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ON THE NUMBER AND RELATIONS OF THE GANG

LION CELLS AND MEDULLATED NERVE
FIBERS IN THE SPINAL NERVES OF FROGS
OF DIFFERENT AGES.

By IRVING HARDESTY. (From the Hearst Anatomical Laboratory of the University of California).

CONTENTS.

I. Introduction. II. Material and Technique. III. General Results of the Enumerations. IV. The Number of Spinal Ganglion Cells and their Relation to the Num

ber of Fibers Connected with the Ganglion. V. Further Observations on the Conditions Determining the Excess of Fi.

bers on the Distal Side of the Spinal Ganglion.
VI. The Gain of Cells and Fibers with the Gain in Weight.
VII. Summary.
VIII. Bibliography.

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That the number of medullated nerve fibers in the spinal nerves increases during the growth of the animal toward maturity is generally accepted. Aside from the evident requirements of growth, the fact has been established on several occasions, at least for the frog and mammal, by direct enumerations of the fibers.

In two papers of some years ago the author ('99 and 'oo) undertook to determine the rate at which the increase of fibers takes place in the frog. It was then found (1) that the increase occurs more rapidly in younger specimens; (2) that in specimens of varying weights, counts of the fibers in transverse sections taken at intervals along the ventral root toward the spinal cord

and along both the dorsal root and the nerve trunk towards the spinal ganglion, showed a gradual increase of fibers at the average rate of nearly 2% per millimeter of length of the roots and of the trunk; (3) in considering the VIth nerves alone of a series of 17 specimens ranging gradually in weight from 5 grams to 79 grams, it was found that in this one pair of spinal nerves there was for each gram of weight gained an average gain of 7.4 fibers in the two nerve roots and 10.4 in the trunk and dorsal branches. As to the numerical relations of the fibers present on the proximal and distal sides of the spinal ganglia, there was always found (4) an appreciable excess of fibers in the sum of the trunk and dorsal branches as compared with the sum of the dorsal and ventral roots. This "distal excess" was found to vary in the different spinal nerves, ranging from 7% to as much as 61% of the sum of the two roots. It was found to be greater in those spinal nerves which have a relatively greater number of fibers in the dorsal branches, but showed neither a regular increase nor decrease with the increase in the size of the specimens.

BIRGE ('82) had previously counted the ventral root fibers of all the spinal nerves of one side of six frogs varying in weight from 1.5 to ili grams and, assuming the fibers of the two sides approximately equal in number, his figures show an average total gain of 51 ventral root fibers for each gram gained in body weight. Hatai ('02 and '03) has since determined the number of medullated fibers in the dorsal and ventral roots of three given spinal nerves of each of four white rats ranging in body weight from 10 to 264 grams. He finds that the number of fibers in the roots of these three nerves is 2.7 times greater at maturity than in the ro gram rat, and that the increase is more rapid in the younger animals. BIRGE, though he did not take into consideration the dorsal branches, obtained noticeable distal excesses of fibers in the several nerves, and he was the first to attach significance to it. Gaule and LEWIN ('96) investigating the sacral nerves of the rabbit, found an excess of fibers on the distal side of the spinal ganglion amounting to as much as 19%, and BÜHLER

('98) mentions a distal excess of 25% in the IXth spinal nerve of the frog

An investigation of the numerical relations of the axones on the two sides of the spinal ganglion naturally suggests an inquiry as to the number of ganglion cells in it and the relation they bear to the numerical arrangements of the fibers. This paper is offered with the hope of contributing something (1) to the knowledge of the relations between the number of the nerve fibers in the dorsal roots and the number of ganglion cells in the spinal ganglia whence the fibers are supposed to arise ; (2) toward throwing further light upon the conditions determining the excess of fibers on the distal side of the spinal ganglion ; and (3) concerning the influence of growth upon the relative number of ganglion cells and the numerical relations of the fibers in the region of the ganglion.

The investigation was begun some time ago when the author was connected with the neurological laboratory of the University of Chicago. It has been allowed to rest on account of the difficulty in obtaining the material required. Rana virescens having been employed in the beginning of this and and the previous papers with which the results of this must be compared, it was thought advisable to continue with this species though it is apparently very rare in California. It finally became necessary that the material from the specimens of the sizes required to complete the work be sent from the laboratory in Chicago, and I desire to thank Professor DONALDSON and Dr. Dunn for their kindness and care in obtaining it for me.

Finally, let it be said that the author realizes that the resuits of an investigation of this kind are valueless unless obtained by most trustworthy procedure.

II. Material and technique. Only three of the spinal nerves of each frog are dealt with. These are the Vth, Vith, and IXth of each of seven frogs varying in weight from 10.4 to 63.4 grams. The Vth was chosen as a typical thoracic nerve and because, being near the lumbar region, its dorsal and ventral roots are long enough to render manipulation less difficult. The VIth nerve in many respects is similar to the thoracic nerves anterior to it,

but at the same time it is often larger than the Vth and in some respects resembles the VIIth which belongs almost entirely to the lumbar distribution. Morever, the VIth was employed in a previous investigation ('oo) and is included in this in order to accumulate data concerning it. The IXth nerve was chosen because it is one of the largest spinal nerves in the frog, and differs from the Vth and Vith in that all the fibers of its trunk are contributed to the lumbar plexus. These three nerves alone are considered, it being assumed that whatever conditions found true for these are approximately true for the remaining spinal nerves. To enumerate the ganglion cells and the nerve fibers in the dorsal and ventral roots and the trunks of the entire ten spinal nerves would have been so arduous a task that the additional information probably to be obtained by such was considered a reward scarcely sufficient for the ordeal.

To obtain a series ranging as gradually and as widely as possible, each specimen was chosen with reference to its body weight. The smallest specimen of Rana virescens obtained for this observation had a body weight of 10.4 grams. Smaller specimens may be obtained at -certain seasons of the year just after the transition from the tadpole stage.

The largest specimen of the series weighed 63-4 grams and was, of course, female, since the males seldom exceed 50 grams while the females have been known to attain a body weight of 80 grams with the ovaries removed.

In each case the animal was chloroformed in the usual way and, if female, the ovaries were removed. It was then weighed and its body weight and sex recorded. The procedure in dissecting, fixing, imbedding, sectioning, etc., was much the same as that followed in the previous investigation and it is there given in detail. The spinal cord was laid bare from the ventral side, the spinal ganglia and dorsal branches carefully exposed and dissected free with the aid of the dissecting microscope. Avoiding pulling and stretching, the dorsal and ventral roots were clipped with fine scissors close up to the spinal cord and the nerve trunk near its junction with the ramus communicans. Then the ganglion with the roots and portion of the nerve trunk attached was laid on a small piece of card-board. There the roots were arranged straight and parallel with each other, and the dorsal branches straightened out parallel with the nerve trunk in order to obtain transverse sections of the two with the same stroke of the knife. In doing this, careful use of the dissecting lens was always advisable lest some of the smaller of the dorsal branches should be overlooked and should remain tangled or doubled back upon themselves, and thus in the sec

tions occasion a double counting of the fibers contained in them. In each case the strip of card-board with the specimen arranged upon it was placed into a vial containing 1% osmic acid and bearing a label stating the date, the weight and sex of the animal, the number of the spinal nerve, and whether taken from the right or left side. After being subjected to the action of the fixing agent for 15 or 20 minutes, the parts become stiffened sufficiently to maintain their arranged relations and may be gently lifted from the card-board that the fluid may penetrate more freely from all sides. The larger nerves were always removed into the fluid. After 12 to 24 hours in osmic acid, the specimen was washed about 12 hours in water frequently changed, and then transferred to 70% alcohol. While washing, an outline sketch of the specimen was made for the purpose of orientation.

Each specimen was embedded in paraffin separately in a small paper box on the side of which was copied the label on the vial in which it was fixed, washed, dehydrated, etc. Transverse sections of the two nerve roots of the trunk and dorsal branches were made of about 4 u in thickness, and those from the required localities were mounted serially. The sections of the nerve roots were taken from about midway their length, while those from the trunk and dorsal branches were taken close up to the peripheral side of the spinal ganglion, yet far enough away to avoid any portion of the ganglion itself being involved. Separate slides were devoted to each of the localities required, and the locality mentioned in their labelling. On approaching the ganglion, from whichever side the sectioning was begun, the thickness of the sections was changed. All sections involving the ganglion were made 9 u thick and special attention was given to mounting them serially.

For counting the nerve fibers the photographic method, devised in the neurological laboratory of the University of Chicago ('99) was employed for every section except an occasional very small twig of the dorsal branches. The mounted sections from a given locality were first carefully looked over with the compound microscope, and one suitable for photographing was picked out, that is, a perfect section and one which high power showed to be flat and evenly adhering to the surface of the slide. Under low power this section was marked by a ring of ink on the cover glass. The photographs were made with the aid of the Zeiss projection apparatus. The magnification employed was from 300 to 500 diameters, varying according to the size of the section None of the sections in this investigation when sufficiently magnified were too large to get on a single plate of the

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