Paper - The development of the olfactory and the accessory olfactory formations in human embryos and fetuses: Difference between revisions

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Humphrey T. [[Paper - The development of the olfactory and the accessory olfactory formations in human embryos and fetuses|The development of the olfactory and the accessory olfactory formations in human embryos and fetuses]] J. Comp. Neurol.
{{Ref-Humphrey1940}}
The Development Of The Olfactory And The Accessory Olfactory Formations In Human Embryos And Fetuses
Tryphena Humphrey
Department of Anatomy, University of Pittsburgh, Pennsylvania
NINE fiGURES
CONTENTS
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 431
Material and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 432
Literature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .- . . . . . . . . 433
Description of material . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 434
The primitive olfactory bulb before the lamination pattern begins to
develop . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 435
The olfactory bulb before regressive changes occur in the accessory
olfactory formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 437
The olfactory bulb after regressive changes appear in the accessory
olfactory formation . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 445
Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 458
Discussion and conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 460
INTRODUCTION
In a recent study of the adult human olfactory bulb the
accessory olfactory formation was found to be absent (Humphrey and Crosby, ’38; Crosby and Humphrey, ’39 a, ’39 b),
but it was stated that a primordial accessory olfactory bulb
would probably be present during embryonic development.
The unusual collection of human embryonic material available in the Department of Anatomy of the University of
Pittsburgh has made it possible to verify this suggestion and
to study the development of both the olfactory and the accessory olfactory formations. The results of this research are
presented in the present paper.
‘ Aided by grants from the Penrose Fund of the American Philosophical Society,
the Carnegie Corporationof New York and the University of Pittsburgh. Publication no. 7, Physiological and morphological studies on human prenatal development.
MATERIAL AND METHODS
The material used consists of serial sections of the brains
of human fetuses varying in size from approximately 14 mm.
to 145 mm. in crown—rump length, or from about 6.5 to 18.5
weeks of menstrual age. The thirteen series described, all but
the last five of which were horizontally sectioned, are listed
chronologically in table 1. The brains of the last five fetuses
were frontally sectioned. All of the fetuses under 11 weeks of
menstrual age were sectioned in toto. The central nervous
TABLE 1
APPROXIMATE
MEN STRUAL STAIN
AGE IN WEE K s *
NO. or FETUS C-R LENGTH
IN COLI£D0'l'ION IN MM.*
P 14+ 6.5 Hematoxylin & eosin
1 16.0 7.0 Erythrosin & toluidin blue
0 22.5 8.0 Activated protargol
22 26.0 8.5 Erythrosin & toluidin blue
19 26.5 8.5 Pyridine silver
33 32.0 9.5 Activated protargol
16 35.0 9.5 Pyridine silver & Lyon ’s blue
34 37.0 10.0 Activated protargol
51 49.0 11.0 Activated protargol
39 61.5 12.0 Activated protargol & erythrosin
37 85.5 14.0 Activated protargol
47 112.0 15.5 Erythrosin & toluidin blue
3 145.0 18.5 Pyridine silver
* All but the first and third fetuses listed in this table are from the series on
which cinematographic records of fetal behavior have been made by Hooker (’36,
et seq.). The approximate menstrual age of each was computed by the use of the
Streeter tables (Streeter, ’20). The material wasprepared by Dr. Ira D. I-Iogg,
Mr. Reinhardt Rosenberg, and Miss Beryl Dimmick.
system was removed from all those over 11 weeks of age and
sectioned separately. The pyridine silver series were stained
according to the method of Ranson. The series made with
activated protargol follow the method of Bodian (’36 and ’37).
The outlines for the drawings and the major structures
therein were made with the aid of a slightly modified Edinger
projection apparatus. The drawings were then completed
with the use of the compound microscope. All computations
of cell size were made with the oil immersion objective. In ach case at least ten or more cells were measured and the
average taken.
LITERATURE
Aside from such general questions of development as gross
appearance, position, relation to the ventricles of the brain,
and development of related fissures, the structure of the olfactory bulb in human embryos has received little attention. An
exception is Von K6lliker’s (1882 and 1883) description of the
bulb in an 8-weeks embryo. The microscopic structure of the
olfactory bulb in adult man, however, except for the absence
of the accessory olfactory formation (Humphrey and Crosby,
’38 ; Crosby and Humphrey, ’39 a, ’39 b), follows in all essentials the lamination pattern characteristic for such mammals
as marsupials, rodents, carnivores, ungulates, insectivores,
chiropteres, and other primates as described by numerous
observers (Ramon y Cajal, ’11; Winkler and Potter, ’11 and
’14; McCotter, ’12; Herrick, ’24; Gurdjian, ’25; Obenchain,
’25; Hines, ’29; Le Gros Clark, ’31; Humphrey, ’36; Young,
’36; Crosby and Humphrey, ’39 b; Fox, ’40, and many others).
The accessory olfactory formation, or accessory olfactory
bulb as it is also termed, is absent not only in adult man, but
also in the bat (Humphrey, ’36), in most of the macaque
material which has been described (although a minute vestigial structure was found in a young macaque; Crosby and
Humphrey, ’39 a, ’39 b), in some reptiles (Crosby, ’17; Ariéns
Kappers, Huber and Crosby, ’36; Crosby and Humphrey,
’39 b), and in birds (Huber and Crosby, ’29 ; Ariéns Kappers,
Huber and Crosby, ’36; Crosby and Humphrey, ’39 b).
The suggestion of the last named authors (Crosby and Humphrey, ’39 b) that an accessory olfactory formation would
probably be present in early human embryos only to disappear
at some later time is supported by the fact that the vomeronasal or J acobson’s nerve (connected centrally with the accessory olfactory bulb; McCotter, ’12 and ’17) is present not only
in early human embryos (Von Kiilliker, 1882 and 1896; Keibel,
’lO; Schaeffer, ’20 and ’28, and many others), but has been demonstrated in a human fetus as old as 6 months (McCotter,
’15) and Jacobson’s organ has been described often in the
adult (Peter, ’01; Mangakis, ’02; Kallius, ’05; Keibel, ’10,
and others). However, the relation of this nerve to any specific part of the olfactory bulb in human embryos has not been
indicated by these authors, although according to von Kolliker
(1882 and 1896), Kallius (’05), and Read (’O8) a branch of
the olfactory nerve goes to Jacobson ’s organ. Likewise Hertwig (’15) observed that the nerve from this organ is connected
with the “Regio olfactoria.”
DESCRIPTION OF MATERIAL
The present account is concerned with the development of
the microscopic configuration of the olfactory and accessory
olfactory formations and not with the entering nerve fibers
or their areas of peripheral distribution. Since recognition
of the accessory olfactory formation itself in the earlier embryos is actually determinable only on the basis of its relationship to the vomeronasal nerve, however, it has been necessary
to refer to it and to the general state of development of the
organ of Jacobson.
N o attempt has been made to study the development of the
nervus terminalis. Nevertheless it should be stated that the
vomeronasal nerve as used here is limited to the fibers passing
from the vomeronasal organ to a special part of the olfactory
bulb (the accessory olfactory formation) and does not include
the nervus terminalis as this term is used by such observers
as McCotter (’12), Huber and Guild (’13), Johnston (’l3 and
’14), Larsell (’18), and Herrick (’31). These two nerves are
so intimately associated in the earlier human embryos studied,
however, that it is not always possible to differentiate them.
In such early embryos the fibers which terminate adjacent to
the area of penetration of the olfactory fila, in a region which
later shows the developing lamination pattern of the accessory
olfactory formation, are considered to be vomeronasal. Those
entering caudal to the developing olfactory bulb are probably part of the nervus terminalis. Such a distinction appears to
be in harmony with the recent observations of Pearson (’40).
The primitive olfactory bulb before the lamination pattern
begins to develop
14+ mm. embryo (6.5 weeks). At this age the vomeronasal
organ is represented by a deep groove extending almost directly medialward into the wall of the developing nasal septum. From the much thicker epithelium of the cephalic
(medial) wall of this groove the fibers of the vomeronasal
nerve extend forward to the forebrain vesicle. At this time
no fibers are contributed from the thinner caudal (lateral)
wall of the developing vomeronasal organ.
Neither the primordial olfactory formation nor the primitive accessory olfactory bulb present any evidences of beginning lamination such as occurs in the adult. Instead the wide
ependymal zone around the ventricle is separated from a narrow, peripherally situated acellular layer by a region of intermediate width which is formed by cells migrating out from
the ependyma. The developing accessory olfactory formation
has the same three regions, but the narrow peripheral zone is
less definite in character and the intermediate region contains
fewer migrating cells. Apparently these three regions just
indicated correspond to the ependymal, mantle, and marginal
layers described elsewhere in the developing neural tube.
16 mm. embryo (7 weeks). The degree of development of
the olfactory areas of the nasal fossa in this embryo is essentially the same as in the preceding one, although the groove
forming the organ of Jacobson is less definite in character.
As in the first embryo, nerve fibers pass centralward only from
the thickened cephalic (medial) wall of the developing Jacobson’s organ (the wall nearer the forebrain). No indication of
lamination is to be found in either the primordial olfactory or
accessory olfactory formations, both of which show no demonstrable advance in differentiation. These two areas of the
primitive olfactory bulb, or pars anterior of the rhinenceph—
alon, are definitely distinguishable from each other only on
the basis of the entering nerve fibers.
22.5 mm. fetus (8 weeks). At 8 weeks of menstrual age
J acobson’s organ had deepened and elongated sufficiently to
appear as a closed—off pocket for a part of its extent, and both
its cephalic (medial) and caudal (lateral) walls have a tall
lining epithelium. Fascicles of the vomeronasal nerve pass to
the rhinencephalon from both the cephalic and caudal surfaces of the Vomeronasal organ in this fetus, as also is the
case in all of the older fetuses in this account which are Seetioned in toto.
The vomeronasal fibers enter the developing olfactory bulb
just medial and slightly dorsal to the fila olfactoria (fig. 1 B).
ABBREVIATIONS
acc.olf.form., accessory olfactory forma- mant.l., mantle layer
tion marg.l., marginal layer
ant.o1f.nuc., anterior olfactory nucleus mit.c.l., mitral cell layer
ep., epeudyma o1f.f., olfactory fila
ep.l., ependymal layer olf.n., olfactory nerve fibers
ext.gran.l., external granular layer olf.v., olfactory ventricle
ext.molec.l., external molecular layer outw.n1it.c., outwandered mitral cells
glom., glomerulus pr.acc.olf.form., primordial accessory
glom.l., glomerular layer olfactory formation
int.gran.l., internal granular layer pr.olf.form., primordial olfactory forint.gran.l.(olf.form.), internal granular mation
layer of olfactory formation vest.acc.olf.form., vestigial accessory
int.molec.l., internal molecular layer ‘olfactory formation
l.gran. +mit.c., layer of granule and \'on1.n., vomerpuasal nerve fibers
mitral cells
 
fig. 1 Drawings to illustrate the location and structure of the primordial
olfactory and accessory olfactory formations in a 22.5 mm. embryo. Activated
protargol preparation. A, 9. horizontal section through the forebrain vesicles and
the developing nasal fossae to show the plane of the sections and the location of
drawing B. X 3. B, the area indicated by the dotted lines in figure A, enlarged to
show the structure of the primordial olfactory and accessory olfactory formations.
X 30.
Even through the center of the primitive olfactory and accessory olfactory formations as yet no indication of beginning
lamination exists. Only the three regions representing the
ependymal, mantle, and marginal layers found elsewhere in the
early development of the neural tube are present in either area,
although a definite advance in development occurs in J acob—
son’s organ (see p.— 436). The differences between the developing olfactory and accessory olfactory formations at this age
are the fewer cells migrating outward from the ependyma, the
slightly lesser width of this zone of migrating cells (mantle
layer), the greater width of the peripheral, acellular region
(marginal zone), and the coarser, more deeply penetrating
nerve fibers in the primitive accessory olfactory bulb as compared with the primordial olfactory formation. At levels
through the middle of the developing accessory olfactory
formation this structure forms a minute elevation on the surface. Von K6lliker’s description (1882, 1883) of the olfactory
lobe at this age also indicates no development of the bulbar
lamination pattern.
The olfactory bulb before regressive changes occur in the
accessory olfactory formation
26 mm. fetus (8.5 weeks). The vomeronasal organ in the
26 mm. human fetus has elongated and deepened sufficiently
to appear as a saccular structure having a definite tube—like
connection with the nasal fossa. In both the developing olfactory and accessory olfactory formations some indications of
beginning layer formation may be seen (fig. 2, B and C). In
each region the wide ependyma and the adjacent area of
neuroblasts proliferating from it are still present about the
ventricle, but toward the surface changes have occurred. In
the olfactory formation (fig. 2 B) numerous cells at the surface form a layer in intimate relation with the entering olfactory fila, which, however, do not extend more deeply. This
peripheral .cellular layer is separated from the deeper portion
of the olfactory formation by a narrow, less cellular lamina.
438 TRYPI-IENA HUMPHREY
Central to the latter is a relatively wide lamina of cells which
is slightly separated from the neuroblasts migrating out from
the ependyma by another less cellular region. Later developmental changes in the olfactory formation indicate that the
glomerular and external granular layers differentiate in the
cellular region of termination of the entering fibers. Thus
this zone forms the anlage for the external granular layer,
the deeper acellular region is the primordial external molecular layer, the adjacent cellular lamina represents the future
fig.2 Illustrations to show the position and morphology of the developing
olfactory and accessory olfactory formations in the 26 mm. fetus. Erythrosin and
toluidin blue preparation. A, a horizontal section through the forebrain to indicate
the plane of the sections and the location of the areas enlarged in B and C. X 3.
B, an enlargement of the region shown within the dotted lines on the left side of
figure A, but at a level 80;; inferior to that of A. This drawing illustrates the
beginning of lamination in the olfactory formation. X 30. C, the area enclosed
in the dotted lines on the right side of figure A, enlarged to illustrate the beginning of lamination in the accessory olfactory formation. X 30.
mitral cell layer, and the neuroblasts still migrating out from
the ependyma form the primordial internal granular layer.
These indications of lamination are present practically all
through the region into which the olfactory fila are entering,
although no difference in the size of the cells in the developing
layers is as yet demonstrable.
The accessory olfactory formation in this fetus retains its
dorsomedial position on the caudal surface of the developing
olfactory bulb; there it again forms a definite eminence (fig.
2 C). As compared with the olfactory formation (fig. 2 B) in the same fetus, the accessory olfactory bulb (fig. 2 C) has
less numerous entering nerve fibers, fewer cells throughout,
and certain minor difierences in the developing lamination
pattern. The vomeronasal fibers are coarser than the olfactory fila and many of them penetrate more deeply, but some
terminate also in the region of the few cells at the surface.
However, the primordial external granular layer in this part
of the olfactory bulb is more deeply situated, so that a relatively acellular zone, with only a few cells among the entering
fibers, is present at the surface. At this time the primitive
external granular layer presents evidences of forming a lamina
separated from the deeper layers in only a small part of the
accessory olfactory formation. In such regionsa still deeper
cell band (the primordial mitral cell layer) may be seen and
this in turn is separated from the neuroblasts still migrating
out (the developing internal granular layer) from the ependyma by a relatively less cellular zone (the region of the
internal molecular layer). In view of the developmental
changes which occur in these regions later it is possible to
state that the Various laminae of the accessory olfactory
formation are represented at this age as indicated by the
names given in parentheses above.
26.5 mm. fetus (8.5 weeks). The accessory olfactory formation in this embryo is essentially the same as that in the preceding one of approximately the same size. A similar degree
of differentiation is present, although in certain respects somewhat less clearly seen due to the difference in staining of the
two series, the pyridine silver impregnation of the series now
under consideration being less favorable for cellular study.
32 mm. fetus (9.5 weeks). At this age, although not in two
older fetuses at least (35 mm. and 37 mm.), the organ of Jacobson no longer opens into the nasal fossa, but is connected with
it only by a solid cord of epithelial cells. According to Schaeffer ( ’20) after 10 weeks of embryonal life Jacobson’s organ
“varies considerably” and “complete degeneration in early
fetal life may ensue.” Both this author (Schaeffer, ’2O and
’28) and Kallius (’05) state, however, that the vomeronasal organ reaches the height of its development in the twentieth
week, an age comparable to that at which a fully differentiated
accessory olfactory formation is also found in the present
study (18.5 weeks old fetus; see p. 456).
The developing olfactory bulb now extends medialward as
well as ventralward. The primitive accessory olfactory formation again occupies its dorsomedial portion and again forms
a slight superficial eminence (fig. 3 B). At this time there is
a degree of differentiation similar to that seen in this region
in the preceding fetus (see p. 438), but for a somewhat greater
portion of the area. The most superficial layer of cells constitutes the external granular layer, a lamina which is more
deeply situated than in the olfactory formation. In this fetus
it is more definitely separated from the deeper layers, however, and consists of cells averaging 6.4;: in their greatest
diameter as compared with 8.9 u for the average diameter of
the cells of the wider and more definite primitive mitral cell
layer. The primordial external molecular, internal molecular,
and internal granular layers are essentially unchanged as
compared with these laminae in the 8.5 weeks old fetus. It is
of interest that in the lateral part of the differentiating accessory olfactory formation the developing mitral cell layer is
wider and more distinct than elsewhere although the primitive
external granular layer noted medially cannot be identified
throughout the same area.
Although having increased greatly i11 both size and ventrafrontal extent as compared with the olfactory formation in
the 26 mm. fetus, the olfactory formation of the 32 mm. fetus
has no marked advance in the differentiation of its lamination
pattern (fig. 3 C). In fact there is even less indication of a
primordial internal molecular layer. Thus the region of the
primitive mitral cell layer is in continuity, for the most part,
with the neuroblasts migrating out from the ependyma to
form the internal granular layer. The cells in the region
allocated to the mitral cell layer, however, are somewhat more
compactly arranged and now average 8.6 u in size as compared
with 7.2 u for tl1ose more deeply located. At this age the fila olfactoria are more intimately associated with the cell layer
at the surface, the external granular layer. Actually the two
appear as a single band of intermingled cells and fibers
although the proportion of cells is greater on the deeper sur—
face. As at all earlier ages considered the developing olfactory formation is much more cellular than is the primitive
accessory olfactory formation in the same fetus.
35 mm. fetus (9.5 weeks). The developing lamination pattern of the accessory olfactory formation is not only more
clear than is that in the preceding fetus but also more extensive. A slight eminence on the surface continues to mark the
fig. 3 Drawings to illustrate the olfactory and accessory olfactory formations
in the 32 mm. fetus. Activated protargol preparation. A, a diagram of a. horizontal section through the entire fetus to indicate the plane of the sections and
the areas enlarged in B and C. X 3. B, an enlargement of the accessory olfactory
formation from the area enclosed in dotted lines in figure A. X 30. C, a drawing
of the olfactory formation from an area comparable to that of figure B, but from
a section 380 u inferior to the plane of that figure. X 30.
outline of the accessory olfactory formation. In its best differentiated part the primordia of those laminae previously
observed in this region are now demonstrated more clearly.
A comparable difference in size (2.5 u) between the cells of the
primordial mitral cell layer and those of the developing external granular layer, observed in the 32 mm. fetus, also is
found here; the former average 8.7 u, the latter 6.2 u in diameter. The apparent decrease in the size of the cells may be due
to the greater shrinkage in the present preparation, which is
stained with pyridine silver.
442 TRYPIIENA HUMPHREY
Except for an increase in size and the marked extension
medially as well as ventrally, the olfactory formation in the
35 mm. fetus has not advanced in development. In fact, the
slightly larger size of the cells forming the primordial mitral
cell layer, as compared with the developing granule cells, cannot be noted here (possibly due to the type of preparation),
although a similar difference in size can be observed in the
developing accessory olfactory formation.
.37 mm. fetus (10 weeks). Although the opening is narrow,
the organ of Jacobson retains its tubular connection with the
nasal fossa in this fetus as well as in that of 35 mm. crownrump length. In other respects it resembles the organ of
Jacobson in the 32 mm. fetus, in which, however, the connection with the nasal fossa has been lost (see p. 439).
The lamination pattern noted in the olfactory and accessory
olfactory formations in the younger fetuses is now much
plainer. The surface eminence formed by the accessory olfactory formation is still demonstrable (fig. 4B). The more
lateral portion of the vomeronasal nerve sends fibers even
farther lateralward near the surface after entering the accessory olfactory formation (fig.' 4 B). In the region nearest the
olfactory formation (fig. 4 C) the mitral cell layer extends for
a considerable distance lateralward to come into relation with
the anterior olfactory nucleus (see p. 444). Although a comparison of figure 4 C with figure 9 B (both of which are drawn
at the same magnification) appears to indicate that the accessory olfactory formation in the 37 mm. fetus is larger than
in the 145 mm. fetus this appearance is probably due in part
at least to differences in the plane of section.
The laminae of the accessory olfactory formation are now
much better differentiated (fig. 4, B and C). The external
granular layer is less compact in nature but wider and more
definitely demonstrable. Again its cells are distinctly smaller
than are those of the mitral cell layer. The average diameter
of the cells in the mitral cell layer is 8.9 u as compared with
7.0;: for those in the external granular layer and 6.7 u for
those in the primitive internal granular layer. Numerous fibers of the Vomeronasal nerve extend directly inward to the
external granular layer; a few pass medially and laterally
among the scattered cells just within the surface. Some mitral
cell processes extend out into the external granular layer also.
The developing external molecular layer is now wider and
definitely separates the external granular and mitral cell
fig.4 Drawings to illustrate the olfactory and accessory olfactory formations
in the 37 mm. fetus. Activated protargol preparation. A, a diagram of a horizontal
section through the fetus to indicate the planeof the sections and the location of
the detailed drawings which follow. X 3. B, an enlargement of the more dorsamedial portion of the accessory olfactory formation, the area shown within the
dotted lines in figure A. X 30. G, the accessory olfactory formation on the same
side of the brain, as seen in a section 100p. inferior to A and B. X 30. D, a
section through the olfactory formation, 480;: inferior to figure C. X 30.
layers. The mitral cell layer itself is the most clearly differentiated of all the laminae. It appears as a relatively wide
band of neurons of larger size than the remainder of the cells
in the accessory olfactory formation. That these cells do not
exhibit the shape typical for mitral cells in the adult is probably due to the relatively small amount of cytoplasm present as yet. In the region of entrance of the vorneronasal fila the
mitral cell layer is broken up and less clear, but a more distinct lamina extends lateralward from this area. Here the
mitral cell layer is thicker and more definite, a condition in
harmony with the greater number of fibers entering this region.
In the more lateral part of the accessory olfactory formation,
however, the developing external granular layer is less clear
than in the more medial portion (fig. 40). Central to the
mitral cell layer the internal molecular layer consists of a
narrow zone in which there are relatively few cells. The
primitive internal granular layer at this age is still represented by almost uniformly scattered small neuroblasts migrating out from the ependyma.
As the mitral cell layer of the accessory olfactory formation
extends lateralward, its clarity in outline decreases and there
appears deep to it a second cell band (fig. 4 C) which becomes
more distinct farther lateralward. The cells in this lamina
average 8.1 u in diameter, a size intermediate between that of
the mitral cells (8.9 H) and the granule cells (6.7 u). From
its position and other relations, this deeply situated cell layer
represents the anterior olfactory nucleus.
The olfactory fila enter the olfactory formation (fig. 4D)
frontally on its ventral, medial, and lateral surfaces. A greater
degree of cellular proliferation than is observed in the accessory olfactory formation continues to exist in the olfactory
formation of this fetus. The highest differentiation is present
in the areas of entrance of the nerve fibers. There is little
change in the lamination pattern as yet as compared with that
in the 32 mm. fetus. No clumping of cells (average size, 7.6 u)
can be observed in the internal granular layer, into which
many neuroblasts are still migrating from the ependyma. The
most definite change is in the mitral cells, some of which are
now beginning to assume a more triangular outline and average 11.0 p in diameter as compared with 8.6 p in the 32 mm.
fetus. They do not form a sharply defined layer either deeply
or superficially, however, and evidence of an internal molecular layer is present only rarely. Developing mitral cells are found either singly or in small groups within the external
molecular layer. Rarely such a cell is present even in the
inner border of the cell layer at the surface of the olfactory
formation, the external granular layer. As yet no glomeruli
have developed.
The olfactory bulb after regressive changes appear in the
accessory olfactory formation
49 mm. fetus (11 weeks). According to Von Kiilliker (1882)
at the age of 3 months the olfactory bulb is directed posteriorly as well as downward but later extends medialward and
fig. 5 Drawings illustrating the olfactory and accessory olfactory formations
in the 49 mm. fetus. Activated protargol preparation. A, an outline drawing to
indicate the plane of the sections and the location of figures B and C. X 3. B, a
detailed drawing of the right accessory olfactory formation from the area enclosed
in dotted line on the left side of figure A. X 30. C, a drawing of the left olfactory
and accessory olfactory formations taken from the area enclosed in dotted lines
on the right side of figure A. X 30.
forward. Such a position for the developing olfactory bulbs
is observed in this and in the next two fetuses studied. In the
fetuses of 15.5 weeks of menstrual age or over the bulb is
directed frontally.
The most striking advance in the development of the olfactory formation (fig. 5 C) occurs in the mitral cell layer.
Although there has been no significant increase in the size of
446 TRYPJIENA HUMPHREY
the mitral cells, 11.3 in in diameter as compared with 11.1 p in
the 37 mm. fetus) and but little more separation from the
internal granular layer of the bulb, the mitral cells are now
arranged in the sectors seen iii the olfactory bulbs of all older
specimens studies (see pp. 447, 449, 452 and 454) and in the
adult (Humphrey and Crosby, ’38; Crosby and Humphrey,
’39 b). Two such sectors, concave toward the surface of the
bulb, are illustrated in figure 5 C. Outwandered mitral cells
are present in the external molecular layer and, more rarely,
at the border of the external granular layer. Glomeruli have
not as yet begun to differentiate.
The accessory olfactory formation (fig. 5, B and C) is about
equally well developed on both sides of the brain and on each
side regressive changes have begun to appear. The mitral cell
layer is more compact and clearly delimited although the
component neurons have not changed significantly in size
(8.4 u as compared with 8.9 u in the preceding fetus). Neither
accessory olfactory formation has an external granular layer
such as is present earlier and glomeruli are not differentiated
as might be expected had further development occurred. Deep
to the mitral cell layer is the anterior olfactory nucleus in
which the slightly smaller cells are more loosely arranged than
are those in the mitral cell layer. 011 both sides of the brain,
although more clearly on the left (fig. 5 C), the medial part
of the mitral cell layer is in intimate relation with the anterior
olfactory nucleus, often appearing either to be a part of it or
to turn deeply to join it.
61.5 mm. fetus (12 weeks). In the 61.5 mm. fetus the olfactory ventricles are still patent throughout although proportionately smaller in size. All the layers of the olfactory
formation (fig. 6 B) cannot be recognized definitely as yet, but
certain advances in lamellar differentiation have taken place.
Among the most noteworthy of these is the greater number of
outwandered mitral cells and the change in the shape of many
mitral cells to the more typical triangular outline of the adult
neuron. l\Ieasurement of the mitral cells of the olfactory
formation now gives an average of 11.5 u, not enough of an increase in size to be significant. Another advance in development is the greater clarity in the arrangement of the mitral
cells in the various sectors which were first noted in the 49 mm.
fetus (see also pp. 452 and 454). The dorsal and ventral sectors are the most prominent, but lateral and medial ones are
also present. A glomerular layer has not yet appeared. The
external molecular layer contains many mitral cells and fewer
cells of the granule type. The internal molecular layer,
mi1.c.I.<————accoIf.form.
   
 
A
oulwmitc.
fig. 6 Illustrations of the olfactory and accessory olfactory formations of the
61.5 mm. fetus. Activated protargol and erythrosin preparation. A, an outline
drawing of a section through the anterior commissure to show the plane of section
of the series and the general location of the detailed figure which follows. X 3.
B, the left olfactory and accessory olfactory formations enlarged from the area
within the dotted lines in figure A. X 30.
although narrow, may be seen in such areas as the middle of
the sectors of the mitral cell layer, but it is not distinguishable
elsewhere. N o clumping of granule cells, such as is present in
the adult, is demonstrable in the internal granular layer and
neuroblasts are still migrating out into it from the ependyma.
The accessory olfactory formation (fig. 6B) of this fetus
exhibits, on the Whole, degenerative rather than developmental
changes. An approximately equivalent degree of differentiation is present on both sides although the difference in the angle at which the two olfactory bulbs are sectioned (see fig.
6A) makes comparison difiicult. Apparently the accessory
olfactory formation on the left side of figure 6A is cut in a
plane approaching the sagittal; the one on the right side,
however, is sectioned more nearly frontally.
The accessory olfactory formation on the left side (fig. 6 B)
is situated at the angle at which the developing olfactory bulb
turns medialward and, at this age, caudalward (although later
frontally; see p. 445). Most of the vomeronasal fila enter it
at this point of turning although some enter both cephalic and
caudal to the area in question. The most clearly developed
part of the accessory formation is situated just dorsal to the
ventricle in figure 6 B. Both ends of the curved band of cells
in this region come into relation with the anterior olfactory
nucleus on its deeper aspect. At the level illustrated the cells
in this layer are larger (8.7 p) at the narrow end of the lamina
and grade off into smaller ones (6.1 u) at the thicker end of
the layer, the part in more intimate contact with the anterior
olfactory nucleus in this figure. Between the two parts is a
zone intermediate as to cell size. Superficial to this cell layer
are uniformly scattered cells, which, although equal in size
(8.9 p) to the larger ones in the cell layer deep to them, have
a different staining reaction. Cells of this same type, which
are present throughout the area of entrance of the vomeronasal fibers in figure 6B, and in the more frontal area into
which fibers penetrate at other levels, obviously belong to the
accessory olfactory formation. At least part of the curved
cell lamina represents the mitral cell layer of the accessory
olfactory bulb, but the portions which pass over into the
anterior olfactory nucleus without any demonstrable change
appear either to have failed to develop sufiiciently to form a
mitral cell layer and to have acquired instead the characteristics of the anterior olfactory nucleus, or to be undergoing
regressive changes such that they are taking on the characteristics of this center. Superficial to the mitral cell layer the
scattered cells probably represent cells of the external granuDEVELOPMENT or HUMAN OLFACTOBY BULB 449
lar layer which have increased in size with the changes in the
mitral layer and become uniformly scattered through the area.
The right accessory olfactory formation resembles the left
in all essentials. The plane of the sections of this olfactory
bulb permits observation of the fact that the vomeronasal fila
enter on the dorsomedial surface and that many of them extend
across the dorsal aspect of the area after entrance. Cells of
the same type as those in the area into which fibers are entering in figure 6 B are scattered along these fibers. At one level,
in the medial part of the field and again less clearly lateralward, these cells are clumped into a mass resembling the
vestigial accessory olfactory formation illustrated in figures
7 C, 7 D, and 8 A for the 85.5 mm. and 112 mm. fetuses.
85.5 mm. fetus (14 weeks). At 14 weeks of menstrual age the
olfactory formation is but little changed as compared with
that of the 12 weeks old fetus. The only significant differences
are the clumping of the granule cells in a Very limited portion
of the peripheral part of the internal granular layer and the
onset of closure of the olfactory ventricle by the growth of
cells into it from the ependyma. The sector arrangement of
the mitral cells first observed in the 11 weeks old fetus is present here also, although less clearly defined in this case than in
either the 12 or 15.5 Weeks old fetuses.
A rudimentary accessory olfactory formation is demonstrable on both sides of the brain. On the left side three different stages in the regression of this structure are present.
Dorsomedially it is possible to trace the vomeronasal nerve
to a very limited area (fig. 7 A) similar to the accessory formation in the 49 mm. fetus; this area is comparable to the medial
part of the mitral cell layer illustrated in figure 5 0. Here,
in addition to the mitral cell layer which is present in the
accessory olfactory formation in the 49 mm. fetus, a layer of
smaller cells representing the external granular layer is situated more superficially and extends farther lateralward. A
second vestige (fig. 7 B) of the accessory olfactory formation
is represented by a single small glomerulus resembling the
left accessory olfactory formation of the 112 mm. fetus (fig.
450 TRYPHENA I-IUMPHREY
8 B). The position of this glornerulus, into relation with which
Vomeronasal fibers can also be traced, is equivalent to the
middle of the mitral cell layer in figure 5 C. It has a frontecaudal extent of approximately 50 u, a mediolateral diameter
of about 70 u, and consists of a single glomerulus with its
accompanying intra— a11d periglomerular cells. None of the
cells, however, have acquired the character of mitral cells.
 
fig. 7 Drawings of the vestigial remains of the accessory olfactory formation
of an 85.5 mm. human fetus. Activated protargol preparation. X 30. A, the best
developed part of the left accessory olfactory formation. This area is comparable
in its location to the most medial portion of the accessory formation illustrated in
figure 5 C. B, vestiges of the accessory olfactory formation in the form of a single
glomerulus located in an area comparable to the middle of the accessory olfactory
formation of figure 5 C. C, the undifferentiated remains of the accessory olfactory
formation found at the region corresponding to the most lateral part of the accessory olfactory formation of figure 50. D, the vestiges of the right accessory
olfactory formation. The area drawn is comparable in position to the middle of
the accessory olfactory formation of figure 5B.
The third vestige (fig. 7 C) of the accessory olfactory formation is located still farther lateralward in a region corresponding to the most lateral portion of the mitral cell layer of the
accessory formation in figure 5 C. It is slightly larger than
the glomerulus just described and resembles the rudiment of
the accessory olfactory formation on the right side of the
brain in this fetus (fig. 7 D) and in the 112 mm. fetus (fig. 8 A).
On the right side, however, the vomeronasal nerve is definitely
in relation with the mass of cells. Toward the more frontal end of the right bulb scattered cells with no particular arrangement also come into relation with the entering vomeronasal
fila. Although a few sections of the right bulb are missing
from the series through this area, such sections are scattered
sufiiciently so that no further representation of the accessory
olfactory formation is believed to be present. In that case it
would appear that the accessory olfactory formation on the
left. side of the brain in this fetus is more clearly represented
than is that on the right side—a situation noted also in the
two older fetuses described (112 mm. and 145 mm.; see pp.
452 and 458).
112 mm. fetus (15.5 weeks). At this age the olfactory ventricle (fig. 8) is still patent in parts of the bulb although
comparatively narrow and in many places partially obliterated. For example, in some sections there are two or three
small openings instead of a single large one and the remainder
of the ventricle is represented either by ependyma or completely obliterated. In other sections only ependymal remains
of the ventricle are present. Caudalward, in the region of the
olfactory crus, a definite, slit—like ventricle is in continuity
with the anterior horn of the lateral ventricle. The olfactory
bulb itself is considerably elongated and flattened with a
clearly developed crus. It is now directed cephalad (see p. 445).
Although the olfactory formation has not yet attained the
degree of differentiation found in that of the 145 mm. fetus,
certain distinct advances in development are present (fig. 8).
The olfactory fila enter the bulb on all surfaces at its most
frontal tip, but in following the series caudally they disappear
progressively from the dorsal, lateral, ventral, and medial
surfaces respectively, remaining farthest caudalward on the
ventromedial aspect as in the macaque (Crosby and Humphrey, ’39 b). Glomeruli are beginning to appear in a few
regions deep to the zone of entering fibers on the ventral surface, but no definite glomerular layer is as yet present. Mitral
cell dendrites can be traced into many of the developing glomeruli identified. Because of the small number of glomeruli
which have developed and the scarcity of granule cells in the region, the external granular layer is still represented primarily by the cells situated deep to the entering fila. The
external molecular layer varies in width with the depth of the
mitral cell layer and contains many cells of both mitral and
granule cell types. The mitral cell layer is more prominent
than in either the 145 mm. fetus or in the adult and exhibits
Very clearly the arrangement into sectors which began to
appear in the 49 mm. fetus. These sectors present a concavity
superficially and are best defined just caudal to the accessory
olfactory formation. In the middle of each sector the mitral
cells are more compactly arranged and the layer is thicker.
At the ends, where two sectors join each other, the mitral cells
are fewer in number and more scattered. The dorsal sector
appears farthest frontalward, extends less far caudally than
any of the other sectors, and its neurons are more loosely
arranged. The mitral cells now average 12.9 n in diameter,
are more definitely triangular in outline, and contain a considerable accumulation of Nissl substance. The layer is usually two or more cells thick with the neurons frequently
arranged in small clumps. The internal molecular layer, like
the external, varies in width and clearness. It contains many
granule cells and a few mitral cells. In the internal granular
layer the cells at the periphery are arranged in little clumps
between which are small fiber bundles. Centrally such an
arraxigement is not present, the granule cells there appearing
uniformly distributed.
Vestiges of the accessory olfactory formation may be recognized on both sides of the brain in spite of the fact that marked
regressive changes have taken place. It is only on the left
side, however, that a discrete area with any organized structure is still retained (fig. 8B). Here the minute vestigial
accessory olfactory formation appears as a tiny eminence
about 170 p in diameter and of approximately the same frontocaudal extent. It is situated near the middle of the area occupied by the dorsal sector of the olfactory formation farther
frontalward, a position comparable to that of the vestigial
accessory olfactory formation in the young macaque (Crosby and Humphrey, ’39 b). Morphologically this accessory olfactory formation consists of one to three glomeruli, deep to
which small mitral cells (10.1 n) and a few granule cells are
intermingled in the form of a limiting layer. A few scattered
granule cells in continuity with the internal granular layer of
the olfactory formation are found beneath the layer of mitral
and granule cells. In structure, then, as well as in location,
this rudimentary accessory olfactory formation is strikingly
similar to the vestigial one found in the young macaque, the
only significant difference being the greater number of mitral
fig.8 Illustrations of frontal sections of the olfactory bulbs of a. 112 mm.
fetus. Erythrosin and toluidin blue preparation. X 30. A, the right olfactory
bulb to show the course of the vomeronasal nerve within its surface and the cells
along it which are the only demonstrable vestiges of the accessory olfactory formation. The illustration of the course of the vomeronasal nerve with the accompanying
neurons was made by combining the features observed in six consecutive 15p.
sections. B, the left olfactory bulb showing a vestigial accessory olfactory formation similar to that observed in the young macaque. Both the plane of the section
and its frontocaudal location differ slightly from those of figure A.
cells present. Recognition of the relationship of the vomeronasal fila to this area is not easy since the fibers have become
incorporated within the surface of the bulb (see also pp. 449
and 454). The compact arrangement and deeper staining
reaction of these fibers, however, make it possible to trace
them from the medial surface of the bulb to a position near the
vestigial accessory olfactory formation, but the discrete nature
of the bundle is lost before it reaches the accessory olfactory
formation itself.
On the right side of the brain the accessory olfactory formation (fig. 8 A) has undergone still greater regressive changes,
so that the presence of any representation of it at all might
be questionable were it not for the relationship with the
vomeronasal fila and the presence of similar vestiges in other
fetuses (see pp. 450, 451, and fig. 7, C and D). The vo1neronasal nerve, however, is more readily followed than on the
left side. It enters the olfactory bulb dorsomedially as a compact bundle (fig. 8A) which extends lateralward just within
the dorsal surface to a point somewhat beyond the middle of
the bulb. Along this nerve bundle, in addition to scattered
cells, there are also two to three little clumps of more deeply
staining neurons in relation with which the nerve apparently
terminates. These scattered neurons and the small cell groups
along the vomeronasal nerve within the olfactory bulb are the
only vestiges of the right accessory olfactory formation,
which is, then, even less well represented than is the left.
145 mm. fetus (18.5 weeks). The lamination pattern of the
olfactory formation in the 18.5 weeks old fetus under consideration is in every respect as well differentiated as is that in
the adult bulb (Humphrey and Crosby, ’38) and in some respects even plainer (see p. 455), although the olfactory bulb
itself is smaller in size (fig. 9, A and B). As in the adult, the
olfactory ventricle is no longer patent in the bulb, but in the
caudal part of the olfactory crus in this fetus it is present and
retains its connection with the lateral ventricle.
The olfactory fila (fig. 9, A and B) surround the frontal tip
of the bulb and enter it on all sides although the dorsal portion of the fibers enter only quite far cephalad. The fila situated lateralward do not extend so far caudalward as do the
ventromedial bundles which are still present caudally after
all others have entered the bulb. Thus the arrangement of the
olfactory fila into the four sectors, noted both in the adult bulb
(Humphrey and Crosby, ’38) and in the preceding fetus, is
also present here. There is a marked infolding of the mitral
cell layer in the ventral, lateral, and medial sectors of the bulb
and the mitral cell layer in the dorsal sector is more clearly defined as is the case also in the olfactory formation of the
pig (Crosby and Humphrey, ’39 b). At this age there are
proportionately fewer outwandered mitral cells present in
the external molecular layer than occur in the adult, so that
the mitral cell layer is more definite. More outwandered mitral
cells are present in this fetus, however, than in the preceding
one. The average size of the mitral cells at this time is 19.2 p.
 
fig.9 Drawings of frontal sections through the olfactory and accessory olfactory formations of a 145 mm. fetus. Pyridine silver preparation. X 30. A, the
left olfactory bulb cephalic to the region of the accessory olfactory formation.
B, the left olfactory bulb at a level through the middle of the accessory olfactory
formation and the frontal tip of the anterior olfactory nucleus. 0, the right
accessory olfactory formation in the region of its better difierentiated cephalic
portion. D, the right accessory olfactory formation at the level of its less well
developed caudal portion.
A glomerular layer is now represented on all aspects of the
olfactory formation, but it is better developed on the ventral
and lateral surfaces (fig. 9, A and B). In regions in which it
is not represented the granule cells deep to the entering fibers
are now markedly increased in number. Both peri- and intraglomerular cells can be distinguished. The granule cells form
a more definite external granular layer than is found in the
adult olfactory bulb. Occasionally an outwandered mitral cell
may be seen in the border of this layer but less often than in
the adult. The external molecular layer contains both outwandered mitral cells, singly or in groups, and granule cells;
the former are more conspicuous although less numerous than
the latter. The internal molecular layer is narrow, poorly de~
fined and often so filled with granule cells as to be difficult to
distinguish at all. The internal granular layer is still represented centrally by closely massed granule cells which are
uniformly arranged. Peripherally, however, the lamellar
arrangement of little clumps of granule cells interspersed
with fiber bundles is present for a wider distance than in the
112 mm. fetus. VVith this preparation it is not possible to
determine what portion of the cells in the internal granular
layer consists of neuroglia.
Laterally, ventrally, and particularly medially (fig. 9A),
isolated and rather sharply circumscribed masses of neurons
are found in the outer part of the internal granular layer, but
not necessarily at the same frontocaudal level. These cells
are intermediate in size between the mitral cells and the granule cells and are of a type similar to those found in the anterior
olfactory nucleus farther caudalward. It seems likely that
these neurons may constitute cell groups in the path of the
olfactory tracts and hence be comparable, at least, to the isolated parts of the bulbar anterior olfactory nucleus described
in the adult human olfactory bulb (Humphrey and Crosby,'38; Crosby and Humphrey, ’41).
The accessory olfactory formation is present and occupies
the same position in relation to both olfactory bulbs, but it
does not have the same clearness of delineation and lamination on the right side (fig. 9, C and D) as is manifested on the left
(fig. 9B). In following this transversely sectioned series of
the brain frontocaudally the accessory olfactory formation
first appears at levels in which the most cephalic part of the
anterior olfactory nucleus is found. Such levels correspond
to those in which the accessory olfactory formation is located
in the macaque (Crosby and Humphrey, ’39 b) and in many
other mammals. Although situated dorsomedially in early
human fetuses, the accessory olfactory formation in the 145
mm. fetus occupies a dorsolateral position, adjacent to the
region which has no olfactory formation at these levels. This
location is just lateral to that suggested as its possible site
by Crosby and Humphrey (’39 b, Y of fig. 6 E) for adult man.
In the fetus under consideration the accessory olfactory
formation of the left olfactory bulb (fig. 9 B) is clearly delineated through its middle portion, although less clear frontally
and still less caudally. ‘All the laminae typical of this structure in other mammals are present. The vomeronasal fila
enter from the medial side of the area. The glomeruli are
small as compared with those of the olfactory formation, but
form more than a single layer, at least through the best developed part of the accessory olfactory formation. The external
granular layer has a few mitral cells adjacent to it and periand intraglomerular cells are present. The external molecular
layer, although definite and often equal in width to this layer
in the olfactory formation, contains more granule cells among
the fibers. The mitral cell layer is wide, as is that of the dorsal
sector of the olfactory formation. The mitral cells are not as
large as are those in the olfactory formation, having attained
an average diameter of only 15.6 u (see p. 455). The internal
molecular layer is indefinite because of the many granule cells
it contains. The internal granular layer consists only of diffusely scattered granule cells which are continuous with those
in the olfactory formation. At the frontal end of the accessory
olfactory formation the mitral cell layer is in continuity with
this layer of the olfactory formation both medially and laterally. Through its caudal part, however, the accessory forma458 TRYP1-IENA HUMPHREY
tion is bordered medially by an area free of olfactory formation
and laterally by a small region in which the continuity
of the mitral cell layer with that in the olfactory formation
is interrupted by a zone containing scattered neurons of both
granule and mitral cell types.
The accessory olfactory formation on the right side (fig. 9,
C and D) of the brain, although having about equal frontecaudal and mediolateral dimensions as that on the left side,
is much less clearly delimited and fails to show a well differentiated lamination pattern. It is more clearly defined toward
its frontal end (fig. 9, 0), however, and in such areas the zone
of entering fila may be bordered either by an area resembling
a large glomerulus or only by a wide band of granule cells
which forms the external granular layer. In a part of the
accessory formation (fig. 9 C) an indefinite mitral cell layer
is distinguishable, but in the remainder of the region (fig. 9D) a
wide layer of granule cells with a few small mitral cells is separated from the internal granular layer of the olfactory formation only by a zone containing a few scattered cells. In many
areas, and especially farther caudalward, the neurons are all
of about equal size although a few larger ones resembling
mitral cells may be present.
SUMMARY
1. The development of the olfactory and the accessory
olfactory formations was studied in thirteen human embryos
ranging from approximately 14 mm. to 145 mm. crown-rump
length, or from about 6.5 to 18.5 weeks of menstrual age.
2. Differentiation within the primordial olfactory and accessory olfactory formations is first observed at approximately
8.5 weeks of menstrual age. In the series of embryos studied,
differentiation in the two regions appeared at the same time.
3. The accessory olfactory formation is represented in embryos from 6.5 to 18.5 weeks of menstrual age, although beginning at 11 weeks regressive changes occur in this region on
one or both sides of the brain.
4. In an 18.5 weeks old fetus a fully differentiated accessory
olfactory formation is present on the left side of the brain
although not on the right and only vestiges of the structure
may remain in still younger fetuses. Such vestiges vary from
a tiny structure consisting of two or three typical glomeruli
with granule and small mitral cells through a single small
glomerulus with no mitral cells to clumps of neurons along
the course of the vomeronasal nerve within the olfactory bulb.
5. The position of the accessory olfactory formation Varies
from dorsomedial with relation to the primitive rhinenceph—
alon to dorsolateral with reference to the olfactory bulb at
18.5 weeks of menstrual age. At intermediate ages vestiges of
the accessory olfactory formation often occupy a middorsal
position.
6. Differentiation of the lamination pattern is apparently
slightly more rapid as Well as more clear cut in the accessory
olfactory as compared with the olfactory formation. In the
latter region, however, there is a greater proliferation of
neuroblasts from the ependyma, particularly in the earlier
stages.
7. From the time differentiation is first observed in the
series of embryos considered, representation of all of the
laminae in the adult olfactory and accessory olfactory formations, with the exception of the glomerular layer, is present.
This layer, which is the last to develop, does not appear until
after lamellar differentiation is present in the superficial part
of the internal granular layer.
8. An arrangement of the mitral cell layer of the olfactory
formation into sectors first appears at 11 weeks of menstrual
age and is demonstrable thereafter in all of the fetuses studied.
9. A difference in the size of the developing mitral and
granule cells is first observed in both the olfactory and the
accessory olfactory formations at 9.5 weeks of menstrual age.
In the olfactory formation there is a progressive increase in
the size of the mitral cells from that time onward. In the
accessory olfactory formation regressive changes are accompanied by failure of the mitral cells to increase further in size.
10. Outwandered mitral cells are definitely present as early
as 11 weeks of menstrual age and occur throughout later stages
of development although they are proportionately less numerous than in the adult. The mitral cell layer of the olfactory
formation, however, is more clearly delineated at 18.5 weeks
of menstrual age than in the adult.
11. Closure of the olfactory ventricle by growth of cells into
it from the ependyma begins first at 14 Weeks of menstrual
age. Except in the caudal part of the crus, closure is complete
at 18.5 Weeks.
12. The anterior olfactory nucleus appears earliest in rela—
tion with the accessory olfactory formation. In at least certain
cases in which regression occurs in the accessory olfactory
formation, some of its mitral cells appear to become a part of
the anterior olfactory nucleus.
DISCUSSION AND CONCLUSIONS
In the series of fetuses considered, differentiation of the
primordial olfactory and accessory olfactory formations begins at the same time, about 8.5 weeks of menstrual age (26 and
26.5 mm. embryos). It is of interest that the beginning of
differentiation occurs at approximately the same age that
fetal activity is first shown in the cinematographic records of
Hooker (’36 and ’39a; fetus With a crown-rump length of
25 mm. or between 8 and 8.5 weeks of menstrual age). This
initiation of differentiation in the rhinencephalon simultaneously With the appearance of fetal activity is in accordance
with the observations of Coghill (’28) on Amblystoma that
“In the early—flexure stage the centers of difierentiation have
become active in the tectum, the epithalamic, thalamic, infundibular, and rhinencephalic regions.”
The accessory olfactory formation, or its vestigial remains,
may be demonstrated in human fetuses up to the age of 18.5
weeks. At this time it may have attained a degree of differentiation comparable to that of the accessory bulb of many other
adult mammals, that is, typical glomerular, external granular,
external molecular, mitral cell, and internal molecular layers may be observed. Before this age is reached, however, and in
fact usually before complete differentiation of all its laminae
is attained, regressive changes have often appeared. As a
result of these changes even the recognition of vestiges of the
accessory olfactory formation may be difficult in some fetuses
although in others a definite, but tiny rudimentary structure
similar to that found in the young macaque is present. From
the material available it is not possible to state whether or not
all vestiges of the accessory olfactory formation may have
disappeared completely in some cases before the age of 18.5
weeks.
Although the accessory olfactory formation may attain a
morphologic pattern typical for this structure in one olfactory
bulb, it may not reach this degree of differentiation in the
other olfactory bulb of the same fetus (18.5 weeks). There
appears to be a tendency for the better developed accessory
olfactory formation to be retained on the left side of the brain
in older fetuses (85.5 mm., 112 mm., and 145 mm.). Although
the number of cases in which any distinct diflerence can be
noted is too few to be significant, it is more than suggestive
of a difference in the rate of development on the two sides of
the brain. It is of interest that Kallius (’05) noted a similar
difference between the left and the right organ of Jacobson
in a 22 weeks old fetus. The left remained fully developed,
but the right had lost its connection with the nasal cavity and
consisted only of a blind pocket.
Throughout its early development the accessory olfactory
formation exhibits a lesser degree of cellular proliferation than
is observed in the olfactory formation. This would indicate
a slight initial lag in development in the accessory olfactory
formation, although later complete differentiation appears
to be present before it is found in the olfactory formation
(see p. 463). In the earliest stages studied, and wherever a
vestige showing definite olfactory structure remains, the accessory olfactory formation appears as an eminence on the surface of the developing olfactory bulb. In the 18.5 weeks old
fetus it occupies a dorsolateral position with relation to the olfactory bulb, although in the early embryos it has a medial
location and at 15.5 weeks the vestigial accessory olfactory
formation occupies a middorsal position. This apparent
change in position is possibly the result of rotation of the bulb
as it turns first caudalward, then frontally, during development.
It is of interest that the history of the accessory olfactory
formation as observed here parallels that of J acobson’s organ
in human embryos. The earliest indications of regression in
the accessory olfactory formation are found at 11 weeks of
menstrual age, while according to Schaeffer (’20) Jacobson’s
organ “varies considerably” after 10 weeks. Since, according
to Schaeffer (’20 and ’28) and Kallius (’05), J acobson’s organ
attains its highest development in the twentieth week in man
(if it does not degenerate earlier) the accessory olfactory
formation might be expected to be fully differentiated at a
similar age. Such is the case in the left accessory olfactory
formation of the oldest fetus observed (18.5 weeks), although
definite dedifferentiation has occurred on the right side in
this fetus as well as in both accessory formations in younger
fetuses (14 and 15.5 weeks old fetuses, for example).
The laminae of the olfactory and accessory olfactory formations develop in a similar manner. first marginal, mantle,
and ependymal layers, such as are found elsewhere in the
neural tube, appear. From the mantle layer cells migrate
outward to form two laminae, separated from each other and
from the neuroblasts still migrating out from the ependyma
by two less cellular zones. The inner of the two cellular bands
forms the mitral cell layer and the neuroblasts which continue
to migrate out from the ependyma become the internal granular layer in both the olfactory and the accessory olfactory
formations. The outer cellular band, which forms the external
granular layer in each case, is situated at the surface just
beneath the entering fila olfactoria in the olfactory formation.
In the accessory olfactory formation it is situated more deeply,
being separated from the surface by a less cellular area. Many
of the vomeronasal fila penetrate to this layer, however, as is
DEVELOPMENT OF HUMAN OLFACTOBY BULB
the case with the olfactory fila. A few cells are also found at
the surface in relation with other entering vomeronasal fibers.
The glomeruli of the olfactory formation develop in relation
with the external granular layer. Whether or not this is true
in the accessory olfactory formation it is not possible to state
definitely, although such appears to be the case. The glomerular layer itself is the last lamina to develop in both the olfactory and the accessory olfactory formations.
A definite explanation for the late development of the
glomerular layer is not possible from a study of the material
available. It may be that, although at least some mitral cell
dendrites extend out into the external granular layer as early
as 10 Weeks of menstrual age, the complex dendritic branching
which constitutes the glomerulus in the adult (cat, Ramon y
Cajal, ’11) does not develop until much later. Perhaps also
the granule cells in this region do not participate in the synaptic relations or develop extensive branching of their dendrites
until later. At least there is an increase in the number of
granule cells deep to the entering fila shortly before the development of the glomeruli. Possibly another factor in the
late appearance of the glomeruli may be that complete differentiation of the other bulbar laminae is essential before
the synaptic relations between mitral cells and olfactory
fibers become concentrated in one region. The substantiation
of any explanation will depend, of course, on a studyof Golgi
preparations.
The primordia of all the laminae in the accessory olfactory
formation are more clear cut than are those in the olfactory
formation in early development. Apparently all the laminae
are represented earliest in the accessory olfactory formation,
since in this structure in the 15.5 weeks old fetus typical
glomeruli are present although glomeruli are only beginning
to develop in the olfactory formation. However, there is not
suflicient evidence on this point.
A difierence in the average size of the mitral and the granule
cells in the accessory olfactory formation appears as early
as 9.5 weeks of menstrual age and increases in clarity in all
464 TRYPHENA I-IUMPHREY
cases in which development has continued in the accessory
olfactory formation. A difference in the size of the developing
mitral and granule cells in the olfactory formation occurs at
the same time, but the mitral cells attain a greater size than
do those in the accessory olfactory formation.
In the olfactory formation indications of a sector arrangement of the mitral cell layer first appears in the 11 Weeks old
fetus. The sectors are most clearly defined at 15.5 Weeks, but
are more definite in the 18.5 Weeks old fetus than in the adult
olfactory formation. In the older fetuses the dorsal sector
is the most distinct and is characterized by a wider mitral cell
layer. It is of interest that not only these sectors of the mitral
cell layer but also the entire lamination pattern are better
developed in the olfactory formation at 18.5 Weeks of menstrual age than in the adult olfactory bulb. Perhaps this is a
repetition of phylogeny since the olfactory formation is relatively better developed in subprimates than in primates. It
also suggests that regression, which usually has progressed so
far in the accessory olfactory formation, is beginning in the
olfactory formation as Well by the time adult life is reached.
The external molecular layer is narrow and indefinite in the
olfactory formation during early development, but at 11 weeks
of menstrual age increases in width. At the same time, with
the more definite character assumed by the mitral cell layer,
outwandered mitral cells are more readily observed, although
they are demonstrable in the 10 Weeks old fetus (fig. 4 D, unlabeled). Although present either singly or in clumps at all
older stages observed, the outwandered mitral cells are not
as numerous as in the adult.
The internal molecular layer in the olfactory formation is
never clearly defined. The internal granular layer first begins
to develop clumps of granule cells interspersed With fibers in
the 14 weeks old fetus. These occur only at the periphery of
the layer at first but in older fetuses extend farther centrally.
In the oldest fetus studied (18.5 weeks), however, the central part of the internal granular layer consists of uniformly distributed cells as in the adult.
The olfactory ventricle remains Widely patent up to 12
weeks of menstrual age. At 14 weeks cells begin to grow into
it from the ependyma and at 15.5 weeks of menstrual age it is
partially obliterated in many places and in a few regions
completely so. At 18.5 weeks the olfactory ventricle is entirely
obliterated except for the connection of the crural part with
the lateral ventricle. .
The earliest representation of the anterior olfactory nucleus
is in relation with the accessory olfactory formation. The
position of this part of the nucleus, which is also its most
cephalic end, is comparable to that at which its frontal tip is
situated in the macaque and in the newborn pig (Crosby and
Humphrey, ’39 b). Several isolated masses of cells of similar
character, situated farther frontalward, appear to be comparable to the isolated cell masses forming the bulbar part of
the anterior olfactory nucleus in the adult (Humphrey and
Crosby, ’38; Crosby and Humphrey, ’41). In some cases
(49 mm. and 61.5 mm. fetuses) the occurrence of regressive
changes in the accessory olfactory formation is accompanied
by a more intimate relation of its mitral cell layer with the
anterior olfactory nucleus in such a manner as to suggest that
with further dedifferentiation this lamina of the accessory
olfactory formation may possibly take on the function of the
higher center. Details of the development of the anterior
olfactory nucleus, however, are not within the scope of the
present paper.
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HOOKER, D. 1936 Early fetal activity in maunnals. Yale J. Biol. and Med.,
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1939b Fetal behavior. Res. Publ. Ass. nerv. ment. Dis., vol. 19,
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HUBER, G. CARL, AND E. C. CROSBY 1929 The nuclei and fiber paths of the avian
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HUBER, G. CARL, AND S. R. GUILD 1913 Observations on the peripheral distribu
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1915 A note on the course and distribution of the nervus terminalis
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1914 An anatomical guide to experimental researches on the cat’s
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Humphrey T. The development of the olfactory and the accessory olfactory formations in human embryos and fetuses. (1940) J. Comp. Neurol. 431-468.

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Humphrey T. The development of the olfactory and the accessory olfactory formations in human embryos and fetuses J. Comp. Neurol.


Humphrey T. The development of the olfactory and the accessory olfactory formations in human embryos and fetuses. (1940) J. Comp. Neurol. 431-468.

The Development Of The Olfactory And The Accessory Olfactory Formations In Human Embryos And Fetuses

Tryphena Humphrey

Department of Anatomy, University of Pittsburgh, Pennsylvania


NINE fiGURES

CONTENTS

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 431 Material and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 432 Literature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .- . . . . . . . . 433 Description of material . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 434

The primitive olfactory bulb before the lamination pattern begins to develop . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 435

The olfactory bulb before regressive changes occur in the accessory olfactory formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 437

The olfactory bulb after regressive changes appear in the accessory olfactory formation . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 445 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 458 Discussion and conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 460

INTRODUCTION

In a recent study of the adult human olfactory bulb the accessory olfactory formation was found to be absent (Humphrey and Crosby, ’38; Crosby and Humphrey, ’39 a, ’39 b), but it was stated that a primordial accessory olfactory bulb would probably be present during embryonic development. The unusual collection of human embryonic material available in the Department of Anatomy of the University of Pittsburgh has made it possible to verify this suggestion and to study the development of both the olfactory and the accessory olfactory formations. The results of this research are presented in the present paper.

‘ Aided by grants from the Penrose Fund of the American Philosophical Society, the Carnegie Corporationof New York and the University of Pittsburgh. Publication no. 7, Physiological and morphological studies on human prenatal development.


MATERIAL AND METHODS

The material used consists of serial sections of the brains of human fetuses varying in size from approximately 14 mm. to 145 mm. in crown—rump length, or from about 6.5 to 18.5 weeks of menstrual age. The thirteen series described, all but the last five of which were horizontally sectioned, are listed chronologically in table 1. The brains of the last five fetuses were frontally sectioned. All of the fetuses under 11 weeks of menstrual age were sectioned in toto. The central nervous

TABLE 1

APPROXIMATE MEN STRUAL STAIN AGE IN WEE K s *

NO. or FETUS C-R LENGTH IN COLI£D0'l'ION IN MM.*

P 14+ 6.5 Hematoxylin & eosin 1 16.0 7.0 Erythrosin & toluidin blue 0 22.5 8.0 Activated protargol 22 26.0 8.5 Erythrosin & toluidin blue 19 26.5 8.5 Pyridine silver 33 32.0 9.5 Activated protargol 16 35.0 9.5 Pyridine silver & Lyon ’s blue 34 37.0 10.0 Activated protargol 51 49.0 11.0 Activated protargol 39 61.5 12.0 Activated protargol & erythrosin 37 85.5 14.0 Activated protargol 47 112.0 15.5 Erythrosin & toluidin blue 3 145.0 18.5 Pyridine silver

  • All but the first and third fetuses listed in this table are from the series on

which cinematographic records of fetal behavior have been made by Hooker (’36, et seq.). The approximate menstrual age of each was computed by the use of the Streeter tables (Streeter, ’20). The material wasprepared by Dr. Ira D. I-Iogg, Mr. Reinhardt Rosenberg, and Miss Beryl Dimmick.

system was removed from all those over 11 weeks of age and sectioned separately. The pyridine silver series were stained according to the method of Ranson. The series made with activated protargol follow the method of Bodian (’36 and ’37).

The outlines for the drawings and the major structures therein were made with the aid of a slightly modified Edinger projection apparatus. The drawings were then completed with the use of the compound microscope. All computations of cell size were made with the oil immersion objective. In ach case at least ten or more cells were measured and the average taken.

LITERATURE

Aside from such general questions of development as gross appearance, position, relation to the ventricles of the brain, and development of related fissures, the structure of the olfactory bulb in human embryos has received little attention. An exception is Von K6lliker’s (1882 and 1883) description of the bulb in an 8-weeks embryo. The microscopic structure of the olfactory bulb in adult man, however, except for the absence of the accessory olfactory formation (Humphrey and Crosby, ’38 ; Crosby and Humphrey, ’39 a, ’39 b), follows in all essentials the lamination pattern characteristic for such mammals as marsupials, rodents, carnivores, ungulates, insectivores, chiropteres, and other primates as described by numerous observers (Ramon y Cajal, ’11; Winkler and Potter, ’11 and ’14; McCotter, ’12; Herrick, ’24; Gurdjian, ’25; Obenchain, ’25; Hines, ’29; Le Gros Clark, ’31; Humphrey, ’36; Young, ’36; Crosby and Humphrey, ’39 b; Fox, ’40, and many others). The accessory olfactory formation, or accessory olfactory bulb as it is also termed, is absent not only in adult man, but also in the bat (Humphrey, ’36), in most of the macaque material which has been described (although a minute vestigial structure was found in a young macaque; Crosby and Humphrey, ’39 a, ’39 b), in some reptiles (Crosby, ’17; Ariéns Kappers, Huber and Crosby, ’36; Crosby and Humphrey, ’39 b), and in birds (Huber and Crosby, ’29 ; Ariéns Kappers, Huber and Crosby, ’36; Crosby and Humphrey, ’39 b).

The suggestion of the last named authors (Crosby and Humphrey, ’39 b) that an accessory olfactory formation would probably be present in early human embryos only to disappear at some later time is supported by the fact that the vomeronasal or J acobson’s nerve (connected centrally with the accessory olfactory bulb; McCotter, ’12 and ’17) is present not only in early human embryos (Von Kiilliker, 1882 and 1896; Keibel, ’lO; Schaeffer, ’20 and ’28, and many others), but has been demonstrated in a human fetus as old as 6 months (McCotter, ’15) and Jacobson’s organ has been described often in the adult (Peter, ’01; Mangakis, ’02; Kallius, ’05; Keibel, ’10, and others). However, the relation of this nerve to any specific part of the olfactory bulb in human embryos has not been indicated by these authors, although according to von Kolliker (1882 and 1896), Kallius (’05), and Read (’O8) a branch of the olfactory nerve goes to Jacobson ’s organ. Likewise Hertwig (’15) observed that the nerve from this organ is connected with the “Regio olfactoria.”

DESCRIPTION OF MATERIAL

The present account is concerned with the development of the microscopic configuration of the olfactory and accessory olfactory formations and not with the entering nerve fibers or their areas of peripheral distribution. Since recognition of the accessory olfactory formation itself in the earlier embryos is actually determinable only on the basis of its relationship to the vomeronasal nerve, however, it has been necessary to refer to it and to the general state of development of the organ of Jacobson.

N o attempt has been made to study the development of the nervus terminalis. Nevertheless it should be stated that the vomeronasal nerve as used here is limited to the fibers passing from the vomeronasal organ to a special part of the olfactory bulb (the accessory olfactory formation) and does not include the nervus terminalis as this term is used by such observers as McCotter (’12), Huber and Guild (’13), Johnston (’l3 and ’14), Larsell (’18), and Herrick (’31). These two nerves are so intimately associated in the earlier human embryos studied, however, that it is not always possible to differentiate them. In such early embryos the fibers which terminate adjacent to the area of penetration of the olfactory fila, in a region which later shows the developing lamination pattern of the accessory olfactory formation, are considered to be vomeronasal. Those entering caudal to the developing olfactory bulb are probably part of the nervus terminalis. Such a distinction appears to be in harmony with the recent observations of Pearson (’40).

The primitive olfactory bulb before the lamination pattern begins to develop

14+ mm. embryo (6.5 weeks). At this age the vomeronasal organ is represented by a deep groove extending almost directly medialward into the wall of the developing nasal septum. From the much thicker epithelium of the cephalic (medial) wall of this groove the fibers of the vomeronasal nerve extend forward to the forebrain vesicle. At this time no fibers are contributed from the thinner caudal (lateral) wall of the developing vomeronasal organ.

Neither the primordial olfactory formation nor the primitive accessory olfactory bulb present any evidences of beginning lamination such as occurs in the adult. Instead the wide ependymal zone around the ventricle is separated from a narrow, peripherally situated acellular layer by a region of intermediate width which is formed by cells migrating out from the ependyma. The developing accessory olfactory formation has the same three regions, but the narrow peripheral zone is less definite in character and the intermediate region contains fewer migrating cells. Apparently these three regions just indicated correspond to the ependymal, mantle, and marginal layers described elsewhere in the developing neural tube.

16 mm. embryo (7 weeks). The degree of development of the olfactory areas of the nasal fossa in this embryo is essentially the same as in the preceding one, although the groove forming the organ of Jacobson is less definite in character. As in the first embryo, nerve fibers pass centralward only from the thickened cephalic (medial) wall of the developing Jacobson’s organ (the wall nearer the forebrain). No indication of lamination is to be found in either the primordial olfactory or accessory olfactory formations, both of which show no demonstrable advance in differentiation. These two areas of the primitive olfactory bulb, or pars anterior of the rhinenceph—

alon, are definitely distinguishable from each other only on the basis of the entering nerve fibers.


22.5 mm. fetus (8 weeks). At 8 weeks of menstrual age J acobson’s organ had deepened and elongated sufficiently to appear as a closed—off pocket for a part of its extent, and both its cephalic (medial) and caudal (lateral) walls have a tall lining epithelium. Fascicles of the vomeronasal nerve pass to the rhinencephalon from both the cephalic and caudal surfaces of the Vomeronasal organ in this fetus, as also is the case in all of the older fetuses in this account which are Seetioned in toto.

The vomeronasal fibers enter the developing olfactory bulb just medial and slightly dorsal to the fila olfactoria (fig. 1 B).

ABBREVIATIONS

acc.olf.form., accessory olfactory forma- mant.l., mantle layer

tion marg.l., marginal layer ant.o1f.nuc., anterior olfactory nucleus mit.c.l., mitral cell layer ep., epeudyma o1f.f., olfactory fila ep.l., ependymal layer olf.n., olfactory nerve fibers ext.gran.l., external granular layer olf.v., olfactory ventricle ext.molec.l., external molecular layer outw.n1it.c., outwandered mitral cells glom., glomerulus pr.acc.olf.form., primordial accessory glom.l., glomerular layer olfactory formation int.gran.l., internal granular layer pr.olf.form., primordial olfactory forint.gran.l.(olf.form.), internal granular mation

layer of olfactory formation vest.acc.olf.form., vestigial accessory int.molec.l., internal molecular layer ‘olfactory formation

l.gran. +mit.c., layer of granule and \'on1.n., vomerpuasal nerve fibers mitral cells


fig. 1 Drawings to illustrate the location and structure of the primordial olfactory and accessory olfactory formations in a 22.5 mm. embryo. Activated protargol preparation. A, 9. horizontal section through the forebrain vesicles and the developing nasal fossae to show the plane of the sections and the location of drawing B. X 3. B, the area indicated by the dotted lines in figure A, enlarged to show the structure of the primordial olfactory and accessory olfactory formations. X 30.


Even through the center of the primitive olfactory and accessory olfactory formations as yet no indication of beginning lamination exists. Only the three regions representing the ependymal, mantle, and marginal layers found elsewhere in the early development of the neural tube are present in either area, although a definite advance in development occurs in J acob— son’s organ (see p.— 436). The differences between the developing olfactory and accessory olfactory formations at this age are the fewer cells migrating outward from the ependyma, the slightly lesser width of this zone of migrating cells (mantle layer), the greater width of the peripheral, acellular region (marginal zone), and the coarser, more deeply penetrating nerve fibers in the primitive accessory olfactory bulb as compared with the primordial olfactory formation. At levels through the middle of the developing accessory olfactory formation this structure forms a minute elevation on the surface. Von K6lliker’s description (1882, 1883) of the olfactory lobe at this age also indicates no development of the bulbar lamination pattern.

The olfactory bulb before regressive changes occur in the accessory olfactory formation

26 mm. fetus (8.5 weeks). The vomeronasal organ in the 26 mm. human fetus has elongated and deepened sufficiently to appear as a saccular structure having a definite tube—like connection with the nasal fossa. In both the developing olfactory and accessory olfactory formations some indications of beginning layer formation may be seen (fig. 2, B and C). In each region the wide ependyma and the adjacent area of neuroblasts proliferating from it are still present about the ventricle, but toward the surface changes have occurred. In the olfactory formation (fig. 2 B) numerous cells at the surface form a layer in intimate relation with the entering olfactory fila, which, however, do not extend more deeply. This peripheral .cellular layer is separated from the deeper portion of the olfactory formation by a narrow, less cellular lamina. 438 TRYPI-IENA HUMPHREY

Central to the latter is a relatively wide lamina of cells which is slightly separated from the neuroblasts migrating out from the ependyma by another less cellular region. Later developmental changes in the olfactory formation indicate that the glomerular and external granular layers differentiate in the cellular region of termination of the entering fibers. Thus this zone forms the anlage for the external granular layer, the deeper acellular region is the primordial external molecular layer, the adjacent cellular lamina represents the future


fig.2 Illustrations to show the position and morphology of the developing olfactory and accessory olfactory formations in the 26 mm. fetus. Erythrosin and toluidin blue preparation. A, a horizontal section through the forebrain to indicate the plane of the sections and the location of the areas enlarged in B and C. X 3. B, an enlargement of the region shown within the dotted lines on the left side of figure A, but at a level 80;; inferior to that of A. This drawing illustrates the beginning of lamination in the olfactory formation. X 30. C, the area enclosed in the dotted lines on the right side of figure A, enlarged to illustrate the beginning of lamination in the accessory olfactory formation. X 30.

mitral cell layer, and the neuroblasts still migrating out from the ependyma form the primordial internal granular layer. These indications of lamination are present practically all through the region into which the olfactory fila are entering, although no difference in the size of the cells in the developing layers is as yet demonstrable.

The accessory olfactory formation in this fetus retains its dorsomedial position on the caudal surface of the developing olfactory bulb; there it again forms a definite eminence (fig. 2 C). As compared with the olfactory formation (fig. 2 B) in the same fetus, the accessory olfactory bulb (fig. 2 C) has less numerous entering nerve fibers, fewer cells throughout, and certain minor difierences in the developing lamination pattern. The vomeronasal fibers are coarser than the olfactory fila and many of them penetrate more deeply, but some terminate also in the region of the few cells at the surface. However, the primordial external granular layer in this part of the olfactory bulb is more deeply situated, so that a relatively acellular zone, with only a few cells among the entering fibers, is present at the surface. At this time the primitive external granular layer presents evidences of forming a lamina separated from the deeper layers in only a small part of the accessory olfactory formation. In such regionsa still deeper cell band (the primordial mitral cell layer) may be seen and this in turn is separated from the neuroblasts still migrating out (the developing internal granular layer) from the ependyma by a relatively less cellular zone (the region of the internal molecular layer). In view of the developmental changes which occur in these regions later it is possible to state that the Various laminae of the accessory olfactory formation are represented at this age as indicated by the names given in parentheses above.

26.5 mm. fetus (8.5 weeks). The accessory olfactory formation in this embryo is essentially the same as that in the preceding one of approximately the same size. A similar degree of differentiation is present, although in certain respects somewhat less clearly seen due to the difference in staining of the two series, the pyridine silver impregnation of the series now under consideration being less favorable for cellular study.

32 mm. fetus (9.5 weeks). At this age, although not in two older fetuses at least (35 mm. and 37 mm.), the organ of Jacobson no longer opens into the nasal fossa, but is connected with it only by a solid cord of epithelial cells. According to Schaeffer ( ’20) after 10 weeks of embryonal life Jacobson’s organ “varies considerably” and “complete degeneration in early fetal life may ensue.” Both this author (Schaeffer, ’2O and ’28) and Kallius (’05) state, however, that the vomeronasal organ reaches the height of its development in the twentieth week, an age comparable to that at which a fully differentiated accessory olfactory formation is also found in the present study (18.5 weeks old fetus; see p. 456).

The developing olfactory bulb now extends medialward as well as ventralward. The primitive accessory olfactory formation again occupies its dorsomedial portion and again forms a slight superficial eminence (fig. 3 B). At this time there is a degree of differentiation similar to that seen in this region in the preceding fetus (see p. 438), but for a somewhat greater portion of the area. The most superficial layer of cells constitutes the external granular layer, a lamina which is more deeply situated than in the olfactory formation. In this fetus it is more definitely separated from the deeper layers, however, and consists of cells averaging 6.4;: in their greatest diameter as compared with 8.9 u for the average diameter of the cells of the wider and more definite primitive mitral cell layer. The primordial external molecular, internal molecular, and internal granular layers are essentially unchanged as compared with these laminae in the 8.5 weeks old fetus. It is of interest that in the lateral part of the differentiating accessory olfactory formation the developing mitral cell layer is wider and more distinct than elsewhere although the primitive external granular layer noted medially cannot be identified throughout the same area.

Although having increased greatly i11 both size and ventrafrontal extent as compared with the olfactory formation in the 26 mm. fetus, the olfactory formation of the 32 mm. fetus has no marked advance in the differentiation of its lamination pattern (fig. 3 C). In fact there is even less indication of a primordial internal molecular layer. Thus the region of the primitive mitral cell layer is in continuity, for the most part, with the neuroblasts migrating out from the ependyma to form the internal granular layer. The cells in the region allocated to the mitral cell layer, however, are somewhat more compactly arranged and now average 8.6 u in size as compared with 7.2 u for tl1ose more deeply located. At this age the fila olfactoria are more intimately associated with the cell layer at the surface, the external granular layer. Actually the two appear as a single band of intermingled cells and fibers although the proportion of cells is greater on the deeper sur— face. As at all earlier ages considered the developing olfactory formation is much more cellular than is the primitive accessory olfactory formation in the same fetus.

35 mm. fetus (9.5 weeks). The developing lamination pattern of the accessory olfactory formation is not only more clear than is that in the preceding fetus but also more extensive. A slight eminence on the surface continues to mark the


fig. 3 Drawings to illustrate the olfactory and accessory olfactory formations in the 32 mm. fetus. Activated protargol preparation. A, a diagram of a. horizontal section through the entire fetus to indicate the plane of the sections and the areas enlarged in B and C. X 3. B, an enlargement of the accessory olfactory formation from the area enclosed in dotted lines in figure A. X 30. C, a drawing of the olfactory formation from an area comparable to that of figure B, but from a section 380 u inferior to the plane of that figure. X 30.

outline of the accessory olfactory formation. In its best differentiated part the primordia of those laminae previously observed in this region are now demonstrated more clearly. A comparable difference in size (2.5 u) between the cells of the primordial mitral cell layer and those of the developing external granular layer, observed in the 32 mm. fetus, also is found here; the former average 8.7 u, the latter 6.2 u in diameter. The apparent decrease in the size of the cells may be due to the greater shrinkage in the present preparation, which is stained with pyridine silver. 442 TRYPIIENA HUMPHREY

Except for an increase in size and the marked extension medially as well as ventrally, the olfactory formation in the 35 mm. fetus has not advanced in development. In fact, the slightly larger size of the cells forming the primordial mitral cell layer, as compared with the developing granule cells, cannot be noted here (possibly due to the type of preparation), although a similar difference in size can be observed in the developing accessory olfactory formation.

.37 mm. fetus (10 weeks). Although the opening is narrow, the organ of Jacobson retains its tubular connection with the nasal fossa in this fetus as well as in that of 35 mm. crownrump length. In other respects it resembles the organ of Jacobson in the 32 mm. fetus, in which, however, the connection with the nasal fossa has been lost (see p. 439).

The lamination pattern noted in the olfactory and accessory olfactory formations in the younger fetuses is now much plainer. The surface eminence formed by the accessory olfactory formation is still demonstrable (fig. 4B). The more lateral portion of the vomeronasal nerve sends fibers even farther lateralward near the surface after entering the accessory olfactory formation (fig.' 4 B). In the region nearest the olfactory formation (fig. 4 C) the mitral cell layer extends for a considerable distance lateralward to come into relation with the anterior olfactory nucleus (see p. 444). Although a comparison of figure 4 C with figure 9 B (both of which are drawn at the same magnification) appears to indicate that the accessory olfactory formation in the 37 mm. fetus is larger than in the 145 mm. fetus this appearance is probably due in part at least to differences in the plane of section.

The laminae of the accessory olfactory formation are now much better differentiated (fig. 4, B and C). The external granular layer is less compact in nature but wider and more definitely demonstrable. Again its cells are distinctly smaller than are those of the mitral cell layer. The average diameter of the cells in the mitral cell layer is 8.9 u as compared with 7.0;: for those in the external granular layer and 6.7 u for those in the primitive internal granular layer. Numerous fibers of the Vomeronasal nerve extend directly inward to the external granular layer; a few pass medially and laterally among the scattered cells just within the surface. Some mitral cell processes extend out into the external granular layer also. The developing external molecular layer is now wider and definitely separates the external granular and mitral cell


fig.4 Drawings to illustrate the olfactory and accessory olfactory formations in the 37 mm. fetus. Activated protargol preparation. A, a diagram of a horizontal section through the fetus to indicate the planeof the sections and the location of the detailed drawings which follow. X 3. B, an enlargement of the more dorsamedial portion of the accessory olfactory formation, the area shown within the dotted lines in figure A. X 30. G, the accessory olfactory formation on the same side of the brain, as seen in a section 100p. inferior to A and B. X 30. D, a section through the olfactory formation, 480;: inferior to figure C. X 30.

layers. The mitral cell layer itself is the most clearly differentiated of all the laminae. It appears as a relatively wide band of neurons of larger size than the remainder of the cells in the accessory olfactory formation. That these cells do not exhibit the shape typical for mitral cells in the adult is probably due to the relatively small amount of cytoplasm present as yet. In the region of entrance of the vorneronasal fila the mitral cell layer is broken up and less clear, but a more distinct lamina extends lateralward from this area. Here the mitral cell layer is thicker and more definite, a condition in harmony with the greater number of fibers entering this region. In the more lateral part of the accessory olfactory formation, however, the developing external granular layer is less clear than in the more medial portion (fig. 40). Central to the mitral cell layer the internal molecular layer consists of a narrow zone in which there are relatively few cells. The primitive internal granular layer at this age is still represented by almost uniformly scattered small neuroblasts migrating out from the ependyma.

As the mitral cell layer of the accessory olfactory formation extends lateralward, its clarity in outline decreases and there appears deep to it a second cell band (fig. 4 C) which becomes more distinct farther lateralward. The cells in this lamina average 8.1 u in diameter, a size intermediate between that of the mitral cells (8.9 H) and the granule cells (6.7 u). From its position and other relations, this deeply situated cell layer represents the anterior olfactory nucleus.

The olfactory fila enter the olfactory formation (fig. 4D) frontally on its ventral, medial, and lateral surfaces. A greater degree of cellular proliferation than is observed in the accessory olfactory formation continues to exist in the olfactory formation of this fetus. The highest differentiation is present in the areas of entrance of the nerve fibers. There is little change in the lamination pattern as yet as compared with that in the 32 mm. fetus. No clumping of cells (average size, 7.6 u) can be observed in the internal granular layer, into which many neuroblasts are still migrating from the ependyma. The most definite change is in the mitral cells, some of which are now beginning to assume a more triangular outline and average 11.0 p in diameter as compared with 8.6 p in the 32 mm. fetus. They do not form a sharply defined layer either deeply or superficially, however, and evidence of an internal molecular layer is present only rarely. Developing mitral cells are found either singly or in small groups within the external molecular layer. Rarely such a cell is present even in the inner border of the cell layer at the surface of the olfactory formation, the external granular layer. As yet no glomeruli have developed.

The olfactory bulb after regressive changes appear in the accessory olfactory formation

49 mm. fetus (11 weeks). According to Von Kiilliker (1882) at the age of 3 months the olfactory bulb is directed posteriorly as well as downward but later extends medialward and


fig. 5 Drawings illustrating the olfactory and accessory olfactory formations in the 49 mm. fetus. Activated protargol preparation. A, an outline drawing to indicate the plane of the sections and the location of figures B and C. X 3. B, a detailed drawing of the right accessory olfactory formation from the area enclosed in dotted line on the left side of figure A. X 30. C, a drawing of the left olfactory and accessory olfactory formations taken from the area enclosed in dotted lines on the right side of figure A. X 30.

forward. Such a position for the developing olfactory bulbs is observed in this and in the next two fetuses studied. In the fetuses of 15.5 weeks of menstrual age or over the bulb is directed frontally.

The most striking advance in the development of the olfactory formation (fig. 5 C) occurs in the mitral cell layer. Although there has been no significant increase in the size of 446 TRYPJIENA HUMPHREY

the mitral cells, 11.3 in in diameter as compared with 11.1 p in the 37 mm. fetus) and but little more separation from the internal granular layer of the bulb, the mitral cells are now arranged in the sectors seen iii the olfactory bulbs of all older specimens studies (see pp. 447, 449, 452 and 454) and in the adult (Humphrey and Crosby, ’38; Crosby and Humphrey, ’39 b). Two such sectors, concave toward the surface of the bulb, are illustrated in figure 5 C. Outwandered mitral cells are present in the external molecular layer and, more rarely, at the border of the external granular layer. Glomeruli have not as yet begun to differentiate.

The accessory olfactory formation (fig. 5, B and C) is about equally well developed on both sides of the brain and on each side regressive changes have begun to appear. The mitral cell layer is more compact and clearly delimited although the component neurons have not changed significantly in size (8.4 u as compared with 8.9 u in the preceding fetus). Neither accessory olfactory formation has an external granular layer such as is present earlier and glomeruli are not differentiated as might be expected had further development occurred. Deep to the mitral cell layer is the anterior olfactory nucleus in which the slightly smaller cells are more loosely arranged than are those in the mitral cell layer. 011 both sides of the brain, although more clearly on the left (fig. 5 C), the medial part of the mitral cell layer is in intimate relation with the anterior olfactory nucleus, often appearing either to be a part of it or to turn deeply to join it.

61.5 mm. fetus (12 weeks). In the 61.5 mm. fetus the olfactory ventricles are still patent throughout although proportionately smaller in size. All the layers of the olfactory formation (fig. 6 B) cannot be recognized definitely as yet, but certain advances in lamellar differentiation have taken place. Among the most noteworthy of these is the greater number of outwandered mitral cells and the change in the shape of many mitral cells to the more typical triangular outline of the adult neuron. l\Ieasurement of the mitral cells of the olfactory formation now gives an average of 11.5 u, not enough of an increase in size to be significant. Another advance in development is the greater clarity in the arrangement of the mitral cells in the various sectors which were first noted in the 49 mm. fetus (see also pp. 452 and 454). The dorsal and ventral sectors are the most prominent, but lateral and medial ones are also present. A glomerular layer has not yet appeared. The external molecular layer contains many mitral cells and fewer cells of the granule type. The internal molecular layer,

mi1.c.I.<————accoIf.form.




A

oulwmitc.

fig. 6 Illustrations of the olfactory and accessory olfactory formations of the 61.5 mm. fetus. Activated protargol and erythrosin preparation. A, an outline drawing of a section through the anterior commissure to show the plane of section of the series and the general location of the detailed figure which follows. X 3. B, the left olfactory and accessory olfactory formations enlarged from the area within the dotted lines in figure A. X 30.

although narrow, may be seen in such areas as the middle of the sectors of the mitral cell layer, but it is not distinguishable elsewhere. N o clumping of granule cells, such as is present in the adult, is demonstrable in the internal granular layer and neuroblasts are still migrating out into it from the ependyma.

The accessory olfactory formation (fig. 6B) of this fetus exhibits, on the Whole, degenerative rather than developmental changes. An approximately equivalent degree of differentiation is present on both sides although the difference in the angle at which the two olfactory bulbs are sectioned (see fig. 6A) makes comparison difiicult. Apparently the accessory olfactory formation on the left side of figure 6A is cut in a plane approaching the sagittal; the one on the right side, however, is sectioned more nearly frontally.

The accessory olfactory formation on the left side (fig. 6 B) is situated at the angle at which the developing olfactory bulb turns medialward and, at this age, caudalward (although later frontally; see p. 445). Most of the vomeronasal fila enter it at this point of turning although some enter both cephalic and caudal to the area in question. The most clearly developed part of the accessory formation is situated just dorsal to the ventricle in figure 6 B. Both ends of the curved band of cells in this region come into relation with the anterior olfactory nucleus on its deeper aspect. At the level illustrated the cells in this layer are larger (8.7 p) at the narrow end of the lamina and grade off into smaller ones (6.1 u) at the thicker end of the layer, the part in more intimate contact with the anterior olfactory nucleus in this figure. Between the two parts is a zone intermediate as to cell size. Superficial to this cell layer are uniformly scattered cells, which, although equal in size (8.9 p) to the larger ones in the cell layer deep to them, have a different staining reaction. Cells of this same type, which are present throughout the area of entrance of the vomeronasal fibers in figure 6B, and in the more frontal area into which fibers penetrate at other levels, obviously belong to the accessory olfactory formation. At least part of the curved cell lamina represents the mitral cell layer of the accessory olfactory bulb, but the portions which pass over into the anterior olfactory nucleus without any demonstrable change appear either to have failed to develop sufiiciently to form a mitral cell layer and to have acquired instead the characteristics of the anterior olfactory nucleus, or to be undergoing regressive changes such that they are taking on the characteristics of this center. Superficial to the mitral cell layer the scattered cells probably represent cells of the external granuDEVELOPMENT or HUMAN OLFACTOBY BULB 449

lar layer which have increased in size with the changes in the mitral layer and become uniformly scattered through the area.

The right accessory olfactory formation resembles the left in all essentials. The plane of the sections of this olfactory bulb permits observation of the fact that the vomeronasal fila enter on the dorsomedial surface and that many of them extend across the dorsal aspect of the area after entrance. Cells of the same type as those in the area into which fibers are entering in figure 6 B are scattered along these fibers. At one level, in the medial part of the field and again less clearly lateralward, these cells are clumped into a mass resembling the vestigial accessory olfactory formation illustrated in figures 7 C, 7 D, and 8 A for the 85.5 mm. and 112 mm. fetuses.

85.5 mm. fetus (14 weeks). At 14 weeks of menstrual age the olfactory formation is but little changed as compared with that of the 12 weeks old fetus. The only significant differences are the clumping of the granule cells in a Very limited portion of the peripheral part of the internal granular layer and the onset of closure of the olfactory ventricle by the growth of cells into it from the ependyma. The sector arrangement of the mitral cells first observed in the 11 weeks old fetus is present here also, although less clearly defined in this case than in either the 12 or 15.5 Weeks old fetuses.

A rudimentary accessory olfactory formation is demonstrable on both sides of the brain. On the left side three different stages in the regression of this structure are present. Dorsomedially it is possible to trace the vomeronasal nerve to a very limited area (fig. 7 A) similar to the accessory formation in the 49 mm. fetus; this area is comparable to the medial part of the mitral cell layer illustrated in figure 5 0. Here, in addition to the mitral cell layer which is present in the accessory olfactory formation in the 49 mm. fetus, a layer of smaller cells representing the external granular layer is situated more superficially and extends farther lateralward. A second vestige (fig. 7 B) of the accessory olfactory formation is represented by a single small glomerulus resembling the left accessory olfactory formation of the 112 mm. fetus (fig. 450 TRYPHENA I-IUMPHREY

8 B). The position of this glornerulus, into relation with which Vomeronasal fibers can also be traced, is equivalent to the middle of the mitral cell layer in figure 5 C. It has a frontecaudal extent of approximately 50 u, a mediolateral diameter of about 70 u, and consists of a single glomerulus with its accompanying intra— a11d periglomerular cells. None of the cells, however, have acquired the character of mitral cells.





fig. 7 Drawings of the vestigial remains of the accessory olfactory formation of an 85.5 mm. human fetus. Activated protargol preparation. X 30. A, the best developed part of the left accessory olfactory formation. This area is comparable in its location to the most medial portion of the accessory formation illustrated in figure 5 C. B, vestiges of the accessory olfactory formation in the form of a single glomerulus located in an area comparable to the middle of the accessory olfactory formation of figure 5 C. C, the undifferentiated remains of the accessory olfactory formation found at the region corresponding to the most lateral part of the accessory olfactory formation of figure 50. D, the vestiges of the right accessory olfactory formation. The area drawn is comparable in position to the middle of the accessory olfactory formation of figure 5B.

The third vestige (fig. 7 C) of the accessory olfactory formation is located still farther lateralward in a region corresponding to the most lateral portion of the mitral cell layer of the accessory formation in figure 5 C. It is slightly larger than the glomerulus just described and resembles the rudiment of the accessory olfactory formation on the right side of the brain in this fetus (fig. 7 D) and in the 112 mm. fetus (fig. 8 A). On the right side, however, the vomeronasal nerve is definitely in relation with the mass of cells. Toward the more frontal end of the right bulb scattered cells with no particular arrangement also come into relation with the entering vomeronasal fila. Although a few sections of the right bulb are missing from the series through this area, such sections are scattered sufiiciently so that no further representation of the accessory olfactory formation is believed to be present. In that case it would appear that the accessory olfactory formation on the left. side of the brain in this fetus is more clearly represented than is that on the right side—a situation noted also in the two older fetuses described (112 mm. and 145 mm.; see pp. 452 and 458).

112 mm. fetus (15.5 weeks). At this age the olfactory ventricle (fig. 8) is still patent in parts of the bulb although comparatively narrow and in many places partially obliterated. For example, in some sections there are two or three small openings instead of a single large one and the remainder of the ventricle is represented either by ependyma or completely obliterated. In other sections only ependymal remains of the ventricle are present. Caudalward, in the region of the olfactory crus, a definite, slit—like ventricle is in continuity with the anterior horn of the lateral ventricle. The olfactory bulb itself is considerably elongated and flattened with a clearly developed crus. It is now directed cephalad (see p. 445).

Although the olfactory formation has not yet attained the degree of differentiation found in that of the 145 mm. fetus, certain distinct advances in development are present (fig. 8). The olfactory fila enter the bulb on all surfaces at its most frontal tip, but in following the series caudally they disappear progressively from the dorsal, lateral, ventral, and medial surfaces respectively, remaining farthest caudalward on the ventromedial aspect as in the macaque (Crosby and Humphrey, ’39 b). Glomeruli are beginning to appear in a few regions deep to the zone of entering fibers on the ventral surface, but no definite glomerular layer is as yet present. Mitral cell dendrites can be traced into many of the developing glomeruli identified. Because of the small number of glomeruli which have developed and the scarcity of granule cells in the region, the external granular layer is still represented primarily by the cells situated deep to the entering fila. The external molecular layer varies in width with the depth of the mitral cell layer and contains many cells of both mitral and granule cell types. The mitral cell layer is more prominent than in either the 145 mm. fetus or in the adult and exhibits Very clearly the arrangement into sectors which began to appear in the 49 mm. fetus. These sectors present a concavity superficially and are best defined just caudal to the accessory olfactory formation. In the middle of each sector the mitral cells are more compactly arranged and the layer is thicker. At the ends, where two sectors join each other, the mitral cells are fewer in number and more scattered. The dorsal sector appears farthest frontalward, extends less far caudally than any of the other sectors, and its neurons are more loosely arranged. The mitral cells now average 12.9 n in diameter, are more definitely triangular in outline, and contain a considerable accumulation of Nissl substance. The layer is usually two or more cells thick with the neurons frequently arranged in small clumps. The internal molecular layer, like the external, varies in width and clearness. It contains many granule cells and a few mitral cells. In the internal granular layer the cells at the periphery are arranged in little clumps between which are small fiber bundles. Centrally such an arraxigement is not present, the granule cells there appearing uniformly distributed.

Vestiges of the accessory olfactory formation may be recognized on both sides of the brain in spite of the fact that marked regressive changes have taken place. It is only on the left side, however, that a discrete area with any organized structure is still retained (fig. 8B). Here the minute vestigial accessory olfactory formation appears as a tiny eminence about 170 p in diameter and of approximately the same frontocaudal extent. It is situated near the middle of the area occupied by the dorsal sector of the olfactory formation farther frontalward, a position comparable to that of the vestigial accessory olfactory formation in the young macaque (Crosby and Humphrey, ’39 b). Morphologically this accessory olfactory formation consists of one to three glomeruli, deep to which small mitral cells (10.1 n) and a few granule cells are intermingled in the form of a limiting layer. A few scattered granule cells in continuity with the internal granular layer of the olfactory formation are found beneath the layer of mitral and granule cells. In structure, then, as well as in location, this rudimentary accessory olfactory formation is strikingly similar to the vestigial one found in the young macaque, the only significant difference being the greater number of mitral



fig.8 Illustrations of frontal sections of the olfactory bulbs of a. 112 mm. fetus. Erythrosin and toluidin blue preparation. X 30. A, the right olfactory bulb to show the course of the vomeronasal nerve within its surface and the cells along it which are the only demonstrable vestiges of the accessory olfactory formation. The illustration of the course of the vomeronasal nerve with the accompanying neurons was made by combining the features observed in six consecutive 15p. sections. B, the left olfactory bulb showing a vestigial accessory olfactory formation similar to that observed in the young macaque. Both the plane of the section and its frontocaudal location differ slightly from those of figure A.

cells present. Recognition of the relationship of the vomeronasal fila to this area is not easy since the fibers have become incorporated within the surface of the bulb (see also pp. 449 and 454). The compact arrangement and deeper staining reaction of these fibers, however, make it possible to trace them from the medial surface of the bulb to a position near the vestigial accessory olfactory formation, but the discrete nature of the bundle is lost before it reaches the accessory olfactory formation itself.


On the right side of the brain the accessory olfactory formation (fig. 8 A) has undergone still greater regressive changes, so that the presence of any representation of it at all might be questionable were it not for the relationship with the vomeronasal fila and the presence of similar vestiges in other fetuses (see pp. 450, 451, and fig. 7, C and D). The vo1neronasal nerve, however, is more readily followed than on the left side. It enters the olfactory bulb dorsomedially as a compact bundle (fig. 8A) which extends lateralward just within the dorsal surface to a point somewhat beyond the middle of the bulb. Along this nerve bundle, in addition to scattered cells, there are also two to three little clumps of more deeply staining neurons in relation with which the nerve apparently terminates. These scattered neurons and the small cell groups along the vomeronasal nerve within the olfactory bulb are the only vestiges of the right accessory olfactory formation, which is, then, even less well represented than is the left.

145 mm. fetus (18.5 weeks). The lamination pattern of the olfactory formation in the 18.5 weeks old fetus under consideration is in every respect as well differentiated as is that in the adult bulb (Humphrey and Crosby, ’38) and in some respects even plainer (see p. 455), although the olfactory bulb itself is smaller in size (fig. 9, A and B). As in the adult, the olfactory ventricle is no longer patent in the bulb, but in the caudal part of the olfactory crus in this fetus it is present and retains its connection with the lateral ventricle.

The olfactory fila (fig. 9, A and B) surround the frontal tip of the bulb and enter it on all sides although the dorsal portion of the fibers enter only quite far cephalad. The fila situated lateralward do not extend so far caudalward as do the ventromedial bundles which are still present caudally after all others have entered the bulb. Thus the arrangement of the olfactory fila into the four sectors, noted both in the adult bulb (Humphrey and Crosby, ’38) and in the preceding fetus, is also present here. There is a marked infolding of the mitral cell layer in the ventral, lateral, and medial sectors of the bulb and the mitral cell layer in the dorsal sector is more clearly defined as is the case also in the olfactory formation of the pig (Crosby and Humphrey, ’39 b). At this age there are proportionately fewer outwandered mitral cells present in the external molecular layer than occur in the adult, so that the mitral cell layer is more definite. More outwandered mitral cells are present in this fetus, however, than in the preceding one. The average size of the mitral cells at this time is 19.2 p.


fig.9 Drawings of frontal sections through the olfactory and accessory olfactory formations of a 145 mm. fetus. Pyridine silver preparation. X 30. A, the left olfactory bulb cephalic to the region of the accessory olfactory formation. B, the left olfactory bulb at a level through the middle of the accessory olfactory formation and the frontal tip of the anterior olfactory nucleus. 0, the right accessory olfactory formation in the region of its better difierentiated cephalic portion. D, the right accessory olfactory formation at the level of its less well developed caudal portion.



A glomerular layer is now represented on all aspects of the olfactory formation, but it is better developed on the ventral and lateral surfaces (fig. 9, A and B). In regions in which it is not represented the granule cells deep to the entering fibers are now markedly increased in number. Both peri- and intraglomerular cells can be distinguished. The granule cells form a more definite external granular layer than is found in the adult olfactory bulb. Occasionally an outwandered mitral cell may be seen in the border of this layer but less often than in the adult. The external molecular layer contains both outwandered mitral cells, singly or in groups, and granule cells; the former are more conspicuous although less numerous than the latter. The internal molecular layer is narrow, poorly de~ fined and often so filled with granule cells as to be difficult to distinguish at all. The internal granular layer is still represented centrally by closely massed granule cells which are uniformly arranged. Peripherally, however, the lamellar arrangement of little clumps of granule cells interspersed with fiber bundles is present for a wider distance than in the 112 mm. fetus. VVith this preparation it is not possible to determine what portion of the cells in the internal granular layer consists of neuroglia.

Laterally, ventrally, and particularly medially (fig. 9A), isolated and rather sharply circumscribed masses of neurons are found in the outer part of the internal granular layer, but not necessarily at the same frontocaudal level. These cells are intermediate in size between the mitral cells and the granule cells and are of a type similar to those found in the anterior olfactory nucleus farther caudalward. It seems likely that these neurons may constitute cell groups in the path of the olfactory tracts and hence be comparable, at least, to the isolated parts of the bulbar anterior olfactory nucleus described in the adult human olfactory bulb (Humphrey and Crosby,'38; Crosby and Humphrey, ’41).

The accessory olfactory formation is present and occupies the same position in relation to both olfactory bulbs, but it does not have the same clearness of delineation and lamination on the right side (fig. 9, C and D) as is manifested on the left (fig. 9B). In following this transversely sectioned series of the brain frontocaudally the accessory olfactory formation first appears at levels in which the most cephalic part of the anterior olfactory nucleus is found. Such levels correspond to those in which the accessory olfactory formation is located in the macaque (Crosby and Humphrey, ’39 b) and in many other mammals. Although situated dorsomedially in early human fetuses, the accessory olfactory formation in the 145 mm. fetus occupies a dorsolateral position, adjacent to the region which has no olfactory formation at these levels. This location is just lateral to that suggested as its possible site by Crosby and Humphrey (’39 b, Y of fig. 6 E) for adult man.

In the fetus under consideration the accessory olfactory formation of the left olfactory bulb (fig. 9 B) is clearly delineated through its middle portion, although less clear frontally and still less caudally. ‘All the laminae typical of this structure in other mammals are present. The vomeronasal fila enter from the medial side of the area. The glomeruli are small as compared with those of the olfactory formation, but form more than a single layer, at least through the best developed part of the accessory olfactory formation. The external granular layer has a few mitral cells adjacent to it and periand intraglomerular cells are present. The external molecular layer, although definite and often equal in width to this layer in the olfactory formation, contains more granule cells among the fibers. The mitral cell layer is wide, as is that of the dorsal sector of the olfactory formation. The mitral cells are not as large as are those in the olfactory formation, having attained an average diameter of only 15.6 u (see p. 455). The internal molecular layer is indefinite because of the many granule cells it contains. The internal granular layer consists only of diffusely scattered granule cells which are continuous with those in the olfactory formation. At the frontal end of the accessory olfactory formation the mitral cell layer is in continuity with this layer of the olfactory formation both medially and laterally. Through its caudal part, however, the accessory forma458 TRYP1-IENA HUMPHREY

tion is bordered medially by an area free of olfactory formation and laterally by a small region in which the continuity of the mitral cell layer with that in the olfactory formation is interrupted by a zone containing scattered neurons of both granule and mitral cell types.

The accessory olfactory formation on the right side (fig. 9, C and D) of the brain, although having about equal frontecaudal and mediolateral dimensions as that on the left side, is much less clearly delimited and fails to show a well differentiated lamination pattern. It is more clearly defined toward its frontal end (fig. 9, 0), however, and in such areas the zone of entering fila may be bordered either by an area resembling a large glomerulus or only by a wide band of granule cells which forms the external granular layer. In a part of the accessory formation (fig. 9 C) an indefinite mitral cell layer is distinguishable, but in the remainder of the region (fig. 9D) a wide layer of granule cells with a few small mitral cells is separated from the internal granular layer of the olfactory formation only by a zone containing a few scattered cells. In many areas, and especially farther caudalward, the neurons are all of about equal size although a few larger ones resembling mitral cells may be present.

SUMMARY

1. The development of the olfactory and the accessory olfactory formations was studied in thirteen human embryos ranging from approximately 14 mm. to 145 mm. crown-rump length, or from about 6.5 to 18.5 weeks of menstrual age.

2. Differentiation within the primordial olfactory and accessory olfactory formations is first observed at approximately 8.5 weeks of menstrual age. In the series of embryos studied, differentiation in the two regions appeared at the same time.

3. The accessory olfactory formation is represented in embryos from 6.5 to 18.5 weeks of menstrual age, although beginning at 11 weeks regressive changes occur in this region on one or both sides of the brain.


4. In an 18.5 weeks old fetus a fully differentiated accessory olfactory formation is present on the left side of the brain although not on the right and only vestiges of the structure may remain in still younger fetuses. Such vestiges vary from a tiny structure consisting of two or three typical glomeruli with granule and small mitral cells through a single small glomerulus with no mitral cells to clumps of neurons along the course of the vomeronasal nerve within the olfactory bulb.

5. The position of the accessory olfactory formation Varies from dorsomedial with relation to the primitive rhinenceph— alon to dorsolateral with reference to the olfactory bulb at 18.5 weeks of menstrual age. At intermediate ages vestiges of the accessory olfactory formation often occupy a middorsal position.

6. Differentiation of the lamination pattern is apparently slightly more rapid as Well as more clear cut in the accessory olfactory as compared with the olfactory formation. In the latter region, however, there is a greater proliferation of neuroblasts from the ependyma, particularly in the earlier stages.

7. From the time differentiation is first observed in the series of embryos considered, representation of all of the laminae in the adult olfactory and accessory olfactory formations, with the exception of the glomerular layer, is present. This layer, which is the last to develop, does not appear until after lamellar differentiation is present in the superficial part of the internal granular layer.

8. An arrangement of the mitral cell layer of the olfactory formation into sectors first appears at 11 weeks of menstrual age and is demonstrable thereafter in all of the fetuses studied.

9. A difference in the size of the developing mitral and granule cells is first observed in both the olfactory and the accessory olfactory formations at 9.5 weeks of menstrual age. In the olfactory formation there is a progressive increase in the size of the mitral cells from that time onward. In the accessory olfactory formation regressive changes are accompanied by failure of the mitral cells to increase further in size.


10. Outwandered mitral cells are definitely present as early as 11 weeks of menstrual age and occur throughout later stages of development although they are proportionately less numerous than in the adult. The mitral cell layer of the olfactory formation, however, is more clearly delineated at 18.5 weeks of menstrual age than in the adult.

11. Closure of the olfactory ventricle by growth of cells into it from the ependyma begins first at 14 Weeks of menstrual age. Except in the caudal part of the crus, closure is complete at 18.5 Weeks.

12. The anterior olfactory nucleus appears earliest in rela— tion with the accessory olfactory formation. In at least certain cases in which regression occurs in the accessory olfactory formation, some of its mitral cells appear to become a part of the anterior olfactory nucleus.

DISCUSSION AND CONCLUSIONS

In the series of fetuses considered, differentiation of the primordial olfactory and accessory olfactory formations begins at the same time, about 8.5 weeks of menstrual age (26 and 26.5 mm. embryos). It is of interest that the beginning of differentiation occurs at approximately the same age that fetal activity is first shown in the cinematographic records of Hooker (’36 and ’39a; fetus With a crown-rump length of 25 mm. or between 8 and 8.5 weeks of menstrual age). This initiation of differentiation in the rhinencephalon simultaneously With the appearance of fetal activity is in accordance with the observations of Coghill (’28) on Amblystoma that “In the early—flexure stage the centers of difierentiation have become active in the tectum, the epithalamic, thalamic, infundibular, and rhinencephalic regions.”

The accessory olfactory formation, or its vestigial remains, may be demonstrated in human fetuses up to the age of 18.5 weeks. At this time it may have attained a degree of differentiation comparable to that of the accessory bulb of many other adult mammals, that is, typical glomerular, external granular, external molecular, mitral cell, and internal molecular layers may be observed. Before this age is reached, however, and in fact usually before complete differentiation of all its laminae is attained, regressive changes have often appeared. As a result of these changes even the recognition of vestiges of the accessory olfactory formation may be difficult in some fetuses although in others a definite, but tiny rudimentary structure similar to that found in the young macaque is present. From the material available it is not possible to state whether or not all vestiges of the accessory olfactory formation may have disappeared completely in some cases before the age of 18.5 weeks.

Although the accessory olfactory formation may attain a morphologic pattern typical for this structure in one olfactory bulb, it may not reach this degree of differentiation in the other olfactory bulb of the same fetus (18.5 weeks). There appears to be a tendency for the better developed accessory olfactory formation to be retained on the left side of the brain in older fetuses (85.5 mm., 112 mm., and 145 mm.). Although the number of cases in which any distinct diflerence can be noted is too few to be significant, it is more than suggestive of a difference in the rate of development on the two sides of the brain. It is of interest that Kallius (’05) noted a similar difference between the left and the right organ of Jacobson in a 22 weeks old fetus. The left remained fully developed, but the right had lost its connection with the nasal cavity and consisted only of a blind pocket.

Throughout its early development the accessory olfactory formation exhibits a lesser degree of cellular proliferation than is observed in the olfactory formation. This would indicate a slight initial lag in development in the accessory olfactory formation, although later complete differentiation appears to be present before it is found in the olfactory formation (see p. 463). In the earliest stages studied, and wherever a vestige showing definite olfactory structure remains, the accessory olfactory formation appears as an eminence on the surface of the developing olfactory bulb. In the 18.5 weeks old fetus it occupies a dorsolateral position with relation to the olfactory bulb, although in the early embryos it has a medial location and at 15.5 weeks the vestigial accessory olfactory formation occupies a middorsal position. This apparent change in position is possibly the result of rotation of the bulb as it turns first caudalward, then frontally, during development.

It is of interest that the history of the accessory olfactory formation as observed here parallels that of J acobson’s organ in human embryos. The earliest indications of regression in the accessory olfactory formation are found at 11 weeks of menstrual age, while according to Schaeffer (’20) Jacobson’s organ “varies considerably” after 10 weeks. Since, according to Schaeffer (’20 and ’28) and Kallius (’05), J acobson’s organ attains its highest development in the twentieth week in man (if it does not degenerate earlier) the accessory olfactory formation might be expected to be fully differentiated at a similar age. Such is the case in the left accessory olfactory formation of the oldest fetus observed (18.5 weeks), although definite dedifferentiation has occurred on the right side in this fetus as well as in both accessory formations in younger fetuses (14 and 15.5 weeks old fetuses, for example).

The laminae of the olfactory and accessory olfactory formations develop in a similar manner. first marginal, mantle, and ependymal layers, such as are found elsewhere in the neural tube, appear. From the mantle layer cells migrate outward to form two laminae, separated from each other and from the neuroblasts still migrating out from the ependyma by two less cellular zones. The inner of the two cellular bands forms the mitral cell layer and the neuroblasts which continue to migrate out from the ependyma become the internal granular layer in both the olfactory and the accessory olfactory formations. The outer cellular band, which forms the external granular layer in each case, is situated at the surface just beneath the entering fila olfactoria in the olfactory formation. In the accessory olfactory formation it is situated more deeply, being separated from the surface by a less cellular area. Many of the vomeronasal fila penetrate to this layer, however, as is DEVELOPMENT OF HUMAN OLFACTOBY BULB

the case with the olfactory fila. A few cells are also found at the surface in relation with other entering vomeronasal fibers. The glomeruli of the olfactory formation develop in relation with the external granular layer. Whether or not this is true in the accessory olfactory formation it is not possible to state definitely, although such appears to be the case. The glomerular layer itself is the last lamina to develop in both the olfactory and the accessory olfactory formations.

A definite explanation for the late development of the glomerular layer is not possible from a study of the material available. It may be that, although at least some mitral cell dendrites extend out into the external granular layer as early as 10 Weeks of menstrual age, the complex dendritic branching which constitutes the glomerulus in the adult (cat, Ramon y Cajal, ’11) does not develop until much later. Perhaps also the granule cells in this region do not participate in the synaptic relations or develop extensive branching of their dendrites until later. At least there is an increase in the number of granule cells deep to the entering fila shortly before the development of the glomeruli. Possibly another factor in the late appearance of the glomeruli may be that complete differentiation of the other bulbar laminae is essential before the synaptic relations between mitral cells and olfactory fibers become concentrated in one region. The substantiation of any explanation will depend, of course, on a studyof Golgi preparations.

The primordia of all the laminae in the accessory olfactory formation are more clear cut than are those in the olfactory formation in early development. Apparently all the laminae are represented earliest in the accessory olfactory formation, since in this structure in the 15.5 weeks old fetus typical glomeruli are present although glomeruli are only beginning to develop in the olfactory formation. However, there is not suflicient evidence on this point.

A difierence in the average size of the mitral and the granule cells in the accessory olfactory formation appears as early as 9.5 weeks of menstrual age and increases in clarity in all 464 TRYPHENA I-IUMPHREY

cases in which development has continued in the accessory olfactory formation. A difference in the size of the developing mitral and granule cells in the olfactory formation occurs at the same time, but the mitral cells attain a greater size than do those in the accessory olfactory formation.

In the olfactory formation indications of a sector arrangement of the mitral cell layer first appears in the 11 Weeks old fetus. The sectors are most clearly defined at 15.5 Weeks, but are more definite in the 18.5 Weeks old fetus than in the adult olfactory formation. In the older fetuses the dorsal sector is the most distinct and is characterized by a wider mitral cell layer. It is of interest that not only these sectors of the mitral cell layer but also the entire lamination pattern are better developed in the olfactory formation at 18.5 Weeks of menstrual age than in the adult olfactory bulb. Perhaps this is a repetition of phylogeny since the olfactory formation is relatively better developed in subprimates than in primates. It also suggests that regression, which usually has progressed so far in the accessory olfactory formation, is beginning in the olfactory formation as Well by the time adult life is reached.

The external molecular layer is narrow and indefinite in the olfactory formation during early development, but at 11 weeks of menstrual age increases in width. At the same time, with the more definite character assumed by the mitral cell layer, outwandered mitral cells are more readily observed, although they are demonstrable in the 10 Weeks old fetus (fig. 4 D, unlabeled). Although present either singly or in clumps at all older stages observed, the outwandered mitral cells are not as numerous as in the adult.

The internal molecular layer in the olfactory formation is never clearly defined. The internal granular layer first begins to develop clumps of granule cells interspersed With fibers in the 14 weeks old fetus. These occur only at the periphery of the layer at first but in older fetuses extend farther centrally.

In the oldest fetus studied (18.5 weeks), however, the central part of the internal granular layer consists of uniformly distributed cells as in the adult.


The olfactory ventricle remains Widely patent up to 12 weeks of menstrual age. At 14 weeks cells begin to grow into it from the ependyma and at 15.5 weeks of menstrual age it is partially obliterated in many places and in a few regions completely so. At 18.5 weeks the olfactory ventricle is entirely obliterated except for the connection of the crural part with the lateral ventricle. .

The earliest representation of the anterior olfactory nucleus is in relation with the accessory olfactory formation. The position of this part of the nucleus, which is also its most cephalic end, is comparable to that at which its frontal tip is situated in the macaque and in the newborn pig (Crosby and Humphrey, ’39 b). Several isolated masses of cells of similar character, situated farther frontalward, appear to be comparable to the isolated cell masses forming the bulbar part of the anterior olfactory nucleus in the adult (Humphrey and Crosby, ’38; Crosby and Humphrey, ’41). In some cases (49 mm. and 61.5 mm. fetuses) the occurrence of regressive changes in the accessory olfactory formation is accompanied by a more intimate relation of its mitral cell layer with the anterior olfactory nucleus in such a manner as to suggest that with further dedifferentiation this lamina of the accessory olfactory formation may possibly take on the function of the higher center. Details of the development of the anterior

olfactory nucleus, however, are not within the scope of the present paper.

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