biophysical properties of cultured human glial cells

BRAIN RESEARCH 279

BIOPHYSICAL PROPERTIES OF CUL TU RED H U M A N GLIAL CELLS

M. C. TRACHTENBERG*, P. L. KORNBLITH AND J. HA, UPTLI**

Neurosurgical Service, Massachusetts General Hospital, Boston, Mass. 02114 (U.S.A.)

(Accepted September 30th, 1971)

INTRODUCTION

Neuroglial nuclei outnumber neuronal nuclei several fold1,41; neuroglial cells constitute almost 5 0 ~ of the volume 29 of the mammalian neocortex. At various times these elements have been implicated, to greater or lesser degrees, in every known or suspected nervous system functiong,11, 28 (see reviews refs. 12, 29, 62, 63). The availability of reliable values for glial membrane biophysical properties could provide a basis for excluding some and testing other of these hypotheses. Such values could help to distinguish between: normal and pathological neuroglia; different glial and glial versus non-glial cell types; anatomically similar glial cells in different species; glial cells in different developmental or physiological states; and similar cells under various stress conditions, e.g. in vivo versus in vitro.

In most physiological investigations correlative cytological detail of the cells studied is not available. Yet for the physiological data to have meaning the cell type must be anatomicallyidentified and the cell membrane and morphometric features studied. It is not possible at this time, for example, to measure the surface area of astrocytes in situ or in most in vitro preparations because of their extensive ramification. But, the surface area of the cell must be accurately known in order to derive reliable membrane specific values (Rm, specific resistance and Cs, specific capacitance). A monolayer culture system permitting both identification and morphometric study was therefore chosen for this correlative investigation.

METHODS

Tissue source and culture conditions

Brain tissue, freed of meninges, was removed by an oblique coronal cut in the frontal portion of the telencephalon of a 14-16 week human embryo. The cut did not

* Current address: Neuropsychology Laboratory, McLean Hospital, Belmont, Mass. 02178 and Department of Neurology, Boston University School of Medicine, Boston, Mass., U.S.A. ** Present address: Surgical University Clinic B, Kantonsspital, Zurich, Switzerland.

Brain Research, 38 (1972) 279-298

280 M.C. TRACHTENBERG et al.

extend to the ventricle. The tissue was divided into 1 mm cubes by sharp dissection and placed in a culture bottle containing the standard medium, described below. The medium always fronted an air interface; no additional oxygen was provided. For the first 4 months the explant was maintained at 34°C. Thereafter the incubator temperature was raised to 37°C. Aside from periodic changes of medium, the tissue was un- disturbed for several months during which time the cells grew out forming a monolayer. The outgrowth was then subcultured (passaged) approximately fortnightly to thin the culture or to provide study samples.

Standard medium consisted of 100 ml nutritive medium F-10 including glutamin (Gibco), 15 ml fetal calf serum and 1 ml penicillin-streptomycin (100 units/ml and 100 #g/ml, respectively). The complete medium had a pH of 7.5 and an osmolarity of 298 mOsM. Cells were subcultured by standard procedures after trypsinization (0.25 ~ trypsin in BSS (Gibco-Solution A) (296 mOsm)).

Microscopy

Sample subcultures were examined by light microscopy following adherence to the coverslip and thereafter until 8 days of age; at that time a majority of ceils in population were dead or dyinglL Light micrographs of the living cells were made using the stopped diaphragm brightfield technique. Surface area was determined by planimetry. These subcultures were then fixed with Zenker's fluid and stained with hematoxylin and eosin, hematoxylin and Giemsa or Jenner and Giemsa. Additional samples, 3-6 days post-subculture age, were prepared for electron microscopy or biophysical study. This period was selected because the cells were in the plateau phase of their growth cycle 15 and had not yet shown degenerative changes. At this stage the number of intermitotic cells was maximal. Also, the cells could be expected to be in ionic equilibrium with the medium again, following the stress of subculturing.

The detailed electron microscopic preparation techniques have been reported ~1. It is sufficient to note here that cells destined for electron microscopy were grown on carbon film coated Leighton tube coverslips, fixed with 0.1 M phosphate buffered 4 ~ glutaraldehyde, embedded in Epon 812 and stained with lead citrate and uranyl acetate.

K - Na determination

Two culture bottles, grown under standard conditions, served, each as one sample, for determination of the K-Na ratio. The methods are detailed by Lees and Shein 3z. The medium was decanted and the cells washed 6 times with Tris-mannitol buffer, pH 7.4, 292 mOsM, while still in the culture bottles. Cell membranes were disrupted by beating with a rubber policeman and the contents scraped from the bottle. The material was dried and then extracted 3 times with 0.75 N HCI. Samples of the extract were analyzed directly for sodium and potassium by atomic absorption spectrophotometry (Perkin-Elmer, model 303).

Brain Research, 38 (1972) 279-298

BIOPHYSICS OF CULTURED HUMAN GLIA 281

Biophysical study

Experiments were conducted in a test chamber containing the standard growth medium at the microscope stage temperature, 26 ± 0.5 °C. Cells adhering to the Leigh- ton tube coverslip were impaled by 2 M potassium citrate filled glass micropipette electrodes of 5-10 Mf~ impedance under microscopic guidance, at 320 ×. Electrodes were selected according to their ability to convey undistorted current pulses as tested before and after each cell impalement. The detailed recording considerations have been described85,46, zB. An Ag-AgC1 wire with or without an agar bridge completed the circuit.

A comparative study employing cultured hamster astrocytes whose culture conditions and morphology have been describedS0, 51 was similarly conducted.

The assumptions and considerations underlying the data analyses have been detaileda5, 56. The membrane voltage response to each current pulse was subjected to 2 or 3 analyses as a check on the efficacy of the measure. Interanalysis differences were quite small. The data for each morphological variant shown in Table I are the averaged mean values for each cell for all suitable current injections, i.e. current values wherein no electrode distortion was observed nor were anomalous deviations seen on the cell's current-voltage plot.

RESULTS

Post-explantation outgrowth: light microscopic observations

As post-explantation outgrowth patterns and morphological development following subculture are at best only poor indicators of cell type, suffice it to note that the outgrowth pattern is clearly similar to that described by Hogue 19 and Nakazawa 39 and that the morphologic development is like that previously reported for astrocytes 34,3s,50. Cytologic examination made periodically during outgrowth and subculture yielded data corresponding to that reported for astrocytes in viv012,13,22, 42 and in vitro 34,~s (among others). Therefore detailed description seems unwarranted. Three morphological variants are seen in the subculture. Most commonly seen are moderate to large sized, obviously single cells, second are cell aggregates and third, small, single cells. Cell aggregates, which we are calling 'contactual' groups, consist of several cells in apparent apposition. The exact boundaries of a given member of a contactual group could not be determined by any light microscopic technique, however electron micrographs do not indicate that the cells are syncytial. Contactual groups most probably arise through the growth of daughter cells, though contacts between fortuitously adjacent cells cannot be excluded. The small cells are probably immediately pre-mitotic or daughter cells. We presume this because the culture has been shown to undergo continuous mitosis 15.

Subeultured cells: electron microscopic characteristics

Figs. 1-3 show the major features of these cells. The most prominent of these

Brain Research, 38 (1972) 279-298

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Brain Research, 38 (1972) 279-298


Fig. 1. Electron micrographs of cultured glial cells during the plateau phase of the culture's mitotic cycle. A, Micrograph of the nucleus and immediate cytoplasmic region illustrating the granular endoplasmic reticulum (ger) with ribosome free areas (arrows); elongate mitochondria (m); inclusion bodies (i); free ribosomes (r); some glycogen (g) and the nucleus (N) with clumping of chromatin (c) beneath the nuclear envelope, x 50,000. All calibration bars equal 0.2 ltm. B, Non-junctional membrane surfaces of two astrocytes (between arrows). Intercellular distance ranges from 20 to 50 nm. The upper cell contains many clusters of free ribosomes (r) while the lower cell exhibits numerous aggregations of glycogen (g). A few vesicles (V) can be seen near the membrane. A small lake is visible at the lower left. × 50,000. C, Distal process containing many vesicles (V), some of the coated variety (arrow). Filaments (f) and free ribosomes may be seen. The membrane (M) is oriented tangentially, x 75,000.


Fig. 2. Detail of filaments and membrane appearance. A, Two filament varieties near each other. Microfilaments (f), 5-6 nm diameter, are situated submembranously and form a loose meshwork some of whose fibers are parallel. Deeper in the cytoplasm parallel arrays of 8-9 nm diameter filaments can be seen (F). M, cell membrane; g, glycogen particles, x 75,000. All calibration bars equal 0.2/tin. B, 8-9 nm diameter filaments deeper in the cytoplasm. Both parallel arrays and a meshwork (row) can be observed. Glycogen particles (g) and free ribosomes (r) appear between adjacent arrays. × 50,000. C, Punctum adherens with microfilament bundles (fs) extending up to the junction. The adjacent membrane area lacks both microfilaments and vesicle formations, x 50,000.

are cytoplasmic filaments and glycogen (Figs. 2 and 3). The cells have little granular

endoplasmic reticulum but the cisternae that are present have wide bores and ribo-

some-free areas (Fig. lA). Numerous cisternae o f the Golgi apparatus and elongated

mi tochondr ia are present. Free ribosomes, often in chains and rosettes (polyribosomes), and glycogen particles are prominent (Fig. 1B). Many membrane bound inclusions

are seen; some of these may be lysosomes or residual bodies. Concentrat ions o f vesicles occur (Fig. IC) at the distal regions o f the cytoplasm while they appear less

frequently in more central regions. These vesicles are present whether or not the cell is juxtaposed to another cell. The plasma and nuclear membranes are o f the usual

dimensions and the nuclear envelope shows clear nuclear pores. The nuclei, which

commonly contain nucleoli, are usually lobulated al though the nuclear membrane does not exhibit folds.

Both 8-9 nm and 5-6 nm diameter filaments exist in these ceils. The larger diameter filaments are similar to those in astrocytes examined in vivo. They are always

located well into the cytoplasm and frequently form either open parallel arrays o r

feltworks (Fig. 2A, B). In some cases the filaments, originating as feltworks, join to

form dense bundles (Fig. 3). Other organelles are excluded f rom regions o f heavy filament concentrations.

Save at their distal reaches cells do not grow on top o f each other. Consequently

Brain Research, 38 (1972) 279-298


Fig. 3. Complex pattern of filaments, 8-9 mn diameter, deep in the cytoplasm. Loose feltworks of filaments (F) aggregate to form dense bundles (FB). × 50,000. Calibration bar equals 0.4 pm.

any cell to cell contacts are restricted to the perimeter of the cells. Extended gap

junctions are not found. Puncta adhaerentia 45 constitute the only clear cell to cell

contacts and inserting into these are cytoplasmic bundles of 5-6 nm diameter filaments (Fig. 2C). At most other locations membranes face each other across an intercellular

cleft 20-50 nm in width. Occasionally, larger intercellular spaces (lakes) occur (Figs.

1B and 2C). Microvilli are absent and thin processes, 20-200 nm in width, rare. Endo-exocytotic vesicles are prevalent along some membrane surfaces but they are

not uniformly distributed, so that they are common in areas of heavy cytoplasmic

vascularity and absent from regions having subsurface bundles of 5-6 nm diameter

filaments. Thereis no microscopic evidence that a cytoplasmic syncytium exists be-

tween the cells.

Discussion of cell identification

Three anatomical features distinguish astrocytes from other cells in the central nervous system: cytoplasmic concentrations of glycogen granules ~6, 8-9 nm diameter filaments 36,a5, and terminal expansions which form capillary and subpial end feet aS. Although tanycytes and ependymal astrocytes also contain 8 nm diameter filaments, the tissue removal procedure mitigates against these cells being in the culture and no cell exhibits their unique features10,21,sL These cultured human cells contain considerable glycogen and numerous filaments of 8-9 nm diameter; end feet are, of course,

Brain Research, 38 (1972) 279-298


absent in monolayer cultures. Overall, the cells closely resemble astrocytes as described in situ 2°,86,37,45,59 though some subtle modifications, likely due to the culture condition,

are evident; the data strongly suggest these cultured human brain cells to be astrocytes. In addition, sister subcultures gave positive evidence of the acidic protein

S-100 when analyzed by a complement fixation technique 33. Similar examination of a number of astrocytomas, grades i-IV, oligodendrogliomas, meningiomas and these fetal cells, all grown under the same culture conditions, indicated S-100 only in the astrocytomas and the fetal ceils. Although one report 24 suggests that oligodendrocytes, the perineuronal satellites of Deiters' nucleus, contain S-100, 25 -40~ of perineuronal satellite cells are astrocytes 6~. This datum requires re-examination in light of the findings of Sotelo and Palay 52 that oligodendrocytes rarely contact the giant cells of Deiters; rather, Deiters' cells are surrounded by astrocytic processes. Finally, Perez et al. 44 demonstrated an increase in S-100 during Wallerian degeneration of rabbit optic nerve. (On the contrary, Wallerian degeneration of peripheral nerve is associated with S-100 decrease as the Schwann cells degenerate43.) The time course of this increase corresponds to that of fibrous astrocyte hypertrophy described by Vaughn and his colleagues 57,5a for the degenerating rat optic nerve and under the conditions described by them oligodendrocytes do not increase their volume. Taken together, these anatomical and biochemical data indicate these fetal cells to be astrocytes.

Cell surface area and volume

Fig. 5 illustrates a typical single cell. This photomicrograph was taken with the diaphragm stopped to enhance edge contrast and thereby facilitate determination of edge contour. It is accompanied by a drawing of the outline of the cell used for determination of the surface area. The ceils are treated as having two equal area faces. The surface contribution of the cell's 2 #m average thickness, determined by observing micrometer readings at the initial pipette resistance increase (vide infra), signifying membrane contact, and at current pulse distortion, signifying contact of the pipette and the glass surface, is considered inconsequential. Measurements of cell thickness in electron micrographs yield a similar thickness value. The extent of membrane surface area increase attributable to thin cell-flaps, which are poorly resolved in the bright field microscope because of their low contrast, is generously estimated from electron micrographs to be no more than 12 ~ in the extreme cases. As the percent error varies from cell to cell it is not considered in the following calculations. Membrane flaps increase in number and size with increased cell size so that more accurate areal measurements are possible for small and medium sized cells. Table I lists the mean surface area for each of the 3 morphological varieties present in the culture.

The two-dimensional environment imposed by a monolayer produces flattened apparently large cells. Eight, large, single, presumably intermitotic ceils have a mean surface of 8,700 sq./zm. Taking the average thickness as 2 #m their volume is 17,400 #m 3. This compares to about 14,150 #m 8 for in situ astrocytes taking their diameter as 30/~m 13 and treating the cells as spheres to maximize volume. (Astrocytes probably 'strive' to maximize surface area.) No exact value for in situ astrocytic volume exists

Brain Research, 38 (1972) 279-298


largely due to the large number of highly convoluted processes. Nuclear dimensions

of the cultured cells are also greater (even allowing a spheric to discoid t ransformat ion)

than that of their in situ counterparts , 13/ tm diameter to 13 #m by 25 #m for cul tured

astrocytes versus 6-10 /zm diameter in brain histological material 13.

Biochemical analysis

Determinat ions of the K - N a ratio on duplicate (stock) subcultures yielded

values of 1.8:1 and 1.1:1. The medium K - N a ratio was 0.04:1, thus it is evident that

the cells could alter this ratio at least 25 fold in favor of potassium.

Biophysical analysis

Cells of all 3 morphological varieties - - large single, small single and contactual

- - were studied by means of an intracellular electrode. A DC offset (and audi tory

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Fig. 4. Biophysical data for a representative cultured human glial cell. A, Membrane voltage responses (dark curves) to intracellularly imposed square wave current pulses (light curves) of 1 4 nA amplitude. Centermost dark trace is the extracellular electrode response to a 1 nA current pulse. B, Virtually perfect match between glial cell membrane voltage response (wavy trace) to a 4 nA current pulse and an exponential curve generated across a parallel resistance-capacitance network (unbroken, lighter trace). C, Semi-logarithmic plot of voltage change per unit time versus time for the data shown in A in response to a 1 nA current pulse. Note abscissa in /~sec. D, Current-voltage plot derived from data in A. Cell response is non-rectifying and linear over the tested range.

Brain Research, 38 (1972) 279-298


Fig. 5. Surface area determination technique. A stopped diaphragm bright field micrograph is shown on the right. On the left appears its outline used in determining the cell's surface area by planimetry. Calibration bar equals 50 itm. Extracellular artifacts have been lightened.

membrane 'click') signalled penetration of the plasma membrane by the electrode. Resting potentials (for all groups) averaged - -7 .7 mV ranging from ---4 to - -12 mV (Table I). Spontaneous activity was not observed nor did regenerative responses occur to either the make or break of intracellular current pulses despite imposed alterations of the membrane resting potential by as much as -4- 25 mV.

Single exponential voltage curves were generated across the cell membrane in response to inward (hyperpolarizing) or outward (depolarizing) going square wave current pulses of 1-4 nA amplitude and 0.5-1.5 msec duration (Fig. 4A). These curves were determined to be exponential by their straight line fit on a log dV/dt vs. t plot (Fig. 4C) and by the near-perfect match obtained with exponentials generated across a parallel resistance-capacitance network (Fig. 4B). Fig. 4A shows a representative series of membrane charging curves. The time constant (~-), as derived from the regression of the log dV/dt vs. t curves to time zero (Fig. 4C), averaged almost 311 /~sec (Table I), a value about 4 ~ that for the feline Betz cell3L The membrane voltage

maxima reached at times much greater than 3, in response to intracellularly delivered, long duration (relative to 3; up to 53), rectangular current pulses are plotted against applied current in Fig. 4D. The astrocytic membrane was non-rectifying and linear

Brain Research, 38 (1972) 279-298


throughout the explored range. The cells' input resistance derived from these curves was low, averaging 2.63 Mf~ (Table I).

Table I shows the resting potential, r and the input resistance for each class of cells studied while the collective means and standard deviations for all the cells are shown below (human). No significant difference exists between the various populations.

On day 3 and early on day 4 post-subculture large single cells constitute the most common cell type. Because many intermitotic and single cells were expected in the population at this time 15 it is thought that these populations are coextensive. The intermitotic condition is that normally encountered in vivo. To forestall errors which may arise from ignorance of the real surface area of each of the cells of a contactual group or from uncertainties of the functional character of the junctions between contactual cells only the data obtained from the first group, i.e. large, single, intermitotic cells can be considered valid for comparison. Table I shows their mean Rm to be almost 326 f~-sq.cm and Cs to be almost 1 #F/sq. cm.

The membrane specific data for the 9 samples from contactual cells are not given for the above noted reasons. However, were these data calculated using the total apparent surface area a surprisingly large and probably spurious range for Cs (and Rm) would be obtained. The greatest Cs values would be no more than 47 ~ that of the mean value for the large single cells given above. In the extreme case Cs would only be 4.4 ~ of this value. No apparent physical reason exists to account for such profound alterations in Cs, nor for those in Rm which must follow because of the homogeneity of z values.

The question, whether significant current transverses the membrane at j unctional regions, assuming them to be functional, can be approached by considering the relative non-junctional membrane (rm) and junctional membrane (rj) resistances. Rm is taken as 326 f2.sq.cm, the mean of the single cell values (Table I) and Rj, the junctional specific resistance, as 100 f~.sq.cm (ref. 25). The surface area is taken as 17,398 sq.#m, the mean surface area of the single cells (Table I). The surface area of the junctional region is calculated to be 20 sq./~m, assuming 100 junctions per cell, each junction the full 2/~m edge thickness in height and 100 nm in length. It can be shown then that rm is about 1.9 M~) while rj is 500 MO, i.e. 266 times greater than rm. This calculation indicates that an insignificant portion of the current crossing the membrane does so via junctional regions. Whether geometrical and/or environ- mental factors alter this bias sufficiently to permit significant current to cross from one cell to another is unknown. Contactual cells were observed in various geometric patterns preventing us from constructing a satisfactory model.

Four small cells were examined. The resting potentials, and input resistances (Table I) do not (in these cases) appreciably differ from those for the other single or the contactual ceils studied. However, because of their small surface area the calculated Rm was quite small and Cs very large. These differences may suggest alteration of the functional state of the membrane. Alternatively membrane thickness might be changed (see Discussion). Because a certain percentage of the cells in the' population are mitosing (cells a priori expected to be small) we initially suspected that these cells

Brain Research, 38 (1972) 279-298


might be pre- or early postmitotic. Cole and colleagues ~-7 report of increased Cs in pre-mitotic cells supports this contention. From currently available biochemical evidence 4s it is reasonable to expect Rm to decrease pre-mitotically.

A number of large, single, monolayered cells explanted from hamster brains and identified as astrocytes 5° were examined in their nutritive medium under the conditions described above. Their anatomy and culture conditions have been described 5°,51. At 26 ± 0.5°C 18 cells had a mean resting potential o f - -28 .2 mV (Table I). Values as great as - -50 mV were observed for some of these cells. The membrane charging curves were exponential; no spontaneous activity was seen nor could regenerative responses be evoked to suitable membrane potential displacements. ~ averaged almost 295 /~sec (Table I). The current-voltage relationship was linear and non-rectifying. The input resistance averaged 5.2 M ~ (Table I), double the mean for the human cells described. Their size, visually adjudged, was considerably smaller than our large single human astrocytes. No accurate determination of the surface area was made.

DISCUSSION

Membrane character and morphometrics

Extremely few microvilli are seen and endo-exocytotic vesicles are not overly abundant. The biophysical derivations based on the assumption of a uniform membrane of given surface area are probably correct. Although there is no clear evidence of gap junctions, we cannot discount their possible presence.

Compared to in situ astrocytes, whose volume is calculated on the basis of light microscopic measurements 16, these cultured astrocytes are about 22 % larger. Wolff 64, in estimating astrocytic volume morphologically from electron micrographs of central nervous tissue, concludes that 'one astrocyte has a volume of about 7,000-18,000 #m a'. However, his estimate includes the thin, sheet-like (20-200 nm wide) processes which, although they are not visible in the light microscope, are absent from our cultured cells. Interestingly enough, sheet-like processes do not appear in ontogeny until synapses develop and Wolff* is of the opinion that they may not develop in the absence of synapses.

One reason for the increased volume may be the reduced K - N a ratio. The values obtained for our cultured astrocytes, 1.8:1 and 1.1:1 may be compared with 9.3:1 for subcultures (6.7:1 for primary cultures) of cultured hamster astrocytes 32 and over 8:1 for in vivo leech glia 40. A consequence of high internal sodium is an increase in cell size as chloride follows sodium into the cell down its electrochemical gradient and water follows to maintain tonicity.

Biophysical data

Resting potential and input resistance values for all the human cells constituted

* Personal communication.

Brain Research, 38 (1972) 279-298


a single population. These values differ significantly from those obtained for the hamster astrocytes (t < 0.001, resting potential and t < 0.02, input resistance). The human and hamster cells differed markedly in intracellular K -N a ratios 32 and in cell size z7. Whatever the causal factor (vide infra) the altered K -N a ratio helps to explain the low resting potential. Differences in culture media may also have contributed to those observations while decrease of 15-10 mV may be attributed to lowered recording temperature z.

Low resting potential brings into question the microelectrode impalement efficacy or suggests other exigencies of the technique. However, neither the greater resting potentials recorded in the hamster cells (up to - -50 mV) nor the higher resting potentials of meningioma cells27, 46 grown and recorded under similar conditions support these inferences. Further, cat neocortical glia examined with the same variety of electrode had resting potentials as great as - -92 mV 55. Also, poorly impaled cells would have lowered T due to the introduced current shunt. No correlation exists between resting potential and "r. The intergroup consistency of r further speaks against leaky impalement. These arguments apply to all of the cells reported.

Cell size directly affects input resistance; an inverse relationship exists with surface area in the absence of junctional complexes or other local membrane spe- cializations. Single (large, intermitotic) human astrocytes were unusually large even allowing for the differences between 2 and 3 dimensional representations; their input resistance was low. Hamster astrocytes were smaller, more closely approximating in vivo dimensions; their input resistance was greater. The decreased K -N a ratio likely contributed to the large size of the fetal human cells for with decreased ionic 'pump' activity cells swell23; chloride and water follow sodium into the cell for electrochemical and osmotic reasons.

The membrane specific data, Cs and Rm, for the large, single cells averaged 0.98 /~F/sq.cm and 325.98 £2.sq.cm, respectively. The accuracy of these values depends firstly, on the accuracy of T, secondly on the accuracy of the surface area measurement and thirdly on the accuracy of the input resistance. (Passive electrical properties are apparently little affected by temperatureZZ.) Cs is dependent upon the first pair, Rm on the second pair of variables. The accuracy of v is most strongly supported by the intragroup, intergroup, intercondition and interspecies value similarity (Table I) 54,56. Some factors capable of influencing T and input resistance were considered above and dismissed. In the absence of Cs alterations (vide infra) the validity of ~ supports the input resistance value, it too displays intragroup consistency. Moreover, Cs is virtually unchanged by the introduction of a parallel capacitance. Regarding surface area, we believe the values presented to be valid on the following bases: (1) absence of microvilli or extensive micropinocytosis; (2) electron microscopic and electrical measurement of cell thickness; (3) edge contrast provided by the stopped diaphragm technique; (4) the restriction to large, single, intermitotic cells for base data. An undetected increase in the membrane surface area would, as an analog of muscle sarcoplasmic reticulum, similarly increase Cs. This membrane condition has not been electron microscopically observed. Two types of membrane changes could alter Cs - - alteration in the dielectric properties of the

Brain Research, 38 (1972) 279-298


membrane or alteration in membrane thickness. The former may be applicable to the pre-mitotic membrane alterations discussed below. The latter has not been seen in

electron micrographs. These data may now be used to examine those obtained from the contactual

and the small cells. The primary question regarding the contactual cells is, do biophysical data indicate the presence of a functional, in this case an electrical, syncytium? If the difficulty in finding gap junctions reflects their scarcity and gap junctions are considered the only sites for intercellular current transfer, then in conjunction with the high ionic permeability of the entire membrane their influence in transferring current will be quite small. The percent current crossing gap junctions is directly related to their surface area and ionic conductance versus that of the remaining membrane. The calculation presented under Results suggests that little of the available current traverses junctional regions. Even if anatomically defined junctions are not considered the only sites for intercellular current transfer the absence of difference in the input resistance between contactual and single cells (means, 2.24 M~2 and 2.24 Mf~, respectively) further indicates that the surface area available for current passage is about the same, i.e. the contactual ceils do not appear electrically 'bigger' than the single cells. Possible conditions under which gap junctions might function to produce a syncytium in vivo have been considered 56, see also ~0,31. Biophysical and anatomical data taken together indicate the absence of an electrical or a physical syncytium in this situation.

Due to their size and relative scarcity, the small cells were a pr ior i thought to correspond to the mitosing population. This belief was reinforced by their high Cs value. A number of different species of marine egg cells have shown dramatic increases in Cs, averaging 3.8 fold, when examined in the fertilized versus the un- fertilized condition 3-7. In addition to a high Cs these cells had low Rm. The studies of Robbins and Micalli 48 suggest that a withdrawal of calcium from the plasma membrane attends mitosis. Such a mechanism could account for the decreased Rm value. Decreased synthetic activity could now free some ATP. Were this ATP available for ion pumping it might help to maintain the resting potential despite the increased permeability. Under conditions of low intermitotic resting potential such maintenance is not unreasonable. Furthermore, decreased size would, with regard to the input resistance, partially overcome the effect of proposed permeability increase. Table I shows the similarity of the resting potential and input resistance of these cells and the intermitotic population. Electron micrographs are not available for the small cells precluding any statements on membrane dimensional alteration, intramem- branous molecular configurational changes are, of course, also possible. Whether Cs and Rm always vary inversely or whether a fortuitous situation exists in this case remains for future elucidation.

It is pertinent to ask how these data relate to currently available literature information.

Brain Research, 38 (1972) 279-298


Reactivity

Astrocytes and astrocyte-like glia (in invertebrates) exhibit no spontaneous or induced regenerative activityS,14,~s,28,z0,3x,56, 60 (see also references in these articles). This was true for both the human and hamster cells although the finding is mitigated somewhat, particularly for the human cells, because of their low resting potential. While non-reactivity is a necessary physiological identification characteristic in vivo

it is ancillary in these anatomically defined populations.

Resting potential

Kuffler and colleagues8, 29-31 have shown neuroglial cells in leeches, amphibians and mammals to have high resting potentials - - up to - -90 inV. Similar values have been found for neocortical glial cells14, 56 (among others). While our glial cells exhibited lower resting potentials, at lowered recording temperature, unusually low resting potentials have previously been recorded from cultured glial cells; Wardell ~0 reported values as low as - -5 mV, Hild et al. 17 as low as --15 mV. Moreover, the reduced N a - K ratio found for the human glial cells, a situation not observed for the hamster cells or for in vivo leech glia 4° (K-Na = 8:1) - - parallels the lowered resting potential.

lnput resistance

Input resistance, depending so heavily on cell size, quality of penetration and membrane permeability, varies greatly. Our human astrocytes averaged 2.63 M~) ranging from 0.8 to 4.6 Mr2 while the hamster astrocytes averaged 5.18 M~ . Hild and Tasaki is reported a range of 0.6-1.7 Mr2 for cultured rat and cat glial cells while a range and mean value of 0,5-10.0 M ~ and 4.2 M~2 were observed by Wardell 6°. For cat neocortical glia Trachtenberg and Pollen 58 reported a mean of 10.49 Mf~ with a range of 4.5-19.6 Mf~ and Krnjevi~ and Schwartz 28 a mean of 48.4 MfL Few speculations can be made from the limited data available save to note that the lower values occur more commonly. The human astrocytes were larger than either the hamster cells or the estimated size of in vivo astrocytes. The increased size would account for the relatively reduced input resistances.

Rectification and time constant

Despite differences in input resistances current-voltage relationships have been found to be linear and non-rectifying2S,a0,31,~6; human and hamster astrocytic membrane behaved similarly. All astrocytes so studied showed exponential membrane charging curves with short z, in vivo ~ , in vitro is and in the present study (Table I). Tasaki ~4 reported T values of less than 0.5 msec; sample data from a number of cultured human astrocytomas revealed z < 0.5 msec*. The similarity of z values

* Unpublished data.

Brain Research, 38 (1972) 279-298


allows that, with constant Cs, Rm is very similar for normal and cultured astrocytes of the neocortex, cerebellum and unidentified brain loci as examined in 4 different mammalian species (the present data and refs. 18,56). The data presented suggest z to be a stable parameter despite variation in resting potential or input resistance. -r, in addition to reactivity characteristics and resting potential can be used to distinguish astrocytes from neurons or mesenchymal cells - - ~ = 5-30 msec for cultured meningioma cells 27,32. Being area independent and therefore less liable to physiological variation than is input resistance z can better indicate faulty electrode impalement. With a determined z and a known Cs not only Rm but effective surface area can be derived for cells available only to microelectrode study.

Membrane specific capacitance and specific resistance

Cole and Curtis 6 have summarized the Cs data for a large number of animal and plant tissues. 'The membrane values capacities extend from 0.4 to 9 /~F/sq.cm. The extreme values may be questioned, and a considerable number of cells lie in the range 1-2 #F/sq.cm'. Cs not only determines the position of the cells on a biological continuum but may provide data on the physiological state of cells. Cs is an indicator of the dielectric constant which in turn is an indicator of the membrane chemical composition. If the dielectric constant is known measurement of the capacitance allows prediction of the membrane thickness 49. The Rm values of 300-600 fbsq.cm compare favorably to those estimated 54,56 or estimateable for glial cells based on their z values (assuming Cs =- 1 /~F/sq.cm). This value of several hundred f~.sq.cm contrasts with an Rm of 3-10.fLsq.cm estimated by Hild and Tasaki is for cultured glial cells or Kuffler and Potter's 31 suggested similarity of glial and neuronal Rm. The minimal Rm value for leech glia is 1000 ~.sq.cm 31. Neuronal membrane Rm has been or can be estimated to be 1000-10,000 D.sq.cm35,47, 53 (based on z SD :- 9.9 msec z5 and Cs = I #F/sq.cm). (Cs = 1.5/~F/sq.cm would yield the more commonly accepted Rm upper limit of 6000 f2.sq.cm.) A low glial Rm means that the specific conductance (the reciprocal of Rm), largely a potassium conductance8, 30, is very great. The physiological significance of a high glial ionic conductance, one which allows rapid transfer of large amounts of potassium, has been discussed 56.

SUMMARY

Biophysical studies were performed on cultured human fetal brain cells identified by morphologic, cytologic and biochemical characteristics as astrocytic glia. Resting potentials were low (,X ---- --7.7 mV) because of the temperature during examination and because of an altered intracellular N a - K ratio attributed to the culture metabolic conditions. Low resting potential appears unrelated to membrane biophysical properties and does not block the cell's ability to divide and grow although the growth rate is reduced 15. The cells displayed no spontaneous electrical activity nor were regenerative responses seen despite large membrane potential displacements. The astrocytic membranes were non-rectifying to imposed current pulses. The glial cell's

Brain Research, 38 (1972) 279-298


time constant averaged 311 #sec, 1/26 that of the feline Betz cell 35 but less than one standard deviation smaller than that found for in vivo feline astrocytes 56. Membrane specific capacitance was found to be about 1 /~F/sq.cm, a value common to many biological membranes. Membrane specific resistance averaged 327 ~-sq.cm, a very low value compared to that of neurons but in accord with calculations from other glial (astrocyte) studies. These biophysical values allow rapid transfer of large amounts of potassium.

ACKNOWLEDGEMENTS

The authors express their gratitude to their various colleagues and particularly to Drs. D. A. Pollen and W. H. Sweet, for encouragement and helpful comment; to Dr. J. E. Vaughn for electron micrographs; to Dr. H. Shein for providing hamster glial cells; to Dr. M. Lees, Dr. E. Moore and Mrs. S. Lipshitz for help with biochemical determinations.

This work was supported by N.I.H. program project grant CA-07368 to Dr. W. H. Sweet and N.| .N.D.B. postdoctoral fellowships 32383-01 and 32383-02 to Dr. M. C. Trachtenberg.

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biophysical properties of cultured human glial cells

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