Brain Research, 121 (1977) 229-243
229
© Elsevier/North-HollandBiomedicalPress, Amsterdam- Printed in The Netherlands
DEVELOPMENT OF AFFERENT LAMINATION IN THE FASCIA DENTATA OF THE RAT
REBEKAH LOY*, GARY LYNCH and CARL W. COTMAN Department of Psychobiology, University of Cali/brnia, lrvine, Calif. 92717 (U.S.A.)
(Accepted June 7th, 1976)
SUMMARY In the present study we examine the development of afferent lamination in the fascia dentata of the postnatal rat, as a first step in determining possible mechanisms controlling synaptic specificity in this system. This analysis is based on degenerationinduced argyrophilia as well as autoradiographic labeling of the entorhinal and commissural/associational afferents. Both methods show that in spite of the immaturity of the neonatal fascia dentata, these afferent systems have already established territorial relationships by 4 days of age which persist into adulthood. At 4 days, the entorhinal projection is restricted approximately to the outer 45/~m of the 80/zm wide molecular layer. The commissural/associational projection occupies approximately the inner 35 #m of the molecular layer. At older ages the commissural/associational zone increases in width very slowly relative to the entorhinal zone. We also discuss these results in relation to potential mechanisms of afferent development and dendritic differentiation.
INTRODUCTION The remarkable specificity of synaptic organization in the mammalian nervous system is nowhere more striking than in the rigorous ordering of afferents within the hippocampal formation. While the organization of this brain system has lent itself to many elegant physiological and anatomical studies, it has not been taken advantage of as a model for the development of neuronal specificity. The fascia dentata (dentate gyrus) is particularly well suited for a study of the development of synaptic organization. The structure develops quite late, with most of the cell proliferation, migration, differentiation, and synaptogenesis occurring post* Present address: Department of Neurosciences, M-008, School of Medicine, Universityof California at San Diego, La Jolla, Calif. 92093, U.S.A.
230 natally in the rat 2,5,6,18-2°,26,29. Also, it is composed of one major cell type, whose somata form a discrete layer, and whose dendrites extend into a uniform molecular layer 2s. Most importantly, the afferents segregate themselves proximo-distally along the dendrites, to terminate in one of 4 sublayers: (l) stratum granulosum; (2) supragranular layer; (3) inner molecular layer; (4) outer molecular layer. The granule cell somata are innervated by a dense, basket-like plexus of axons originating from interneurons of the infragranular hilar region 2s. These, in turn, appear to be innervated by monoaminergic fibers originating in the locus coeruleus 22 and brain stem raphe z3, as well as by collaterals of the mossy fiber axons of the granule cells zv. The supragranular sublayer, a thin region beginning at the very top of the stratum granulosum, receives cholinergic afferents which originate within or pass through the medial septal nucleus 21,25. The inner molecular layer receives two overlapping afferents, the commissural 3,1°,27 and associational 10,31,34 systems. These are fibers which originate ipsilateral (associational) and contralateral (commissural) in the pyramidal cell field CA3c or in adjacent cells of the fascia dentata hilus 1°. The outer molecular layer receives sparse projections from the septum 25, contralateral entorhinal cortex 9, brain stem22, 23, and, presumably, from infrequent local interneurons 2s. The dominant input to the outer molecular layer is from the ipsilateral entorhinal cortex, a projection which travels via the perforant path 4,~1,12,27,32,3a. The distribution of this afferent is further subdivided : axons from the medial entorhinal area occupy the most proximal portion of the outer molecular layer; those from the lateral entorhinal cortex innervate the remaining distal portions of the outer molecular layer; those from an intermediate area probably innervate a position between the
~ OML
-I00% E+ fewS
E -57%I
S -0%
Fig. 1. Schematic diagram of fascia dentata afferent organization in the adult rat. Abbreviations: A, associational; C, commissural; DG, fascia dentata (granule cell layer and hilus); E, entorhinat; H, Ammon's horn (pyramidal cell layer); 1ML, inner molecular layer; OML, outer molecular layer; S, septal ; SGL, supragranular layer. Percentages to right of inset represent proportion of molecular layer, measured from the superficialaspect of the stratum granulosum to the obliterated hippocampal fissure, occupied by each afferent system.
231 lateral and medial perforant paths 11,1g,3g,83. Thus, even within a given afferent, there is evidence of precise lamination to a target area (Fig. 1). The study of the development of these afferents should lead us closer to an understanding of the mechanisms underlying the orderly patterning of inputs within the adult structure. The following is a report of one aspect of this process: the timing and arrangement of two major afferent systems as they arrive in the fascia dentata of the neonatal rat. METHODS The material used in this study was prepared by two separate methods. Both employed Sprague-Dawley rats raised from birth in standardized litters of 6-8 pups each. Of a total of 46 animals, 32 were used for Fink-Heimer analysis. Under ether anesthesia, 16 received unilateral aspirated lesions of the entorhinal cortex and 16 received unilateral hippocampal ablations at varying ages between postnatal days 4 and 26. After allowing 18-36 h (most commonly 20 h) survival time, animals were perfused under Nembutal anesthesia with 10 ~ formalin in 0.9 ~o saline. The brains were stored in 20 ~ sucrose-10 ~ formalin at 4 °C for up to one month, then sectioned coronally or horizontally on a freezing microtome at 35/~m. Sections were collected serially and stored at 4 °C in 5 ~ formalin, or stained immediately by one or both of the Schneider30 and LeonardX4,15 modifications of the Fink-Heimer8 method for demonstrating degenerating axon preterminals and terminal elements. Additional sections were mounted on slides directly after sectioning and stained with cresyl violet. Measurements of widths of terminal fields in silver-stained sections were corrected for unavoidable shrinkage and stretching of sections during the silver staining procedures, thereby allowing a more standard comparison of individual brains and tissue sections. This was accomplished by measuring molecular layer widths directly from cresyl violet-stained sections. The boundaries of terminal fields were measured by visually averaging the distance between the molecular layer zone of maximal terminal degeneration for a given projection and the relatively clear zone containing few, if any, degeneration products. These terminal fields were measured as percentages of the total width of the molecular layer for at least 6 points in each silver-stained brain at a rostral level where the two limbs of the fascia dentata first appear continuously along the medial crest of granule cells. The means of these percentages were then converted into micrometer values based on the measurement of the entire layer in corresponding Nissl-stained sections. This correction procedure assumes that any "non-specific" stretching or shrinkage of sections during staining will be uniform throughout the width of the molecular layer. Measurements for adult values were taken from similarly prepared cresyl violet sections and Fink-Heimer sections prepared for earlier studies. The remaining 14 rats each received a total of 4.0 pCi [aH]proline (SchwartzMann) in 0.2/zl 0.9 ~ saline injected stereotaxically through a Hamilton microliter syringe over a period of 15-20 min. At 4 days of age, 3 animals received unilateral injections in the hippocampus and 3 in the entorhinal cortex; at 10 days, 4 animals received injections in each of these brain areas.
232 250-
. . . . . ipl
/
spl
u~
.,i
~200.
150U 100-
504
;
8
1'o
,4
2'~
odo,,
DAYS OF AGE
Fig. 2. Postnatal growth of the fascia dentata molecular layer. Width of the suprapyramidal (solid line) and infrapyramidal (broken line) limb molecular layers measured from formalin-fixed, cresyl violetstained sections through the rostral hippocampal formation. Following a survival time of 36 h, animals were anesthetized with Nembutal and perfused with 1 0 ~ formalin in 0.9 ~ saline. The brains were stored one week at 4 °C in sucrose-formalin, then sectioned at 20 # m on a freezing microtome. Serial sections approximately 100 # m apart were mounted on slides, defatted in xylene, rehydrated and coated with K o d a k nuclear track emulsion, then stored dry at 4 °C for 3 weeks. Slides were developed using K o d a k D-19, fixed, and counterstained lightly with cresyl violet. Terminal fields were measured as for the F i n k - H e i m e r sections. In several cases, grain counts at 5 ~ m intervals were made across the molecular layers of the suprapyramidal and infrapyramidal limbs of the fascia dentata to determine relative afferent density and potential measurement variability. RESULTS Measurements of the width of the suprapyramidal* molecular layer of the rostral fascia dentata from the most superficial granule cells to the hippocampal fissure reveal an increase from 80 # m at postnatal day 4 (P4) to 122 # m at P10. Further expansion of the layer is somewhat slower: at P26 it measures 169 # m ; at adulthood it measures 249 #m. The molecular layer of the infrapyramidal limb increases from 45 # m to 108 # m between P4 and P10, and at adulthood measures 205 # m (Fig. 2 and Table I). Fink-Heimer The 16 animals with entorhinal cortex ablations received variable degrees of damage to the retrohippocampal areas, including some damage to the subiculum in several subjects. Maximal and minimal damage at 3 dorso-ventral levels are represented schematically in horizontal sections at ages P4, P10 and P26 in Fig. 3. * The terminology supra- and infrapyramidal limbs is used throughout, as suggested by Angevine1, to avoid confusion due to convolution of the structure. The suprapyramidal limb at all levels of the septo-temporal axis is nearest the hippocampal fissure in rodents, and corresponds variously to lower limbTM,external arm or leaf7,18, lateral crus x2,34, ectal arm2, and dorsal blade or leaf 1°,~7 of other investigators.
233 TABLE I Abbreviations: CV = cresyl violet; FH = Fink-Heimer; AR -- autoradiography. Measurements represent means ± S.E.M. Days of age
4
Method
CV FH
Widthmolecular layer
Afferent zone*
Suprapyramidal limb (#m)
Entorh#~al
81 d: 3
Infrapyramidal limb (l~m) 45 ± 2
53 -4- 2~o (43 4- 2 ktm)
AR 10
CV FH
Adult
* 37 4- 3 (30 4- 2/~m)
122 ± 2
108 4- 4
AR 26
Commissural
CV FH
169 4- 7
CV FH
249 4- 3
67 4- 4 ~ ( 82 4- 5/~m)
33 4- 1 (40 4- 1 #m)
66 4- 2 ~ ( 80 4- 3 #m)
33 4- 1 (40 4- 1/tm)
73±1~ (123 4- 2/~m)
28 4 - 1 ~ (47 4- 2#m)
73 4- 1~o (183 i 3 #m)
27 ~ 1 (68 4- 3 ,urn)
173 4- 5 205 ± 4
* Terminal zone not detectable above background levels. ** Measured as per cent of suprapyramidal limb molecular layer. I n silver-stained sections from brains o f animals receiving e n t o r h i n a l cortex lesions at P4 (n ----4), degeneration products appear sparse, b u t are c o n c e n t r a t e d * * in the outer 53 ~o (43 # m ) of the molecular layer of the s u p r a p y r a m i d a l limb (Fig. 4A). A t P6 (n ---- 2) a n d P8 (n = 1) the p a t t e r n appears similar to the 4-day pattern. At P10 (n = 4) degeneration products appear in definitely l a m i n a t e d patterns in the infrap y r a m i d a l as well as the s u p r a p y r a m i d a l limb of the fascia dentata. A t this age the zone of restricted e n t o r h i n a l terminals in the outer molecular layer of the s u p r a p y r a m i d a l limb of the rostral fascia d e n t a t a is 82/zm, or 67 ~ of a total molecular layer 122 # m wide (Fig. 4B). There follows a n increase of a b o u t 4 0 / ~ m in the width of this e n t o r h i n a l terminal area to 123/~m, or 73 ~o of the fascia d e n t a t a molecular layer at P26 (n ---- 2 at P26; n = 3 at P14-22) (Fig. 4C).
** It should be noted that quantification of the size of terminal fields revealed by degenerationinduced argyrophilia is more difficult and correspondingly less certain than in the adult. This is due in part to an apparent trailing-off of terminal density towards the area of juxtaposition between the commissural and entorhinal zones at younger ages, but it is also due to a relatively high background, which is unavoidable in nearly all sections showing any successfully stained, degeneration-related products. This background may be due to the "less selective" nature of the Leonard modification of the Fink-Heimer stain used throughout, since the traditional step for suppression of normal fibers must be omitted for the young brains. Alternately, it may be due in part to an increased argyropbilia of the young tissue; that is, somata of granule cells often appear darkly stained, and the degeneration (transneuronal?), or increased sensitivity to silver, Of their dendrites may result in particles staining in the molecular layer extending through the true zone of afferent termination.
Fig. 3. Entorhinal cortex lesions in 3 age groups. Representative horizontal sections illustrate maximal (heavy broken lines) and minimal (light broken lines) entorhinal cortex damage at 3 dorsal-ventral levels in animals lesioned for Fink-Heimer degeneration analysis at 4, 10 and 26 days of age.
Fig. 4. Photomicrographs of terminal degeneration in suprapyramidal limb of rostrai fascia dentata following entorhinal cortex lesions at 3 ages. Stratum granulosum is towards bottom of figure. Fissure and zone of entorhinal terminals are indicated by arrows. A: 4 days (E4-2FH). B: 10 days (E10-1FH). C: 26 days (E26-1FH).
235
Fig. 5. Photomicrographs of terminal degeneration in suprapyramidal limb of rostral fascia dentata following contralateral hippocampal lesions at two ages. Stratum granulosum is towards bottom of figure. Fissure and zone of commissural terminals are indicated by arrows. A: 10 days (C10-1FH). B: 26 days (C26-1FH).
Total unilateral hippocampal ablation was attempted in 16 animals. Damage to dorsal areas was complete in every case. While a variable amount of ventral tissue remained in most of these animals, the fimbria and ventral commissure were completely severed in all cases, assuring that all hippocampal commissural fibers were interrupted. None of the brains of animals receiving unilateral hippocampal lesions at P4 (n = 4), P6 (n = 3) or P8 (n = 2) show any degeneration argyrophilia in the molecular layer of the contralateral fascia dentata. This does not appear to be a general failure of the staining procedures, however, as all brains do contain well-stained degeneration products in the contralateral stratum oriens and stratum radiatum of hippocampal subfields CA3a-b. By P10 (n = 4), a band of terminals 40 # m wide, 33 ~ of the molecular layer, stains in the inner molecular layer immediately suprajacent to the granule cells of the suprapyramidal limb of the rostral fascia dentata (Fig. 5A). A P26 this terminal band is 28 ~ of the molecular layer, or 47 # m wide (Fig. 5B), an increase of only 7/~m between ages PI0 and P26 (n -- 3 at P14-22). Measurement of adult brains indicates that the commissural terminal field occupies 68/~m (27 ~o) of the fascia dentata molecular layer at older ages. These data are summarized in Table I.
236 ,oo.
80
Q
-
O
., __.
:E
o," ~o~
4o.
.
,,,...
O..o
,o_..
I
I 20
I
I 40
I
I 60
I
I 80
I
I I00
I
I I 120
I 140
GRAINS
Fig. 6. Plot of grain density through the molecular layer of the fascia dentata suprapyramidal limb contralateral and ipsilateral to CA3c injection at 4 days. Counts were prepared by plotting onto graph paper, by the use of camera lucida, individual silver grains in 50 #m strips at 5/tin intervals through the suprapyramidal limb of the fascia dentata molecular layer slightly rostral to the injection site (see Fig. 8a) in subject E4-2AR. Top of stratum granulosum is at 0 on ordinate; fissure is at 80 pm. A background count of two grains per 5/~m × 50/~m area due to "physical", non-physiological causes was determined by counting an area not overlying the brain section. This was subtracted from each interval count before plotting. Note that in both ipsilateral (open circles) and contralaterat (filled circles) molecular layers the maximal grain density appears nearest the stratum granulosum, and that the density is above a physiological background (0-3 grains per 5 #m × 50/~m area, as determined by measurements taken over a remote brain area, as per Gottlieb and Cowan z°) for approximately 20-30 /~m. The higher apparent "background" in the outer 70% of the molecular layer ipsilateral to the injection site may be due to non-specific diffusion of the label, or to other factors.
40
o
2o
u
2
4
6
8
I0
12
14
16
18
20
22
24
GRAINS
Fig. 7. Plot of grain density through the molecular layer of the infrapyramidal limb of the fascia dentata contralateral and ipsilateral to CA3c injection at 4 days. The procedure for plotting and determining background levels are as described in Fig. 6. Measurements are taken from the same sections as in Fig. 6, slightly rostral to the injection site. Note that lamination of the commissural projection (filled circles) is weaker than in the suprapyramidal limb (Fig. 6), and that there is no apparent lamination of the associational projection (open circles).
Autoradiography A n a l y s i s o f the b r a i n s p r e p a r e d for a u t o r a d i o g r a p h y reveals a slightly different p a t t e r n o f afferent d e v e l o p m e n t . T h a t is, while n o c o m m i s s u r a l t e r m i n a l field stains in the fascia d e n t a t a m o l e c u l a r layer at 4 d a y s b y the F i n k - H e i m e r m e t h o d , a d i s t i n c t b a n d o f t r a n s p o r t e d label a p p e a r s in the i n n e r 37 %, or 30/zm, o f the m o l e c u l a r layer o f
237
A
E4-2
¢
Fig. 8. Representative injection sites revealing commissural/associational and entorhinal projections into the fascia dentata at 4 and 10 days. A: subject E4-2AR - - heavy cell labeling (black area) is confined to the temporal stratum granulosum and hilus, while moderate cell labeling (hatched area) extends to retrohippocampal cortex. B: subject E10-3AR - - heavy cell labeling is localized in dorsomedial entorhinal cortex with moderate labeling in adjacent retrohippocampal area. C: subject H10-1AR - - heavy cell labeling is localized to stratum granulosum, hilus, and CA3c-CA4 region of the rostral fascia dentata, with moderate cell labeling in Ammon's horn extending from temporal subiculum to rostral CA3a-CA2. the suprapyramidal limb (Fig. 6), but not the infrapyramidal limb (Fig. 7), contralateral to the proline injections in 3 subjects at this age. In all 3 of these animals the injections reached into the CA3c-hilus region of A m m o n ' s horn as well as into the stratum granulosum of fascia dentata (Fig. 8A). In contrast, two other animals received injections of C A 3 a - b regions; these show no evidence of transported label into the contralateraJ fascia dentata, but do reveal a projection into the hippocampus proper. This agrees with Gottlieb and Cowan's 10 localization of the origin of commissural projections into the fascia dentata of the adult rat. In addition, all three 4-day animals, which show a commissural projection to the fascia dentata, also reveal the presence of the ipsilateral associational system from CA3c-hilus into the suprapyramidal molecular layer (Fig. 6), in accordance with
238
~
IOO--
2
4
6
8
IO
12
14
16
18
20
22
GRAINS
Fig. 9. Plot of grain density through the molecular layer of the suprapyramidal limb of the fascia dentata ipsilateral to entorhinal cortex injection at 10 days. The procedure for plotting and determining background levels is as described in Fig. 6. Subject EI0-3AR.
100i-
~: so o
60
40-m
20-m m
I
II
II
I
I
IIII
GRAINS
Fig. 10. Plot of grain density through the molecular layer of the infrapyramidal limb of the fascia dentata ipsilateral to entorhinal cortex injection at 10 days. The procedure for plotting and determining background levels is as described in Fig. 6. Subject and brain sections same as in Fig. 9.
239
%o
10
20
30
40
SO
60
GRAINS
Fig. 11. Plot of grain density through the molecular layer of the suprapyramidal limb of the fascia dentata ipsilateral and contralateral to CA3c injection at 10 days. The procedure for plotting and determining background levels is as described in Fig. 6. Subject, HI0-1AR. Injection site depicted in Fig. 8C. ]psilateral (open circles) and contralateral (closed circles) plots taken from same section, slightly rostral to the injection site.
ioo m
°
'°:!!
.o,
,.oll ° 40
~----.~%
GRAINS
Fig. 12. Plot of grain density through the molecular layer of the infrapyramidal limb of the fascia dentata ipsilateral and contralateral to CA3c injection at I0 days. The procedure for plotting and determining background levels is as described in Fig. 6. Subject, H10*IAR, same as Fig. 11. Measurements taken from same brain sections as Fig. 11. Zimmer's 34 studies in adult rats. The projection resulting f r o m the two caudal injections (Fig. 8A) appears considerably more extensive than the single case in which this system was labeled following a rostral injection. While in b o t h instances the pattern is laminated and restricted to approximately the inner 30 # m (37 ~ ) o f the fascia dentata molecular layer, the suprapyramidal cell field is more nearly innervated along its entire length in the f o r m e r cases, although at this age the lamination is not well defined in the infrapyramidal limb (Fig. 7). The terminal fields potentially labeled by the injections into the medial ento-
240
40pm ~/.//y//>
,/////,/. .,///~//,
q I 4 DAY
10 DAY
T.
I
26 DAY
:1 ADULT
Fig. 13. Schematic summary of dendritic growth and afferent lamination in the developing fascia dentata. Relative terminal zones in the suprapyramidal limb of the fascia dentata molecular layer as given in Table I are represented at 4 ages. Entorhinal terminal zone is represented by hatching; commissural/associational terminal zone is represented by stippling. The zones of juxtaposition between these two terminal fields have been matched at the various ages to compare their relative growth. Note that the commissural/associational terminal field increases relatively little, particularly if the later developing "clear " zone suprajacent to the stratum granulosum (the supragranular zone of Fig. 1) is taken into consideration. This may represent terminals of axons originating in the medial septal nucleus, which may begin arriving in the molecular layer around postnatal day 10, although afferents from other sources may be partially responsible for this apparent removal of commissural fibers from the immediate supragranular zone. The lighter stippling of the more distal commissural/ associational zone in the adult figure is based on observations of Gottlieb and Cowan 1° that a dense commissural zone of 30 ttm is bounded by a zone of lighter termination continuing an additional 20 #m into the molecular layer. All measurements were taken from the Fink-Heimer series except the commissural zone at 4 days, for which the autoradiographic measurement was used. See text for details.
rhinal cortex were n o t a p p a r e n t a b o v e the r a t h e r high b a c k g r o u n d , p r o b a b l y due to the coincidental labeling o f cells in the t e m p o r a l fascia d e n t a t a in these 4-day animals. A t 10 days, however, the t e r m i n a l field in the d e n t a t e a p p e a r s well l a m i n a t e d a n d restricted to the o u t e r 66 ~o (80/zm) o f the m o l e c u l a r layer in b o t h s u p r a p y r a m i d a l and infrap y r a m i d a l limbs (Figs. 9 a n d 10) following e n t o r h i n a l cortex injections (Fig. 8B). The p a t t e r n s o f c o m m i s s u r a l a n d a s s o c i a t i o n a l axons a n d t e r m i n a l s in the 10d a y a n i m a l s are essentially similar to those outlined in 4 - d a y - o l d r a t s : p r o j e c t i o n s to the fascia d e n t a t a after injections in C A 3 c - h i l u s (Fig. 8C) o c c u p y the inner 33 ~,, (40 # m ) o f the s u p r a p y r a m i d a l l i m b m o l e c u l a r l a y e r (Fig. 11), a n d a p p e a r l a m i n a t e d also in the i n f r a p y r a m i d a l l i m b (Fig. 12). T h e o v e r l a p o f c o m m i s s u r a l a n d e n t o r h i n a l projections, as c a l c u l a t e d f r o m the grain counts in Figs. 9 a n d 11 is less t h a n 10 # m even at 10 days o f age; this is c o m p a r a b l e to t h a t m e a s u r e d in a d u l t s ( M a t t h e w s , C o t m a n a n d Lynch, u n p u b l i s h e d observations). These d a t a are i n c l u d e d with those o f the F i n k - H e i m e r study in T a b l e I.
241 DISCUSSION By all available measures, the hippocampal formation of the neonatal rat is extremely immature. At 4 days after birth the molecular layer of the suprapyramidal fascia dentata is only one-third the width it will attain at adulthood, and the infrapyramidal limb is even less well developed. At this age the majority of granule cells are still migrating into the stratum granulosumz,29, and morphological and biochemical differentiation have only just begun2, 7,1s-~°,26. In light of this overall youthful condition it seems remarkable that at 4 days of age the newly arriving entorhinal and commissural/associational afferents have already established territorial relationships that seem to persist into adulthood. At 4 days, the entorhinal projection, as revealed by the Fink-Heimer method, is restricted to the outer 40-45 #m of the molecular layer. This lamination is probably not as sharp as in the adult and is present only in the suprapyramidal limb of more rostral hippocampal sections. The commissural projection appears by the autoradiographic analysis also to be laminated at 4 days, being restricted to the inner 30-35/~m of the suprapyramidal limb. The Fink-Heimer data, however, suggest that this projection may be more immature than the entorhinal, as the terminals of the former do not stain by this method until 10 days of age. The present study does not allow conclusions as to the establishment of the lamination prior to 4 days of age. One possibility is that the two systems co-mingle within the young molecular layer until this age in the suprapyramidal limb (or even older in the later developingl,Z, ~9 infrapyramidal limb); around 4 days of age, the systems would then have to segregate themselves to produce the patterns described at this and older ages. The present Fink-Heimer analysis suggests, however, that entorhinal afferent arrival may precede that of the commissural/associational system, thereby occupying alone the full width of the very young molecular layer. The commissural/ associational system then appears to enter the molecular layer as a relatively established axon bundle about 30 #m wide. If this interpretation is correct, the entorhinal terminals may reorganize by moving outward on the dendrites or by removing themselves altogether from the inner molecular layer. Alternately, the dendrites may grow selectively in the inner molecular layer, beginning at the point of entry of the commissural/associational axons, the free end of the suprapyramidal limb. This growth would carry the entorhinal axons into a position more distal to the cell bodies than that which they occupy in the youngest animals, leaving new dendritic surface for the commissural/associational afferents to contact. Beginning at about 10 days of age a separate afferent system, probably originating in the medial septal nucleus, appears to enter the supragranular zone of the molecular layer, perhaps inducing growth of the dendrites in this zone to accommodate the new arrival. This interpretation of a later arriving population is consistent with earlier studies of Mellgren20 and Matthews et al. 18, in which the mature pattern of acetylcholinesterase staining in the fascia dentata does not appear until around the sixteenth postnatal day, at which time a relatively intense zone appears immediately suprajacent to the stratum granulosum. Since continued expansion of the molecular
242 layer from day 26 onward is reflected mainly as increased width in the zone of entorhinal terminals, further dendritic e l o n g a t i o n most likely occurs in this more distal region. This s u m m a r y is schematized in Fig. 13. While the present study does not provide unequivocal evidence for afferent induction of dendritic growth, as proposed in other b r a i n systems13, 24, it seems at least reasonable to propose that e l o n g a t i o n occurs at several points along the dendrites d u r i n g development. T h a t is, it is more likely that dendritic growth is correlated with, if n o t regulated by, the sequentially arriving afferents, t h a n that the relationships measured are m a i n t a i n e d by a shifting of the terminal p o p u l a t i o n s to a c c o m m o d a t e new arrivals. O f course, the conclusive d e t e r m i n a t i o n of this hypothesis m u s t await studies analyzing the afferent p a t t e r n prior to 4 days of age as well as the growth of dendrites at various postnatal ages, in order to determine the true relationship of afferent ingrowth a n d dendritic differentiation.
REFERENCES l Angevine, J. B., Time of neuron origin in the hippocampal region. An autoradiographic study in the mouse, Exp. Neurol., Suppl. 2 (1965) 1-70. 2 Bayer, S. A. and Altman, J., Hippocampal development in the rat : cytogenesis and morphogenesis examined with autoradiography and low-level X-irradiation, J. comp. Neurok, 158 (1974) 55-80. 3 Blackstad, T. W., Commissural connections of the hippocampal region in the rat, with special reference to their mode of termination, J. comp. NeuroL, 105 (1956) 417-538. 4 Blackstad, T. W., On the termination of some afferents to the hippocampus and fascia dentata: an experimental study in the rat, Acta anat. (Basel), 35 (1958) 202-214. 5 Cotman, C. W., Taylor, D. and Lynch, G., Ultrastructural changes in synapses in the dentate gyrus of the rat during development, Brain Research, 63 (1973) 205-213. 6 Crain, B., Cotman, C., Taylor, D. and Lynch, G., A quantitative electron microscopic study of synaptogenesis in the dentate gyrus of the rat, Brain Research, 63 (1973) 195-204. 7 Das, G. D. and Kreutzberg, G. W., Postnatal differentiation of the granule cells in the hippocampus and cerebellum: a histochemical study, Histochemie, 10 (1967) 246-260. 8 Fink, R. P. and Heimer, L., Two methods for selective silver impregnation of degenerating axons and their synaptic endings in the central nervous system, Brain Research, 4 (1967) 369-374. 9 Goldowitz, D., White, W. F., Steward, O., Cotman, C. and Lynch, G., Anatomical evidence for a projection from the entorhinal cortex to the contralateral dentate gyrus of the rat, Exp. NeuroL, 47 (1975) 433-441. l0 Gottlieb, D. I. and Cowan, W. M., Autoradiographic studies of the commissural and ipsilateral association connections of the hippocampus and dentate gyrus of the rat. I. The commissural connections, J. comp. Neurol., 149 (1973) 393-422. l I Hjorth-Simonsen, A., Projection of the lateral part of the entorhinal area to the hippocampus and fascia dentata, J. comp. Neurok, 146 (1972) 219-232. 12 Hjorth-Simonsen, A. and Jeune, B., Origin and termination of the hippocampal perforant path in the rat studied by silver impregnation, J. comp. Neurol., 144 (1972) 215-232. 13 Kornguth, S. E. and Scott, G., The role of climbing fibers in the formation of Purkinje cell dendrites, J. comp. Neurol., 146 (1972) 61-82. 14 Leonard, C. M., A method for assessing stages of neural maturation, Brain Research, 53 0973) 412-416. 15 Leonard, C. M., Degeneration argyrophilia as an index of neural maturation : studies on the optic tract of the golden hamster, J. comp. Neurol., 156 (1974) 435-458. 16 Lorente de N6, R., Studies on the structure of the cerebral cortex. II. Continuation of the study of the ammonic system, J. PsyehoL Neurol. (Lpz.), 46 (1934) 113-177. 17 Lynch, G. and Cotman, C. W., The hippocampus as a model for studying plasticity in the adult brain. In R. L. Isaacson and K. H. Pribram (Eds.), The Hippocampus, Vol. 1, Structure and Development, Plenum, New York, 1975, pp. 123-154.
243 18 Matthews, D. A., Nadler, J. V., Lynch, G. S. and Cotman, C. W., Development of cholinergic innervation in the hippocampal formation of the rat. I. Histochemical demonstration of acetylcholinesterase activity, Develop. Biol., 36 (1974) 130-141. 19 Mellgren, S. I., Distribution of succinate-, a-glycerophosphate-, NADH-, and NADPH dehydrogenases (tetrazolium reductases) in the hippocampal region of the rat during postnatal development, Z. ZellJbrsch., 141 (1973) 347-373. 20 Mellgren, S. I., Distribution of acetylcholinesterase in the hippocampal region of the rat during postnatal development, Z. Zellforsch., 141 (1973) 375-400. 21 Mellgren, S. I., and Srebro, B., Changes in acetylcholinesterase and distribution of degenerating fibres in the hippocampal region after septal lesions in the rat, Brain Research, 52 (1973) 19-36. 22 Moore, R. Y., Monoamine neurons innervating the hippocampal formation and septum: organization and response to injury. In R. L. Isaacson and K. H. Pribram (Eds.), The Hippocampus, Vol. 1, Structure and Development, Plenum, New York, 1975, pp. 215-237. 23 Moore, R. Y. and Halaris, A. E., Hippocampal innervation by serotonin neurons of the midbrain raphe in the rat, J. comp. NeuroL, 164 (1975) 171-184. 24 Morest, D. K., The growth of dendrites in the mammalian brain, Z. Anat. Entwiekl.-Gesch., 128 (1969) 290-317. 25 Mosko, S., Lynch, G. and Cotman, C. W., The distribution of septal projections to the hippo.campus of the rat, J. comp. Neurol., 152 (1973) 163-174. 26 Nadler, J. V., Matthews, D., Cotman, C. W. and Lynch, G. S., Development of cholinergic innervation in the hippocampal formation of the rat. II. Quantitative changes in choline acetyltransferase and acetylcholinesterase activities, Develop. Biol., 36 (1974) 142-154. 27 Raisman, G., Cowan, W. M. and Powell, T. P. S., The extrinsic afferent, commissural and association fibres of the hippocampus, Brain, 88 (1965) 963-996. 28 Ram6n y Cajal, S., The Structure of .4mmon's Horn (L. M. Kraft, Transl.), Thomas, Springfield, 111., 1968. 29 Schlessinger, A. R., Cowan, W. M. and Gottlieb, D. I., An autoradiographic study of the time of origin and the pattern of granule cell migration in the dentate gyrus of the rat, J. comp. Neurol., 159 (1975) 149-176. 30 Schneider, G. E., Two visual systems, Science, 163 (1969) 895-902. 31 Segal, M. and Landis, S., Afferents to the hippocampus of the rat studied with the method of retrograde transport of horseradish peroxidase, Brain Research, 78 (1974) 1-15. 32 Steward, O., Topographical organization of the projections from the entorhinal area to the hippocampal formation of the rat, J. eomp. NeuroL, 167 (1976) 285-314. 33 Van Hoesen, G. W. and Pandya, D. N., Some connections of the entorhinal (area 28) and perirhinal (area 35) cortices in the rhesus monkey. IlL Efferent connections, Brain Research, 95 (1975) 39-59. 34 Zimmer, J., lpsilateral afferents to the commissural zone of the fascia dentata, demonstrated in de-commissurated rats by silver impregnation, J. comp. Neurol., 142 (1971) 393-416.
229
© Elsevier/North-HollandBiomedicalPress, Amsterdam- Printed in The Netherlands
DEVELOPMENT OF AFFERENT LAMINATION IN THE FASCIA DENTATA OF THE RAT
REBEKAH LOY*, GARY LYNCH and CARL W. COTMAN Department of Psychobiology, University of Cali/brnia, lrvine, Calif. 92717 (U.S.A.)
(Accepted June 7th, 1976)
SUMMARY In the present study we examine the development of afferent lamination in the fascia dentata of the postnatal rat, as a first step in determining possible mechanisms controlling synaptic specificity in this system. This analysis is based on degenerationinduced argyrophilia as well as autoradiographic labeling of the entorhinal and commissural/associational afferents. Both methods show that in spite of the immaturity of the neonatal fascia dentata, these afferent systems have already established territorial relationships by 4 days of age which persist into adulthood. At 4 days, the entorhinal projection is restricted approximately to the outer 45/~m of the 80/zm wide molecular layer. The commissural/associational projection occupies approximately the inner 35 #m of the molecular layer. At older ages the commissural/associational zone increases in width very slowly relative to the entorhinal zone. We also discuss these results in relation to potential mechanisms of afferent development and dendritic differentiation.
INTRODUCTION The remarkable specificity of synaptic organization in the mammalian nervous system is nowhere more striking than in the rigorous ordering of afferents within the hippocampal formation. While the organization of this brain system has lent itself to many elegant physiological and anatomical studies, it has not been taken advantage of as a model for the development of neuronal specificity. The fascia dentata (dentate gyrus) is particularly well suited for a study of the development of synaptic organization. The structure develops quite late, with most of the cell proliferation, migration, differentiation, and synaptogenesis occurring post* Present address: Department of Neurosciences, M-008, School of Medicine, Universityof California at San Diego, La Jolla, Calif. 92093, U.S.A.
230 natally in the rat 2,5,6,18-2°,26,29. Also, it is composed of one major cell type, whose somata form a discrete layer, and whose dendrites extend into a uniform molecular layer 2s. Most importantly, the afferents segregate themselves proximo-distally along the dendrites, to terminate in one of 4 sublayers: (l) stratum granulosum; (2) supragranular layer; (3) inner molecular layer; (4) outer molecular layer. The granule cell somata are innervated by a dense, basket-like plexus of axons originating from interneurons of the infragranular hilar region 2s. These, in turn, appear to be innervated by monoaminergic fibers originating in the locus coeruleus 22 and brain stem raphe z3, as well as by collaterals of the mossy fiber axons of the granule cells zv. The supragranular sublayer, a thin region beginning at the very top of the stratum granulosum, receives cholinergic afferents which originate within or pass through the medial septal nucleus 21,25. The inner molecular layer receives two overlapping afferents, the commissural 3,1°,27 and associational 10,31,34 systems. These are fibers which originate ipsilateral (associational) and contralateral (commissural) in the pyramidal cell field CA3c or in adjacent cells of the fascia dentata hilus 1°. The outer molecular layer receives sparse projections from the septum 25, contralateral entorhinal cortex 9, brain stem22, 23, and, presumably, from infrequent local interneurons 2s. The dominant input to the outer molecular layer is from the ipsilateral entorhinal cortex, a projection which travels via the perforant path 4,~1,12,27,32,3a. The distribution of this afferent is further subdivided : axons from the medial entorhinal area occupy the most proximal portion of the outer molecular layer; those from the lateral entorhinal cortex innervate the remaining distal portions of the outer molecular layer; those from an intermediate area probably innervate a position between the
~ OML
-I00% E+ fewS
E -57%I
S -0%
Fig. 1. Schematic diagram of fascia dentata afferent organization in the adult rat. Abbreviations: A, associational; C, commissural; DG, fascia dentata (granule cell layer and hilus); E, entorhinat; H, Ammon's horn (pyramidal cell layer); 1ML, inner molecular layer; OML, outer molecular layer; S, septal ; SGL, supragranular layer. Percentages to right of inset represent proportion of molecular layer, measured from the superficialaspect of the stratum granulosum to the obliterated hippocampal fissure, occupied by each afferent system.
231 lateral and medial perforant paths 11,1g,3g,83. Thus, even within a given afferent, there is evidence of precise lamination to a target area (Fig. 1). The study of the development of these afferents should lead us closer to an understanding of the mechanisms underlying the orderly patterning of inputs within the adult structure. The following is a report of one aspect of this process: the timing and arrangement of two major afferent systems as they arrive in the fascia dentata of the neonatal rat. METHODS The material used in this study was prepared by two separate methods. Both employed Sprague-Dawley rats raised from birth in standardized litters of 6-8 pups each. Of a total of 46 animals, 32 were used for Fink-Heimer analysis. Under ether anesthesia, 16 received unilateral aspirated lesions of the entorhinal cortex and 16 received unilateral hippocampal ablations at varying ages between postnatal days 4 and 26. After allowing 18-36 h (most commonly 20 h) survival time, animals were perfused under Nembutal anesthesia with 10 ~ formalin in 0.9 ~o saline. The brains were stored in 20 ~ sucrose-10 ~ formalin at 4 °C for up to one month, then sectioned coronally or horizontally on a freezing microtome at 35/~m. Sections were collected serially and stored at 4 °C in 5 ~ formalin, or stained immediately by one or both of the Schneider30 and LeonardX4,15 modifications of the Fink-Heimer8 method for demonstrating degenerating axon preterminals and terminal elements. Additional sections were mounted on slides directly after sectioning and stained with cresyl violet. Measurements of widths of terminal fields in silver-stained sections were corrected for unavoidable shrinkage and stretching of sections during the silver staining procedures, thereby allowing a more standard comparison of individual brains and tissue sections. This was accomplished by measuring molecular layer widths directly from cresyl violet-stained sections. The boundaries of terminal fields were measured by visually averaging the distance between the molecular layer zone of maximal terminal degeneration for a given projection and the relatively clear zone containing few, if any, degeneration products. These terminal fields were measured as percentages of the total width of the molecular layer for at least 6 points in each silver-stained brain at a rostral level where the two limbs of the fascia dentata first appear continuously along the medial crest of granule cells. The means of these percentages were then converted into micrometer values based on the measurement of the entire layer in corresponding Nissl-stained sections. This correction procedure assumes that any "non-specific" stretching or shrinkage of sections during staining will be uniform throughout the width of the molecular layer. Measurements for adult values were taken from similarly prepared cresyl violet sections and Fink-Heimer sections prepared for earlier studies. The remaining 14 rats each received a total of 4.0 pCi [aH]proline (SchwartzMann) in 0.2/zl 0.9 ~ saline injected stereotaxically through a Hamilton microliter syringe over a period of 15-20 min. At 4 days of age, 3 animals received unilateral injections in the hippocampus and 3 in the entorhinal cortex; at 10 days, 4 animals received injections in each of these brain areas.
232 250-
. . . . . ipl
/
spl
u~
.,i
~200.
150U 100-
504
;
8
1'o
,4
2'~
odo,,
DAYS OF AGE
Fig. 2. Postnatal growth of the fascia dentata molecular layer. Width of the suprapyramidal (solid line) and infrapyramidal (broken line) limb molecular layers measured from formalin-fixed, cresyl violetstained sections through the rostral hippocampal formation. Following a survival time of 36 h, animals were anesthetized with Nembutal and perfused with 1 0 ~ formalin in 0.9 ~ saline. The brains were stored one week at 4 °C in sucrose-formalin, then sectioned at 20 # m on a freezing microtome. Serial sections approximately 100 # m apart were mounted on slides, defatted in xylene, rehydrated and coated with K o d a k nuclear track emulsion, then stored dry at 4 °C for 3 weeks. Slides were developed using K o d a k D-19, fixed, and counterstained lightly with cresyl violet. Terminal fields were measured as for the F i n k - H e i m e r sections. In several cases, grain counts at 5 ~ m intervals were made across the molecular layers of the suprapyramidal and infrapyramidal limbs of the fascia dentata to determine relative afferent density and potential measurement variability. RESULTS Measurements of the width of the suprapyramidal* molecular layer of the rostral fascia dentata from the most superficial granule cells to the hippocampal fissure reveal an increase from 80 # m at postnatal day 4 (P4) to 122 # m at P10. Further expansion of the layer is somewhat slower: at P26 it measures 169 # m ; at adulthood it measures 249 #m. The molecular layer of the infrapyramidal limb increases from 45 # m to 108 # m between P4 and P10, and at adulthood measures 205 # m (Fig. 2 and Table I). Fink-Heimer The 16 animals with entorhinal cortex ablations received variable degrees of damage to the retrohippocampal areas, including some damage to the subiculum in several subjects. Maximal and minimal damage at 3 dorso-ventral levels are represented schematically in horizontal sections at ages P4, P10 and P26 in Fig. 3. * The terminology supra- and infrapyramidal limbs is used throughout, as suggested by Angevine1, to avoid confusion due to convolution of the structure. The suprapyramidal limb at all levels of the septo-temporal axis is nearest the hippocampal fissure in rodents, and corresponds variously to lower limbTM,external arm or leaf7,18, lateral crus x2,34, ectal arm2, and dorsal blade or leaf 1°,~7 of other investigators.
233 TABLE I Abbreviations: CV = cresyl violet; FH = Fink-Heimer; AR -- autoradiography. Measurements represent means ± S.E.M. Days of age
4
Method
CV FH
Widthmolecular layer
Afferent zone*
Suprapyramidal limb (#m)
Entorh#~al
81 d: 3
Infrapyramidal limb (l~m) 45 ± 2
53 -4- 2~o (43 4- 2 ktm)
AR 10
CV FH
Adult
* 37 4- 3 (30 4- 2/~m)
122 ± 2
108 4- 4
AR 26
Commissural
CV FH
169 4- 7
CV FH
249 4- 3
67 4- 4 ~ ( 82 4- 5/~m)
33 4- 1 (40 4- 1 #m)
66 4- 2 ~ ( 80 4- 3 #m)
33 4- 1 (40 4- 1/tm)
73±1~ (123 4- 2/~m)
28 4 - 1 ~ (47 4- 2#m)
73 4- 1~o (183 i 3 #m)
27 ~ 1 (68 4- 3 ,urn)
173 4- 5 205 ± 4
* Terminal zone not detectable above background levels. ** Measured as per cent of suprapyramidal limb molecular layer. I n silver-stained sections from brains o f animals receiving e n t o r h i n a l cortex lesions at P4 (n ----4), degeneration products appear sparse, b u t are c o n c e n t r a t e d * * in the outer 53 ~o (43 # m ) of the molecular layer of the s u p r a p y r a m i d a l limb (Fig. 4A). A t P6 (n ---- 2) a n d P8 (n = 1) the p a t t e r n appears similar to the 4-day pattern. At P10 (n = 4) degeneration products appear in definitely l a m i n a t e d patterns in the infrap y r a m i d a l as well as the s u p r a p y r a m i d a l limb of the fascia dentata. A t this age the zone of restricted e n t o r h i n a l terminals in the outer molecular layer of the s u p r a p y r a m i d a l limb of the rostral fascia d e n t a t a is 82/zm, or 67 ~ of a total molecular layer 122 # m wide (Fig. 4B). There follows a n increase of a b o u t 4 0 / ~ m in the width of this e n t o r h i n a l terminal area to 123/~m, or 73 ~o of the fascia d e n t a t a molecular layer at P26 (n ---- 2 at P26; n = 3 at P14-22) (Fig. 4C).
** It should be noted that quantification of the size of terminal fields revealed by degenerationinduced argyrophilia is more difficult and correspondingly less certain than in the adult. This is due in part to an apparent trailing-off of terminal density towards the area of juxtaposition between the commissural and entorhinal zones at younger ages, but it is also due to a relatively high background, which is unavoidable in nearly all sections showing any successfully stained, degeneration-related products. This background may be due to the "less selective" nature of the Leonard modification of the Fink-Heimer stain used throughout, since the traditional step for suppression of normal fibers must be omitted for the young brains. Alternately, it may be due in part to an increased argyropbilia of the young tissue; that is, somata of granule cells often appear darkly stained, and the degeneration (transneuronal?), or increased sensitivity to silver, Of their dendrites may result in particles staining in the molecular layer extending through the true zone of afferent termination.
Fig. 3. Entorhinal cortex lesions in 3 age groups. Representative horizontal sections illustrate maximal (heavy broken lines) and minimal (light broken lines) entorhinal cortex damage at 3 dorsal-ventral levels in animals lesioned for Fink-Heimer degeneration analysis at 4, 10 and 26 days of age.
Fig. 4. Photomicrographs of terminal degeneration in suprapyramidal limb of rostrai fascia dentata following entorhinal cortex lesions at 3 ages. Stratum granulosum is towards bottom of figure. Fissure and zone of entorhinal terminals are indicated by arrows. A: 4 days (E4-2FH). B: 10 days (E10-1FH). C: 26 days (E26-1FH).
235
Fig. 5. Photomicrographs of terminal degeneration in suprapyramidal limb of rostral fascia dentata following contralateral hippocampal lesions at two ages. Stratum granulosum is towards bottom of figure. Fissure and zone of commissural terminals are indicated by arrows. A: 10 days (C10-1FH). B: 26 days (C26-1FH).
Total unilateral hippocampal ablation was attempted in 16 animals. Damage to dorsal areas was complete in every case. While a variable amount of ventral tissue remained in most of these animals, the fimbria and ventral commissure were completely severed in all cases, assuring that all hippocampal commissural fibers were interrupted. None of the brains of animals receiving unilateral hippocampal lesions at P4 (n = 4), P6 (n = 3) or P8 (n = 2) show any degeneration argyrophilia in the molecular layer of the contralateral fascia dentata. This does not appear to be a general failure of the staining procedures, however, as all brains do contain well-stained degeneration products in the contralateral stratum oriens and stratum radiatum of hippocampal subfields CA3a-b. By P10 (n = 4), a band of terminals 40 # m wide, 33 ~ of the molecular layer, stains in the inner molecular layer immediately suprajacent to the granule cells of the suprapyramidal limb of the rostral fascia dentata (Fig. 5A). A P26 this terminal band is 28 ~ of the molecular layer, or 47 # m wide (Fig. 5B), an increase of only 7/~m between ages PI0 and P26 (n -- 3 at P14-22). Measurement of adult brains indicates that the commissural terminal field occupies 68/~m (27 ~o) of the fascia dentata molecular layer at older ages. These data are summarized in Table I.
236 ,oo.
80
Q
-
O
., __.
:E
o," ~o~
4o.
.
,,,...
O..o
,o_..
I
I 20
I
I 40
I
I 60
I
I 80
I
I I00
I
I I 120
I 140
GRAINS
Fig. 6. Plot of grain density through the molecular layer of the fascia dentata suprapyramidal limb contralateral and ipsilateral to CA3c injection at 4 days. Counts were prepared by plotting onto graph paper, by the use of camera lucida, individual silver grains in 50 #m strips at 5/tin intervals through the suprapyramidal limb of the fascia dentata molecular layer slightly rostral to the injection site (see Fig. 8a) in subject E4-2AR. Top of stratum granulosum is at 0 on ordinate; fissure is at 80 pm. A background count of two grains per 5/~m × 50/~m area due to "physical", non-physiological causes was determined by counting an area not overlying the brain section. This was subtracted from each interval count before plotting. Note that in both ipsilateral (open circles) and contralaterat (filled circles) molecular layers the maximal grain density appears nearest the stratum granulosum, and that the density is above a physiological background (0-3 grains per 5 #m × 50/~m area, as determined by measurements taken over a remote brain area, as per Gottlieb and Cowan z°) for approximately 20-30 /~m. The higher apparent "background" in the outer 70% of the molecular layer ipsilateral to the injection site may be due to non-specific diffusion of the label, or to other factors.
40
o
2o
u
2
4
6
8
I0
12
14
16
18
20
22
24
GRAINS
Fig. 7. Plot of grain density through the molecular layer of the infrapyramidal limb of the fascia dentata contralateral and ipsilateral to CA3c injection at 4 days. The procedure for plotting and determining background levels are as described in Fig. 6. Measurements are taken from the same sections as in Fig. 6, slightly rostral to the injection site. Note that lamination of the commissural projection (filled circles) is weaker than in the suprapyramidal limb (Fig. 6), and that there is no apparent lamination of the associational projection (open circles).
Autoradiography A n a l y s i s o f the b r a i n s p r e p a r e d for a u t o r a d i o g r a p h y reveals a slightly different p a t t e r n o f afferent d e v e l o p m e n t . T h a t is, while n o c o m m i s s u r a l t e r m i n a l field stains in the fascia d e n t a t a m o l e c u l a r layer at 4 d a y s b y the F i n k - H e i m e r m e t h o d , a d i s t i n c t b a n d o f t r a n s p o r t e d label a p p e a r s in the i n n e r 37 %, or 30/zm, o f the m o l e c u l a r layer o f
237
A
E4-2
¢
Fig. 8. Representative injection sites revealing commissural/associational and entorhinal projections into the fascia dentata at 4 and 10 days. A: subject E4-2AR - - heavy cell labeling (black area) is confined to the temporal stratum granulosum and hilus, while moderate cell labeling (hatched area) extends to retrohippocampal cortex. B: subject E10-3AR - - heavy cell labeling is localized in dorsomedial entorhinal cortex with moderate labeling in adjacent retrohippocampal area. C: subject H10-1AR - - heavy cell labeling is localized to stratum granulosum, hilus, and CA3c-CA4 region of the rostral fascia dentata, with moderate cell labeling in Ammon's horn extending from temporal subiculum to rostral CA3a-CA2. the suprapyramidal limb (Fig. 6), but not the infrapyramidal limb (Fig. 7), contralateral to the proline injections in 3 subjects at this age. In all 3 of these animals the injections reached into the CA3c-hilus region of A m m o n ' s horn as well as into the stratum granulosum of fascia dentata (Fig. 8A). In contrast, two other animals received injections of C A 3 a - b regions; these show no evidence of transported label into the contralateraJ fascia dentata, but do reveal a projection into the hippocampus proper. This agrees with Gottlieb and Cowan's 10 localization of the origin of commissural projections into the fascia dentata of the adult rat. In addition, all three 4-day animals, which show a commissural projection to the fascia dentata, also reveal the presence of the ipsilateral associational system from CA3c-hilus into the suprapyramidal molecular layer (Fig. 6), in accordance with
238
~
IOO--
2
4
6
8
IO
12
14
16
18
20
22
GRAINS
Fig. 9. Plot of grain density through the molecular layer of the suprapyramidal limb of the fascia dentata ipsilateral to entorhinal cortex injection at 10 days. The procedure for plotting and determining background levels is as described in Fig. 6. Subject EI0-3AR.
100i-
~: so o
60
40-m
20-m m
I
II
II
I
I
IIII
GRAINS
Fig. 10. Plot of grain density through the molecular layer of the infrapyramidal limb of the fascia dentata ipsilateral to entorhinal cortex injection at 10 days. The procedure for plotting and determining background levels is as described in Fig. 6. Subject and brain sections same as in Fig. 9.
239
%o
10
20
30
40
SO
60
GRAINS
Fig. 11. Plot of grain density through the molecular layer of the suprapyramidal limb of the fascia dentata ipsilateral and contralateral to CA3c injection at 10 days. The procedure for plotting and determining background levels is as described in Fig. 6. Subject, HI0-1AR. Injection site depicted in Fig. 8C. ]psilateral (open circles) and contralateral (closed circles) plots taken from same section, slightly rostral to the injection site.
ioo m
°
'°:!!
.o,
,.oll ° 40
~----.~%
GRAINS
Fig. 12. Plot of grain density through the molecular layer of the infrapyramidal limb of the fascia dentata ipsilateral and contralateral to CA3c injection at I0 days. The procedure for plotting and determining background levels is as described in Fig. 6. Subject, H10*IAR, same as Fig. 11. Measurements taken from same brain sections as Fig. 11. Zimmer's 34 studies in adult rats. The projection resulting f r o m the two caudal injections (Fig. 8A) appears considerably more extensive than the single case in which this system was labeled following a rostral injection. While in b o t h instances the pattern is laminated and restricted to approximately the inner 30 # m (37 ~ ) o f the fascia dentata molecular layer, the suprapyramidal cell field is more nearly innervated along its entire length in the f o r m e r cases, although at this age the lamination is not well defined in the infrapyramidal limb (Fig. 7). The terminal fields potentially labeled by the injections into the medial ento-
240
40pm ~/.//y//>
,/////,/. .,///~//,
q I 4 DAY
10 DAY
T.
I
26 DAY
:1 ADULT
Fig. 13. Schematic summary of dendritic growth and afferent lamination in the developing fascia dentata. Relative terminal zones in the suprapyramidal limb of the fascia dentata molecular layer as given in Table I are represented at 4 ages. Entorhinal terminal zone is represented by hatching; commissural/associational terminal zone is represented by stippling. The zones of juxtaposition between these two terminal fields have been matched at the various ages to compare their relative growth. Note that the commissural/associational terminal field increases relatively little, particularly if the later developing "clear " zone suprajacent to the stratum granulosum (the supragranular zone of Fig. 1) is taken into consideration. This may represent terminals of axons originating in the medial septal nucleus, which may begin arriving in the molecular layer around postnatal day 10, although afferents from other sources may be partially responsible for this apparent removal of commissural fibers from the immediate supragranular zone. The lighter stippling of the more distal commissural/ associational zone in the adult figure is based on observations of Gottlieb and Cowan 1° that a dense commissural zone of 30 ttm is bounded by a zone of lighter termination continuing an additional 20 #m into the molecular layer. All measurements were taken from the Fink-Heimer series except the commissural zone at 4 days, for which the autoradiographic measurement was used. See text for details.
rhinal cortex were n o t a p p a r e n t a b o v e the r a t h e r high b a c k g r o u n d , p r o b a b l y due to the coincidental labeling o f cells in the t e m p o r a l fascia d e n t a t a in these 4-day animals. A t 10 days, however, the t e r m i n a l field in the d e n t a t e a p p e a r s well l a m i n a t e d a n d restricted to the o u t e r 66 ~o (80/zm) o f the m o l e c u l a r layer in b o t h s u p r a p y r a m i d a l and infrap y r a m i d a l limbs (Figs. 9 a n d 10) following e n t o r h i n a l cortex injections (Fig. 8B). The p a t t e r n s o f c o m m i s s u r a l a n d a s s o c i a t i o n a l axons a n d t e r m i n a l s in the 10d a y a n i m a l s are essentially similar to those outlined in 4 - d a y - o l d r a t s : p r o j e c t i o n s to the fascia d e n t a t a after injections in C A 3 c - h i l u s (Fig. 8C) o c c u p y the inner 33 ~,, (40 # m ) o f the s u p r a p y r a m i d a l l i m b m o l e c u l a r l a y e r (Fig. 11), a n d a p p e a r l a m i n a t e d also in the i n f r a p y r a m i d a l l i m b (Fig. 12). T h e o v e r l a p o f c o m m i s s u r a l a n d e n t o r h i n a l projections, as c a l c u l a t e d f r o m the grain counts in Figs. 9 a n d 11 is less t h a n 10 # m even at 10 days o f age; this is c o m p a r a b l e to t h a t m e a s u r e d in a d u l t s ( M a t t h e w s , C o t m a n a n d Lynch, u n p u b l i s h e d observations). These d a t a are i n c l u d e d with those o f the F i n k - H e i m e r study in T a b l e I.
241 DISCUSSION By all available measures, the hippocampal formation of the neonatal rat is extremely immature. At 4 days after birth the molecular layer of the suprapyramidal fascia dentata is only one-third the width it will attain at adulthood, and the infrapyramidal limb is even less well developed. At this age the majority of granule cells are still migrating into the stratum granulosumz,29, and morphological and biochemical differentiation have only just begun2, 7,1s-~°,26. In light of this overall youthful condition it seems remarkable that at 4 days of age the newly arriving entorhinal and commissural/associational afferents have already established territorial relationships that seem to persist into adulthood. At 4 days, the entorhinal projection, as revealed by the Fink-Heimer method, is restricted to the outer 40-45 #m of the molecular layer. This lamination is probably not as sharp as in the adult and is present only in the suprapyramidal limb of more rostral hippocampal sections. The commissural projection appears by the autoradiographic analysis also to be laminated at 4 days, being restricted to the inner 30-35/~m of the suprapyramidal limb. The Fink-Heimer data, however, suggest that this projection may be more immature than the entorhinal, as the terminals of the former do not stain by this method until 10 days of age. The present study does not allow conclusions as to the establishment of the lamination prior to 4 days of age. One possibility is that the two systems co-mingle within the young molecular layer until this age in the suprapyramidal limb (or even older in the later developingl,Z, ~9 infrapyramidal limb); around 4 days of age, the systems would then have to segregate themselves to produce the patterns described at this and older ages. The present Fink-Heimer analysis suggests, however, that entorhinal afferent arrival may precede that of the commissural/associational system, thereby occupying alone the full width of the very young molecular layer. The commissural/ associational system then appears to enter the molecular layer as a relatively established axon bundle about 30 #m wide. If this interpretation is correct, the entorhinal terminals may reorganize by moving outward on the dendrites or by removing themselves altogether from the inner molecular layer. Alternately, the dendrites may grow selectively in the inner molecular layer, beginning at the point of entry of the commissural/associational axons, the free end of the suprapyramidal limb. This growth would carry the entorhinal axons into a position more distal to the cell bodies than that which they occupy in the youngest animals, leaving new dendritic surface for the commissural/associational afferents to contact. Beginning at about 10 days of age a separate afferent system, probably originating in the medial septal nucleus, appears to enter the supragranular zone of the molecular layer, perhaps inducing growth of the dendrites in this zone to accommodate the new arrival. This interpretation of a later arriving population is consistent with earlier studies of Mellgren20 and Matthews et al. 18, in which the mature pattern of acetylcholinesterase staining in the fascia dentata does not appear until around the sixteenth postnatal day, at which time a relatively intense zone appears immediately suprajacent to the stratum granulosum. Since continued expansion of the molecular
242 layer from day 26 onward is reflected mainly as increased width in the zone of entorhinal terminals, further dendritic e l o n g a t i o n most likely occurs in this more distal region. This s u m m a r y is schematized in Fig. 13. While the present study does not provide unequivocal evidence for afferent induction of dendritic growth, as proposed in other b r a i n systems13, 24, it seems at least reasonable to propose that e l o n g a t i o n occurs at several points along the dendrites d u r i n g development. T h a t is, it is more likely that dendritic growth is correlated with, if n o t regulated by, the sequentially arriving afferents, t h a n that the relationships measured are m a i n t a i n e d by a shifting of the terminal p o p u l a t i o n s to a c c o m m o d a t e new arrivals. O f course, the conclusive d e t e r m i n a t i o n of this hypothesis m u s t await studies analyzing the afferent p a t t e r n prior to 4 days of age as well as the growth of dendrites at various postnatal ages, in order to determine the true relationship of afferent ingrowth a n d dendritic differentiation.
REFERENCES l Angevine, J. B., Time of neuron origin in the hippocampal region. An autoradiographic study in the mouse, Exp. Neurol., Suppl. 2 (1965) 1-70. 2 Bayer, S. A. and Altman, J., Hippocampal development in the rat : cytogenesis and morphogenesis examined with autoradiography and low-level X-irradiation, J. comp. Neurok, 158 (1974) 55-80. 3 Blackstad, T. W., Commissural connections of the hippocampal region in the rat, with special reference to their mode of termination, J. comp. NeuroL, 105 (1956) 417-538. 4 Blackstad, T. W., On the termination of some afferents to the hippocampus and fascia dentata: an experimental study in the rat, Acta anat. (Basel), 35 (1958) 202-214. 5 Cotman, C. W., Taylor, D. and Lynch, G., Ultrastructural changes in synapses in the dentate gyrus of the rat during development, Brain Research, 63 (1973) 205-213. 6 Crain, B., Cotman, C., Taylor, D. and Lynch, G., A quantitative electron microscopic study of synaptogenesis in the dentate gyrus of the rat, Brain Research, 63 (1973) 195-204. 7 Das, G. D. and Kreutzberg, G. W., Postnatal differentiation of the granule cells in the hippocampus and cerebellum: a histochemical study, Histochemie, 10 (1967) 246-260. 8 Fink, R. P. and Heimer, L., Two methods for selective silver impregnation of degenerating axons and their synaptic endings in the central nervous system, Brain Research, 4 (1967) 369-374. 9 Goldowitz, D., White, W. F., Steward, O., Cotman, C. and Lynch, G., Anatomical evidence for a projection from the entorhinal cortex to the contralateral dentate gyrus of the rat, Exp. NeuroL, 47 (1975) 433-441. l0 Gottlieb, D. I. and Cowan, W. M., Autoradiographic studies of the commissural and ipsilateral association connections of the hippocampus and dentate gyrus of the rat. I. The commissural connections, J. comp. Neurol., 149 (1973) 393-422. l I Hjorth-Simonsen, A., Projection of the lateral part of the entorhinal area to the hippocampus and fascia dentata, J. comp. Neurok, 146 (1972) 219-232. 12 Hjorth-Simonsen, A. and Jeune, B., Origin and termination of the hippocampal perforant path in the rat studied by silver impregnation, J. comp. Neurol., 144 (1972) 215-232. 13 Kornguth, S. E. and Scott, G., The role of climbing fibers in the formation of Purkinje cell dendrites, J. comp. Neurol., 146 (1972) 61-82. 14 Leonard, C. M., A method for assessing stages of neural maturation, Brain Research, 53 0973) 412-416. 15 Leonard, C. M., Degeneration argyrophilia as an index of neural maturation : studies on the optic tract of the golden hamster, J. comp. Neurol., 156 (1974) 435-458. 16 Lorente de N6, R., Studies on the structure of the cerebral cortex. II. Continuation of the study of the ammonic system, J. PsyehoL Neurol. (Lpz.), 46 (1934) 113-177. 17 Lynch, G. and Cotman, C. W., The hippocampus as a model for studying plasticity in the adult brain. In R. L. Isaacson and K. H. Pribram (Eds.), The Hippocampus, Vol. 1, Structure and Development, Plenum, New York, 1975, pp. 123-154.
243 18 Matthews, D. A., Nadler, J. V., Lynch, G. S. and Cotman, C. W., Development of cholinergic innervation in the hippocampal formation of the rat. I. Histochemical demonstration of acetylcholinesterase activity, Develop. Biol., 36 (1974) 130-141. 19 Mellgren, S. I., Distribution of succinate-, a-glycerophosphate-, NADH-, and NADPH dehydrogenases (tetrazolium reductases) in the hippocampal region of the rat during postnatal development, Z. ZellJbrsch., 141 (1973) 347-373. 20 Mellgren, S. I., Distribution of acetylcholinesterase in the hippocampal region of the rat during postnatal development, Z. Zellforsch., 141 (1973) 375-400. 21 Mellgren, S. I., and Srebro, B., Changes in acetylcholinesterase and distribution of degenerating fibres in the hippocampal region after septal lesions in the rat, Brain Research, 52 (1973) 19-36. 22 Moore, R. Y., Monoamine neurons innervating the hippocampal formation and septum: organization and response to injury. In R. L. Isaacson and K. H. Pribram (Eds.), The Hippocampus, Vol. 1, Structure and Development, Plenum, New York, 1975, pp. 215-237. 23 Moore, R. Y. and Halaris, A. E., Hippocampal innervation by serotonin neurons of the midbrain raphe in the rat, J. comp. NeuroL, 164 (1975) 171-184. 24 Morest, D. K., The growth of dendrites in the mammalian brain, Z. Anat. Entwiekl.-Gesch., 128 (1969) 290-317. 25 Mosko, S., Lynch, G. and Cotman, C. W., The distribution of septal projections to the hippo.campus of the rat, J. comp. Neurol., 152 (1973) 163-174. 26 Nadler, J. V., Matthews, D., Cotman, C. W. and Lynch, G. S., Development of cholinergic innervation in the hippocampal formation of the rat. II. Quantitative changes in choline acetyltransferase and acetylcholinesterase activities, Develop. Biol., 36 (1974) 142-154. 27 Raisman, G., Cowan, W. M. and Powell, T. P. S., The extrinsic afferent, commissural and association fibres of the hippocampus, Brain, 88 (1965) 963-996. 28 Ram6n y Cajal, S., The Structure of .4mmon's Horn (L. M. Kraft, Transl.), Thomas, Springfield, 111., 1968. 29 Schlessinger, A. R., Cowan, W. M. and Gottlieb, D. I., An autoradiographic study of the time of origin and the pattern of granule cell migration in the dentate gyrus of the rat, J. comp. Neurol., 159 (1975) 149-176. 30 Schneider, G. E., Two visual systems, Science, 163 (1969) 895-902. 31 Segal, M. and Landis, S., Afferents to the hippocampus of the rat studied with the method of retrograde transport of horseradish peroxidase, Brain Research, 78 (1974) 1-15. 32 Steward, O., Topographical organization of the projections from the entorhinal area to the hippocampal formation of the rat, J. eomp. NeuroL, 167 (1976) 285-314. 33 Van Hoesen, G. W. and Pandya, D. N., Some connections of the entorhinal (area 28) and perirhinal (area 35) cortices in the rhesus monkey. IlL Efferent connections, Brain Research, 95 (1975) 39-59. 34 Zimmer, J., lpsilateral afferents to the commissural zone of the fascia dentata, demonstrated in de-commissurated rats by silver impregnation, J. comp. Neurol., 142 (1971) 393-416.
