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Chemical studies of the satellite cells of the squid giant nerve fiber
Experimental
1
Cell Research 20, l-1 1 (1960)
CHEMICAL
STUDIES
OF THE SATELLITE
SQUID GIANT NERVE ROSE
ROBYNS
Department
of Biology,
FIBER’
COELHO,z JOAN WRIGHT M. BLAIR BOWERS Massachusetts
Institute
CELLS OF THE
of Technology,
GOODMAN
and
Cambridge, Mass.,
U.S.A.
Received June 28, 1959
PEHIPHEHALnerve axons are enclosed over most of their lengths by a monolayer of satellite (Schwann) cells. The physiological function of these cells is poorly understood except for their role in the morphogenesis of myelin, which they form by wrapping the axon in many turns of their infolded double surface membrane [a]. In unmyelinatetl fibers, e.g. in invertebrate fibers and in vertebrate C fibers, the satellite cells are not thus differentiated though they apparently serve a role vital to the normal functioning of these fiber types also. Electron microscope studies suggest that the satellite cells, at least in lobster fibers, may be morphodynamically active as in the formation of mitochondria-like particulates [3, 41. The subject of axon-satellite cell relationship has been reviewed by Schmitt and Geschwind [14]. Attention has been drawn to the possible role of the satellite-cell layer in solute exchange between axon and extracellular space, hence also in impulse proof their circumaxonal disposition [lx, 13, l-11. pagation, 1)~ virtue Because of the intimate structural relationship between axon and satellite cells because of the very high surface-to-volume ratio it has so far been impossible to obtain evidence regarding satellite-cell metabolism in unaltered fibers. \C’ith the squid giant-fiber a technique has been developed by which the gaseous metabolism of the sheath may be determined after removal of axoplasm by slitting the sheath in the axis of the fiber. Under these atlmittedly grossly abnormal conditions the satellite cells continue to respire for hours. Their approximate respiratory rate can be estimated from values from intact fibers, slit-sheath preparations and pcriaxonal connective tissue (from 1 These studies were aided by a research grant (B-24) from the National lnstitute of Neurological Diseases and Blindness, of the National Institutes of Health, U.S. Public Health Service; by a contract between the Office of Naval Research, Department of the Navy, and the Massachusetts Institute of Technology (NR-119-100); and by grants from the Trustees under the Wills of Charles A. and Marjorie King, and from the Lou and Gene Marron Foundation. 2 Public Health Research Fellow of the National Institute of Neurological Diseases and Blindness. Present address: Children’s Medical Center, Boston 15, Massachusetts. I-
603704
Experimental
Cell Research 20
2
Rose Robyns Coelho, Joan Wright Goodman and M. Blair Bowers
which to assess the significance of the fibrocytes in the axon sheath). As reported briefly in this paper, the results demonstrate that the satcllitc cells account for almost half the respiration of the entire fiber.
METHODS The giant nerve fibers of Loligo pealii, obtained from squid brought to the M.I.T. laboratories in the manner previously described by Maxfield [9], were dissected from the mantle muscle and transferred to chilled filtered sea water for removal of loose connective tissue and small nerve fibers. This dissection was accomplished as rapidly as possible, but with every effort to avoid traumatization of fiber constituents. Slitting of sheaths was accomplished by insertion of sharp iridectomy scissors into the fiber and cutting the cleaned axon-sheath longitudinally. Quick cleaning of the fibers after dissection was found necessary for the fiber and slit sheaths to respire in the fashion described below. The slit sheaths were washed by gentle shaking in sea water kept at 15°C. This process removes axoplasm and extracellular fluids. Natural and artificial sea water, filtered through VC millipore filters, served both as a medium for cleaning and slitting fibers and for suspending fibers and slit sheaths The composition of the two artificial saline media for measurement of respiration. used is given in Table I. TABLE I. Composition Solution
ofnrtificid
saline me&r
used in respiration
NaCl
KC1
CaCl,
Mgso,
MgCl,
H,UO,
I
355
17.0
11.4
40
29.0
17.6
II
475
10.0
10.7
28.6
26.7
1.0
KaHCO,
2.5
memuremenfs. W’O,
2.0x 10 3
All values are expressed in mM/l.
Oxygen consumption was measured in slightly modified Scholander-type microrespirometersl In each respirometer vessel were placed either three of four intact ligated and cleaned giant fibers of about 10 mg wet weight or six slit-sheaths of about 6 mg wet weight. These specimens were suspended in 0.2 ml sea water or artificial saline. Temperature was maintained at 14°C. For comparison and calibration several runs were made in Warburg respirometers fitted with small vessels; of necessity more fibers and sheaths were required for this less sensitive method. Rates of respiration were found similar in the two methods when the shaking rate of the Scholander vessels was maintained in the range of 130 -170 per minute. Higher shaking rates reduced respiratory rates of slit sheaths presumably due to mechanical injury. 1 These microrespirometers were constructed by Mr. \I’. Strovink of this Department. The manometer plungers were activated by precision micrometers to obtain maximum accuracy of volume measurement. Dow Corning 200 fluid was used as manometer fluid instead of kerosencmineral oil because it was found to move more uniformly in the capillaries. Experimenfal
Cell Research 20
Studies of satellite cells of squid nerue fiber After measurement of respiration, the fibers or slit sheaths were removed from the vessels, washed briefly in distilled water and homogenized in 2 ml of 10 per cent trichloracetic acid. Determinations of protein concentrations were performed on 0.5 ml of the hornogenate in duplicate by the methods of Lowry [8]. Similar analyses were made on uncontaminated axoplasm especially extruded for the purpose; the fact that the results agreed well with those obtained by Koechlin [7] constitutes evidence of the reliability and consistency of the techniques. The collagen content of specimens was estimated from hydroxyproline values, determined by the methods of Neuman and Logan [IO], on the assumption that squid collagen contains 10 per cent hydroxyproline. Samples for analysis were hydrolyzed in 1 ml of 6 11’ HCI by autoclaving for 5 hours at 15 pounds pressure. The method was demonstrated to account quantitatively for the hydroxyproline in purified calfskin collagen.
Estimation
of Partition
of Fiber Constituents
If the contribution of the satellite cells to the metabolism of the intact fiber is to he estimated, data must be obtained concerning the fraction of the average preparation of intact fibers and slit sheaths that is represented 1)~: (1) satellite cells, (2) other metabolizing structures, chiefly fibrocytes and small nerve fibers in the sheath (herein called “pcriasonal tissue”), and (3) non-mctaholizing material, chiefly collagen and inorganic salts. The pertinent approximate information is oldainetl by consideration of three compartments: (1) intact fihrrs cleaned in the usual fashion, (2) slit sheath preparations, (3) pcriaxonal tissue ohtaineci during the routine process of cleaning fibers after dissection from the mantle muscle. Histological esamination sho~~ti periasonal tissue to contain primarily collagen filwrs, fihrocytes and a relatively small fraction of small (ca 10 p) nerve fihcrs. Slit sheaths were found to contain, besides the axon surface membrane and the surrounding monolayer of satellite cells, closely adhering layers of connective tissue \vhich contained, crnhccltied in the collagen fibers anti ground suhstance, fibrocytrs roughly eclualling in number the satellite cells, as judged by nuclear counts in OsO,-fixed lx-eparations.1 13wause of the srnall size of the samples, xveighing in the \yct state \\-as difficult anti inaccurate. ,Ilore reliance \vas placed on the protein content. The partition of protein, as rneasuretl in per cent of the dry weight 1)~ the method of Lo\\-ry [Xl, was: Intact fibers, ‘26; Slit sheaths, 26; l’eriaxonal tissue, 33; and Reference collagen (calf skin), 35. 1 WC are grateful of squid nerve.
to bliss Olive Fletcher
and Dr. Norman
Geschwind
for the histological
Experimental
studies
Cell Research
20
4
Rose Robyns Coelho, Joan Wright Goodman and M. Blair Bowers The cellular (Total
protein
is roughly
protein
equal to:
in a sample) - (collagen
protein)
X 0.35.
Corrected thus for collagen it was estimated that the protein content per unit of dry weight was in per cent: Intact fibers, 28; Slit sheaths, 15; and Periaxonal tissue, 31. These values, though not of high accuracy in themselves, permit interconversion of “cell protein” values and “cell dry weight” values. From such data, together with those on axoplasm (amino acid content, etc.) and hydroxyproline values, the figure of 7.9 per cent nitrogen per unit of dry weight emerged for intact fibers. This is in good agreement with the nitrogen values obtained experimentally by Koechlin [7] of 8 per cent. For slit sheaths the value of 9 per cent nitrogen per unit of dry weight was obtained, whereas the value obtained from micro-Kjeldahl analysis was 11 per cent. The source of this discrepancy cannot yet be determined because little is known of the chemical composition of the sheath. The weight ratio of the three compartments (axoplasm, sheath cells and periaxonal tissue) can be calculated from micro-Kjeldahl and hydroxyproline values on weighed samples of intact fibers and slit sheaths. It was found that the average value of slit-sheath collagen was 46 per cent. The nitrogen contents of the three compartments were in per cent dry weight: Axoplasm,l 5.8; Intact fiber,2 8.0; and Slit sheath,2 11.3. From these figures it may readily be shown that axoplasm accounts for 60 per cent of the dry weight of intact fibers, and collagen accounts for 18 per cent. The non-collagenous component represents 22 per cent of the dry weight. In terms of dry weight of non-collagenous material this means that the cellular constituents in the sheath account for 27 per cent and axoplasm for 73 per cent. EXPERIMENTAL
RESULTS
Under the conditions described, intact fibers and slit sheaths consume oxygen at a relatively constant rate for several hours after the beginning of Typical data are shown in Fig. 1; in Table II are sumthe experiment. marized the data for experiments in which natural sea water was used as suspension medium. As may be seen in Fig. 1, the rate of oxygen uptake of slit sheaths is not constant for the first fifteen minutes. It seems unlikely that more than about 1 Results of Koechlin [7]. 2 Kindly determined in this laboratory Experimental
Cell Research 20
by Dr. G. IIeffner.
5
Sfudies of satellite cells of squid nerve fiber
five minutes is required for temperature equilibration anti this was allowed before taking the first measurements. It seems more probable that the initial high rate is clue to the trauma to the sheath cells in the slitting operation. Only the linear portion of the curve was usecl in estimating respiratory rates. The slit-sheath preparations are, not unexpectedly, highly sensitive to damaging procedures in the precertain, no\v only partially understood,
Minutes Fig. l.-Respiration of slit sheaths and whole fibers in filtered sea water. Ordinates: mm3 O,/mg dry weight of collagen-free material. 0, sheaths; 0, axons.
TABLE
II. Respiratory
rates of intact fibers, slit shenths in filtered nnturul sea writer.
(mm3 O,/mg dry weight
Tissue Fibers Sheaths Periaxonal tissue
of collagen-free
rind perinxonnl
tissue
material/hour.)
Average respiratory rate
Number of experiments
Standard deviation
7.88 13.50 7.80
IO 22 6
2.26 4.55 3.40
Experimental
Cell Research 20
6
Rose Robyns Coelho, Joan Wright Goodman and M. Blair Bowers
parative technique. Thus of thirty-fire experiments in all, the slit sheaths in thirteen experiments (not included in the table) showed no apparent respiration. In most of these cases the source of the excessive damage during the preparation of the specimens or the poor condition of the squid after the 40 30
s
20 IO 0 O\-”30 0” s 20 s 0) I;: IO 0
30 20 IO 0
0
200
400
m MK/kg
600
800
1,000
wet weight
Fig. 2.-Potassium content of slit sheaths, whole fibers and periaxonal S, sheaths; F, fibers; and C.T., periaxonal tissue. Abscissa: mM K/kg wet weight of collagen-free material.
tissue.
eighty-mile trip by truck from the fishing grounds to the laboratory could be demonstrated. In the latter connection the necessity of good aeration and of maintaining a low temperature (ca 14°C) in the sea water circulating in the tanks was established. Indeed, the metabolic behavior of the slit-sheath preparation proved a sensitive criterion of the success of all steps in the technique from the catching and transportation of the squid to the cleaning Experimental
Cell Research 20
Studies of satellite cells of squid nerve fiber
7
and slitting of the giant fiber. As might be expected, the spread in the data from slit sheaths is greater than for intact fibers. The mean respiratory rates obtained by the Scholander and Warburg methods are identical in the case of fibers. The value obtained for sheaths by the Warburg method is lower (8 mm3 O,/mg collagen-free material/hour) than that found using Scholander microrespirometers. ‘This is not surprising since 30 slit sheaths had to be collected before the experiment could be started, which corresponded to a delay of 7 hours between the killing of the squid and the termination of the experiment. Altogether 6 experiments have been done using the Warburg technique. The respiratory rates for intact fibers are also reasonably similar, considering the differences in techniques, to those obtained by Connellp [l] using the oxygen electrode at 16°C. From the average collagen content of dissected fibers it is possible to convert Connelly’s average figure of 68 mm3 O,/gm wet weight/hr into the corresponding figure in terms employed by us, namely mm3 O,/mg dry weight of non-collagenous material/hr. In this case, Connelly’s figure becomes 5.0, as compared with our own figure of 7.9. Efect
of Saline Medium
on Oxygen Consumption
The data in Table III show that the respiration of intact fibers-and of periaxonal tissue-is not substantially different in artificial sea water. However, the effect upon the respiration of slit sheaths is as dramatic as it is unexplained. Out of twelve experiments, the usual rate of respiration of slit sheaths in the artificial saline was demonstrated in only two experiments. In the remaining ten experiments the preparations either did not respire at all or stopped respiring within minutes of the beginning of experiment. ‘I’ABLE
III.
Respirrctory
rates of intact fibers, slit sheaths in artificinl media.
(mm3 O,/mg dry weight
Tissue Fiber Sheaths Periaxonal Tissue Altogether in the Table.
12 runs were
of collagen-free
and pericwonrrl
material/hour.)
Average respiratory rate
Number of experiments
7.30 15.00 7.1
8 2 2
done on sheaths;
10 showed
tissue
no respiration
and were not included
Experimental
Cell Research 20
Rose Robyns Coelho, Joan Wrighf Goodman and M. Blair Bowers
8
Because of the possible significance of the unidentified factor-possibly an oligodynamic effect-in the respiratory chain, efforts mere made to characterize the factor. These efforts were only partially successful. It appears that the factor is not diffusible because dialysis of artificial sea water against natural sea -\vater did not prevent the respiratory inhibition. Roiling or autoclaring natural sea Tvater had no eBect. The natural sea \vater used showed a broad absorption band centered at about 2660 A as measured in the Cary spectrophotometer. Possibly organic material, such as nucleotide derivatives or amino acids, mav be present which serve as a factor or cofactor for satellite-cell metabolism.
Efict
of Respiratory
Inhibitors
‘L,h-DNP profoundly influences the ionic fluxes of squid giant fibers [a]. Sodium efflux and potassium influx are greatly reduced while sodium influx and potassium efflux are little effected. Accordingly it would be interesting to determine the effect of this substance and of other inhibitors, such as azide and cyanide, on the metabolism of slit sheaths as compared with intact fibers. From the data in Table IV it will be seen that I)NP has little efrect on the respiratory rate of intact fibers or of connective tissue cells, but inhibits that of slit sheaths by about a third in concentrations of 10-5 to 10e4 ~11. Sodium azide has a similar effect: little influence on intact fibers in 10-5 to 10-4 M concentration, but inhibition of slit sheaths which was complete at lo-4 111concentration. TABLE
IV. Effect
of
2,bdinitrophenol
and sodium
azide
Average Number of experiments
(mm3 O,/mg Inhibitor
Intact
fibers
dry weight
on respiratory
respiratory
rates
of collagen-free
Slit sheaths
rates.
material/hour) Periaxonal
See Table I
Unpoisoned
7.88
13.5
7.8
7
2,4-DNP (1 x IO-6 to 1 x IO-4 M)
7.1
8.3
7.3
2
1 x 10-s M NaX,
8.4
-
3
1 x 10-4 M NaN,
Experimental
Cell Research 20
5.9
0
tissue
Studies of satellite cells of squid nerve fiber Potassium
Concentration
in Satellite
Cells
So data are available concerning the electrolyte concentrations in satellite cells and their variation with function and \vith environmental changes. Only a modest heginning has been made in these experiments toward obtaining such information. The electrolyte pattern, like oxygen consumption, is a valuable index of the state of cells; its determination \vas therefore important in the present experiments. If the potassium concentration of the cells in the slit-sheath preparation remains characteristically high (like that of axoplasm) despite the trauma of the preparation and the two hours of shaking in the rcspirometer, it may he presumed that information obtained from such preparations may not he entirely artifactitious. It remains for future work to broaden the scope so as to throw light on the possible role of the satellite cells in trophic and excitatory processes of the nerve fiber. The potassium concentrations \vere determined by flame photometry on supernates from trichloracctic acid homogenates of the various preparations after respiration runs. The values obtained lvere: 3.53 anti 272 mM K/kg wet Aveight, calculated on the collagen-free basis, for slit sheaths anti intact fibers, respectively. The spread in these figures, for intact fibers as well as for slit sheaths was rather wide. It has long been known that the concentrations of K, Sa and Cl in axoplasm of giant fibers change as the fibers are allowed to stand after dissection. As the normal specific permeability is slowly lost, I<+ is reduced while Na+ and Cl- are increased. In the present experiments the fibers were not of uniform length or diameter and the time elapsed between dissection and homogenization in trichloracetic acid varied from 1 to 5 hours. n’hen allowance is made for such factors, the values for intact figures agree well with those given by Keynes and Lewis [6] for comparable post-dissection standing. The higher potassium concentration in the slit sheaths as compared with intact fibers anti periaxonal tissue is considered significantly greater than the experimental error. \\‘hen it is remembered that slit sheaths contain substantial amounts of periaxonal tissue-type cells having lo\\- potassium concentration, the true potassium figure for the satellite cells of the slit sheath hecomes even higher. Considerations of this sort have particular pertinence to the problem of potassium movement hetlveen axon and extracellular space. Potassium values for periaxonal tissue averaged only 175 mM/kg Ivet weight, calculated on the collagen-free basis. If this figure does in fact reflect Experimental
Cell Research 20
10
Rose Robyns Coelho, Joan Wright Goodman and M. Blair Bowers
the content of the cellular constituents and small nerve fibers in the samples, it would appear that they tend to lose potassium on standing faster than do intact giant fibers and slit sheaths. Treatment of such “connective tissue” with DNP (10-S M for 2 hours) caused no significant change in potassium concentration (181 mM K), but similar treatment of slit sheaths reduced the potassium concentration about 40 per cent (from 353 to 216 mnl).
DISCUSSION
Although the satellite cells in the slit-sheath preparations were studied under conditions highly abnormal to them because of the trauma of dissection and exposure to abnormal saline media (having high Na and low K content), and although the present experiments, extending over two summer seasons, require extension and confirmation, certain conclusions may be drawn with a fair degree of conviction. Among these the following seem most pertinent: The oxidative metabolism and the potassium concentration-and their sensitivity to DNP and azide are such as to support the view that these cells are centers of very active biochemical and possibly physiological processes. Electron microscopic evidence [3, 12, 13, 141 particularly concerning the high development of an intracytoplasmic membrane system, and an intimate and possibly causal relationship to mitochondria (which would be consistent with the suggestion that the sheath depends more than does the axoplasm on oxidative processes, especially those of the citric acid cycle [ll 1) support this view. These cells also have a very high surface to volume ratio; indeed they are so thin (ca 1 p or 0.2 per cent of the axon thickness in squid giant fibers and ca 0.1 ,u< 1 per cent of the fiber thickness in lobster fibers) that they have for the most part been ignored by physiologists. From the present raw data it would appear that the cells in the slit-sheath preparations constitute about 27 per cent of the dry weight of the average cleaned fiber, but have a respiration equal to 45 per cent of that of intact fibers (all calculations being on the collagen-free basis). The manner in which these metabolically active cells function with respect to the axon and its surface membrane in rest and activity, in growth, injury and repair are not illuminated further by these experiments. However, further investigations along these lines and in combination with isotopic tracer methods should prove fruitful.
Experimental
Cell Research 20
Studies of satellite cells of squid nerve fiber
11
SUMMARY
The respiration of squid giant nerve fibers and of the satellite (Schwann) cell layer surrounding the axon surface membrane has been measured bp a sheath-slitting technique, using a modification of the Scholander microrespirometers. Estimation of the contribution of the satellite cells was facilitated by determination of wet and dry weights, total protein and c,ollagcn content of intact fibers, slit sheaths and dissected periaxonal tissue. The oxygen consumption averaged 13.5, 7.9 and 7.8 mm3 O,/mg dry weight/hour for slit sheaths, intact fibers and periaxonal tissue, respectively, all calculated on the collagen-free basis. The sheath cells are responsible for about 4,? per cent of the respiration of the intact fiber though contributing only about 27 per cent of the dry weight. Some evidence for the presence of a factor or co-factor in sea water, not present in saline media artificially prepared from pure chemicals, is described. IXnitrophenol and azide have little effect on intact fibers, but strongly inhibit the respiration of slit sheaths in 10-s to 10-4 ‘12 concentration. The potassium concentration remains high in slit sheaths as compared with intact fibers and periaxonal tissue even after several hours of shaking. DSP reduces the concentration in slit sheaths substantially. These experiments form part of the program of chemical and structural studies of squid nerve performed during the summers of 1956 and 1957; we are greatly indebted to Professor Francis 0. Schmitt and to the other members of the “squid team” for assistance and encouragement throughout the work. REFERENCES C. M., Biol. Bull. 103, 315 (1952). 1. CONNELLY, B. B., Exptl. Cell Research 7, 558 (1954). 3. GEREN, B. B. and SCMITT, F. D., Proe. NatI. Acad. Sci. 40, 863 (1954). Intern. Union Biol. Sci. Ser. B 21, 251 (1955). 4. -4. L. and KEYNES, R. D., J. Physiol. 128, 28 (1955). 5. HODGKIN, 6. KEYNES, R. D. and LEWIS, P. R., J. Physiol. 114, 151 (1951). B. A., J. Biophys. Biochem. Cytol. 25, 511 (1955). 7. KOECHLIN, N. J., J. Biol. Chem. 184, 299 (1950). 8. LOWRY, 0. H. and ROSENBROURGH, M., .J. Gen. PhysioL 37, 201 (1953). 9. MAXFIELD, 10. NEUYAN, R. E. and LOGAN, hf. A., .I. Biol. Chem. 184, 299 (1950). 11. ROBERTS, N. R., COELHO, R. R., LOWRY, 0. H. and CRAWFORD, E. .J., J. Seurochem. 3, 109 (1958). 12. SCHMITT, F. O., in Metabolism of the Nervous System, p. 35. D. Richter, Ed. Pergamon Press, New York, 1957. 13. SCHMITT, 12. O., Exptl. Cell Research Suppl. 5, 33 (1958). N., Progr. in Biophys, 8, 165 (1957). 14. SCHMITT, F. 0. and GESCHWIND, 15. SCHOLANDER, P. F., CHEFF, C. L., ANDREWS, J. R. and WELLSH, D. F., J. Gen. Physiol. 35, 375 (1952).
2. GEREN,
Experimental
Cell Research 20
1
Cell Research 20, l-1 1 (1960)
CHEMICAL
STUDIES
OF THE SATELLITE
SQUID GIANT NERVE ROSE
ROBYNS
Department
of Biology,
FIBER’
COELHO,z JOAN WRIGHT M. BLAIR BOWERS Massachusetts
Institute
CELLS OF THE
of Technology,
GOODMAN
and
Cambridge, Mass.,
U.S.A.
Received June 28, 1959
PEHIPHEHALnerve axons are enclosed over most of their lengths by a monolayer of satellite (Schwann) cells. The physiological function of these cells is poorly understood except for their role in the morphogenesis of myelin, which they form by wrapping the axon in many turns of their infolded double surface membrane [a]. In unmyelinatetl fibers, e.g. in invertebrate fibers and in vertebrate C fibers, the satellite cells are not thus differentiated though they apparently serve a role vital to the normal functioning of these fiber types also. Electron microscope studies suggest that the satellite cells, at least in lobster fibers, may be morphodynamically active as in the formation of mitochondria-like particulates [3, 41. The subject of axon-satellite cell relationship has been reviewed by Schmitt and Geschwind [14]. Attention has been drawn to the possible role of the satellite-cell layer in solute exchange between axon and extracellular space, hence also in impulse proof their circumaxonal disposition [lx, 13, l-11. pagation, 1)~ virtue Because of the intimate structural relationship between axon and satellite cells because of the very high surface-to-volume ratio it has so far been impossible to obtain evidence regarding satellite-cell metabolism in unaltered fibers. \C’ith the squid giant-fiber a technique has been developed by which the gaseous metabolism of the sheath may be determined after removal of axoplasm by slitting the sheath in the axis of the fiber. Under these atlmittedly grossly abnormal conditions the satellite cells continue to respire for hours. Their approximate respiratory rate can be estimated from values from intact fibers, slit-sheath preparations and pcriaxonal connective tissue (from 1 These studies were aided by a research grant (B-24) from the National lnstitute of Neurological Diseases and Blindness, of the National Institutes of Health, U.S. Public Health Service; by a contract between the Office of Naval Research, Department of the Navy, and the Massachusetts Institute of Technology (NR-119-100); and by grants from the Trustees under the Wills of Charles A. and Marjorie King, and from the Lou and Gene Marron Foundation. 2 Public Health Research Fellow of the National Institute of Neurological Diseases and Blindness. Present address: Children’s Medical Center, Boston 15, Massachusetts. I-
603704
Experimental
Cell Research 20
2
Rose Robyns Coelho, Joan Wright Goodman and M. Blair Bowers
which to assess the significance of the fibrocytes in the axon sheath). As reported briefly in this paper, the results demonstrate that the satcllitc cells account for almost half the respiration of the entire fiber.
METHODS The giant nerve fibers of Loligo pealii, obtained from squid brought to the M.I.T. laboratories in the manner previously described by Maxfield [9], were dissected from the mantle muscle and transferred to chilled filtered sea water for removal of loose connective tissue and small nerve fibers. This dissection was accomplished as rapidly as possible, but with every effort to avoid traumatization of fiber constituents. Slitting of sheaths was accomplished by insertion of sharp iridectomy scissors into the fiber and cutting the cleaned axon-sheath longitudinally. Quick cleaning of the fibers after dissection was found necessary for the fiber and slit sheaths to respire in the fashion described below. The slit sheaths were washed by gentle shaking in sea water kept at 15°C. This process removes axoplasm and extracellular fluids. Natural and artificial sea water, filtered through VC millipore filters, served both as a medium for cleaning and slitting fibers and for suspending fibers and slit sheaths The composition of the two artificial saline media for measurement of respiration. used is given in Table I. TABLE I. Composition Solution
ofnrtificid
saline me&r
used in respiration
NaCl
KC1
CaCl,
Mgso,
MgCl,
H,UO,
I
355
17.0
11.4
40
29.0
17.6
II
475
10.0
10.7
28.6
26.7
1.0
KaHCO,
2.5
memuremenfs. W’O,
2.0x 10 3
All values are expressed in mM/l.
Oxygen consumption was measured in slightly modified Scholander-type microrespirometersl In each respirometer vessel were placed either three of four intact ligated and cleaned giant fibers of about 10 mg wet weight or six slit-sheaths of about 6 mg wet weight. These specimens were suspended in 0.2 ml sea water or artificial saline. Temperature was maintained at 14°C. For comparison and calibration several runs were made in Warburg respirometers fitted with small vessels; of necessity more fibers and sheaths were required for this less sensitive method. Rates of respiration were found similar in the two methods when the shaking rate of the Scholander vessels was maintained in the range of 130 -170 per minute. Higher shaking rates reduced respiratory rates of slit sheaths presumably due to mechanical injury. 1 These microrespirometers were constructed by Mr. \I’. Strovink of this Department. The manometer plungers were activated by precision micrometers to obtain maximum accuracy of volume measurement. Dow Corning 200 fluid was used as manometer fluid instead of kerosencmineral oil because it was found to move more uniformly in the capillaries. Experimenfal
Cell Research 20
Studies of satellite cells of squid nerue fiber After measurement of respiration, the fibers or slit sheaths were removed from the vessels, washed briefly in distilled water and homogenized in 2 ml of 10 per cent trichloracetic acid. Determinations of protein concentrations were performed on 0.5 ml of the hornogenate in duplicate by the methods of Lowry [8]. Similar analyses were made on uncontaminated axoplasm especially extruded for the purpose; the fact that the results agreed well with those obtained by Koechlin [7] constitutes evidence of the reliability and consistency of the techniques. The collagen content of specimens was estimated from hydroxyproline values, determined by the methods of Neuman and Logan [IO], on the assumption that squid collagen contains 10 per cent hydroxyproline. Samples for analysis were hydrolyzed in 1 ml of 6 11’ HCI by autoclaving for 5 hours at 15 pounds pressure. The method was demonstrated to account quantitatively for the hydroxyproline in purified calfskin collagen.
Estimation
of Partition
of Fiber Constituents
If the contribution of the satellite cells to the metabolism of the intact fiber is to he estimated, data must be obtained concerning the fraction of the average preparation of intact fibers and slit sheaths that is represented 1)~: (1) satellite cells, (2) other metabolizing structures, chiefly fibrocytes and small nerve fibers in the sheath (herein called “pcriasonal tissue”), and (3) non-mctaholizing material, chiefly collagen and inorganic salts. The pertinent approximate information is oldainetl by consideration of three compartments: (1) intact fihrrs cleaned in the usual fashion, (2) slit sheath preparations, (3) pcriaxonal tissue ohtaineci during the routine process of cleaning fibers after dissection from the mantle muscle. Histological esamination sho~~ti periasonal tissue to contain primarily collagen filwrs, fihrocytes and a relatively small fraction of small (ca 10 p) nerve fihcrs. Slit sheaths were found to contain, besides the axon surface membrane and the surrounding monolayer of satellite cells, closely adhering layers of connective tissue \vhich contained, crnhccltied in the collagen fibers anti ground suhstance, fibrocytrs roughly eclualling in number the satellite cells, as judged by nuclear counts in OsO,-fixed lx-eparations.1 13wause of the srnall size of the samples, xveighing in the \yct state \\-as difficult anti inaccurate. ,Ilore reliance \vas placed on the protein content. The partition of protein, as rneasuretl in per cent of the dry weight 1)~ the method of Lo\\-ry [Xl, was: Intact fibers, ‘26; Slit sheaths, 26; l’eriaxonal tissue, 33; and Reference collagen (calf skin), 35. 1 WC are grateful of squid nerve.
to bliss Olive Fletcher
and Dr. Norman
Geschwind
for the histological
Experimental
studies
Cell Research
20
4
Rose Robyns Coelho, Joan Wright Goodman and M. Blair Bowers The cellular (Total
protein
is roughly
protein
equal to:
in a sample) - (collagen
protein)
X 0.35.
Corrected thus for collagen it was estimated that the protein content per unit of dry weight was in per cent: Intact fibers, 28; Slit sheaths, 15; and Periaxonal tissue, 31. These values, though not of high accuracy in themselves, permit interconversion of “cell protein” values and “cell dry weight” values. From such data, together with those on axoplasm (amino acid content, etc.) and hydroxyproline values, the figure of 7.9 per cent nitrogen per unit of dry weight emerged for intact fibers. This is in good agreement with the nitrogen values obtained experimentally by Koechlin [7] of 8 per cent. For slit sheaths the value of 9 per cent nitrogen per unit of dry weight was obtained, whereas the value obtained from micro-Kjeldahl analysis was 11 per cent. The source of this discrepancy cannot yet be determined because little is known of the chemical composition of the sheath. The weight ratio of the three compartments (axoplasm, sheath cells and periaxonal tissue) can be calculated from micro-Kjeldahl and hydroxyproline values on weighed samples of intact fibers and slit sheaths. It was found that the average value of slit-sheath collagen was 46 per cent. The nitrogen contents of the three compartments were in per cent dry weight: Axoplasm,l 5.8; Intact fiber,2 8.0; and Slit sheath,2 11.3. From these figures it may readily be shown that axoplasm accounts for 60 per cent of the dry weight of intact fibers, and collagen accounts for 18 per cent. The non-collagenous component represents 22 per cent of the dry weight. In terms of dry weight of non-collagenous material this means that the cellular constituents in the sheath account for 27 per cent and axoplasm for 73 per cent. EXPERIMENTAL
RESULTS
Under the conditions described, intact fibers and slit sheaths consume oxygen at a relatively constant rate for several hours after the beginning of Typical data are shown in Fig. 1; in Table II are sumthe experiment. marized the data for experiments in which natural sea water was used as suspension medium. As may be seen in Fig. 1, the rate of oxygen uptake of slit sheaths is not constant for the first fifteen minutes. It seems unlikely that more than about 1 Results of Koechlin [7]. 2 Kindly determined in this laboratory Experimental
Cell Research 20
by Dr. G. IIeffner.
5
Sfudies of satellite cells of squid nerve fiber
five minutes is required for temperature equilibration anti this was allowed before taking the first measurements. It seems more probable that the initial high rate is clue to the trauma to the sheath cells in the slitting operation. Only the linear portion of the curve was usecl in estimating respiratory rates. The slit-sheath preparations are, not unexpectedly, highly sensitive to damaging procedures in the precertain, no\v only partially understood,
Minutes Fig. l.-Respiration of slit sheaths and whole fibers in filtered sea water. Ordinates: mm3 O,/mg dry weight of collagen-free material. 0, sheaths; 0, axons.
TABLE
II. Respiratory
rates of intact fibers, slit shenths in filtered nnturul sea writer.
(mm3 O,/mg dry weight
Tissue Fibers Sheaths Periaxonal tissue
of collagen-free
rind perinxonnl
tissue
material/hour.)
Average respiratory rate
Number of experiments
Standard deviation
7.88 13.50 7.80
IO 22 6
2.26 4.55 3.40
Experimental
Cell Research 20
6
Rose Robyns Coelho, Joan Wright Goodman and M. Blair Bowers
parative technique. Thus of thirty-fire experiments in all, the slit sheaths in thirteen experiments (not included in the table) showed no apparent respiration. In most of these cases the source of the excessive damage during the preparation of the specimens or the poor condition of the squid after the 40 30
s
20 IO 0 O\-”30 0” s 20 s 0) I;: IO 0
30 20 IO 0
0
200
400
m MK/kg
600
800
1,000
wet weight
Fig. 2.-Potassium content of slit sheaths, whole fibers and periaxonal S, sheaths; F, fibers; and C.T., periaxonal tissue. Abscissa: mM K/kg wet weight of collagen-free material.
tissue.
eighty-mile trip by truck from the fishing grounds to the laboratory could be demonstrated. In the latter connection the necessity of good aeration and of maintaining a low temperature (ca 14°C) in the sea water circulating in the tanks was established. Indeed, the metabolic behavior of the slit-sheath preparation proved a sensitive criterion of the success of all steps in the technique from the catching and transportation of the squid to the cleaning Experimental
Cell Research 20
Studies of satellite cells of squid nerve fiber
7
and slitting of the giant fiber. As might be expected, the spread in the data from slit sheaths is greater than for intact fibers. The mean respiratory rates obtained by the Scholander and Warburg methods are identical in the case of fibers. The value obtained for sheaths by the Warburg method is lower (8 mm3 O,/mg collagen-free material/hour) than that found using Scholander microrespirometers. ‘This is not surprising since 30 slit sheaths had to be collected before the experiment could be started, which corresponded to a delay of 7 hours between the killing of the squid and the termination of the experiment. Altogether 6 experiments have been done using the Warburg technique. The respiratory rates for intact fibers are also reasonably similar, considering the differences in techniques, to those obtained by Connellp [l] using the oxygen electrode at 16°C. From the average collagen content of dissected fibers it is possible to convert Connelly’s average figure of 68 mm3 O,/gm wet weight/hr into the corresponding figure in terms employed by us, namely mm3 O,/mg dry weight of non-collagenous material/hr. In this case, Connelly’s figure becomes 5.0, as compared with our own figure of 7.9. Efect
of Saline Medium
on Oxygen Consumption
The data in Table III show that the respiration of intact fibers-and of periaxonal tissue-is not substantially different in artificial sea water. However, the effect upon the respiration of slit sheaths is as dramatic as it is unexplained. Out of twelve experiments, the usual rate of respiration of slit sheaths in the artificial saline was demonstrated in only two experiments. In the remaining ten experiments the preparations either did not respire at all or stopped respiring within minutes of the beginning of experiment. ‘I’ABLE
III.
Respirrctory
rates of intact fibers, slit sheaths in artificinl media.
(mm3 O,/mg dry weight
Tissue Fiber Sheaths Periaxonal Tissue Altogether in the Table.
12 runs were
of collagen-free
and pericwonrrl
material/hour.)
Average respiratory rate
Number of experiments
7.30 15.00 7.1
8 2 2
done on sheaths;
10 showed
tissue
no respiration
and were not included
Experimental
Cell Research 20
Rose Robyns Coelho, Joan Wrighf Goodman and M. Blair Bowers
8
Because of the possible significance of the unidentified factor-possibly an oligodynamic effect-in the respiratory chain, efforts mere made to characterize the factor. These efforts were only partially successful. It appears that the factor is not diffusible because dialysis of artificial sea water against natural sea -\vater did not prevent the respiratory inhibition. Roiling or autoclaring natural sea Tvater had no eBect. The natural sea \vater used showed a broad absorption band centered at about 2660 A as measured in the Cary spectrophotometer. Possibly organic material, such as nucleotide derivatives or amino acids, mav be present which serve as a factor or cofactor for satellite-cell metabolism.
Efict
of Respiratory
Inhibitors
‘L,h-DNP profoundly influences the ionic fluxes of squid giant fibers [a]. Sodium efflux and potassium influx are greatly reduced while sodium influx and potassium efflux are little effected. Accordingly it would be interesting to determine the effect of this substance and of other inhibitors, such as azide and cyanide, on the metabolism of slit sheaths as compared with intact fibers. From the data in Table IV it will be seen that I)NP has little efrect on the respiratory rate of intact fibers or of connective tissue cells, but inhibits that of slit sheaths by about a third in concentrations of 10-5 to 10e4 ~11. Sodium azide has a similar effect: little influence on intact fibers in 10-5 to 10-4 M concentration, but inhibition of slit sheaths which was complete at lo-4 111concentration. TABLE
IV. Effect
of
2,bdinitrophenol
and sodium
azide
Average Number of experiments
(mm3 O,/mg Inhibitor
Intact
fibers
dry weight
on respiratory
respiratory
rates
of collagen-free
Slit sheaths
rates.
material/hour) Periaxonal
See Table I
Unpoisoned
7.88
13.5
7.8
7
2,4-DNP (1 x IO-6 to 1 x IO-4 M)
7.1
8.3
7.3
2
1 x 10-s M NaX,
8.4
-
3
1 x 10-4 M NaN,
Experimental
Cell Research 20
5.9
0
tissue
Studies of satellite cells of squid nerve fiber Potassium
Concentration
in Satellite
Cells
So data are available concerning the electrolyte concentrations in satellite cells and their variation with function and \vith environmental changes. Only a modest heginning has been made in these experiments toward obtaining such information. The electrolyte pattern, like oxygen consumption, is a valuable index of the state of cells; its determination \vas therefore important in the present experiments. If the potassium concentration of the cells in the slit-sheath preparation remains characteristically high (like that of axoplasm) despite the trauma of the preparation and the two hours of shaking in the rcspirometer, it may he presumed that information obtained from such preparations may not he entirely artifactitious. It remains for future work to broaden the scope so as to throw light on the possible role of the satellite cells in trophic and excitatory processes of the nerve fiber. The potassium concentrations \vere determined by flame photometry on supernates from trichloracctic acid homogenates of the various preparations after respiration runs. The values obtained lvere: 3.53 anti 272 mM K/kg wet Aveight, calculated on the collagen-free basis, for slit sheaths anti intact fibers, respectively. The spread in these figures, for intact fibers as well as for slit sheaths was rather wide. It has long been known that the concentrations of K, Sa and Cl in axoplasm of giant fibers change as the fibers are allowed to stand after dissection. As the normal specific permeability is slowly lost, I<+ is reduced while Na+ and Cl- are increased. In the present experiments the fibers were not of uniform length or diameter and the time elapsed between dissection and homogenization in trichloracetic acid varied from 1 to 5 hours. n’hen allowance is made for such factors, the values for intact figures agree well with those given by Keynes and Lewis [6] for comparable post-dissection standing. The higher potassium concentration in the slit sheaths as compared with intact fibers anti periaxonal tissue is considered significantly greater than the experimental error. \\‘hen it is remembered that slit sheaths contain substantial amounts of periaxonal tissue-type cells having lo\\- potassium concentration, the true potassium figure for the satellite cells of the slit sheath hecomes even higher. Considerations of this sort have particular pertinence to the problem of potassium movement hetlveen axon and extracellular space. Potassium values for periaxonal tissue averaged only 175 mM/kg Ivet weight, calculated on the collagen-free basis. If this figure does in fact reflect Experimental
Cell Research 20
10
Rose Robyns Coelho, Joan Wright Goodman and M. Blair Bowers
the content of the cellular constituents and small nerve fibers in the samples, it would appear that they tend to lose potassium on standing faster than do intact giant fibers and slit sheaths. Treatment of such “connective tissue” with DNP (10-S M for 2 hours) caused no significant change in potassium concentration (181 mM K), but similar treatment of slit sheaths reduced the potassium concentration about 40 per cent (from 353 to 216 mnl).
DISCUSSION
Although the satellite cells in the slit-sheath preparations were studied under conditions highly abnormal to them because of the trauma of dissection and exposure to abnormal saline media (having high Na and low K content), and although the present experiments, extending over two summer seasons, require extension and confirmation, certain conclusions may be drawn with a fair degree of conviction. Among these the following seem most pertinent: The oxidative metabolism and the potassium concentration-and their sensitivity to DNP and azide are such as to support the view that these cells are centers of very active biochemical and possibly physiological processes. Electron microscopic evidence [3, 12, 13, 141 particularly concerning the high development of an intracytoplasmic membrane system, and an intimate and possibly causal relationship to mitochondria (which would be consistent with the suggestion that the sheath depends more than does the axoplasm on oxidative processes, especially those of the citric acid cycle [ll 1) support this view. These cells also have a very high surface to volume ratio; indeed they are so thin (ca 1 p or 0.2 per cent of the axon thickness in squid giant fibers and ca 0.1 ,u< 1 per cent of the fiber thickness in lobster fibers) that they have for the most part been ignored by physiologists. From the present raw data it would appear that the cells in the slit-sheath preparations constitute about 27 per cent of the dry weight of the average cleaned fiber, but have a respiration equal to 45 per cent of that of intact fibers (all calculations being on the collagen-free basis). The manner in which these metabolically active cells function with respect to the axon and its surface membrane in rest and activity, in growth, injury and repair are not illuminated further by these experiments. However, further investigations along these lines and in combination with isotopic tracer methods should prove fruitful.
Experimental
Cell Research 20
Studies of satellite cells of squid nerve fiber
11
SUMMARY
The respiration of squid giant nerve fibers and of the satellite (Schwann) cell layer surrounding the axon surface membrane has been measured bp a sheath-slitting technique, using a modification of the Scholander microrespirometers. Estimation of the contribution of the satellite cells was facilitated by determination of wet and dry weights, total protein and c,ollagcn content of intact fibers, slit sheaths and dissected periaxonal tissue. The oxygen consumption averaged 13.5, 7.9 and 7.8 mm3 O,/mg dry weight/hour for slit sheaths, intact fibers and periaxonal tissue, respectively, all calculated on the collagen-free basis. The sheath cells are responsible for about 4,? per cent of the respiration of the intact fiber though contributing only about 27 per cent of the dry weight. Some evidence for the presence of a factor or co-factor in sea water, not present in saline media artificially prepared from pure chemicals, is described. IXnitrophenol and azide have little effect on intact fibers, but strongly inhibit the respiration of slit sheaths in 10-s to 10-4 ‘12 concentration. The potassium concentration remains high in slit sheaths as compared with intact fibers and periaxonal tissue even after several hours of shaking. DSP reduces the concentration in slit sheaths substantially. These experiments form part of the program of chemical and structural studies of squid nerve performed during the summers of 1956 and 1957; we are greatly indebted to Professor Francis 0. Schmitt and to the other members of the “squid team” for assistance and encouragement throughout the work. REFERENCES C. M., Biol. Bull. 103, 315 (1952). 1. CONNELLY, B. B., Exptl. Cell Research 7, 558 (1954). 3. GEREN, B. B. and SCMITT, F. D., Proe. NatI. Acad. Sci. 40, 863 (1954). Intern. Union Biol. Sci. Ser. B 21, 251 (1955). 4. -4. L. and KEYNES, R. D., J. Physiol. 128, 28 (1955). 5. HODGKIN, 6. KEYNES, R. D. and LEWIS, P. R., J. Physiol. 114, 151 (1951). B. A., J. Biophys. Biochem. Cytol. 25, 511 (1955). 7. KOECHLIN, N. J., J. Biol. Chem. 184, 299 (1950). 8. LOWRY, 0. H. and ROSENBROURGH, M., .J. Gen. PhysioL 37, 201 (1953). 9. MAXFIELD, 10. NEUYAN, R. E. and LOGAN, hf. A., .I. Biol. Chem. 184, 299 (1950). 11. ROBERTS, N. R., COELHO, R. R., LOWRY, 0. H. and CRAWFORD, E. .J., J. Seurochem. 3, 109 (1958). 12. SCHMITT, F. O., in Metabolism of the Nervous System, p. 35. D. Richter, Ed. Pergamon Press, New York, 1957. 13. SCHMITT, 12. O., Exptl. Cell Research Suppl. 5, 33 (1958). N., Progr. in Biophys, 8, 165 (1957). 14. SCHMITT, F. 0. and GESCHWIND, 15. SCHOLANDER, P. F., CHEFF, C. L., ANDREWS, J. R. and WELLSH, D. F., J. Gen. Physiol. 35, 375 (1952).
2. GEREN,
Experimental
Cell Research 20