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Ultracentrifugation of rat spinal ganglion cells with special reference to neurofibrillae.

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T'LTRACEXTRIFUGATION O F RAT XPIXAL GAXG-
LION
cmr,s, WITH
sPEcIhrJ REFERESCX
TO N I':TTR( )FIRRTLTAE
€1. W. T3EA3IS A K D €1. \V. KIlISIIENBLTT
Zoologicul Lccboiutoiy, Stntc U n r n o s i f y of Iowa, Torrn C / t ! /
ONE PL.4TE ( F O U R FIGURES)
Siiice tlie discoveiy of iieurofibrillae by Reniak in 1843
(Parker, '%), investigator*s have not been able to agree as to
whether or iiot they are present as such in living nerve cells.
It lias been lield by Bayliss ( ' X ) and others tliat there is no
actual proof of tlieir existcncc in the living state and that
they are in all probability artifacts, the typically fibrillar appearance being brought about by the fixative tlii.ough a coagnlation of some substance in the cell of non-fibrillar character.
For it long wliile the chief arguiiieiit against tlie existence
of neurofibrillae was the fact that 11iey could not be convincingly tlenions t rat ed in living nerve cells, notmitlis t anding
the fact that the first observations of Remak on neurofibrillae
~ e r rriade
c
on the living nerve cells of tlie crayfish. However,
in 1927 Bozlci. was able t o show clearly the presence of rieurofibi-illae in tlie living neurones of tlic jellyfish, aiicl de RCnTTi
('32) saw them in living ciwstaceaii nerve cells. They were
first clcai.ly shown to exist in the living condition i n vertebrates when Weiss and Wang ( ' 3 6 ) puhlished pliotomicrog:.r*aplisof embryonic chick ganglion cells, cultivated in vitro,
sliowing distinct paranuclear threads in the cytoplasm. Tlicse
observations have in a general way been confirmed by Levi
and l\leyer ( ' 3 7 ) .
Foi. a review of the literature on this general subject the
readel. is 1-efcrrcd to tlie woi*ks of I'ai.kei* ( '29)' TTTciss and
Kaiig ( '36), and IToei.i* ( '36).
95
96
H. W. BEAMS A4ND H. W. KIKSHENBLIT
I n view of the fact that the ultracentrifuge had been used
to study certain structures in nerve cells, such as Nissl bodies
(Beams and King, '35), Golgi apparatus (Muliyil, '35 ; Brown,
'36), and mitochondria (Brown, '36), it occurred to us that
it might likewise be useful in a study of the neurofibrillae
with reference to the question of their reality in the spinal
ganglion cells of adult rats. Furthermore, our results may
also be considered a check on the observations of Ingvar ( '23),
who displaced the neurofibrillae with a n ordinary clinical
centrifuge, under conditions which he admits were most unfavorable, since many of the cells had undergone considerable
disintegration and cytolysis.
The spinal ganglia of the albino r at were used in this study,
the animals being about 25 days old when sacrificed. They
were killed either by exposure to the fumes of ether o r by
having their necks broken. Sections of the spinal column
were immediately removed and split along the dorsal side.
They were then placed in Ringer's or isotonic sodium chloride
solution and the spinal ganglia quickly dissected out. Some
of the ganglia were kept a s controls, while the remainder were
placed in the rotor of the ultracentrifuge together with a
quantity of the isotonic salt solution sufficient to cover them.
The ganglia were then ultracentrifuged for 30 minutes
at a speed generating a force of about 400,000 times gravity.
Control ganglia were kept in the isotonic salt solution while
the centrifugation was in progress, and both controls and
centrifuged ganglia were fixed immediately after completion
of the centrifugation.
Neurofibrillae were denionstrated by the protargol-copper
method of Bodian ( '37). I n most cases it was found advisable
to follow reduction and washing with a second impregnation
in fresh protargol-copper solution although it was possible
to secure a distinct, though somewhat fainter prepar at'ion
of neurofibrillae by a single impregnation. All sections mere
toned with gold chloride.
F o r a description of the ultracentrifuge the reader is
referred to Beams, Weed and Pickels ( '33).
U L T R A C E N T R I F U G A T I O N O F S P I N A L GANGLIA
97
I n the normal nerve cell (fig. 1) the neurofibrillae appear
as a network of relatively fine threads, completely enclosing
the nucleus, and distributed throughout the cytoplasm at all
levels of focus. The fibrillae do not appear to extend beyond
the cell wall but in those cells in which the nerve process can
be seen leaving the cell body, the neurofibrillae collect into a
bundle, losing their disordered appearance, and form a cable
of distinct threads in which no clear anastomoses are apparent.
In cells ultracentrifuged a t 400,000 times gravity f o r 30
minutes the neurofibrillae are seen to be concentrated at the
centrifugal end of the cell (figs. 2, 3, 4). The threads of the
fibrillae nearest the 'centripetal ends of some cells appear
frayed as though they may have been torn away from the cell
wall.
It is not an easy matter t o determine whether o r not the
neurofibrillae in centrifuged ganglion cells have been displaced t o the centrifugal end of the cell because of their
greater density or because they have been dragged there by
the displacement of the nucleus and the Nissl bodies through
the cytoplasm during the centrifuging. The former view seems
more probable in view of the fact that Ingvar ('23) found in
centrifuged nerve cells that the nucleus retained its original
position while neurofibrillae were displaced t o the centrifugal
end of the cell. From our work it seems probable that considerable change in the physical state of the nerve cells used by
Ingvar must have taken place since we were never able to
displace the neurofibrillae a t forces comparable to those used
by him.
The cytoplasm of vertebrate nerve cells, particularly the
spinal ganglion cells, is obviously of a hiphly viscous nature.
This is substantiated by the fact that browmian movement is
usually not observed (review by de R h y i , '32) and that
microdissection studies indicate their physical nature to be
that of a rather solid gelatinous structure. The fact that
such high centrifugal force is necessary to displace the various
cytoplasmic elements would also support this view.
98
H. W. BEAMS A N D H. \Ir.1iIBSHEJSBLIT
We were unable to clearly displace tlie Golgi apparatus of
the spinal ganglion cells by centrifuging at 900,000 times
gravity for 30 minutes. This, of course, may be due to the
i*elatively slight difference in specific gravity between the
Qolgi apparatus and surrounding cytoplasm.
However, the fact that neurofibrillae may be displaced, distorted, and torn by ultracentrifugation of living iierve cells
(it has been shown that cells a r e not killed by this strength
of centrifugal force-Beams
and King, ' 3 5 ) argues for a
definite structure of the neurofibrillae in the living cell compalable to that demonstrated by the classical histological
methods, rather than suggesting that they are a n artifact.
LITERATURE CITED
B ~ Y L I S SW.
, M. 1927 Piinciples of General Physiology, p. 397.
B ~ a x s H.
, W., BND R. L. KING 1933 Effects of ultracentrifuging on the spinal
ganglion cells of the rat. J. Conip. Neur., vol. 61, p. 175.
B e k ~ r x J.
, W., A. J. WEEDA N D E. G . PICKELS
1933 The ultracentrifuge. Science,
vol. 78, p. 338.
1936 A new nictliod for staining nerve fibers and nerve endings i n
H O D I ~ ND.
,
mounted paraffin sections. Anat. Ree., vol. 65, p. 89.
BOZLER,
E. 1927 Untersuchungen uber das Ncrvensystrni d r r Coelenteraten.
Zeit. Vergl. Physiol., vol. 6, p. 235.
BROWN,
It. H. J. 1936 The effect of ultracentrifuging vertebrate neuiones.
Quart. J. Micr. Sci., vol. 79, p. 73.
HOERR,K. W. 1936 ('xtologie:il studies hy the A1tm:inn Gersh freezing-di:ing
niethod. Anat. Rec., vol. 66, p. 81.
I S G V ~S.R ,1923 Centiifugation of the nervous system. Arch. Neurol. and
Psychiatr., vol. 10, p. 267.
T,xvI, G., AND €1. MEYER 1937 Die Struktur der lchenden Keuronen. Anat. Anz.,
vol. 83, p. 401.
MULIYIL,J. A. 193,i The effect of ultracentrifuging on the spinal ganglion cells
of eertain Oithoptera. &it. f. Zellforsch. und Mikr. Anat., rol. 23,
p. 627.
P ~ R K FG.R€1.
, 1929 The neurofibril hypothesis. Quart. Rev. Riol., vol. 4, p. 153.
DE R ~ N Y IG, . S. 1931 The structure of eells in tissue a s revealed by microdissection. V. The physical properties of nerve cclls of the f r o g (Rana
pipiens). J. Comp. Neur., vol. 53, p. 497.
~ _ _ 1932
_
Architecture of the nerve cell as ie\ealcd 1)y miclodissettion.
Special Cytology, vol. 3, (edited by E. V. Cowdry).
WEISS,P., AND H. WANG 1936 Pv'eurofihrils in living ganglion cclls of the cllick
cultivated in vitio. A4nat. Rec., vol. 67, p. 105.
PLATE
PLATE 1
E X P L A N I T I O K O F FIGURES
All figures are photomicrographs of rat spiiinl ganglion cells stained by the
protargol-copper method. Figures 2, 3 and 1 were centrifuged at 400,000 times
gravity f o r 30 minutes. The direction of the centrifugal force in all cases except
figure 1 is approximately toward the bottom of the page.
1 Shows a control nerve cell with normal distribution of neurofibrillae.
2, 3 and 4 are cells showing the effects of centrifuging upon the nuclei and
neurofibrillae.
100
U L T R A C E N T R I F U G A T I O N O F SPINAL GANGLIA
H. W . B E A M S AND H. U
'
. ICIlIS€IIZXBLIT
101
PLATE 1
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