JOURNAL OF COMPARATIVE NEUROLOGY 190:115-134 (1980)

Development of the Hippocampal Region in the Rat II. Morphogenesis During Embryonic and Early Postnatal Life

SHIRLEY A. BAYER Laboratory ofDevelopmental NeurobiJJlogy, Department ofBiJJlogical Sciences, Purdue University, West Lafayette, Indiana 47907

ABSTRACT Morphogenesis of the entire hippocampal region was examined in normal rats from embryonic (E) day EIO to E22 and on postnatal (P) days PI, P7, and P21, and was correlated with autoradiographic datings (Bayer, '79c). This region is postulated to form from two connected primordia in the telencephalon, easily recognizable on E16. One lies in the dorsomedial wall and generates a portion of the subiculum, Ammon's horn and the dentate gyrus. The other lies in the dorsoposterior wall and generates the entorhinal cortex and part of the parasubiculum and presubiculum. A cortical plate appears in the presumptive entorhinal region on E17; a cell-sparse fibrous zone appears in the middle of the cortical plate on E19. Lamination in the entorhinal cortex proceeds from lateral to medial and from deep to superficial, with the thickening oflayer III being the last to appear on E22. Lamination in the subiculum starts on E18, but the distinction between superficial laminae in the para- and presubiculum cannot be made until E22. The stratum pyramidale is small on E20 in presumptive Ammon's horn, then greatly lengthens between E21-PI. In early dentate gyrus development, cells migrate from the neuroepithelium near the outgrowth of the fimbria and adjacent to the choroid plexus toward a subpial location. The ectal limb ofthe granular layer appears at the two extremes of the dentate gyrus on E20; theendallimb appears perinatally and develops rapidly to become morphologically similar to the ectal limb by P7.

A volumetric analysis of growth in Ammon's horn (including a portion of the subiculum), dentate gyrus, fimbria, and fornix was made from El5 to P2I. The neuroepithelium increases to a peak volume on E18 and disappears by PI. The subependymal zone becomes distinct on E18, reaches a peak volume on E20 and disappears by P7. Rapid rates of growth in the stratum oriens and strata radiatum, lacunosum-moleculare occur between E16-E17 and between E22-PI. The pyrami­dallayer grows rapidly between E18-E19 (presumptive subicular pyramids) and between E22-PI (presumptive Ammon's horn pyramids). Growth rates of the dentate hilus are rapid prenatally and decline postnatally, while dentate granular and molecular layers maintain high postnatal growth rates. The fimbria and fornix have early (E18-EI9) and late (E21-E22) spurts of growth.

To accurately locate regions ofprimitive migratory and mitotic cells within each lamina of Ammon's horn and the dentate gyrus, the number of cells surviving a single exposure to 200 R X-rays in embryonic brains (EI5-E22) were compared with controls. The neuroepithelium increases its radioresistance from E15-E21 and reaches control levels by E22; the subependymal zone is highly radiosensitive throughout development. In contrast, radioresistance in the stratum oriens and dentate hilus declines and reaches a low point on E20. Over 7rP!o of the cells in the strata pyramidale, radiatum, lacunosum-moleculare, and dentate granular layer are radioresistant throughout development.

0021-9967/80/1901-0115$03.50 © 1980 ALAN R. LISS, INC.

116 SA BAYER

The characteristic folds of the adult mamma­lian hippocampus (including part of the sub­iculum, Ammon's horn, and the dentate gyrus) develop from a slight curvature in the primitive telencephalon, one of the earliest recognizable landmarks in the cerebral cortex (Hines, '22). The ease of its recognition may account for sev­eral descriptive studies of its morphogenesis in a variety of species (man: Hines, '22; Filimonoff, '47; Macchi, '51; Humphrey, '66a, b, '67; opossum and pig: Tilney, '38; bat: Brown, '66; rat: Bayer and Altman, '74; Schlessinger et aI., '75; mouse: Stanfield and Cowan, '79). Cytodifferentiation studies. with the Golgi method have been used in the goat (Godina and Barasa, '64), rabbit (Stensaas, '67a-e, '68), and rat (Minkwitz and Holz, '75; Minkwitz, '76). Taken as a whole, these studies show that the hippocampus develops in basically the same pattern in all mammals. Embryonic develop­ment in the remainder of the hippocampal re­gion (entorhinal cortex, presubiculum, parasubiculum) has been only briefly described by Filimonoff ('47) and Macchi ('51).

Additional methods of low-level X­irradiation and 3H-thymidine autoradiography allow a more complete interpretation of devel­opmental patterns. Low-level X-irradiation (200 R) can be used to locate primitive migra­tory and mitotic cells in the developing nervous system (Hicks and D'Amato, '66; Altman et aI., '68; Altman and Nicholson, '71; Bayer and Altman, '74); if animals are allowed to survive a short time after exposure (6 hours), the pyk­notic fragments of the cells destroyed by irradi­ation are still present, and their locations indi­cate regions of cell migration and/or prolifera­tion. Similarly, if animals are killed within hours after injections oPH-thymidine, zones of mitotic cells can be identified by the uptake of label (Angevine, '65; Stanfield and Cowan, '79). As survival time is progressively lengthened, labelled neuroblasts can be "followed" to their final destination in the hippocampus, as A1tman and Das ('65a, b, '66) and Altman ('66) have shown in the rat dentate gyrus, and Stan­field and Cowan ('79) in the mouse hippocam­pus. Multiple 3H-thymidine injections during development and survival to adulthood can be used to quantify the proportion of neurons formed during specific time blocks, as Bayer and Altman ('74) showed in the rat dentate gyrus.

The first paper of this series (Bayer, '80) gave a detailed chronology of the gradients of neurogenesis in the rat hippocampal region based on 3H-thymidine autoradiography. The

present study applies that information to the interpretation of morphogenesis and laminar development in the entire rat embryonic hip­pocampal region. A volumetric analysis of growth in Ammon's horn (including a portion of the subiculum), dentate gyrus, fimbria, and fornix and the proportion of mature cells in each of their respective cellular laminae, as revealed by X-irradiation, is also correlated with the pattern of neurogenesis.

MATERIALS AND METHODS

Purdue-Wistar pregnant females were used with the day of sperm-positivity designated as embryonic (E) day E 1; birth normally occurs on E23. During the afternoons from E10-E22, em­bryos were removed from two or more control (undisturbed) and experimental pregnant females. The experimental group was exposed to a single dose of 200 R X-rays from a GE Maxitron unit (300kVp; half value layer, 2.4 mm copper) six hours before killing. In addi­tion, groups of three male pups were also killed on the afternoons of postnatal (P) days PI, P7, and P21. Either the entire embryo (ElO-E12), and head (E13-E22), or the brain (P1-P21), was kept in Bouin's fixative for 24 hours, then transferred to 10% neutral formalin. Brains of embryos from E16-E22 were dissected from the head with the aid of an American Optical stereomicroscope after fixation. All specimens were embedded in paraffin. Serial sections (6p.m) were prepared in the coronal (all age groups) and horizontal (embryonic age groups) planes. One set of sections was stained with cresyl-violet, the other with hematoxylin and eosin. Table 1 gives a complete list of the con­trol and experimental embryonic brains avail­able for analysis.

TABLE 1. Brains available for analysis

Controls X-Ray

Age Coronal Horizontal Coronal Horizontal

EIO 11 17 Ell 8 29 EI2 3 9 EI3 3 I 5 3 EI4 3 2 4 4 EI5 6 I 2 5 EI6 6 2 5 2 EI7 8 3 6 4 EI8 4 I 6 2 EI9 6 I 6 2 E20 6 2 6 3 E2I 8 2 5 2 E22 6 5 6 4

117 HIPPOCAMPAL MORPHOGENESIS

To qualitatively analyze the development of the hippocampal region three-dimensionally in both normal and experimental groups, a series of photomicrographs were prepared of repre­sentative brains from E15-E22 in the coronal and horizontal planes. The photographs were taken at regular intervals and were mounted serially in strips. The individual strips were used to simultaneously view morphological changes within the hippocampal region at var­ious levels for each age. Two or more ages were simultaneously compared at homologous levels when several strips were used.

In three coronally sectioned control brains from E15-E22 and from PI, P7, and P21, each section containing the hippocampus (sub­iculum, Ammon's horn, and dentate gyrus; sim­ilar to the photos in Fig. 4) was drawn with the aid of a Zeiss microprojector. The total area and areas of the stratum oriens, stratum pyra­midale, strata radiatum and lacunosum-mo­leculare, dentate hilus, dentate granular layer, dentate molecular layer, fimbria, and fornix were also measured with a Summagraph X-Y digitizer interfaced to a Wang 2200 com­puter. The areal data of the zones measured were graphed for each animal: sectional areas were plotted on the Y-axis, the distance be­tween sections on the X-axis; areas under curves were digitized to determine volumes. Figure 5 shows both sectional area curves and resulting volumetric determinations for the total hippocampus. Homologous sections at an­terior, intermedia te and posterior levels through the hippocampus were selected for each normal and experimental brain cut in the coronal plane (Table 1). In Ammon's horn, dor­sal (solid lines, Fig. 4) and ventral (dashed lines, Fig. 4) strips of tissue (25 (Lm wide) were first divided into zones (neuroepithelium, sub­ependymal zone, stratum oriens, stratum pyramidale, strata radiatum and lacunosum­moleculare); in the dentate gyrus, a single strip (dotted lines, Fig. 4) was divided into the granu­lar layer and hilus. The number of cell nuclei lying at least 500/0 within the strip was counted separately for each zone. Strips from experi­mental brains were subdivided in the same way and surviving (non-pyknotic) cells were counted. Age and treatment differences in cell density were examined with an analysis ofvar­iance; the Scheffe test was used to identify sig­nificant differences between group means. Dif­ferences in radiosensitivity between dorsal and ventral strips and between anterior, interme­diate, and posterior strips were statistically analyzed on paired samples by the sign test

(Conover, '71); the rationale for use of this statistic was given in the previous paper (Bayer, '80).

RESULTS

The position of the hippocampal primordium in the forebrain

On E14, the neuroepithelium in the dor­somedial wall of the telencephalon begins to curve into the lateral ventricle. This curvature becomes more pronounced on E15, and by El6 is a distinguishing feature of the cortical sheet. Figure 1 shows the hippocampal region primordia in both horizontal (Fig. IA) and co­ronal (Fig. IB, C) planes. The hippocampal primordium,1 which will form the subiculum, Ammon's horn, and the dentate gyrus, is dor­sally continuous with the neocortex throughout its length (Fig. IB, C). Anteroventrally, it joins the septal region; at the foramen of Monro and, posteroventrally, it joins the choroid plexus. Posteriorly, it blends with the primordium of the entorhinal cortex (Fig. IA). The anterior border of the entorhinal cortex primordium blends with a more mature telencephalic area, the lateral neocortex primordium. Both hip­pocampal region primordia have a thick zone of neuroepithelium surrounded by a layer of less densely packed cells. It is interesting to note that the S-shaped curve in the adult hippocam­pal region is already apparent in the shape of its horizontally sectioned neuroepithelium (Fig. IA).

Laminar development in the retrohippocampal cortex

Entorhinal cortex The primordium of the entorhinal cortex is

difficult to delineate in coronal sections; con­sequently, this structure was studied only in the horizontal plane. Figure 2 shows middle­level horizontal sections through the hip­pocampal region from EI6-E22. Throughout development, the entorhinal cortex shows a lateraf-to-medial gradient of laminar matura­tion. On El6 (sections in Fig. IA and 2 are the

I In this study, the amount of cortex designated as the hippocampal primordium (Fig. IB, Ie, 4, 8) during early embryonic development (up to E17) is considerably smaller than that assigned in earlier em­bryological studies (Hines, '22; Humphrey, '66a, b, '67). Often, the entire hippocampal primordium was designated as the dentate primor­dium alone, and the future Ammon's hom extended farther into the dorsomedial cortical wall (what would be considered part of the neocor­tical primordium in this study). In the light of more recent :JH_ thymidine autoradiographic studies (see Bayer, '80, for a review), the relatively late neurogenesis in Ammon's horn and the exceptionally delayed appearance of the dentate granule cells indicate that their primordia should be quite small in the early telencephalon.

118 SA BAYER

A bbreuiations

ah, Ammon's horn primordium AB, Ammon's horn CAl, field CAl of Ammon's horn pyramidal cells CA3, field CA3 of Ammon's hom pyramidal cells CO, cortical plate CP, choroid plexus dg, dentate gyrus primoridum DG, dentate gyrus ec, entorhinal cortex primordium EC, entorbinal cortex EL, ectal limb of dentate granular layer EN, endallimb of dentate granular layer FI, fimbria FM, foramen of Monro gl, dentate granular layer primordium GL, granular layer H, dentate hilus

HIPPOCAMPAL 8 REGION

PRIMORDIA

hi, hippocampal primordium HP, hypothalamus Inc, lateral neocortical primordium nc, neocortical primordium NE, neuroepithelium PA, parasubiculum PO, preoptic area PR, presubiculum SE, subependymal zone SO, stratum oriens SP, stratum pyramidale SR, stratum radiatum ST, striatum TH, thalamus TZ, transitory zone WM, white matter

Fig. 1. Brains of E16 embryos in horizontal (A) and coronal (B, C) planes; directional arrows (A, anterior; P, posterior; M, medial; L, lateral; D. dorsal; V, ventral) in A and B give section orientation (paraffin, 6 !Lm, hematoxylin and eosin, bar = 0.5 rom). The section in A grazes the dorsal portion of the striatum (asterisk); approximate locations of the coronal sections are indicated in A. The two primordia that will form the hippocampal region (hi and ec) are in the dorsomedial and dorsoposterior telencephalic walls.

, .,

INTERMEDIATE� HORIZONTAL� SECTIONS�

Fig. 2. Horizontal sections at an intennediate level between the dorsal and ventral extremes of the hippocam­pal region from El6 to E22, the day before birth (paraffin, 6 ",m, hematoxylin and eosin; bar = 0.5 mm; directional arrows give section orientation). El6 section is same as Figure lAo Changes in entorhinal cortex (EC), subiculum (SU), Anunon's horn (AH), and dentate gyrus (DG) can be followed; see text for further details.

same), the zone of cells adjacent to the on E 17. It is separated from the neuro­neuroepithelium is thicker laterally than me­ epithelium by a transitory zone. Scattered dially; a cell-sparse zone (arrow, Fig. 2) is lo­ mitotic figures are found in this zone, indi­cated superficially throughout the primordium cating a subependymal germinal matrix; and will become layer I. A cortical plate devel­ other cells here are spindle-shaped and appear ops deep to layer I in the lateral entorhinal area to be migratory. On E18, the cortical plate is

120 S.A. BAYER

"

-' B. E21; \ ... , , '.- .c.' .~~

,rC.E19 .... '" ~.: -1. D.E20

I· . 1:;.". .-'.",

Fig. 3. Laminar developmental stages in the entorhinal cortex, high magnification views ofareas surrounding dotted lines in E19 (C)-E22 (A) sections, Figure 2 (bar = 0.1 mm).

AN

T�

su

::c ~

I a:: ;F

I ~ o <

;)

t'l ~

o L

+M

V

I

I

E16

~

E22

F

ig.

4.

Cor

onal

sec

tion

s of

a p

orti

on o

f th

e su

bicu

lum

(SU

), A

mm

on's

ho

m (

AH

), de

ntat

e gy

rus

(OG

), an

d fi

mbr

ia (

FI)

at

ante

rior

(A

NT

),in

tenn

edia

te(I

NT

),an

dpo

ster

ior(

PO

ST

)le

vels

from

E16

,E18

,E20

, and

E22

. Pla

cem

ento

find

ivid

uals

ecti

ons

corr

espo

nds

to t

he

angu

lart

ilto

fth

ehi

ppoc

ampu

sin

the

brai

n.D

irec

tion

alar

row

sgi

veor

ient

atio

n(p

araf

fin,

6 jJ.

m,h

emat

oxyl

inan

deo

sin;

bar

=0.

5m

m);

.....

t-.

:lE

16 a

nter

ior

and

post

erio

r se

ctio

ns a

re th

e sa

me

as F

igur

e 1B

, C. A

real

lim

its

of th

ese

sect

ions

(as

teri

sks

give

th

e lo

cati

ons

ofth

e cu

toff

.....

� li

nes)

are

sim

ilar

to th

ose

used

for

tota

l hi

ppoc

ampa

l vo

lum

etri

c de

tenn

inat

ion

(Fig

. 5)

. Sol

id l

ines

in

E22

giv

e lo

cati

ons

ofdo

rsal

str

ips�

an

alyz

ed q

uant

itat

ivel

y in

Fig

ure

10;

dash

ed l

ines

, ve

ntra

l st

rips

; do

tted

lin

es,

dent

ate

gyru

s sa

mpl

ing

stri

ps.�

122 SA BAYER

present throughout the entorhinal area and a cell-sparse fibrous zone, the future white mat­ter, appears beneath it. By E19, the lateral part of the cortical plate bifurcates into a central cell-sparse zone (IV, the future lamina dessi­cans) surrounded by superficial (II, III) and deep (V-VI) cell-rich laminae. From E19-E22, these laminae become larger, while the neuroepithelium and transitory zones become smaller; Figure 3 shows high magnification views of the entorhinal cortex in the areas indi­cated by dotted lines in E19-E22 sections. The neuroepithelium becomes progressively thin­ner at the base of the entorhinal cortex from E19-E21 (Fig. 3B, D) and by E22 is no longer present (Fig. 3A). The transitory zone is thick

on E19 and E20, thins out laterally by E21, and is reduced on E22; this zone disappears in the early postnatal period. The white matter is characterized by a network of fine fibers through which move radially migrating spindle-shaped cells. The cortical plate be­comes increasingly thicker and more lami­nated; on E19 and E20, layer IV is thin with laminae II, III slightly thinner than laminae V-VI. On E21, layer IV is indistinct (Fig. 3B), presumably due to the many spindle-shaped cells throughout it and in layers V-VI; cell is­lands (layer II) appear above layer III. By E22, more cells are added to layer II, layer III is thick and densely packed, and layers IV and V-VI are again distinct. Radially oriented spindle­

,0 ,

Fig. 5. Three-dimensional graph of the data for hippocampal (Ammon's horn, including a part of subiculum; dentate gyrus; fimbria and fornix) growth from E15·P21. Lines along base are mean areas (mm2

) of each section throughout the antero-posterior (A, anterior; P, posterior) extent of the hippocampus in three coronally sectioned brains from normal animals. Area under the sectional area curve for each animal was determined to calculate volume (mm'); data, with mean and standard deviation, are plotted along side wall. Note that scales are logarithmic for both area and volume. Volumetric expansion spans five orders of magnitude (0.00437 mm' on E15 and 32.1 mm' on P21); most rapid growth is between E16 and E17.

123 HIPPOCAMPAL MORPHOGENESIS

shaped cells are still seen in the white matter, layers V-VI, and IV on E22, indicating that cells continue to migrate into the cortex.

Parasubiculum and presubiculum

On E18 and E19, the cortical plate curves sharply anterior toward the subiculum proper and primordial Ammon's horn (Fig. 2). By E20, the bifurcation of the cortical plate ceases to progress medially, signalling the boundary of the entorhinal cortex; the adjacent wedge­shaped non-bifurcated cortex (asterisk, Fig. 2) can now be more accurately delineated as the para- and presubiculum. The two structures cannot be distinguished in the superficial lamina until E22 (Fig. 2). Most of the neurons deep to the para- and presubiculum have been generated by the morning ofE18 (Bayer, '79c); presumably, these cells migrate into the corti­cal plate during E18 and E19, before they can be accurately separated from the cells destined for the entorhinal cortex. The superficial neurons in the para- and presubiculum are ex­ceptionally late (not finished until E20; Bayer, '80), correlating with their delayed appearance in the outer lamina.

Morphological development and volumetric expansion in the hippocampus

From E15 through P21, expansion of Am­mon's horn (including a portion of the sub­iculum), the dentate gyrus, and the fimbria­fornix were analyzed volumetrically (see Mate­rials and Methods). Sections in Figure 4 show typical examples of the structures measured. The cell-sparse zone underlying the pia be­comes wider in the region of the presumptive subiculum proper (not the para- and pre­subiculum) and Ammon's horn. The asterisks in Figure 4 mark the point at which the hip­pocampus was delineated from the rest of the cortex. A three-dimensional' reconstruction of the data for the total hippocampus (Fig. 5) shows graphs of individual section areas on the base; the area under these curves was meas­ured to determine the volume, which is graph­ically represented on the side wall. The most rapid rate of growth occurs between E16 and E17, when the hippocampus increases by 19000/0. From E18 through PI, the daily rate of increase ranges from 66% (E18-E19) to 29% (E19-E20). Between PI and P7, the rate of in­crease is approximately 26% daily; between P7

w C)<{

COMPOSITION OF THE DEVELOPING HIPPOCAMPUS

P21 [:I?~9?~{:J;:;:;:;::::SP:~:::;:;:Jn••.· ·uS~>" ·><~LMdW

P7 [}~:~:}~:}~:}}J:::;~;:;:;:;:;:;:::;:::::;:;:;:V"" ..«~

P1 [~{{{{:I~:I~{:~J: ::::>::1 ···· .. ··········.<mJEJ I E22 rn~:~:}~{:~:~:~:?~:}~{{:}]::::·:·::·· :t .. ·····..« .. >!.m I E21 ~:::::::::::::~:~:}}~:}~:}~{::::n::::··· ::;::: .. :;.;::::;:;! ..... ·<>·········.m. I

E20 ~sE=it}~{{{{:)}~:}~{:}~{:I: ::::::::::.::::j •••••••• > .mm I t i. i. i. "E19 ·....... ~:::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::l.~.:::::::·::r.············.·....... I� ~ · •••••• 0 0 0 •• 0.OJ

E18 ............. [~:~:}~:~:~:~:~:~:~:~I:~:~:~{:~:~:~:::~:~:~:~:~:~:[] .•...• >•• '•.:=JI"""""" ..................................�E17 ....: :.: : :.................. ::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::�

...................... ..�·�E16 ........................·..... .. .. ........................................ ::::::::::::::::::::::::::::::::::::::::::: .

::::

E15

10 20 30 40 50 60 70 80 90 100

PERCENT OF TOTAL VOLUME�

Fig. 6. Relative volume taken up by components of the hippocampus from E15-P21. As the neuroepithelium (NE) shrinks in relative volume, laminae in Anunon's horn (SO, SP, SR) and the dentate gyrus (H, GL, MO) expand.

124 SA BAYER

and P21, approximately 120/0 daily. Volumetric expansion was also determined separately for the components of the hippocampus: germinal matrices, Ammon's horn, dentate gyrus, and fimbria-fornix.

Genninal matrices

Both the neuroepithelium and subepen­dymal zone are present during embryonic de­velopment and disappear by PI (neuro­epithelium) or during the early postnatal period (subependymal zone). The neuro­epithelium has its most rapid rate of growth 0300%) between EI6-EI7, reaches a peak volume (0.115 mm3) on E18, and declines rapidly thereafter (Fig. 7A). During early de­velopment, the neuroepithelium makes up 78% (EI5), 67% (EI6) and 49% (EI7) of the total hippocampal volume (Fig. 6). On E18, when the neuroepithelium is largest in absolute volume, relative volume is 28%; by E22, it occupies only 3% of the total volume.

The subependymal zone is never a prominent feature of the hippocampus. On E17, a very small zone (.0005 mm3) can be seen in anterior coronal sections in some animals; by E18, the zone is present throughout the length of the hippocampus, larger toward the subiculum than toward Ammon's horn (drawing, Fig. 7A; arrow, Fig. 2). The most rapid rate of growth 0420/0) occurs between E18 and E19; volume peaks on E20 (0.098 mm3), and declines there­after. On E19, the subependymal zone is most prominent and takes up 14% of the total vol­ume; by E22, only 3% (Fig. 6).

Subiculum and Ammon's horn

Since the boundary between the subiculum and Ammon's horn is not recognizable during embryonic life, no distinction was made be­tween them. The cortex designated as hip­pocampus in Figure 4 contains the entire Am­mon's horn, but only part of the subicular com­plex (the subiculum proper, not the para- or presubiculum). Consequently, the volumetric data reported refer primarily to changes within Ammon's horn.

On E15, a zone of less densely packed, ran­domly oriented cells lie adjacent to the tightly packed, radially oriented neuroepithelium; the zone appears similar on E16 (Fig. 2, 4, 8A). Cells destined for other locations in the hip­pocampus migrate through this area during embryonic and early postnatal life, but a small population of early-forming neurons (EI5-EI7, Bayer,'80) will become permanent residents.

The most rapid rate of growth (3387%) occurs between E16-E17 (Fig. 7C, solid line), when large numbers of cells leave the neuro­epithelium in the region of the presumptive subiculum (Fig. 2). Embryonic growth rate slows (13%) between EI9-E20, at the same time as the pyramidal layer rapidly increases in vol­ume. Another spurt of growth occurs between E22-Pl, when approximately 57% of the vol­ume is added per day; this may be related to the growth of basal pyramidal cell dendrites. The growth rate slows to 19% daily increase be­tween PI-P7, 100/0 between P7-P21. Absolute volume on P21 is 5.67 mm:J• The stratum oriens is most prominent between EI7-EI9, when it occupies between 36-40% of the total volume; relative volume is reduced gradually thereafter to 18% on P21 (Fig. 6).

By E16, a superficial cell-sparse zone, the continuation of cortical layer I, appears in the hippocampus. The few cells located here are presumably the early-forming neurons of the strata radiatum and lacunosum-moleculare (Fig. 2,4, 8A; Bayer, '80). The most rapid rate of growth (2103%) occurs between E16-E17 (Fig. 7C, dotted line). The rate slows to 61% between E20-E21, then spurts between E21­E22 000%) and E22-Pl (approximately 96% daily). The latter spurt may be related to pyramidal cell apical dendritic growth. Be­tween PI-P7, the volume increases by 35% per day, between P7-P21 by 14%. Absolute volume on P21 is 12.14 mm3 . The relative volume (Fig. 6) of the strata radiatum and lacunosum­moleculare continually increases from 7% (EI6) to 38% (P21).

The pyramidal layer does not appear until E18, when a thin lamina can be distinguished in the region of the subiculum (Fig. 2, 4) as a continuation of the cortical plate. The pyrami­dal layer thickens in the neighborhood of the subiculum on E19 and E20, and becomes prom­inent in Ammon's horn from E21 on (compare EI9-E22, Fig. 2). The most rapid rate ofgrowth (275%) is between EI8-EI9; this slows to 57% between E21-E22 and spurts again (820/0 daily) between E22-Pl (Fig. 7C, dashed line). The two growth spurts may be related to the sequential times oforigin between subicular and ammonic pyramidal cells (Bayer, 80). Between PI-P7, volume increases by approximately 23% daily, by 10% daily between P7-P21. Absolute volume on P21 is 5.6 mm3

. The relative volume (Fig. 6) of the stratum pyramidale increases from 4% on E18 to 220/0 at PI; it declines slightly to 17% on P21.

HIPPOCAM.PAL MORPHOGENESIS 125

Dentate gyrus� near the outgrowth of the choroid plexus (ar­rows, Fig. BA) to a subpial area in the concavity

Although the dentate gyrus is the last hip­ formed by the neuroepithelium of Ammon's pocampal structure to appear, its origin can be horn and the subiculum. This migratory followed from early developmental stages. On stream is more distinct on E17 (Fig. BB). Dur­E16, cells migrate from the neuroepithelium ing E1B and E19, cells accumulate in a ball-like

.2 .----A-.G-E-R-M-I-NA-L-Z-O-N-ES---'---S-.F-IM-S-RI-A-a=--=F=-=-O-=R-:-:N----:-"IX-:-----------:-.-t -, 2

.1 "� ~ I "

.5 ~ - NEUROEPITHELIUM · -- SUBEPENDYMALO'. ZONE

05

r l I \� I \�

.01 I \� I \� I \�

.005� 05I \ I \

r<l I r<)\E� EI \E� EI \�

I \�W 001 )f-l-..u....L.Lw..JL..L.J'--_--'- ""--+'---L.L.J....L.L.L.L..L.~_--l. -'-_i( 01 W

~ 20 C. AMMON'S HORN D. DENTATE GYRUS 20 ~ :J 10� 10 :J-l .....� -l

~ 50� ._a 5 ~

1.0� 1.0-_......­fIJ - STRATUM ORIENS .5� 5r- ......

~--Py!j,r:~1~~E . / O-HILUS

LACUNOSUM·MOLECULARE 0--.... STRATA RADIATUM, ,/ / r /

.1� .--GRANULAR LAYER .1

/ ~m"-MOLECULAR LAYER 05.05

f .01 01

005� 005I~I~ .001 '-i-trrnrnlTl---r-------,------l"TTT"TTTTT~---r------_......J 001

E15 E20 Pl P7 P21 E15 E20 Pl P7 P21

AGE Fig. 7. Absolute volumes (rom') of subdivisions within germinal zones (A). fimbria and fornix (considered together in B),

Anunon's horn (C) and dentate gyrus (D)determined as described for entire hippocampus (Fig. 5). Shaded areas in drawing (A. E19; B. E22; C, E21; D. P7) indicate regions quantified. For further discussion, see text.

126 SA BAYER

A .f

B .•. .. .'

I

. • <

.t.,

Fig. 8. Cells migrating (arrows) from the neuroepithelium (NE) adjacent to the choroid plexus (CP) toward the future site of the dentate gyrus (dg) on E16 (A) and E17 (B). Solid lines indicate pial surface; directional arrows give orientation (coronal paraffin sections, 6 !'om, hematoxylin and eosin; bar = 0.1 mrn).

subpial mass (outlined by dotted lines in Fig. 2 and 4), the dentate primordium. Since it pre­sumably contains mainly the early-forming hilar cells (Bayer, '80), the primordial volume on E18 and E19 is called the hilus (Fig. 7D, solid line). The most rapid growth rate (139%) ofthe hilus is between E18-E19; it slows to 47% between E20-E21, then spurts (88%) between E21-E22. Between E22-P1, daily volumetric increase is 300/0,190/0 between P1-P7, and 11% between P7-P21. Absolute volume on P21 is 1.68 mm3. Relative volume (Fig. 6) increases from 4% (E18) to 100/0 (E22), then gradually declines to 5% (P21).

On E20, the ectal limb of the granular layer (arrows, Fig. 4) appears superficial to the hilus in the anterior and posterior dentate gyrus but not in intermediate horizontal (Fig. 2) and co­ronal (Fig. 4) sections. On E21 and E22, the ectal limb of the granular layer becomes more distinct throughout the dentate gyrus; the

endallimb is smaller, but develops rapidly in the first few days after birth. The most rapid rate of growth (3920/0) of the granular layer occurs between E20-E21 and continually de­clines thereafter (Fig. 7D, solid line); however, the growth rate remains high during the post­natal period: 500/0 daily volumetric increase be­tween P1-P7, 21% between P7-P21. Absolute volume on P21 is 1.47 mm3 . Relative volume (Fig. 6) is always low; 0.3% (E20) to 5% (P21). The molecular layer is not present until P1 (Fig. 7D, dotted line). Volume increases daily by 64% between P1-P7, by 28% between P7­P21. Absolute volume on P21 is 4.58 mm:l . Rela­tive volume (Fig. 6) continually increases from 4% (P1) to 12% (P2l).

Fimbria and fornix

Fibers of the presumptive fimbria-fornix sys­tem appear in a few anterior coronal sections in some animals on E17. On E18, the fimbria and

127 HIPPOCAMPAL MORPHOGENESIS

. ,'. ~. '"

........ . .... . ~ :. ". -. )';. ,;;[.. .. .... 'j

"-:."' ,,. . .--' .. '

;; .

~. . ,

Fig. 9. The hippocampus at E20 six hours after a single exposure to 200 R X-rays. Small, dark particles (arrow) are pyknotic remains of cells killed. Notice that the neuroepithelium (NE), subependymal zone (SE), stratum oriens (SO), and dentate hilus (H) have many pyknotic fragments, while the stratum pyramidale (SP), and strata radiatum and lacunosum-moleculare (SR) have few. Strips indicate regions where packing density in control and X-irradiated brains were quantified for Figure 10 (coronal paraffin section, 6 J.Lm, hematoxylin and eosin; bar = 0.25 nun; directional arrows give orientation).

fornix are larger in the anterior hippocampus (Fig. 4); the ventral hippocampal commissure crosses the midline on E19. The volumetric ex­pansion curve (Fig. 7B) is unique in that it shows two very high growth spurts: 1290/0 in­crease between EI8-EI9; 128% increase be­tween E21-E22. (Between E20-E21 the volume increases by only 4%.) During the postnatal period, volume increases daily by 13% between PI-P7, by 8% between P7-P21. Absolute vol­ume on P21 is 1.81 mm:J

• Relative volume (Fig. 6) reaches a peak after the second growth spurt on E22 (13%) and declines to 6% on P21.

Maturation of hippocampal cell layers as determined by X-irradiation

Areas of differentiating cells in the 'neuroepithelium and subependymal zone, or conversely, pockets of immature cells within the laminae of the developing hippocampus, are difficult to identify in normal material. Since mitotic and primitive migratory cells in the irrunature rat brain are killed by a rela­tively low X-ray exposure (200 R) that spares differentiating cells (Hicks and D'Amato, '66; Altman et al., '68; Altman and Nicholson, '71; Bayer and Altman, '74), X-inadiation was used to more accurately locate regions of immature cells in the hippocampus. An intermediate-

level coronal section of the E20 hippocampus six hours after a single exposure to 200 R X­irradiation shows the pyknotic remains of cells killed as small-dark particles (Fig. 9, similar to that in Fig. 4). Many cells are killed in the dorsal neuroepithelium and subependymal zone bordering the subiculum, some cells are killed in the ventral neuroepithelium border­ing Arrunon's horn. Most cells are killed in the dorsal stratum oriens, while some surviving cells can be seen in the ventral stratum oriens. Few cells are killed throughout the pyramidal layer and strata radiatum, lacunosum­moleculare. Many cells are also killed in the dentate hilus. The degree of maturity of each layer and location (strips) indicated in Figure 9 was quantified by counting the number of cells surviving X-irradiation exposure in the three coronallevels2 shown in Figure 4 (see Materials and Methods). For each layer quantified, the number of cells per 150 JLm2 in control and X-irradiated brains were compared with an

2 The anterior section was the second to show 8 definite dentate gyrus between EI8-E22; between EI5-E17, the first to show a continuation with the choroid plexus at the foramen of Monro (Fig. lB). Between E18·E22, the posterior section was the last to show a definite pyramidal layer; between E15·E17. the last to show a thinner neuroepithelium at the ventral border of the hippocampus. The intermediate level was the middle section between anterior and posterior levels.

128 SA BAYER

analysis of variance (Table 2 lists F ratios); the Scheffe test was used to locate differences be­tween means. The degree of radiosensitivity (Fig. 10) is expressed as a percentage of surviv­ing cells. For each layer, this was determined by dividing the number of cells per 150 fLm2 in irradiated brains by the mean number of cells per 150 fLm2 in control brains and multiplying by 100; squares indicate significant (p<.05) de­creases from control levels. In all regions stud­ied, there were no differences between anterior, intermediate, and posterior levels; Figure 10 shows combined data for all levels.

Neuroepitheliurn

Although the neuroepithelium in the ventral strips (line VAH in drawing, Fig. 10) is signifi­cantly more radioresistant (p<.OOOl, sign test) than in dorsal strips (line DAH in drawing, Fig. 10), both sampled areas showed significant age and treatment effects as well as an age by treatment interaction (Table 2). X-irradiation significantly reduces cells below control levels until E21; irradiated and control groups are the same on E22. The proportion of surviving cells increases from 47% (E15) to 73% (E21) in the ventral sample, from 14% (E15) to 73% (E21) in the dorsal sample.

Subependymal zone

The subependymal zone (Fig. lOB) also shows significant effects of age, treatment, and age by treatment interaction (Table 2). Packing density decreases in controls from a peak 5.3 (E19) to 4.0 (E22) cells per 150 fLm2. Through­out development, X-irradiation significantly reduces cell number below control levels; radioresistance decreases during development to reach a low point on E21.

Stratum oriens

Comparisons between stratum oriens sam­ples showed that ventrally located cells are sig­

nificantly more radioresistant (p<.OOOl, sign test) than those located dorsally (Fig. 10C), as in the neuroepithelium (Fig. lOA). Both dorsal and ventral samples show significant age, treatment, and age by treatment interaction during development (Table 2). In the dorsal stratum oriens, packing density reaches a peak level on E18 (3.4 cells/150 fLm2) and decreases rapidly thereafter (1.3 cells per 150 fLm2, E22); the ventral stratum oriens packing density peaks on E19 (2.7 cells per 150fLm2) and de­creases to its lowest point on E22 (1.35 cells per 150 fLm2). X-irradiation significantly reduces cell number in dorsal samples throughout de­velopment; ventral samples are also reduced, but not significantly on E17, E18, and E22. Both locations are least radioresistant on E20 (Fig. 10C).

Strata radiatum, lacunosum-moleculare

There were no differences between dorsal and ventral samples, and the data in Figure 10D are combined. Age, treatment, and age by treatment interaction are significant. Control packing density decreases between E19 (1.65 cells per 150 fLm 2) and E22 (0.9 cells per 150 fLm2). X-irradiation has a biphasic effect; the population is significantly reduced on E16 and E17, radioresistant on E18, reduced on E19 and E20, and radioresistant on E21-E22. The level of survival never goes below 7Cf'!o, indicating that most of these cells have begun to differ­entiate.

Stratum pyramidale

Dorsal and ventral samples are combined in Fig. 10D, since there were no significant differ­ences between them. Age and treatment effects are significant (Table 2). Control packing den­sity significantly decreases between E18 (2.85 cells per 150 fLm2) and E22 (1.95 cells per 150 fLm2). X-irradiation slightly, but significantly, reduces cells on E18 and E19. As indicated by

Fig. 10. Proportion of cells surviving a single exposure to 200 R X·rays six hours before killing throughout the anteropos­terior extent of the neuroepithelium (A), subependymal zone (B), stratum oriens (C), strata pyramidale, radiatum, and lacunosum-moleculare (D), and dentate gyrus (E). For each structure, the percentage of surviving cells in each X-irradiated brain cut in the coronal plane (Table 1) was calculated with the following formula:

# cells/150 iLm ' (X·irradiated) x 100

X # cells/150 iLm' (controls)

means, with standard deviation, are plotted; squares indicate significant reduction (p<.05) from control levels. Anterior, intermediate, and posterior coronal sections, similar to those in Figure 4, were analyzed in three locations: l) dorsal Anunon's horn (line DAR, drawing; solid lines, Fig. 4); 2) ventral Ammon's horn (line VAH, drawing; dashed lines, Fig. 4); 3) dentate gyrus (line DC, drawing; dotted line, Fig. 4). There were no differences between anterior, intermediate, and posterior levels; all graphs show combined data. The neuroepithelium (A) and stratum oriens (C) are more radioresistant in VAR than in DAR samples. The stratum pyramidale and strata radiatum, lacunosum-moleculare (D) are similar in both DAH and VAR samples, and their graphs show combined data. See text for further details.

---

A. N

EU

RO

EP

ITH

ELI

UM

~

10

0

/+-__1

___

1-....

. 1

..... ~

50

//

~

..... -.

J ~

(?

~

> :> 10

§5 ~

B. S

UB

EP

EN

DY

MA

L ZO

NE

==

§'00

u a:

llJ

0.

..

50

10

E15

E

I6

E17

E18

E

19

E2

0

-D

AH

---

VA

H

E21

E

22

C. S

TRA

TUM

OR

IEN

S D

(f

)

10

0-.

J llJ

U

'''t t//

I -.J

,/

a:

"t/

a ~

50

~

~

-D

AH

� aJ

---V

AH

� Ei

VA

H

~

llJ

g 10

0..

. ~,--'-

---1

.5m

m

E. D

EN

TATE

D,

AM

MO

N'S

HO

RN

(E

xcep

t S

To

rie

nw

G

YR

US

r-­

~

,~-vr-ll

l

T

ST

RA

TA

R

AD

IAT

UM

.O

� ~-HILUS

-LA

CU

NO

SU

M M

OLE

CU

LAR

E

'� ~

(f)

1 -.

J t'"

-.J

:s:

"J lO

Ow

~U

(?

mS

TR

AT

UM

PY

RA

MID

ALE

� •

---G

RA

NU

LA

R L

AY

ER

Z

a:>

~

:> z

a:

t<:l

::>

r:n(f

) en

50

L!­ a ~

z llJ

u a:

llJ

10

0...

E15

E

16

E17

AG

E

E18

E

19

E2

0

E21

E

22

E

18

E19

E

20

E

21

E2

2

Fig

ure

10

I--'

r.

:l

'!l

130 SA BAYER

TABLE 2. F ratWs from an analysis of variance between control and X-irradiated groups

Degrees of freedom

Neuroepithelium Dorsal 7,1 Ventral 7,1

Subependymal zone 5,1

Stratum oriens Dorsal 6,1 Ventral 6,1

Pyramidal cells 4,1 Stratum radiatum 6,1 Dentate hilus 4,1 Dentate granular

layer 2,1

·p<.OOI. • p <.05.

the high level of survival, most of these cells have probably begun to differentiate.

Dentate gyrus

The dentate gyrus was sampled along line DG in the drawing of Figure 10. The hilus shows significant age, treatment, and age by treatment interaction effects. Control packing density significantly decreases between E20 (2.85 cells per 150 J1.m 2 ) and E22 (2.2 cells per 150 J1.m 2). The population of cells in the hilus is initially radioresistant (E18, Fig. 10E); X­irradiation effectively reduces cell number dur­ing the rest of embryonic development, espe­cially between E20-E22. On the other hand, the dentate granule cells are always radioresistant (Fig. 10E), indicating that those which accumu­late in the granular layer have already begun to differentiate.

DISCUSSION

The neuroepithelium The neuroepithelium is prominent as long as

active neurogenesis occurs throughout the hip­pocampal region. In the entorhinal cortex, neurons originate mainly between E15-E17 (Bayer, '80); the entorhinal neuroepithelium declines from E18 on <Fig. 2-3). In the subicu­lar complex, Ammon's horn, and dentate hilus, neurons originate mainly between E16-E19 (Bayer, '80), during the time when the hip­pocampal neuroepithelium rapidly expands and reaches its peak volume on E18 (Fig. 7A). Changes in radiosensitivity of the hippocampal neuroepithelium also correlate with neuronal

Main Effects

Age X-irradiation� effect effect Interaction�

23.368* 440.38 * 13.376* 17.087* 149.107* 4.372* 19.077* 436.602* 2.724·

54,317* 404.282* 7.793* 29,426* 130.062* 5.454* 15.953* 21.987* 1.347 28.843· 17.746* 3.688· 13.278· 156.723· 4.204.

.023 .307 1.512

production; X-irradiation kills more cells dur­ing times of maximal neuronal production (up to E18, Fig. 10) than later; but it does not be­come radioresistant until E22, after virtually all neurons (except dentate granule cells) have been produced (Bayer, '80). A similar pattern was reported in the septal region (Bayer, '79b). Yet, the cells adjacent to the lateral ventricle in the hippocampal region continue to be mitoti­cally active after neuronal production, as indi­cated by 3R-thymidine uptake during the postnatal period in a short survival auto­radiographic study (Altman et a!., '68) and by labelled ependymal cells in animals injected with oR-thymidine postnatally (Altman and Bayer, unpublished observations). Since the ependymal cells remain in situ, the cells pro­ducing them are in a stationary germinal zone. Few cells in such zones are killed by 200 R X-irradiation (Altman et a!., '68). This may ex­plain radiosensitivity differences between dor­sal and ventral neuroepithelial samples. The ventral neuroepithelium, closer to the fimbria and choroid plexus, may produce pro­portionally more ependymal cells than neurons and is more radioresistant; the dorsal neuroepithelium may produce proportionally more neurons than ependymal cells and is more radiosensitive <Fig. lOA).

The subependymal zone

The sllbependymal zone is never prominent in the hippocampal region. Mitotic figures are always sparse, and probably most of the densely packed cells are primitive neuroblasts

131 HIPPOCAMPAL MORPHOGENESIS

migrating to their respective locations. Both mitotic and primative migratory cells are known to be radiosensitive, as indicated by the data of Figure lOB and previous studies of Altman and his coworkers (Altman et aI., '68; Altman and Nicholson, '71; Bayer and Altman, '74). However, cells remain here postnatally, after lamination is nearly complete throughout the hippocampal region (dentate granular layer excepted), and may be the source of inter­neurons and glial cells-the majority of which are produced postnatally throughout the hip­pocampus (Bayer and Altman, '74).

Entorhinal cortex

The lateral to medial laminar maturation in the entorhinal cortical plate correlates with a similar neurogenetic gradient (Bayer, '80). Deep laminaE;) are thicker than superficial laminae on E20 (Fig. 3D) corresponding with the deep to superficial neurogenetic gradient; neurons in layers II and IV appear before the prominent expansion of layer IlIon E22 (Fig. 3A, B), in agreement with the later neuro­genesis of layer III cells (Bayer, '80). The last neurons are produced on E18, but the cortical plate is slow to mature; spindle-shaped cells in the white matter on E22 (Fig. 3A) indicate that cells are still migrating into the cortex at this late stage. The thick transitory zone ofE18-E21 (Fig. 2, 3) suggests that most neuroblasts re­main here, delaying their radial migTation, possibly by as long as five days. The signifi­cance of this delay is not known, but it also occurs in the neocortex (Altman and Bayer, in preparation). Two bands of fibers appear dur­ing development (Filimonoff, '47; Macchi, '51). The first appears beneath the cortical plate on E18 and may be composed ofaxons growing from cells within the plate as well as afferent fibers. The endopiriform nucleus sends affer­ents to the lateral entorhinal cortex via the deep cortical white matter (Krettek and Price, '77) and originates early in telencephalic de­velopment (Bayer, in preparation). Fibers in layer IV appear a day later (E19, Fig. 2, 3C) and may be thalamic afferents from the nucleus reuniens (Herkenham, '78) and the anterior nuclear complex (Domesick, '69, '72, '73) which form on E16 and E 17 (Altman and Bayer, '79a, b).

Subiculum and Ammon's horn

The cell layer beginning adjacent to the en­torhinal cortex and extending into the hip­pocampus on E 18 is probably composed of deep cells throughout the subicular complex, formed

between E15-E17 (Bayer, '80). The continual thickening of the pyramidal layer in the sub­iculum on E19 and E20 correlates with the later formation (E17-E18, Bayer, '80) of super­ficial cells. Finally, the late appearance of the superficial cells in the para- and presubiculum (not distinguishable until E22, Fig. 2) is in agreement with the later neurogenesis of these cells (E17-E19, Bayer, '80).

In Ammon's horn, the stratum pyramidale is not prominent until E21 (Fig. 2, 3) correlating with the later neurogenesis of these cells (mainly between E17-E19; Bayer, '80). The increase in stratum oriens radiosensitivity from E19-E21 (Fig. 10C) occurs at a time when pyramidial cells would be expected to migrate through this region. The greater radiosensitiv­ity in the dorsal stratum oriens is probably due to proportionally more pyramidal cells being produced by the subjacent neuroepithelium, and thereby migrating through this area, than the proportionally fewer pyramidal cells pro­duced by the neuroepithelium subjacent to the ventral stratum oriens (see above). Higher cell packing density in the dorsal stratum oriens of normal animals and the slightly later forma­tion ofpyramidal cells in field CAl (Fig. 2) than in field CA3 (Bayer, '80) support this hypo­thesis. By the time cells accumulate in the pyramidal layer, the higher level of radioresis­tance (Fig. 10D) indicates that differentiation has begun. Primitive processes grow from cell bodies within the pyramidal layer as soon as it can be recognized (Godina and Barasa, '64; Stensaas, '67c, d; Minkwitz and Holz, '75). Be­tween E21 and PI, the pyramidal layer ex­pands rapidly (Fig. 7C), mainly in length rather than in depth. As it grows toward the fimbria, well developed at this late stage (Fig. 2, 4) further forward linear expansion is thwarted, and the pyramidal layer takes a sharp deflection toward the ball-like dentate gyrus. Afferent fibers terminate in well­defined laminae on the apical dendrites of the pyramidal cells (Hjorth-Simonsen, '72; Hjorth-Simonsen and Jeune, '72; Steward, '76). The lining up of cell bodies assures that their apical dendrites are in register, allowing each portion of dendrite to be exposed to the same field of afferent fibers.

The large cells in the stratum oriens arid strata radiatum and lacunosum-moleculare originate between E15-E17 (Bayer, '80), and their production may be partly responsible for the early spurts in volumetric expansion of these two laminae (Fig. 7C). Both laminae also contain some radioresistant cells in embryonic

132 SA BAYER

life (Fig. lOC, D); differentiating cells were found here at early developmental stages in the rabbit hippocampus (Stensaas, '67a, b) and would be radioresistant. The spurts in vol­umetric expansion between E22-Pl (Fig. 8C), first in the strata radiatum and lacunosum­moleculare, next in the stratum oriens, corre­lates with the earlier growth of apical and later growth of basal pyramidal cell dendrites (Godina and -Barasa, '64; Stensaas, '67c-e; Minkwitz and Holz, '75; Minkwitz, '76).

Dentate gyrus

The stream of cells migrating toward the dentate gyrus from the neuroepithelium adja­cent to the choroid plexus is prominent in man (Humphrey, '66a, b, '67), less obvious in the rat where it can be delineated only during early development (E16 and E17, Fig. 8). From E18 on, fimbrial growth forces migrating cells to move across it and through the stratum oriens to get to the dentate gyrus. Many of these cells are also destined for the pyramidal layer, but the rapid volumetric expansion of the dentate hilus between E18-E20 (Fig. 7E) indicates that cells continue to move in and accumulate here (Bayer and Altman, '74; Schlessinger et a1., '75; Stanfield and Cowan, '79). Presumably, many of these are the early-forming large cells in the hilus and molecular layer (between E16-E18, Bayer, '80), which begin to differentiate dur­ing early development (Stensaas, '67c) and be­come radioresistant (Fig. WE). An increasingly larger proportion of hilar cells becomes radiosensitive during late embryonic (Fig. 10E) and early postnatal (Bayer and Altman, '74) life; mitotic figures are commonly seen, and many of these cells incorporate 3H-thymidine within a short time after injection (Angevine, '65; Altman and Das, '65a, b, '66; Stanfield and Cowan, '79), The exceptionally late neuro­genesis of the dentate granule cells (mainly postnatal, Bayer and Altman, '74; Bayer, '80; Schlessinger et a1., '75) occurs after the hip­pocampal neuroepithelium becomes the primi­tive ependyma (approximately on E22, Fig. lOA). These observations suggest that some of the cells migrating from the neuroepithelium into the hilus establish a secondary germinal matrix, the source of the granule cells. Thus, the point at which the migratory stream ofcells leaves the neuroepithelium may be termed a "germinal trigone." One prong is the hip­pocampal neuroepithelium, a second is the pre­cursors of the dentate granule cells, a third is the germinal source ofthe choroid plexus which grows from its point of attachment to the neu­

roepithelium (Altman, unpublished observa­tions). Such a trigone has also been described in the development of the secondary matrix of the cerebellar cortex, the external germinal layer (Altman and Bayer, '78).

The morphological appearance of the granu­lar layer follows the three neurogenetic gra­dients reported by Bayer ('80). The granular layer first appears on E20 at the two extremes of the dentate gyrus (Fig. 4); initially, the ectal limb is more prominent than the endal limb (Fig. 2). The layer thickens throughout its ex­tent postnatally and by P7, both limbs are homogeneous (drawing, Fig. 7D). As the granule cells migrate radially from the hilus, the hilus itself is pushed into a V-shape by the rapidly invaginating Ammon's horn on E22-Pl (Fig. 2 and Bayer and Altman, '74); the sharp fold of the granular layer may simply reflect this early distortion. By the time cells accumu­late in the granular layer, they are differentiat­ing as suggested by the increased level of radioresistance (Fig. 10E) and by the Golgi studies of Godina and Barasa ('64) and Sten­saas ('67c--e, '68).

Fimbria

The double spurt of volumetric expansion in the fimbria and fornix (Fig. 7B) may be related to differential times of maximal growth in sep­arate fiber systems. Septal afferents from the medial and diagonal band nuclei travel to the hippocampus via the fimbria and fornix (Swan­son and Cowan, '76; Meibach and Siegel, '77a, and others). Most of these cells are formed on E14-E16 (Bayer, '79a), and the early fimbria and fornix may be composed mainly of their axons. The spurt of growth between E18-E19 may be due to the addition ofaxons from sub­icular pyramidal cells which form mainly on E16-E17 (Bayer, '80), and are destined to ter­minate in the septum and mammillary body (Meibach and Siegel, '75, '77b; Swanson and Cowan, '75, '77, and others). The spurt of growth between E2l-E22 may be due to the addition ofaxons from ammonic pyramidal cells which form mainly on E17-E19 (Bayer, '80), and are destined to terminate in the sep­tum (Meibach and Siegel, '77; Swanson and Cowan, '77, and others).

ACKNOWLEDGMENTS

The author wishes to thank Joseph Altman for adviee and encouragement, Ann Cook for the histological material, Kathy Shuster for preparation of the figures, and Mary Ward for typing the manuscript. Figure 6 was designed

133 HIPPOCAMPAL MORPHOGENESIS

"­

by Peter Miller. Keith Perry, Ronald Bradford, Doug Ross, and Jon Raux assisted in the com­puter analysis. This research was supported by the National Science Foundation, Grant # BNS77-12622.

LITERATURE CITED

Altman, J. (1966) Autoradiographic and histological studies of postnatal neurogenesis. II. A longitudinal investigation of the kinetics, migration and transformation of cells in­corporating tritiated thymidine in infant rats, with special reference to postnatal neurogenesis in some brain regions. J. Compo Neurol., 128:431-474.

Altman, J., and S.A. Bayer (1978) Prenatal development of the cerebellar system in the rat. I. Cytogenesis and his­togenesisofthe deep nuclei and cortex of the cerebellum. J. Compo Neurol., 179:23-48.

Altman, J., and S.A. Bayer (1979a) Development of the diencephalon in the rat. IV. Quantitative study of the time of origin of neurons and the internuclear chronological gradients in the thalamus. J. Compo Neurol.,188:455-472.

Altman J., and S.A. Bayer (l979b) Development of the diencephalon in the rat. V. Thymidine-radiographic ob­servations on internuclear and intranuclear gradients in the thalamus. J. Compo Neurol., 188:473-500.

Altman, J., and G.D. Das 0965a) Postnatal origin of mic­roneurons in the rat brain. Nature, 207:953-956.

Altman, J., and G.D. Das (l965b) Autoradiographic and his­tological evidence of postnatal hippocampal neurogenesis in rats. J. Compo Neurol., 124:319--336.

Altman, J., and G.D. Das (1966) Autoradiographic and his­tological studies of postnatal neurogenesis. I. A longitudi­nal investigation of the kinetics, migration and transfor­mation of cells incorporating tritiated thymidine in neo­nate rats, with special reference to postnatal neurogenesis in some brain regions. J. Compo Neurol., 126:337-390.

Altman, J., W.J. Anderson, and K.A. Wright (1968) Differ­ential radiosensitivity of stationary and migratory primi­tive cells in the brains of infant rats. Exp. Neurol., 22:52-74.

Altman, J., and J.L. Nicholson (1971) Cell pyknosis in the cerebellar cortex of infant rats following low level X­irradiation. Rad. Res., 46:476-489.

Angevine, J.B. (1965) Time of neuronal origin in the hip­pocampal region. Exp. Neurol., Suppl. 2, pp. 1-70.

Bayer, S.A. (1979a) The development of the septal region in the rat. I. Neurogenesis examined with 3H-thymidine au­toradiography, J. Compo Neurol., 183:89-106.

Bayer, S.A. (l979b) The development of the septal region in the rat. II. Morphogenesis in normal and X-irradiated em­bryos. J, Compo Neurol., 183:107-120.

Bayer, S.A. (1980) The development of the hippocampal region in the rat. I. Neurogenesis examined with "H­thymidine autoradiography, J. Compo Neurol. (this issue).

Bayer, S,A., andJ. Altman (1974) Hippocampal development in the rat: cytogenesis and morphogenesis examined with autoradiography and low-level X-irradiation. J. Compo Neurol.,158:55-80.

Brown, J.W. (1966) Some aspects of the early development of the hippocampal formation in certain insectivorous bats. In: Evolution of the Forebrain: Phylogenesis and on­togenesis of the forebrain. R Hassler and H. Stephan, eds. Stuttgart: Georg Thieme Verlag, pp, 92-103,

Conover, W.J. (1971)� Practical Nonparametric Statistics. New York: John Wiley.

Domesick, V.B. (1969) Projections from cingulate cortex in the rat. Brain Res., 12:296-320.

Domesick, V.B. (1972) Thalamic relationships of the medial cortex in the rat. Brain Behav. Evol., 6:457-483.

Domesick, V.B. (1973) Thalamic projections in the cingulum bundle to the parahippocampal cortex of the rat. Anat. Rec., 175:308,

Filimonoff, LN. (1947) A rational subdivision of the cerebral cortex, Arch. Neurol. Psychol.,58:296-311.

Godina, G., and A. Barasa (1964) Morfogenesi ed istogenesi della formazione Ammonica. Z. Zellforsch., 63:327-355.

Herkenham, M. (1978) The connections of the nucleus reu­niens thalami: evidence for a direct thalamo-hippocampal pathway in the rat. J. Compo Neurol., 177:589-610.

Hicks, S.P., and C.J. D'Amato (1966) Effects of ionizing radi­ation on mammalian development. In: Advances in Teratology. D.H.M. Woollam, ed. London: Logos Press, pp. 196-243.

Hines, M. (1922) Studies in the growth and differentiation of the telencephalon in man. The fissura hippocampi. J. Comp, Neurol., 34:73-171.

Hjorth-Simonsen, A., and B. Jeune (1972) Origin and termi­nation ofthe hippocampal perforant path in the rat studied by silver impregnation. J. Compo Neurol., 144:215-231.

Hjorth-Simonsen, A. (1972) Projection of the lateral part of the entorhinal area to the hippocampus and fascia dentata. J. Compo Neurol., 146:219-232.

Humphrey, T, (1966a) The development of the human hip­pocampal formation correlated with some aspects of itS phylogenetic history. In: Evolution of the forebrain: Phylogenesis and ontogenesis of the forebrain. R. Hassler and H. Stephen, eds. Stuttgart: Georg Thime Verlag, pp. 104-115.

Humphrey, T. (1966b) Correlations between the develop­mentofthe hippocampal formation and the differentiation of the olfactory bulbs. Ala. J. Med. Sci., 3:235-269.

Humphrey, T. (1967) The development of the human hip­pocampal fissure. J. Anat., 101:655-676.

Krettek, J.E., and J.L. Price (1977) Projections from the amygaloid complex and adjacent olfactory structures to the entorhinal cortex and to the subiculum in the rat and cat. J. Compo Neurol., 172:723-752.

Macchi, G. (1951) The ontogenetic development of the olfac­tory telencephalon in man. J. Comp, Neurol., 95:245-305.

Meibach, RC" and A. Siegel (1975) The origin of fornix fibers which project to the mammillary bodies in the rat: a horseradish peroxidase study. Brain Res., 88:508-512.

Meibach, R.C., and A. Siegel (1977a) Efferent connections of the septal area in the rat: an analysis using retrograde and anterograde transport methods. Brain Res., 119:1-20.

Meibach, R.C., and A. Siegel (1977b) Efferent connections of the hippocampal formation in the rat. Brain Res., 124: 197-224.

Minkwitz, H. (1976) Zur Entwicklung del' Neuronenstruktur des Hippocampus wahrend der par und postnatalen On­togenese del' Albinoratte. 1. Mitteilung: Neurohis­tologische darstellung del' Entwicklung langaxoniger Neurone aus den Regionen CA3 und CA4. J. Hirnforsch., 17:213-231.

Minkwitz, H., and L. Holz (1975) Die Ontogenetische Ent­wicklung von Pyramidenneuronen aus dem Hippocampus (CAl) der Ratte. J. Hirnforsch., 16:37-54.

Schlessinger, A.R, W.M. Cowan, and D.I. Gottlieb (1975) An autoradiographic study of the time of origin and the pat­tern of granule cell migration in the dentate gyrus of the rat. J. Compo Neurol., 159: 149-176.

Stanfield, B.B., and W,M. Cowan (979) The development of the hippocampus and dentate gyrus in normal and reeler mice. J. Compo Neurol., 185:423-460.

Stensaas, L.J. (1967a) The development of hippocampal and dorsolateral pallial regions of the cerebral hemisphere in fetal rabbits, I. Fifteen millimeter stage, spongioblast morphology. J. Compo Neurol., 129:59-70.

134 SA BAYER

Stensaas, L.J. (1967b) The development of hippocampal and dorsolateral pallial regions of the cerebral hemisphere in fetal rabbits. II. Twenty millimeter stage, neuroblast mor­phology. J. Compo Neurol., 129:71-84.

Stensaas, L.J. (1967c) The development of hippocampal and dorsolateral pallial regions of the cerebral hemisphere in fetal rabbits. III. Twenty-nine millimeter stage, marginal lamina. J. Compo Neurol., 130: 149-162.

Stensaas, L.J. (1967d) The development of hippocampal and dorsolateral pallial regions of the cerebral hemisphere in fetal rabbits. IV. Forty-one millimeter stage, intermediate lamina. J. Compo Neurol., 131:409-422.

Stensaas, L.J. (1967e) The development of hippocampal and dorsolateral pallial regions of the cerebral hemisphere in fetal rabbits. V. Sixty millimeter stage, glial cell morphol­ogy. J. Compo Neurol., 131:423-436.

Stensaas, L.J. (1968) The development of hippocampal and dorsolateral pallial regions of the cerebral hemisphere in

fetal rabbits. VI. Ninety millimeter stage, cortical differ­entiation. J. Compo Neurol., 132:93-108.

Steward, O. (1976) Topographic organization of projections from the entorhinal area to the hippocampal formation of the rat. J. Compo Neurol., 167:285-314.

Swanson, L.W., and W.M. Cowan (1975) Hippocampo­hypothalamic connections: origin in subicular cortex, not in Ammon's hom. Science, 189:303-304.

Swanson, L.W., and W.M. Cowan (1976) Autoradiographic studies of the development and connections of the septal area in the rat. In: The Septal Nuclei. J. W. DeFrance, ed. Plenum, New York, pp. 37-64.

Swanson, L.W., and W.M. Cowan (1977) An autoradio­graphic study of the organization of the efferent connec­tions of the hippocampal formation in the rat. J. Compo Neurol., 172:49-84.

Tilney, F. (1938) The hippocampus and its relation to the corpus callosum. Bull. Neurol. Inst. N.Y., 7:1-77.