Determination of Mass of Nerve-cell Components
by HOLGER HYDEN 1
From the Department of Histology, Medical School, Goteborg
T H E method for determination of mass in biological material described by
Engstrom & Lindstrom (1950) is founded on the fact that the absorption of
X-radiation at 8-10 A is proportional to the mass of the biological substance. In
elaborating this method for quantitative analysis of nerve-cells and their components (Brattgard & Hyden, 1952) we found it important to consider the following sources of error:
1. The reference system.
2. The photographic process and the photometry.
3. The treatment of the material.
1. The reference system. The error in the reference system was investigated
and a new method was elaborated at the Histological Department in Goteborg
for the production of the thin cellulose foils which constitute the reference system
(Brattgard & Hallen, 1952; Brattgard & Hyden, 1952; Hallen, 1953). We found,
however, that this gravimetric method for the control of the mass per unit surface
of the reference system involved too great an error. Therefore an interferometric
method was worked out (Hallen & Ingelstam, 1952). This method determines the
mass of the reference system with an accuracy of + 1 per cent.
2. The photographic process and the photometry. It was found that density
measurements on a secondary magnification of the radiogram (the Lippmannfilm) had a standard deviation of + 36 per cent. (Brattgard & Hyden, 1952, and
unpublished), and this procedure had to be abandoned. Therefore we constructed
a special microphotometer (Brattgard, 1952; Brattgard & Hyden, 1952; Hyden,
1952; Bourghardt etal, 1953). The light intensity over a surface corresponding
to 3-4/x2 in the primary picture is measured by means of a multiplier tube and
recorded automatically. The radiogram can be magnified 800 times. The construction and the results of the tests will be published elsewhere. The error of the
film and the photometry was determined experimentally and found to be + 1 5
per cent.
3. The treatment of the material. It is recommended to use fresh, untreated
material (Brattgard & Hyden, 1952; Hyden, 1952). The fixation and the additional treatment in the cytological procedure cause losses of mass in the cells
(Table 1). If fixed material is used, microchemical determinations must be carried
out simultaneously (Brattgard, 1952). Note that the fixation treatment dissolves
1
Author's address: Histologiska Institutionen, Medicinska Hogskolan, Goteborg C, Sweden.
[J. Embryol. exp. Morph. Vol. 1, Part 3, pp. 315-317, September 1953]
316
H. HYDEN—DETERMINATION OF MASS IN NERVE-CELLS
relatively more substance from the nucleus of the nerve-cell than from the cytoplasm (see Table 1). This agrees well with the data obtained from other types of
cells.
TABLE 1
Effect of fixation on the mass of the nerve cell.
Motor nerve-cells from adult rabbit
The thickness of the sections is comparable in both cases. The values are corrected
and determined optically.
Fresh frozen cells
Carnoy fixed
cells
Total mass
10-9 mg.//*2
After lipid
extraction
10-9 mg.//*2
Cytoplasm
0-71 ±0056
O-35±OO35
034±0017
Nucleus .
0-48±0-016
O-3O±OO37
013 ±0008
10-9 mg.//x2
The method used by us involves a chemical fractionation of the cell substance.
The lipids are extracted by means of chloroform. By digestion with ribonuclease
a reproducible fraction is obtained, which we studied chemically. For the time
being we call this the 'nucleoprotein fraction'. For details see Brattgard (1952)
and Brattgard & Hyden (1952).
Differentiation in nerve-cells. The effect of the removal of the periphery on
the differentiation of motor neuroblasts in terms of the total amount of dry weight
was studied. Neuroblasts and young nerve-cells of chick embryos were investigated. The somatopleur of the embryos had been removed at 60-70 hours' incubation. This material was kindly placed at our disposal by Dr. Karl Mottet, Dept.
of Physiology, Yale University. The material was fixed in Carnoy's solution. The
effect of the fixation procedure on the material was studied (Brattgard, 1952;
Brattgard & Hyden, unpublished). Table 2 gives an example of the retardation in
differentiation in terms of mass and also approximately per cell cytoplasm.
TABLE 2
Chick embryo
Total development: 6 days. Development after operation: 3 days.
Mass
mg. x 10-9//x2
Mass per cell cytoplasm
mg. x 10"9
Control side
Operated side
Young nervecells
Corresponding
cells
0-65 ±0050
0-35±0040
123
10
H. H Y D E N — D E T E R M I N A T I O N O F MASS I N N E R V E - C E L L S
317
REFERENCES
ALLFREY, V., STERN, H., MIRSKY, A. E., & SAETREN, H. (1952). / . gen. Physio!. 35, 529.
BOURGHARDT, S., BRATTGARD, S-O., HYDEN, H., JIEWERTZ, B., LARSSON, S., & PHILHAGE, L. (1953).
(in the press).
BRATTGARD, S-O. (1952). Acta radiol. Suppl. 96.
& HALLEN, O. (1952). Biochim. Biophys. Acta, 9, 488.
& HYDEN, H. (1952). Acta radiol. Suppl. 94.
ENGSTROM, A., & LINDSTROM, B. (1950). Biochim. Biophys. Acta, 4, 351.
HALLEN, O. (1953). Exp. Cell Res. (in the press).
& INGELSTAM, E. (1952). Exp. Cell Res. 3, 248.
HYDEN, H. (1952). Die Chemie und der Stoffwechsel des Nervengewebes. Berlin-GottingenHeidelberg: 3. Colloq. Gesell. Physiol. Chemie, Mosbach 1952.