in a trans-port of extracellular material into the intracellular

to a wooden stick (f) which was fastened to a piston(g) moving in a barrel (h). By regulating the outflowof air from the barrel, the speed of descent of the tissuecould be controlled.

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compartment (37). There is evidence that thewater and electrolyte distribution in central nerv-ous tissue is labile and that transport of extracellu-lar material into the intracellular compartmentoccurs on relatively slight provocation. For in-stance, asphyxiation of central nervous tissue re-sults within minutes in a marked increase of itsimpedance, suggesting that electrolytes have dis-appeared from the extracellular space (36-38, 41).The latter conclusion is supported by the obser-vation with the light microscope of an asphyxialtransport of chloride and water into dendrites(35-38, 42, 43) and into certain glial elements ofthe cerebellum (fibers of Bergmann, 36). Com-parable impedance changes and a similar transportof chloride were observed during spreading de-pression (37, 42). It was postulated that thetransport of electrolytes into the intracellularcompartment is caused by an increase in perme-ability of the cell membranes for sodium andperhaps for other inorganic ions like chloride andpotassium. If it is also assumed that the membraneremains impermeable for large intracellular or-ganic ions, then Donnan forces can be expected tocause a transport of extracellular sodium chlorideinto the intracellular compartment. Water willhave to accompany the electrolyte transport tomaintain osmotic equilibrium.

If short periods of asphyxiation or the functional

changes occurring during spreading depression

can produce appreciable shifts of electrolytes and

water into the intracellular compartment, it is not

inconceivable that the strong stimulation and

chemical alterations caused by the application of

fixatives result in a similar material transfer. In

the present investigation, it was attempted to pre-

serve the water distribution as present in the

living tissue by the use of a freeze-substitution

technique.

METHODS

The method of freeze-substitution used in the presentstudy has been described in detail elsewhere (39).The tissue was frozen rapidly by bringing it intocontact with a heavy silver plug, polished mirrorsmooth and cooled to the temperature of liquidnitrogen under reduced pressure (about -207°C).The silver surface was protected against the con-densation of water and air by a stream of cold dryhelium gas flowing over it. The tissue to be frozen waslowered onto the polished silver surface by a guidingsystem which made it possible to let it descend

through the cold helium at a controlled velocity (30to 40 cm/sec). The apparatus is schematized in Fig. 1.

The cerebellar vermis of white mice anesthetizedwith urethane (0.25 to 0.3 cc of a 20 per cent solution)was used exclusively in this investigation. This tissuewas chosen because spreading depression, whichcauses similar, although less pronounced, electrolyteand water movements as asphyxiation (37, 42), isnot readily elicited in the cerebellum by slightmechanical stimuli as is the case in the cerebralcortex (9, 27, 36). In order to minimize the mechani-cal disturbance of the tissue, the cerebellum was notremoved from the animal, but the whole head wasplaced on the carrier of the guiding system. A suitablyshaped brass strip was introduced into the mouthand kept in place by closing the jaws tightly on thestrip with a ligature circling the head and passingunder the zygomatic arcs. The strip was fastened tothe carrier at such an angle that the vermis becameparallel with the surface of the silver plug. This partof the procedure was carried out while respirationand circulation were intact. The body was thensevered from the head with a scissors' cut immediatelycaudal to the cerebellum. Two cuts were madethrough the lateral regions of the occipital bone, andthe part of the skull covering the cerebellum waslifted off. Blood and fluid on the cerebellum wereremoved by gently touching the surface with tissuepaper (Kleenex) moistened with Ringer's solution.The carrier was then lowered on the freezing surface.The tube, which carried the silver plug at its lowerend (Fig. 1, c), was filled with liquid nitrogen. Thehead was kept in there for 2 to 3 minutes and thentransferred to a Dewar vessel filled with liquid ni-trogen. The cerebellum was flattened on contact withthe silver surface, but the cerebellar vermis andhemispheres could be recognized after fixation by thecharacteristic gyration.

The tissue was then subjected to substitutionfixation at -85°C in a 2 per cent osmium tetroxidesolution in acetone for 2 days. In some experimentsit was shown that after that time most of the molec-ular layer can be rubbed with a wad of cotton fromthe cerebellum kept at low temperature (-80°C).This indicates that a layer of 50 to 100 thick be-comes substituted in 2 days. The head was warmed inthree steps (-25°C, 4°C, and room temperature)while still in the substitution medium. The vermis wasisolated and cut into blocks, each consisting of asingle folium. These were kept for 2 to 3 hours in re-peatedly changed acetone, then in propylene oxide,and finally embedded in Maraglas (10). All sectionswere cut on an LKB Ultrotome at right angles withthe folia. Sections about 1 .u thick were stained withmethylene blue and Azure II (28) for examinationby light microscopy. Thin sections for electron micros-copy were stained with lead citrate (26) or withuranyl acetate (13) followed by the lead stain of

VAN HARREVELD, CROWELL, AND MALHOTRA Extracellular Space in CN Tissue 119

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Millonig (21). Electron micrographs were taken witha Philips EM-200.

The study of the -p-thick sections made it possibleto discard unsuitable blocks. The flattening of thetissue on contact with the silver surface can beexpected to cause distortion of the tissue structure.By accepting only blocks of which the thick sectionsshowed the normal position and direction of recog-nizable tissue elements in the molecular layer, areaswere selected which had first contacted the silverand thus were frozen before they could be disturbedmechanically. Only a narrow layer of tissue, oftenabout 10 /i thick, yields electron micrographs whichdo not exhibit ice crystals. Blocks from which sectionsexamined with the light microscope showed icecrystals in the molecular layer could be discarded,since experience had shown them to be unsuitablefor electron microscopy. Also, blocks in which thepia was unusually thick, covered with a large bloodvessel, or was separated from the cortex by anappreciable space were discarded, since such surfacestructures reduce the ice crystal-free region of thecerebellar tissue which can be used for electronmicroscopy.

RESULTS

In poorly frozen preparations, ghosts of ice crystalsare recognizable as light areas, distinctly andsharply separated from each other by dark lines,which represent the protoplasm concentrated bythe formation of crystals consisting of pure H2 0.

In well frozen preparations the surface layer is freefrom such crystals, but in deeper layers ice crystalsare always present especially in the nucleus andcytoplasm of cells, and in large dendrites. Fig. 9demonstrates ice crystals in a cell body; Fig. 2shows the crystals in a dendrite. Fig. 9 also shows

the damage that results from the formation of icecrystals: the cellular inclusions generally get dis-organized and sometimes even lose their clearidentity. Also, the nuclear membrane in this figureis severely distorted. Still deeper, the ice crystalsbecome larger and more uniformly distributedthroughout the tissue. In such regions, the organi-zation of the tissue and of the individual cellularelements is unsuitable for electron microscopy.

In electron micrographs without recognizableghosts of ice crystals, the criteria now in generaluse for evaluation of satisfactory fixation (Palade,22; Palay et al., 23) are easily met with. Suchmicrographs show sharply delimited, continuouscell membranes, clearly identifiable membranouscellular inclusions, (e.g., endoplasmic reticulum,mitochondria, Golgi apparatus, synaptic vesicles),and uniformly fine ground cytoplasm (see Figs. 5,6, 8, 10, 15, 16). The tissue which meets the aboverequirements and is uniformly satisfactorily pre-served is always found in the superficial part ofthe molecular layer immediately beneath the pia.An estimate of the depth of this layer from the

Abbreviations

a, Axonas, Astrocyteb, Fiber of Bergmannc, Collagen fibersd, Dendriteds, Dendritic spineer, Endoplasmic reticulumg, Glia

ga, Golgi apparatusm, Mitochondrionn, Nucleusp, Piapb, Process of Bergmann fiberps, Presynaptic endings, Synapse

FIGURE 2 Micrograph of a large field of the cerebellar molecular layer frozen 8 minutesafter decapitation. The dendrite (d) in the upper part of the figure is a continuation of theone seen in the lower part, as indicated by the mitochondria in the overlap. The dendriteis characterized by the presence of spines (ds). The pia (p) of the cerebellum is shown inthe upper part of the figure. Immediately underneath the pia are end-feet of Bergmannfibers (b). The profiles of axons (a) are present in groups throughout the micrograph(axon fields). Numerous synapses (s) are recognizable by the vesicles in the presynapticterminals. Many relatively large structures which show few inclusions are scatteredamongst the axon fields; these are difficult to identify. They may be parts of glia or ofdendrites. Ghosts of ice crystals are recognizable as light areas surrounded by relativelyelectron-opaque material in the lower part of the dendrite. Note the absence of appre-ciable extracellular space. (Lead citrate; the calibration line indicates A).


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electron micrographs gives a figure of approxi-mately 10 /. The limitations of the method restrictthis investigation to a study of the very surface ofthe molecular layer of the vermis.

Identification of Cellular Elements in ElectronMicrographs

The identification of cellular elements in electronmicrographs of central nervous tissue made by thefollowing authors has been adopted in the presentpaper. Gray (14) has made a careful study of thefine structure of the cerebellum, with appropriatecontrol by light microscopy. Palay and his col-leagues (23) have published excellent electronmicrographs of the cerebellum and have pointedout criteria for the identification of some of thecellular elements, such as oligodendrocytes andfibrous astrocytes. More recently, Schultz (31) hasalso provided some data for identification of themacroglial elements.

All electron micrographs published in thepresent paper have been produced from sectionscut at right angles to the folia so that they are inthe plane of the dendritic arborization of thePurkinje cells. This plane cuts the fibers of Berg-mann longitudinally. These fibers run straightupward from cell bodies situated in the layer ofPurkinje cells and form end-feet on the surface ofthe molecular layer (33). The axon branches of thegranular cells are cut at right angles. The dendritesof the Purkinje cells can be identified in theelectron micrographs by the presence of character-istic "thorns" or "spines" on their surface, whichserve as postsynaptic processes for axodendriticsynapses (14). Examples of such "spines" areshown in Figs. 2 to 4. Often the section passesonly through the spines, and the relationship withthe main dendritic branch is not obvious. They

can then be identified only when associated withpresynaptic endings characterized by synapticvesicles (Figs. 2, 3, 5, 10 to 14). A dendrite can bedistinguished from a fiber of Bergmann by the factthat the latter forms an end-foot, which runs im-mediately underneath and parallel to the pia(Figs. 3, 6, 7, 13). Dendrites stop short of the pia.The distinction between the two may become un-certain if the fiber of Bergmann cannot be followedright up to the pia. To facilitate identification,electron micrographs have been selected whichshow the formation of end-feet by fibers of Berg-mann. If the cytoplasm of a Bergmann fiber iscompared with that of a dendritic branch lyingclose by, it is noticed that the mitochondria aremore numerous in the dendrite (Figs. 3, 6, and 13).Moreover, there appear to be more elements of theendoplasmic reticulum dispersed in the cytoplasmof the dendrites than in fibers of Bergmann.

The fibers of Bergmann give out branches alongtheir length which invade the surrounding tissue.These branches do not look like the well definedand club-like spines of the dendrites, but are moreirregular (Fig. 7). The electron micrographs shownumerous glial elements scattered throughout thetissue. These show few cellular inclusions and may,at least in part, be sections of the branches ofBergmann fibers (Figs. 2 to 6, 11, 14, 15).

The end-foot of the fiber of Bergmann shown inFigs. 7 and 8 exhibits a special arrangement of theelements of the endoplasmic reticulum enclosedwithin a membrane-delimited, rounded structure.It does not seem to have been described before inthe available literature on the molecular layer ofthe cerebellum and has been very infrequently seenduring the present study. In ultrathin sections, theelements of the endoplasmic reticulum, consti-tuting this organelle, appear either as sausage-like

FIGURE 3 Micrograph of an area of the asphyxiated molecular layer similar to that inFig. 2. A fiber of Bergmann (b) is seen forming an end-foot under the pia (p). Anotherfiber of Bergmann is seen between the dendrite (d) on the left and the Bergmann fiberforming the end-foot. The micrograph exhibits the superficial 16 of the molecular layer.Ghosts of ice crystals begin to appear in the deepest regions only. (Lead citrate; calibra-tion line indicates 1 A).

FIGURE 4 A detail of Fig. (right upper quadrant) is shown at higher magnification. Astructure, probably a dendritic spine (ds), is present which seems to form a synapse. Nar-row clefts are present between adjacent cellular elements. Where three axon or other cellprocesses meet, triangular spaces (arrows) are found. An irregular glial structure (g) issurrounded by axon fields. (Lead citrate; calibration line indicates 1 u).


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cisternae or circular-to-oval profiles, packed to-gether in no well defined order. The membranethat forms the limiting boundary of this organelleand the membranes of the endoplasmic reticulumenclosed in it have the usual unit membranestructure (Fig. 8), and are of about equal thickness(~ 75 A). This organelle has a superficial re-semblance to the "growth spiral" described byRobertson (30), consisting of a single spirallywrapped cisterna of the endoplasmic reticulum inthe cytoplasm of Schwann cells in developingmouse sciatic nerve.

In addition to the fibers of Bergmann, dendritesof the Purkinje cells, and pre- and postsynapticstructures, numerous profiles of unmyelinatedaxons of the granular cells appear in the electronmicrographs of the molecular layer of the cere-bellum. These are seen as small (about 0.2 indiameter) rounded, or polygonal structures whichgenerally appear in groups, axon fields (Figs. 2, 3,5, 10 to 13, 16).

The identification of various cell bodies inelectron micrographs of the cerebellar molecularlayer is often difficult, because many sections passthrough only small regions of cytoplasm, whichmay poorly represent the entire cell body. Noneof the cell bodies illustrated in Figs. 9, 10, and 12seems to be that of a neuron. A vertebrate peri-karyon is generally characterized by the presenceof aggregates of elements of the endoplasmicreticulum in association with clusters of ribosomesthat constitute Nissl bodies; and these micro-graphs do not exhibit such an organization. Thecell body shown in Fig. 9 appears to be an astro-cyte rather than oligodendrocyte because thecytoplasm is not densely packed with ribosomes,

which is a characteristic of the oligodendrocytes(23, 31). Moreover, oligodendrocytes have anarrow rim of cytoplasm and dense nuclearchromatin (31). The cell body shown in Fig. 10 isdifficult to identify; the section has not passedthrough the nucleus, but the appearance and sizeof the cytoplasm suggest that it may be an astro-cyte. The small fraction of cytoplasm seen in Fig.12 may also be that of an astrocyte.

A survey of the electron micrographs of thecerebellar molecular layer produced by the freeze-substitution technique shows that the quality ofpreservation of fine structural details of cellularelements is comparable to that seen by routinefixation with OSO4. However, the mitochondriafrequently show an unusual feature inasmuch asthe usual, about 100 A, gap between the outerand inner limiting membranes and within thecristae is not seen. The mitochondrial membranesare represented by three dense lines separated bytwo pale lines of about equal width. The totalthickness is about 120 A; and they resemble thetight junctions of epithelial cells (8) and also theexternal compound membrane in Schwann cellsdescribed by Robertson (30). This appearance ofthe mitochondria has been seen not only in thecerebellum, but also in the exocrine cells of thepancreas and in liver cells. This will be discussedin more detail in a future paper (Malhotra andVan Harreveld, J. Ultrastruct. Research, 1965, inpress).

Differences between Asphyxiated and Non-As-phyxiated Tissue in Electron Micrographs

In part of the experiments, the cerebellum wasfrozen between 25 and 30 seconds after decapi-

FIGURE 5 This micrograph shows the paucity of extracellular space in the molecularlayer frozen 8 minutes after decapitation. Note the presence of tight junctions (arrows)between adjacent cellular elements. The large structure on the left is probably a dendrite(d). An invagination of a dendritic spine (ds) into a presynaptic structure (ps) is seen.(Lead citrate; calibration line indicates 1 ).

FIGURE 6 In this figure of molecular layer asphyxiated for 8 minutes, a fiber of Bergmann(b), and a dendrite (d) can be compared. The fiber of Bergmann forms an end-foot underthe pia (p) and shows a process (pb) on the left. The dendrite forms a synapse directly witha presynaptic structure (ps). Mitochondria and elements of the endoplasmic reticulumare more numerous in the dendrite than in the Bergmann fiber. The identity of the ho-mogeneous, rounded, membrane-delimited bodies in the end-foot of the fiber of Bergmannis not obvious; these may be lipid inclusions. (Lead citrate; calibration line indicates 1p).


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tation. It is known that, during such a period ofoxygen deprivation due to circulatory arrest, theimpedance of the tissue does not increase markedly(36). According to the concept discussed in theIntroduction, this would indicate that no ap-preciable shifts of electrolytes and water have takenplace during this period. The water distribution inthe tissue at the moment of freezing would,therefore, represent that of the living cerebellum.In other experiments, the tissue was frozen 8minutes after circulatory arrest. Such a period ofasphyxiation causes a major (40 to 70 per cent)increase in tissue impedance, which would indicatethat an appreciable part of the extracellularelectrolytes accompanied by water has moved intothe intracellular compartment. It was, therefore,of interest to compare the extracellular space ofcerebellum asphyxiated for 8 minutes with that ofcerebellar tissue deprived of oxygen for less than30 seconds in electron micrographs obtained by thefreeze-substitution technique.

Figs. 2 to 10 are electron micrographs of thesuperficial parts of the molecular layer of thevermis of mice which had been decapitated about8 minutes before freezing. The general appearanceof the various cellular structures is essentiallysimilar to that seen in comparable electron micro--graphs of cerebellar tissue prepared by ex situ or byperfusion fixation with OsO4 (Gray, 14; Palay et

al., 23; Smith, 33). The cells, dendrites, axons,presynaptic structures, and processes of glial cellsare all closely packed and clearly defined, givingthe impression of uniformly satisfactory fixation.A study of suitably stained sections at high magnifi-cation showed in many preparations tight junctions(zonula occludens) between adjacent cellular ele-ments. These junctions shown in Fig. 5 exhibit afive-layered structure typically seen in epithelial

cells (8). The total thickness of the membranes atthe junctions is about 150 A. Similar tight junctionshave been described recently by Karlsson andSchultz (16) in mammalian central nervous tissuefixed by perfusion with glutaraldehyde or form-aldehyde and subsequent treatment with OsO 4. Inmicrographs of other blocks (Fig. 4) the totalthickness of the apposed membranes is slightlylarger (up to about 250 A), and no middle denseline formed by fusion of the outer lamellae of theapposing membranes is seen; an actual narrowcleft may be present in these instances. Such prepa-rations show small but definite, often triangularareas of extracellular space in which three cellularelements meet (Fig. 4). The synaptic gap wasfound to be of the order of 250 A; a similar valuehas been reported by Gray (14) in ex situ Os0 4-

fixed cerebellar tissue.Figs. 11 to 17 are electron micrographs of

cerebellar molecular layer frozen within 30 secondsof decapitation. There are consistent differences ascompared with the micrographs of 8-minuteasphyxiated cerebellum. The most striking differ-ence is the presence of appreciable extracellularspace between the axons, which were never tightlypacked as in the asphyxiated preparations (Figs.11 to 17). These spaces are usually uniformlydispersed amongst the axons. However, in a fewinstances, rather large extracellular lakes werepresent in the axon fields (Fig. 12). Many axonstouch each other over only small areas. Withhigher magnification, such areas of contact showedin a few instances the tight junction structure(Fig. 17); but in most preparations no middledense line was apparent (Fig. 16).

The clefts between the other cellular elementsof the molecular layer are similar to those foundin preparations fixed with OsO 4 either ex situ or by

FIGURE 7 This figure shows an unusual formation of the endoplasmic reticulum en-closed by a membrane in the end-foot of a fiber of Bergmann (b) described in detail in thetext. Processes of the Bergmann fibers (pb) are shown. The preparation is of 8-minutesasphyxiated cerebellum. (Lead citrate; calibration line indicates 1,u).

FIGURE 8 The typical unit membrane appearance in the endoplasmic reticulum (arrows)and in the limiting membrane (double arrow) of the structure shown in Fig. 7. (Leadcitrate; calibration line indicates 0.1 g).

FIGusRE 9 Nucleus (n) and cytoplasm of a cell identified as an astrocyte in asphyxiatedcerebellum. This cell was situated some distance from the surface and shows ghosts of icecrystals and deformed cytoplasmic inclusions. (Lead citrate; calibration line indicates

1p].

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perfusion. In most electron micrographs, the totaldistance between the inner lamellae of the ap-posing cell membranes of glia and pre- or post-synaptic structures is of the order of 200 to 250 A(Figs. 15 and 16). However, in a few preparationstight junctions were also observed between thesestructures (Fig. 17). In these instances, the distancebetween the inner lamellae was smaller (150 A).The synaptic cleft in non-asphyxiated tissue wasof the order of 250 to 300 A (Fig. 11), which issimilar to the value in asphyxiated material.

Blocks of 16 mouse cerebellums frozen within 30seconds after decapitation were examined. Theresulting electron micrographs consistently showedan appreciable extracellular space in the axonfields. With a modification of the Chalkley method(18), it was attempted to make a quantitativeestimate of this space. In 6 suitable electron micro-graphs the extracellular space varied between 18.1and 25.5 per cent of the tissue volume, with amean of 23.6 per cent. Similar determinations onmicrographs of 8-minute asphyxiated preparationsyielded a value of less than 6 per cent.

With the light microscope, a transport ofchloride into the fibers of Bergmann and dendritesof Purkinje cells was observed and a swelling of theBergmann fibers was demonstrated (36). In the1-u-thick sections of Maraglas-embedded materialof asphyxiated cerebellum, swollen Bergmannfibers were visible as lightly stained structuresrunning from the surface of the cerebellum intothe molecular layer. Also, the dendrites of Purkinjecells appeared swollen in this material. In similar

sections of non-asphyxiated cerebellum, the mo-lecular layer was more or less homogeneous, andBergmann fibers and dendrites could be identifiedonly with some difficulty since they did not standout as lighter-colored structures. These differencesare not as obvious in the electron micrographs ofasphyxiated and non-asphyxiated material, but inFigs. 6 and 7 the fibers of Bergmann look definitelyswollen.

DISCUSSION

Methods of fixation, which give excellent preser-vation of the fine structure of central nervoustissue, have been described by Palay et al. (23)and by Bodian and Taylor (4). The quality offixation is judged by the preservation of finecytological and anatomical details and is generallydescribed as satisfactory, if the criteria adopted byPalade (22) and by Palay et al. (23) are met with.In the absence of any direct evidence on the finestructure of the living tissue, one is forced to makeuse of indirect criteria for the evaluation of electronmicrographs. From the exhaustive discussion andtenable arguments put forth by Palay et al. (23),one is reasonably well convinced that modernelectron microscopy reveals as life-like a represen-tation of the central nervous system as is possiblewith current technical procedures. One cannot besure, however, that the micrographs obtained inthis way faithfully represent each and everyfeature of the living tissue. As mentioned above,there are discrepancies between estimates of theamount of extracellular space based on biochemi-

FIGetE 10 This micrograph shows the cytoplasm of a cell identified as an astrocyte inthe molecular layer of an 8-minute asphyxiated preparation. This cell was located closeto the surface and shows well preserved cytoplasmic inclusions: e.g., endoplasmic reticu-lum (er), mitochondria (m), and Golgi apparatus (ga). The uniformly dispersed minutegranules in the cytoplasm are presumably ribosomes. Surrounding the cell are the usualnerve and glia elements of the molecular layer. (Uranyl acetate and lead; calibrationline indicates 1 A).

FIGURE 11 Micrograph of a part of the molecular layer of a cerebellum frozen within 30seconds after decapitation. The end-foot of a Bergmann fiber (b) is present on the left. Adendrite (d) extends through the field. Axons (a), synapses (s), and glia (g) can be easilydistinguished. Note that the axons are not closely packed as in the asphyxiated prepara-tions, but that the axon fields exhibit abundant extracellular space. The axons contact eachother only over small areas. The synaptic cleft in the large synapse (s) is obliterated at onepoint (arrow). A similar appearance of the synaptic clefts is also seen in Fig. 12. The insetof Fig. 11 shows at high magnification the usual structure of a synapse in the molecularlayer of a preparation frozen within 30 seconds of decapitation (Uranyl acetate andlead; calibration line indicates 0.1 u).


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cal and physiological findings and the results ofelectron microscopy. A search for special methodsdesigned to give information on this specific pointseems, therefore, desirable.

The electron micrographs produced by themethod of rapid freezing and substitution fixationused in the present investigation meet the criteriaof satisfactory fixation. The cell membranes aresharply represented and are generally continuous.The fine structure of cellular organelles seems tobe preserved as well as with any other method offixation. Similar observations have been made onother tissues such as liver (39), and more recentlyon exocrine cells of pancreas (Malhotra and VanHarreveld, J. Ultrastruct. Research, 1965, inpress) and on dorsal spinal roots (Malhotra andVan Harreveld, Anat. Rec., 1965, in press).Exactly the same method of freeze-substitutionwas applied to cerebellum frozen within 30 sec-onds of decapitation and to tissue asphyxiated for8 minutes. It would seem, therefore, that thedifferences in extracellular space observed in thesetissues are due to the state of the tissue at the timeof freezing. If the micrographs are taken at facevalue, they indicate that in cerebellar tissueshortly deprived of its oxygen supply, there is anappreciable amount of extracellular materialwhich disappears during a prolonged period ofasphyxiation.

The above conclusion implies that the method offreeze-substitution used in the present investigationpreserves the water distribution in the tissue morefaithfully than the usual chemical fixation. Thefollowing considerations seem to be pertinent inthis respect. Instantaneous freezing will obviouslypreserve the water distribution. When the tissue isfrozen slowly, however, the formation of crystalsof pure ice will concentrate the solutes. The in-creased osmoconcentration may attract water fromregions in which no ice is formed. If ice forms

uniformly and at the same rate throughout thetissue, no water transfer can be expected. In thepresent experiments, the rate of freezing wasprobably high and uniform in the surface layersof the tissue, since no evidence of ice crystal for-mation was found. In the deeper parts, small icecrystals are often discernible especially in cells anddendrites, indicating that freezing in these layersmay not have been uniform. Although the condi-tions for the above-mentioned water transportwere thus more favorable in the deeper regions,no significant differences between the amounts ofextracellular space in surface and deep layers arenoted in the micrographs shown in Figs. 2 and 12.This may be taken as an indication that watermovement due to unequal freezing has no im-portant effect in the tissue layers considered in thepresent investigation.

The membranes in regions which do not exhibitobvious ghosts of ice crystals are well preserved.This may indicate that the membranes do notsustain severe mechanical damage during theprocess of freezing. The action of acetone as afixative seems to be lost at low temperature(Baker, 3). Also, osmium tetroxide has no apparenteffect on the tissue at a temperature of -85°Csince the color of the tissue remains unchangedafter 1 to 2 days in the substitution fluid. Nochemical fixation thus seems to occur duringsubstitution. The tissue was kept for about an hourat -25°C before it was warmed to 4C, andfinally to room temperature. Fixation by OsO4occurs at -25 0 C, as shown by the blackening ofthe tissue which, in this way, is chemically fixedbefore it is brought to room temperature. Sincethe membranes are apparently not seriously af-fected by freezing and during substitution fixation,it would seem possible that the mechanism for theelectrolyte and water transport, postulated tocause the paucity of extracellular space in routinely

FIGURE 12 A large field of the cerebellar molecular layer frozen within 30 seconds of de-capitation, comparable to Fig. 2, which exhibits a similar field of asphyxiated tissue.There is some overlap of the upper and lower part of the figure. The left upper corner ofthe figure shows a nucleus (n) probably of a fibrocyte which may contain minute icecrystals. Collagen fibers (c) are visible in the pia. Most of the other structures underneaththe end-feet of Bergmann fibers (b) are either profiles of axons (a) or synapses (s). No ob-vious ice crystals are found even in the deepest region of the nervous tissue shown here.The fraction of the cytoplasm visible in the lower right corner is probably of an astrocyte(as). Note the presence of abundant extracellular space amongst the axons (Uranyl acetateand lead; calibration line indicates 1 ).


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fixed preparations, is potentially still operativeafter the substitution medium has replaced the icein the tissue. The conditions under which substitu-tion occurs may prevent such events, however.The amount of water in acetone in equilibriumwith ice diminishes as the temperature is lowered.The water content of acetone under these condi-tions was determined from the nuclear magneticresonance of the water protons and of the naturallyoccurring C13 in acetone. In Fig. 18 A, the watercontent of acetone is plotted against the tempera-ture. At the temperature of substitution (-85°C)the water content is about 2 per cent. If a frozenpreparation is placed in 100 per cent acetone atthis temperature, the solvent will proceed to dis-solve ice from the tissue. The water released locallydiffuses into the surrounding acetone, and in thisway the plane of substitution slowly penetratesdeeper and deeper into the tissue. At no time,however, will the water concentration rise above2 per cent, and the ice in the tissue is in this wayreplaced directly by 98 per cent acetone. Thesolubility of the tissue constituents in this solventis likely to be quite different from that in water.For instance, Fig. 18 B shows the solubility ofsodium chloride in acetone in equilibrium with iceat various temperatures. The solubility of NaCl at-85°C is very low (0.0018 per cent). The mecha-nism postulated to account for the transport ofextracellular electrolytes and water into the intra-cellular compartment during asphyxiation andspreading depression (see Introduction) and per-haps also during routine fixation with OsO4would, therefore, be greatly retarded and perhapsprevented by the relative insolubility of NaC inthe substituting acetone at -85C. Other tissuecomponents may be soluble in acetone, and con-centration differences across cell membranes maycause osmotic adjustments between the extra- andintracellular compartments. There is, therefore,no guarantee that the distribution of the acetonein the tissue is a faithful representation of the

original water distribution. The mean extracellu-lar volume determined in the electron micro-graphs may, for this reason, not accurately repre-sent the magnitude of the extracellular space.

It follows from the above discussion that thefaithful preservation of the water distribution inthe living tissue by the method of freeze-substi-tution may not readily be achieved. The consistentdifference in the extent of extracellular space incerebellum frozen within 30 seconds and after8 minutes of asphyxiation must, however, asmentioned above, be ascribed to differences inthe tissue at the moment of freezing. Althoughperhaps other explanations are possible, the mostsimple and direct one would be that these findingsare the expression of differences in water distri-bution which are preserved by the method offreeze-substitution. Such an assumption wouldmake it unnecessary to assume an unusually highsodium chloride content of glia elements, sincemuch of this compound would find a place in theextracellular space. It would, furthermore, give aready explanation of the low impedance of centralnervous tissue. Finally, the differences in extra-cellular space found in tissue deprived of oxygenfor 30 seconds and for 8 minutes are in goodagreement with the asphyxial impedance changesand chloride and water transport reported pre-viously.

In electron micrographs of non-asphyxiatedcerebellar tissue subjected to substitution fixationat low temperature the spaces between glial,dendritic, and presynaptic structures are similarto those found in tissue fixed with OsO4 eitherex situ or by perfusion. Also, the width of thesynaptic gap is similar. There is, however, con-siderable space between the fibers in the axonfields in contrast to the findings in routinely fixedtissue. In general, this space is rather uniformlydistributed between the axons. Whether largerspaces as shown in Fig. 12 are real cannot bedecided, especially since processes such as em-

FIGURES 13 and 14 Micrographs of superficial molecular layer of the cerebellum frozenwithin 30 seconds after decapitation. In Fig. 13, two Bergmann fibers (b) are shown form-ing end-feet under the pia (p); a dendrite (d) with two synaptic contacts (s) is exhibited onthe left. A comparison with electron micrographs from tissue frozen 8 minutes after de-capitation will show that there is considerably more extracellular space in the axon fields.In Fig. 14, a triangular extracellular space (arrow) is present on both sides of the synapse(s), between the pre- and postsynaptic structures and the surrounding glia (g) (Leadcitrate; calibration lines indicate 1 u).


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134 THE JOURNAL OF CELL BIOLOGY. VOLUME 25, 1965

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A

FIGURE 18 A, The water content (volume percent) of acetone in equilibrium with ice is plottedon the ordinate; the temperature on the abscissa.B, The solubility of NaC1 (weight per 100 cc) inacetone in equilibrium with ice is plotted on theordinate; the temperature on the abscissa.

B

bedding may well have displaced such apparentlyloosely connected cellular elements as the axonsseem to be. In asphyxiated tissue, the spaces be-tween the tissue elements, including those in theaxon fields are narrow, often resulting in tightjunctions, as has been observed after fixation byperfusion with glutaraldehyde and formaldehyde(16).

The technical assistance of Mrs. J. Pagano is grate-fully acknowledged.

This investigation was supported in part by agrant from the National Science Foundation(G-21531).

Received for publication, January 30, 1964.

(References start on p. 136)

FIGURES 15 to 17 Molecular layer frozen within 30 seconds of decapitation shown athigh magnification. Fig. 15 shows a dendrite (d) running from left to right containing twolong mitochondria. There is ample extracellular space present in the axon fields (a). Fig.16 shows an axon field immediately underneath the end-foot of a Bergmann fiber (b). Thepreparation shown in Fig. 17 perhaps contains minute ice crystals. The membranes be-tween the cellular elements show tight junctions (arrows) at many places. (Fig. 15, leadcitrate; calibration line indicates 1 u. Fig. 16, uranyl acetate and lead; calibration lineindicates 0.1 p. Fig. 17, lead citrate; calibration line indicates 0.1 At).


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VAN HARREVELD, CROWELL, AND MALHOTRA Etracellular Space in CN Tissue 137

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