Talk:Book - Contributions to Embryology Carnegie Institution No.41

By Burton D. Myers,

Professor of Anatomy in the Indiana University.

With six figures.

Through the kindness of Professor von Monakow, it has been my privilege to study fifteen sets of serial sections of brains of different developmental stages, selected from very extensive collections in the Institute of Brain Anatomy at Zurich. This study has special reference to certain features of cerebellum. The following report is confined to a brief description of observations on the growth of the Purkinje cells, the growth of the molecular laj'er of the cerebellar cortex, and the ratio of medullary to cortical zones in cerebellum. For the sake of conciseness, each subject will be considered separately throughout all stages of development.

Purkinje Cells

In the many investigations on the cerebellum that have been published during the past twenty years, in only a few instances has attention been directed to the Purkinje cells. An examination of the literature covering this Umited field reveals the fact that investigators have interested themselves for the most part with the relations, internal structure, and histogenesis of these cells. Popoff (1895) came to the conclusion that they arise exclusively from the deepest cells of the outer nuclear layer. Omer (1899), in a study of the Purkinje cells in the sheep and guinea pig, found that they are derived from non-granular cells of ill-defined contour in the outer nuclear layer. Cajal (1907) directed attention to displaced Purkinje cells, annular terminations around the cell-bodies, and neurofibrils in the proto- plasmic aborizations of the cells. No attention has been given by these authors to the determination of the portion of the cerebellum in which the Purkinje cells first make their appearance, or to the possible bearing this may have upon the problem of what, in the cerebellum, is phylogenetically old and what is phylogenet- ically new. Furthermore, no determination has ever been made as to the number of Purkinje cells that are to be found at the different stages of development, nor have the questions which this point might help to solve received consideration. It was to this untouched field, therefore, that the present investigation was directed.

The Purkinje cells are first definitely demonstrable at the sLxth month of intra- uterine life. The cortex of the cerebellar hemisphere of a fetus of this age is shown in figure 1, a drawing with a projection apparatus in which the greatest care has been taken to show every cell of the field in position. A few Purkinje cells are seen along the rather sharp line of demarcation between the nuclear and molecular layers.

The cells have reached their greatest development in the flocculus. Those of the vermis arc more uniformly developed than in any other portion of the cerebellum, though the stage of their develoi)mcnt is not so advanced as it is in the depth of the floccular fissures. It should be mentioned that in a fetus of 6 months the Purkinje cells in the depth of a fissure show a devclojiment markedly beyond that of cells more superficially placed. This difference is illustrated in figure 2, a drawing of the contour of the flocculus; a and h indicate the positions in which the corresponding groups of cells are found. The cells in the depth of the fissure (a) show a denser protoplasm, a more definite contour, and better developed protoplasmic processes and nucleus than the more superficially placed cells (b). The protoplasm of cells b, having no definite boundaries, merges into the surrounding protoplasmic mass. It is less dense, hence the cells appear larger than cells a. This same difference is shown in figure 3 for the hemisphere. In the contour drawing a and h indicate the position.s of the cell-groups a and b. A comparison of the two figures shows how far the Purkinje cells of the flocculus are in advance of those of the hemisphere.

Fig. 1. — Drawing of the cerebellar cortex in which each cell is represented in the field drawn. The most prominent feature of figure 1 is the transitory outer nuclear layer, which occupies a most superficial position in the molecular layer. It disappears at different periods in different animals, corresponding to the age at which myelinization in the cerebellum becomes pronounced and locomotion acquired. Tliis outer nuclear layer is fjrobably absorbed by the inner nuclear layer.

In the seventh, as in the sixth month of |)ivnatal life, tlie Purkinje cells of the vermis show a development in advance of those of the hemisphere. Likewise in the new-born the cells of the flocculus arc; by far the largest, while the cells of the vermis are larger than those of the hemisphere. In both vermis and flocculus the protoplasmic processes are well developed. The Purkinje cells are more numerous in the former than in the latter. An average field in the vermis shows 36 cells, in the flocculus 22, and in the hemisphere 45.

Fig. 2. — A contour drawing of the flocculus. In this drawing a and b indicate the positions in which the cell groups a and b are found. The cells in the depth of the fissure o show a denser cell protoplasm, more definitecontour, and better developed protoplasmic processes and nucleus, than the cells of b, more superficially placed.

In the infant 16 days old the Purkinje cells in these three structures have maintained their relative number and size, being largest and least numerous in the flocculus, smallest and most numerous in the hemispheres, and in the vermis occupying an intermediate position as to size and number between the two extremes. An average field in the flocculus shows 19.6 cells (Zeiss Oc. 4, Obj. A. A.), in the vermis 27, and in the hemispheres 34. Upon the theory that the actual number of Purkinje cells in the cortex of a child of 16 days is the same as in a fetus of 7 months, we may regard the decrease in the number per field as inversely proportional to the increase in the growth of the cortex.

In table A each number is the average of 20 different fields (Zeiss Oc. 4, Obj. A. A.). A study of this table shows that although at 3 months the number of cells per field is still greatest in the hemispheres, and greater in the vermis than in the flocculus, the ratio is approaching 1:1.

Table A.

Fig. 3. — A contour drawing of the cerebellar hemisphere; a and 6 indicate the position of the cell leroups a and b. The same difference noted between cell group a, deeply placed, and cell group ft, superficially placed in the flocculus (fig. 2), arc noted in this hemisphere also.

Age.

Number of cells per field.

Flocculus.

Vermis.

Hemisphere.

22

19.6

18

15

14.6

36

27 20 17.6 16.9

45 34 21 16 15.6

16 davs

If we may regard the number of cells per field as an index of the relative growth of these different parts of the cerebellum, we are led to the conclusion that the cortex of the cerebellar hemisphere increases 100 per cent during the first 3 months of postnatal fife, while the vermis undergoes an increase of 80 per cent, and the flocculus an increase of 20 per cent. These percentages are indicative of the relatively greater maturity of the flocculus at birth. At the age of 2 years the number of cells per field is only a Uttle below that of the brain of 3 months, representing a uniform growth of about 30 per cent in the hemispheres, 20 per cent in the flocculus, and 20 per cent in the vermis. It will be observed that the number of cells in the three structures (flocculus 15, vermis 17.6, and hemisphere 16) corresponds almost exactly to the respective number found in the adult (14.6, 16.9, and 15.6); from which fact we may conclude that the cerebellum of a child of 2 years has nearly reached its full development.

Before taking up a discussion of table B let us note briefly the bearing of the results above enumerated. The development of the Purkinje cells in the flocculus, beginning early and progressing more rapidly than in the vermis, is very unexpected from the old point of view; ?". e., that the vermis is the phylogenetically old and the honiispheros the phylogcMietically new portion of tlie cerebellum. It affords valual)le evidence, however, in favor of the view recently expressed by Edinger, that the vermis and flocculus are both i)hylofj;enetically old. Inasmuch as both sides of the fissura uvulo-nodularis show like develojinK'nt of the Purkinje cells, and as the portion of the cerebellum across this fissure from the flocculus is the representative of the paraflocculus, it suggests the possibility of the paraflocculus, as well as the flocculus, belonging to the paleo-cerebellum.

In the series studied it was possible to first determine the number of Purkinje cells per field in the new-born; from this time on the number remains constant, the apparent decrease being proportionate to the actual increase in surface.

Table B. — Microcephalism.

Age.

Number of cells per field, Zeiss Oc. 4 Obj. A. A.

Floceiilus.

Vermis.

Hemisphere.

6.2 13.8 14.4

9.6

11.4 14.. 5

10.5 11. S 15

Table B deals with microcephalics of various ages, in which the number of Purkinje cells per field was determined as for table A. Upon comjiaring these specimens with those of corresponding ages given in table A, it will be observed that in the cerebellum of the microcephaUc child of 22 months the numl)er of Purkinje cells is about 50 per cent of the normal, as represented by the 2-year-old child given in the preceding table. In the 2-year-old microcejihalic the number of cells in the vermis and hemisphere is about 70 per cent of the normal, while the number in the flocculus is about 90 per cent. In the adult microcephalic the number of cells per field is practically normal.

In each of these cases, however, we are deaUng with a cerebellum actually smaller than normal, with a total cortical area much less than normal; so that in every case the actual number of Purkinje cells must be below that of the normal cerebellum. In the first two specimens the reduction is due not merely to the actual decrease in cortex, for even where the cortex is present there is a relative reduction of 30 to 50 per cent per field. In the adult microcei)halic this decrease in number is directly projiortional to the decrease in cerebellar cortex, inasmuch as the number of cells per field is jiractically normal. This suggests the possibility of a delayed development of Purkinje cells. In microcephalism the cerebrum, as well as the cerebellum, is too small. It is po.ssible that failure of development of the latter is secondary and due to an inadequately stimulating influence from the cerebral cortex.

Growth of the Molecular Layer of the Cerebellar Cortex

Upon examination of the literature I find that lierliner (1905) is the only investigator who has attempted to determine by measurements the rate of development of the cerebellum at prenatal and postnatal stages. In projection drawings of mesial sections of a series of cerebella this writer shows that the superficial folding proceeds most intensively during the second half of the prenatal period and the first 3 months of postnatal life. In order to secure more accurate results and a numerical expression of the relation, he measured the periphery of a mesial section in a series of cerebella of different ages, by means of a cyclometer on contour drawings of about 7 magnifications. These measurements show that the period of most rapid growth is from the fifth month of intrauterine to the fourth month of postnatal life.

---

Table C.

Age.

AveraRc thickness of molecular layer expressed in microns.

Flocculus.

Vermis.

Hemisphere.

Fetus, sixth month. Fetus, seventh month

106 134 157 167 2.58 295 300

88 97 167 169 262 314 317

86 92 110 123 210 305 311

Child, 16 days

Child, 3 months

Child, 2 years

Adult

The average thickness, recorded in table C, represents an average of 20 different measurements in each of the specimens enumerated. Measurements were made with the ocular micrometer and reduced to microns. Care was taken to select places for measurement where the cortex was vertically cut. It will be noted, from an examination of this table, that although before birth the molecular layer of the flocculus shows a greater thickness than that of the hemispheres or the vermis, from birth on the greatest thickness of this layer is found uniformly in the vermis. In certain instances, as the 16-day-old child, the thickness of the molecular layer of the vermis and of the flocculus is so nearly the same that the difference is negligible.

Table D. — Percentage of growth of molecular layer.

Flocculus.

Vermis.

Hemisphere.

From sixth month to birth ....

From birth to 3 months

From .3 months to 2 years

p.ct. 50 64 14

88

p.ct. 90 57 20

88 1

p.ct. 30 91 46

177

As with the Purkinje cells, so with the development of the molecular layer, the period of greatest general growth is between birth and the third month of postnatal Ufe. Table D, based upon the preceding, gives the growth in percentages for different periods. It is apparent that the period of most rapid growth in thickness of the molecular layer of the vermis is from the sixth month of intrauterine life to birth; while for the flocculus and hemispheres it is from birth to the third month, though for this period the percentage increase in the thickness of the molecular layer of the vermis is nearly as great as that of the flocculus. It is evident, therefore, that the period of most rapid growth of the layer as a whole is from the sixth month of intrauterine life to the third month of postnatal hfe. This result corresponds vcrj'^ closely to that of Berliner, as given above. We have, therefore, in the tliickness of the molecular layer an index of the stage of development of the cerebellum, an intlex which will be ajiplied in the consideration of the microcei)halics.

The greater thickness of the molecular layer of the flocculus from the sixth month of antenatal to the tliird month of i)ostnatal hfe is additional evidence in favor of Edinger's view that the flocculus, as well as the vermis, is phylogenetically old. It is interesting to note that between birth and two years of age the percentage increase in thickness of this layer of the flocculus and vermis is the same, and just 50 per cent of that in the hemispheres for the same period (see table D).

As in the development of the Purkinje cells, the adult condition of the molecular layer of the cerebellar cortex is practically reached in the second year, the growth thereafter being only 1 to 2 per cent.

Table E. — Average thickness of molectdar layer.

Age.

Flocculus.

Vermis.

Hemispheres.

MicrocoiilKilic child with spina bifida . Mirniccph-ilio child

1 year.

161

205

277

268 / L.219 \ 1 R. 259 /

208 310 273 371

342

161

294

237

334 / L. 105 to 292 \ R. 408

Mimxi-pluilic child

Microcephalic adult

Adult with hemiatrophic cerebellum .

2 years. 49 years.

20 years

The brains reported in table E were all without pathological change except for their small size. A comparison of this table with table C shows that here, in some instances, we have a very great deviation from the normal. In some of the microcephalic specimens, as in the 1-year-old child with spina bifida, tliis deviation may be interpreted as an arrest of development, inasmuch as in tliis brain four layers of cells in the outer nuclear layer in the vermis and five to six in the hemisphere still I^ersist. This is a condition which, according to Biach, is normal in a child of 6 weeks. The thickness of the molecular layer of vermis and hemisphere, it will be noted, is about midway between the normal for a child of 10 days and that of a child of 3 months. We have, therefore, a persistence of two conditions — the number of cellular layers in the outer nuclear layer and the thickness of the molecular layer. Each may be considered as an index of development and both sjjcak for an arrest of development in this brain at about the sixth week of postnatal life. This same arrest of development was observed in another case of microcephalism not included in the table.

In the other instances of microcephalism there is no indication of the persistence (jf a condition normal at an earlier period of dcvelo])ment. The outer nuclear layer is entirely absent. The tliickness of the molecular layer in some parts, as in the flocculus of the 22-months-old child, is very much below the normal; in other parts, as in the vermis and hemispheres of the adult, it is very much above the normal. In these cases the deviation from normal must be attributed, not to arrest in development, but to an atypical development and an under development as a whole.

In the case of hemiatrophy, the only point of interest Ls that we have in the vermis and right hemisphere a very marked hypertrophy secondary to the atrophy in the left hemisphere and also to an extent in the flocculus of both sides.

Outer Nuclear Layer

The most prominent feature Of figure 1 is the transitory outer nuclear layer, which at this stage of development (sixth month of fetal life) occupies a most superficial position in the molecular layer. This outer molecular layer has recently been studied in Obersteiner's laboratory by Biach, and also by Lowy. Biach studied the time of disappearance of the layer in the human brain, and found a gradual decrease in the number of layers of cells until the whole disappeared, about the eleventh month. Lowy's study is comparative. He directs attention to the disappearance of the outer nuclear layer in different animals at very different periods, corresponding to the ages at which myelinization in the cerebellum becomes pronounced and locomotion is acquired. He gives a very satisfactory review of the various opinions which have been advanced as to what becomes of this outer nuclear layer, whether it goes to help form the inner nuclear layer or the Purkinje cells, constitutes a dejiot for rciiifoifoment of other layers, or disappears in part. The most general view is that of Cajal, that the disappearance of the outer nuclear layer represents merely a change of position.

Fig. 4. — Cross-section of the cerehellum of an embryo of 6 months, from the cortex of which figure 1 is drawn.

Fig. 5. — Two drawings have been superimposed for convoniehoe of comparison. The outline drawing is from a fetus of 7 months, while the heavy, continuous line drawing is of a 16-day-old child. Figures 5 and 6 show the tremendous increase in cerebellar cortex at the time the outer nuclear layer is disappearing.

In the human brain these cells are disappearing at a time when, as is easily seen in figures 5 and 6, the increase in cerebellar surface is very great. The number of cells in the outer nuclear layer, seen in figure 1, is very striking; but when we compare figure 4 (a cross-section of the cerebellum of a 6-months fetus from the cortex of which figure 1 is taken) with figure 5, it is evident that we have at 16 days a surface at least 10 times that of the 6-months fetus. Tliis means that this outer nuclear layer would furnish to the nuclear layer proper, as present in the child of 16 days, one layer of cells with as much space between adjacent cells as is found between alternate cells in the outer nuclear layer of the 6-months fetus. Physically, the absorption of the outer by the inner nuclear layer is very easy, and it would seem unnecessary to postulate the total disappearance of any of these cells.

Fig. 6. — Contour drawing of one-half of the cereljellum of a child 2 years of age.

Thickness of Medulla and Cortex

If, in our study of the growth of the cortex, we make a comparison of the thickness of the medullary and cortical portions of the cerebellum, we ascertain that a relation of about 1 : 1 is maintained, as shown in table F.

Table F. — Thickness of cortical ami medullary portions of the cerebellum.

In each of the series in which measurements are recorded sections were selected at a level just below the place in which the corpus restiforme passes over into the cerebellum. Measurements were made in each section along the line ab, figure 5, passing from the base of the floccular peduncle to meet the surface of the cerebellar cortex at right angles.

In the sixth and seventh months, though medullation is very slight, the medullary and cortical zones are very clearly defined in well-stained sections. The ratio of cortical to medullary field in these series is 3.5 : 4.5. By birth the ratio of 1:1 has been estabUshed between cortical and medullary zones, and this is maintained up to and in the adult. It will be observed that the first two years represent a growth of 100 per cent in thickness of cortical and medullary zones, and that one-third of this growth takes place in the first 3 months.

Though the molecular layer increases in thickness only 1 or 2 per cent after the second year, there is an increase of 15 per cent in the thickness of the cortical zone from the second year to the adult stage.

Age.

Cortex.

Medulla.

Fetus, sixth month . . seventh month

mm.

3.5

3.5

6

6

7

8 12 14

mm.

4.5

4.5

8

6

7

8 12 14

Child, 10 days

.3 weeks

3 months

2 years

Adult

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