MICROSCOPIC STUDIES 
ON THft 
Central Nervous System 
OF 
ilefitiles anil H^afracftfotis* 
THE SPINAL COED OF THE FROG—RANA 
PIPIENS, RANA HALECINA. 
John J. Mascm. M. D 
CHICAGO; 
J. J. Spalding & Co., Peintebs, 158 Clabk St.1 
1880. Microscopic Studies on the Central Nervous 
System of Reptiles and Batrachians. 
John J. Mason; M. D 
ARTICLE I. THE SPINAL CORD OF THE FROG RANA PIPIENS, 
RANA lIALECINA. 
IT is not intended in these articles to give, in detail, an ana- 
tomical description of the nervous system of this class of 
animals. So far as the anourous group of batrachians is con- 
cerned, one could hardly effect such a purpose better than by 
translating the works of either Reissner * or Stieda,f which 
together with those of Wyman;}; and Ecker§ are in the hands 
of most comparative anatomists. Only in writing of species, 
the nervous system of which may not previously have been 
studied, will a full description be attempted, the main object 
being to present from time to time facts observed by the au- 
thor, and regarded by him as supplementary. The form of 
the spinal cord and especially that of its enlargements; the 
nuclei of the nerve cells, and variations in their shape, size, 
etc., in the same individual; the number of ganglionic bodies 
in the spinal cord, and their relations to the roots of the spinal 
nerves, and the differences, if any, which may be determined 
by sex : these, among others, seem to me to be subjects of 
[Reprinted from the Journal of Nervous and Mental Disease, Jan., 1880.] 
*l)er Ban des Centralen Nervensystems dev Ungeschwdnzten Batrachier. 
Dorpat, 18G1. 
f Studien ueber das Central Nervensystem der Wirbelthiere. Leipzig, 1870. 
X Anatomy of the Nervous System of liana pipiens. Washington, 1853. 
%lcones Physiologicce, 1851-59. Licpzig. “ Die Anatomic des Frosches 
des physiologischen Thieres, istfur den Physiologen, kaum minder wichtig, 
als die Anatomie des Menschen.” Mason—Central Nervous System of Reptiles, etc. 
much interest, many of which can he examined remarkably 
well in cold-blooded animals. 
Before describing the method of making preparations which 
I employ, a few features of the process of Stieda will be no- 
ticed. This observer places the entire brains of small animals, 
first in a solution of 80-90 per cent, alcohol, which has been 
tinted yellow by iodine. As soon as the piece feels firm to the 
touch (one to four days according to the size of the specimen) 
it is placed in a dark yellow solution of bichromate of potash, 
care being taken to use a large excess of the solution. After 
a time varying from three weeks to three months, the hard- 
ened part is placed in strong ammoniacal carmine, and is 
removed after from one to five days, placed in alcohol, and 
after all excess of carmine has been removed, is ready for 
section. 
By this process Stieda has been able to prepare sections in 
a long unbroken series, of the brains of mice, frogs, etc., sav- 
ing thereby the risks of several transfers. 
My own method is the old one, except in regard to the 
solutions for hardening. A two per cent, solution of bichro- 
mate of potash has given me excellent preparations, also the 
solution of Clarke, which consists of a solution (1-800) of 
chromic acid, to each ounce of which a grain of bichromate of 
potash is added. So far I prefer to stain after cutting. At 
least two transfers may be avoided by using a siphon tube, to 
remove alcohol or water, and with the flattened spoons of Se- 
guin, there is but little danger of injuring the sections. After 
the piece has remained from three to five weeks in the two per 
cent, bichromate solution, which it is well to renew every two 
weeks at least, it is placed in Ivan off’s modification of Muller’s 
fluid, which consists of one or more parts of sulphate of soda, 
until ready for section, when it is transferred to alcohol for a 
few minutes. 
The membranes of the cord ought not to be removed 
until just before making the sections, or until the part is 
thoroughly hardened, otherwise deformity will surely result, 
showing itself in an oval instead of circular central canal. 
When an unbroken series is not desired, it is better to make 
sections with the membranes on. After cutting, the sec- Mason—Central Nervous System of Reptiles, etc. 
3 
tions are stained in Beale’s glycerine and water solution 
of ammoniacal carmine, for which I often substitute the 
borax carmine of J. W. S. Arnold, after adding to it some 
ammonia, which rather improves the color. Transparency is 
effected by oil of cloves, after absolute alcohol has expelled 
the water, and the mounting is done in Canada balsam dis- 
solved in chloroform. A short piece of copper wire hammered 
flat at one end and bent at right angles is a good substitute for 
the steel spoon, as it can be quickly made of any size, and its 
outline changed by scissors when desired. 
The sections of the alligator’s spinal cord, which were 
shown with photo-micrographs at the last annual meeting of 
the American Neurological Association, measured about 9-10 
mm. through the brachial and crural enlargements, and were 
made from specimens hardened by the same process as that 
recommended by Seguin* for the human cord. 
To prepare isolated nerve-cells, there is perhaps no better 
method than that employed by Karabanowitsch, viz.; macera- 
tion for forty eight hours in a weak solution of bichromate of 
potash (2-100) mixed, equal parts, with a (1-100) solution of 
caustic soda and ammoniacal carmine. 
It is possible, however, to make beautiful preparations of the 
nerve-cells of the frog, by simple agitation, with some teasing, 
in a drop from a solution of glycerine, water and carmine. 
Agitation in osmic acid is another common means of isolation, 
but the preceding methods are generally to be preferred. 
The large cellular structures which form such a prominent 
group in the inferior horns of grey matter are composed of 
large, sharply defined nuclei, surrounded by protoplasmic 
masses which, under the action of certain re agents, look as if 
they were composed of fibrillae, which unite in bundles to form 
what are called the cell processes. The nuclei seem to be 
but slightly affected by these re-agents, but by prolonged 
action, seem to be compressed by the surrounding mass. In 
the cells of medium size the nuclei are rarely if ever changed 
from the spherical form or circular appearance. These nuclei 
which contain a distinct nucleolus, are not too numerous to be 
counted in the frog. For example, in an unbroken series of 
Striker'ii Handbook, p. 64(5, 4 
Mason—Central Nervous System of Reptiles, etc. 
twenty-four sections, from the middle of the brachial enlarge- 
ment of Rana halecina, I counted in both inferior horns 540 
large and medium-sized nuclei, and in an unbroken series of 
twenty-four sections from the middle of the crural enlarge- 
ment I counted in botli horns 390 nuclei. Reissncr * esti- 
mated the length of the brachial enlargement as standing to 
CD CD o 
that of the crural enlargement in the ratio of 6-10, and this 
proportion is true of the American species, as would be sup- 
posed. It seems fair to conclude therefore, that while the 
crural enlargement in frogs has a smaller transverse diameter 
than that of the brachial enlargement, still, by its greater 
length it contains as many if not more ganglion cells than the 
latter. It is then, perhaps, the larger of the two swellings 
and corresponds, as it ought, with the larger size of the poste- 
rior extremities. 
The preponderance of the lumbar enlargement in birds, and 
the equality of the two which I have observed in the alligatorf 
and in several species of lizards can undoubtedly be explained, 
by regarding the amount of grey matter, or possibly the num- 
ber of nerve-cells, as the surer indication of importance of 
function in different regions of the cord. 
The large crural nerve cells, as well as their nuclei, are 
larger than those of the brachial region. 
This fact I have established by numerous measurements, 
which may be condensed into the following average diameters 
for liana halecina: Brachial nuclei, long diameter, 7; crural 
nuclei, long diameter, 8; brachial nuclei, short diameter, 
6.5; crural nuclei, short diameter, 7.5. The numbers denote 
divisions of the micrometer eye-piece, each division with the 
objective used representing .002 mm. 
After a thorough comparison of sections from the brachial 
region of twenty large specimens of liana pipiens, of which 
seven were males, I have been unable to detect any difference 
either in the arrangement, size or structure of the elements, 
that could reasonably be referred to as explaining the remark- 
* Loc. cit.—Length of intumescenlia anterior, 6 mm.; that of intumes- 
centia posterior lu mm. 
•(■See i; Transactions of American Neurological Association,” in preceding 
number of the Journal, Mason—Central Nervous System of Reptiles, etc. 
5 
able and purely reflex energy displayed by the male frog dur- 
ing the embrace of copulation.* The group of cells with 
nuclei of medium size, described by me in the New York 
Medical Journal, of December last, is present in the “pars 
media” of cords from both sexes in three species which I 
have examined with especial reference to this point. 
The distribution of the inferior root fibres in the inferior 
horns of grey matter, among and to the ganglion cells. 
Wyman,f whose memoir appeared at the time Ecker was 
preparing his leones Physiological for publication, states, page 
20 : “After the most careful examination, I have not detected 
any direct connection- between these caudate appendages and 
nerve tubes;” while the latter anatomist prints an illustration 
after Kiipfer £ which represents, in a cross section made 
through the middle of the brachial enlargement, four large 
nerve cells, with processes running downwards and outwards, 
as far as the periphery, and two other large cells, from the 
same group, with processes continuous with the superior 
(posterior) roots! 
Such an exaggerated illustration could never have been 
made by photography. 
Eeissner, § ten years later, in a work than which there are 
few more perfect memoirs, writes: “In regard to the inferior 
roots, I must premise, that the spinal cord of anourous batra- 
chians, which have been at my disposal, is very little suited 
for observing the entrance and course of the fibres in the grey 
substance. * * * * As before stated, a nerve cell may 
lie near the point of entrance of a bundle, and send one of 
its processes into the same. (Fig. M.) * * * * The 
remainder of the nerve root fibres which run upwards or out- 
wards in the grey matter, unite either wholly or in part with 
large nerve cells.” 
Stieda, | after contending for the true cell structure of the 
protoplasmic mass which contains the nucleus, and the struc- 
* Goltz.—Beitrdge zur Lehre von den Functionen dev Nervencentren deg 
Froaches. Berlin, 1809. 
f Loc. cit. 
\ Dias, de Medullas Spinalis Textura in Uanis. Dorpat, 1854, 
§ Loc. cit. pp. 19, 20. 
| Loc. cit. p. 150, et seq. Mason—Central Nervous System of Reptiles, etc. 
6 
tureless state of the axis cylinder, against the views of those 
who believe in their fibrillary composition, enquires: “How 
is it now with the connection between nerve-cells and nerve- 
fibres? * * * * From my observations on fresh as well 
as on hardened ganglia of the spinal nerves in fishes, amphi- 
bia, warm-blooded animals and man, the union is of this 
nature: the axis cylinder of the nerve fibre is the direct con- 
tinuation of the cell substance;” and two pages further on, 
“ It seems to me impossible to see such a connection in sec- 
tions of the brain and spinal cord; it can only be done with 
the help of isolating methods, as we are taught by the latest 
observations of Koschewnikoff.”* It will be observed that 
although Stieda has not seen such a connection in a section, 
he nevertheless considers it as a fact, while Eeissner, in 1864, 
claimed to have seen it; and it was figured by bicker and 
Kiipfer, Dean f has also a plate showing, in the rabbit, a 
nerve process joining a nerve-fibre of an anterior root, and 
passing out as far as the periphery. The frequency with 
which I have obtained sections from the brachial enlargement 
of the frog, which show in the clearest manner what becomes 
of by far the greater part of the inferior root fibres of this 
region, leads me to give here my manner of procedure. 
In the first place, I must assert, contrary to the opinion of 
Heissner given above, that I know of no other animal so well 
suited, and for so many reasons, as is the frog, for the study 
of this point. It is true that the root-fibres in each section are 
comparatively few; but this seems to me an advantage rather 
than an impediment, and if more nerve-fibres are desired, it 
is quite easy to obtain them by making more sections. The 
supply of is ample; and their spinal cords almost come 
out of the spinal canal of themselves, so easy is their removal, 
when the operation is done upon the abdominal side. As the 
chief cause of failure lies in the division by the razor of the 
nerve-tuhe somewhere between the cell and the periphery of 
the cord, it follows that, c&teris paribus, the smaller the 
diameter of the cord, the greater the chance of success. In 
* Archivfur Microsk. Anat. Bel. Y. 18G9. ' 
f J. Dean.—Microscopic Anatomy of the Lumbar Enlargement of the Spinal 
Cord. 1861. Mason—Central Nervous System of Reptiles, etc. 
7 
the brachial enlargement, many fibres from the inferior roots 
enter the cord at right angles, and remain in the same vertical 
plane while describing a curve laterally, the convexity of which 
lies towards the inferior median fissure. This is why longitu- 
dinal sections are useless for showing this connection ; while 
a series of transverse sections, made in the section cutter, in- 
cluding the roots of the second pair of nerves, will often 
show, in well stained specimens, the greater portion, if not 
all, the root fibres, passing through the white substance, and, 
after entering the grey substance, branching outwards among 
the cells. Most of the fibres lose themselves among the larger 
external group of cells, while a few are seen to unite with the 
upper group. In several instances I have traced a connection 
between cell process and nerve-fibre, with so much certainty, 
confirming the observation by using a binocular instrument, 
that I am forced to believe not only in the possibility, but the 
facility of demonstrating in this way an important fact. 
It is always advisable to submit the transparent section to 
microscopic examination before it is covered, as the weight of 
the cover is often sufficient to sever fibres. Good objectives, 
of considerable power, can be used in studying uncovered 
preparations; and 1 have succeeded in obtaining satisfactory 
photographs of some of these. 
1. The central canal of the spinal cord of frogs is more 
nearly cylindrical in shape than has been generally supposed. 
The oval contour is not seen in cross sections below the 
second pair of nerves, when the membranes are not removed 
before hardening. 
2. The nuclei of the large nerve-cells are more generally 
oval in form than are those of the smaller cells, I have con- 
firmed this in a few fresh preparations only. It is possible 
that the re-agonts employed have a different effect upon the 
two classes of nuclei, but it seems more reasonable to con- 
clude that they have a different form anatomically. 
3. The nerve-cells of the crural enlargement are as abundant 
as those of the brachial enlargement, if not more so. Their 
nuclei are larger, as are also the surrounding masses of pro- 
toplasm or cell bodies. 
CONCLUSIONS. 8 
Mason—Central Nervous System of Reptiles, etc. 
4. No difference in structure can be made out in the upper 
portion of the cord, corresponding with the sexual function 
in the male. The long-continued and violent tonic spasm of 
the anterior extremities, must be explained by local hyper- 
semia influencing the same structures as those which exist in 
the female. 
5. The relation which is generally believed to exist be- 
tween the so-called motor-cells and the inferior (anterior) 
roots, can be demonstrated in the frog more readily than in 
any other animal.