A quantitative electron microscopic study of synaptogenesis in the dentate gyrus of the rat

Brain Research, 63 (1973) 195-204 © ElsevierScientificPublishingCompany,Amsterdam- Printed in The Netherlands

195

A QUANTITATIVE ELECTRON MICROSCOPIC STUDY OF SYNAPTOGENESIS IN THE DENTATE GYRUS OF THE RAT

BARBARA CRAIN, CARL COTMAN, DWAN TAYLOR AND GARY LYNCH

Department of Psychobiology, University of California at Irvine, Irvine, Calif. 92664 (U.S.A.} (Accepted May 22nd, 1973)

SUMMARY

Synapse development was studied by electron microscopy in the molecular layer of the dentate gyrus of the rat at 4 different ages: 4, 11, 25, and at least 90 days after birth. At 4 days after birth, less than 1 ~o of the synapses seen in the adult are present. Even by 11 days the total number of synapses is still less than 5 ~ of those in the molecular layer of the adult. Synaptogenesis is most active between 4 and 11 days when the total number of synapses approximately doubles every day. Between 4 days and adult the total number of synapses increases by nearly 100-fold in the dentate gyrus.

INTRODUCTION

The molecular layer of the rat's dentate gyrus, with its orderly segregation of afferent terminals into well-defined lamina, is admirably suited for studying synaptogenesis. The sources of the incoming fibers and the synaptic organization of the area are well known in the adult animal; and since most cell proliferation occurs postnatally2, the dentate gyrus can be easily observed at nearly every stage of its development. The dentate gyrus of the adult rat in sections at right angles to the axis of the hippocampus resembles a horseshoe curved around the regio inferior pyramidal cells of the hippocampus (Fig. 1). The cell bodies of the dentate gyrus, localized in the granular layer, send all their dendrites branching out towards the periphery of the dentate to form the molecular layer; their axons converge in the polymorphic layer beneath the granule cells and course into the regio inferior of the hippocampus as the mossy fibers. While the outer two-thirds of the molecular layer receives its extrinsic input from the entorhinal cortexg, 14, the one-third closest to the granule cell bodies receives innervation from the contralateral hippocampus 5,11. Associational fibers from the ipsilateral

196

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hippocampus also terminate in this latter zone 19. In addition, various types of interneurons in the molecular and polymorphic layers also have their axons, dendrites and synapses throughout the molecular and granular layers lz. Recently we observed that experimental lesions of the entorhinal cortex of either developing or adult rats result in an abnormal organization of synapses in the dentate gyrus, the exact nature of which depended on the age of the rat when the entorhinal lesion was performed 7,12,13. Both the extent of the reorganization and the magnitude of the synaptic change is more dramatic following lesions in young rats. The basis for the difference in postlesion adjustments between developing and adult rats is not known, but must reside in a structural or chemical property peculiar to either the developing or adult system. Both the number of synapses formed at a particular time and the relationships of the different developing fiber systems to the emergent granule cell population would be expected to be important. In the present study we examine the number and type of synapses in the molecular layer of the dentate gyrus in order to determine the extent of synaptogenesis at various developmental states. According to autoradiographic determinations of granule cell birth datesZ, 3, there exist dorsal-ventral and possibly temporo-septal gradients in cell age. It was, therefore, necessary to select a common locus to be used in comparing the different animals. We chose a point in the ventral leaf of the anterior dentate gyrus. A few sections from the more mature dorsal leaf were also examined for comparison. MATERIALS AND METHODS

Normal Sprague-Dawley rats obtained from Horton Laboratories in Oakland, California were sacrificed 4, 11, 25, and at least 90 days after birth. Each was anesthetized with Nembutal and perfused through the heart with a solution of 0.5 ~ glutaraldehyde, 4 . 0 ~ paraformaldehyde, 0 . 5 4 ~ dextrose, and 0.1 M sodium phosphate buffered to pH 7.416. The brains were removed and postfixed for 1 h in the perfusion medium before being sectioned coronally (150/zm sections) with a Sorvall tissue chopper. Each section was placed in 0.1 M phosphate buffer (pH 7.4), and the dorsal and ventral leaves of the dentate gyrus were dissected out of the anterior sections with the aid of a dissecting microscope. The location is illustrated in Fig. 1. After all the sections were cut, they were postfixed in 1 ~ osmium tetroxide in Caulfield buffer6 for 1-2 h and block-stained in 0 . 5 ~ uranyl acetate in Kellenberger buffer 1° overnight. The next day the samples were dehydrated in a graded ethanol solution (35 ~ , 5 0 ~ , 7 0 ~ , 100 ~o). After a final dehydration in propylene oxide, samples were embedded in Epon-Araldite in small dishes so they could be oriented. Sections were cut perpendicular to the granule cell layer, stained with lead citrate 17, and photographed with a Zeiss electron microscope. A series of consecutive photographs was taken at a primary magnification of 8000 × from the edge of the granular layer out to the exposed surface of the dentate gyrus (for ventral leaf sections) or to the hippocampal fissure (for dorsal leaf sections). Each picture covered 60 sq.~m. Animals used for volume estimations of the relative volume of the molecular

SYNAPTOGENESISIN DENTATEGYRUS

197

DLM

G

VLM

Fig. 1. Organization of the anterior hippocampal formation in the rat viewed in a coronal section. The 3 major parts of the hippocampal formation are the regio superior (S), regio inferior (I), and the dentate gyrus (DG). The pyramidal cells (P) are the major neurons in the regio superior and in the regio inferior. In the dentate gyrus the granule cells (G) extend their dendrites into the molecular layer. The molecular layer, which is made up of a dorsal (DLM) and ventral (VLM) leaf, receives its major projections from the entorhinal cortex and contralateral and ipsilateral pyramidal cells. The dorsal leaf of the molecular layer is separated from the regio superior by the hippocampal fissure (F). The samples of the molecular layer used for this study were taken from the areas indicated by the blocks.

layer were perfused intracardially with 10% formalin. A series of coronal sections from the anterior region of the dentate were sectioned (50/zm) on a freezing microtome and stained with cresyl violet. Relative volumes at different ages were obtained by reconstructing a series of sections in that portion of the septo-temporal axis from which the electron microscopic sections were taken; the sum of the individual units gave the total volume. Relative areas of synaptic boutons were computed by cutting these out from prints and weighing the individual boutons. RESULTS Synaptic endings were identified by the presence of 5 or more synaptic vesicles of approximately 40 nm in diameter and a synaptic cleft of 20-30 nm, and were classified according to the appearance of their pre- and postsynaptic membrane specializations. Although many types of synapses were seen, all of them were placed into one of two classes: asymmetric synapses, in which the postsynaptic density is 2 or 3 times as thick as the presynaptic density, and symmetric synapses with pre- and postsynaptic densities of equal width. Symmetric type synapses included those with their postsyn-

Fig. 2. Examples of neonatal synapses which fall into 3 different categories, are as follows. A: those synapses in the asymmetric category have an asymmetric synaptic junctional contact zone in which the postsynaptic density (arrows) gives the synaptic complex an asymmetric appearance; B: a class of synapses was identified in which the synaptic complex was not well enough defined to place into either the symmetric or asymmetric category. Most of the synapses in this category appeared to have been sectioned in an oblique or tangential plane to the synaptic complex (arrow); C: the symmetric class included two types of synapse. The first type is a synapse with a symmetric contact zone which corresponds to the Gray type 11 synapse where neither the pre- nor postsynaptic specializations (arrows) are electron dense (C-I). The second type is a synapse with a symmetric junctional contact zone in which both the pre- and postsynaptic plasma membrane specializations (arrows) are of approximately equal electron density (C-2). Scale bars represent 1 /ml.

SYNAPTOGENESIS IN DENTATE GYRUS

199

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w

0

I I0

i 20

i ~0

klu#

DAYS AFTER BIRTH

Fig. 3. The increase in volume of the molecular layer of the anterior dentate gyrus during development.

aptic densities of the type classified as Gray type II s as well as those with thick postsynaptic densities and a correspondingly dense presynaptic density. Synaptic contact regions which were poorly defined were not placed in either asymmetric or symmetric types, but were included in the total number of synapses. Several examples of our use of this classification are illustrated in Fig. 2A-C. A single presynaptic ending which exhibited more than one area of synaptic contact was counted as one synapse. Processes containing synaptic-like vesicles (PSVs) but without recognizable contact regions were seen more often than all types of identified synapses combined. PSVs were classified as a separate category although these structures may be synapses sectioned through the 'synaptic bag' in a plane excluding the contact region proper 4. As expected, the molecular layer of the anterior dentate gyrus expands and differentiates dramatically between birth and adulthood. Based on light microscopy calculations, the molecular layer in the anterior region increased its total volume over 25 times (Fig. 3). The density of synapses in the ventral leaf rose over 100 times between 4 days after birth to adulthood (Table I). The greatest rate of increase occurred between 4 days and 11 days after birth when the density of synapses increased approximately 20 times. Between 25 days and adult, there is essentially no change in density. In the dorsal leaf at both 4 and 11 days the synaptic density is higher (Table I) than in the ventral leaf. The variations in synapse number, both between animals and between different samples from the same animal, were small and in no case except for 4 day values in the dorsal leaf did the variation in the synaptic density vary more than 7 70 from the mean (Table I). In general the change in density of processes containing vesicles, but without a visible synaptic contact, paralleled that observed for presynaptic boutons with an identifiable contact. The total increase in synapses in anterior sections of ventral leaf, computed by taking into account the increase in total volume of this area, rose approximately 1000-2000 times between 4 days and adult (Fig. 4.) Again, the largest rate increase was observed between 4 days and 11 days when the number of synapses at a minimum doubles on the average every day. Synapses in the dorsal leaf increase approximately 300-fold between 4 days and adult. The proportion of synapses with asymmetric and symmetric contacts varies at different ages with symmetric contacts being prevalent in young animals and asym-

200

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TABLE I CHANGES IN SYNAPTIC DENSITY AND PER CENT OF SYMMETRIC ( S ) AND ASYMMETRIC ( A ) JUNCTIONAL CONTACTS I N THE DENTATE GYRUS DURING DEVELOPMENT

Age (days)

Animal

ventral leaf 4-1 4-2 dorsal leaf 4-1 4-2

Synapses/ 100 sq./zm*

Class ~4S

oJ /o A

0.24 0.25

67

33

1.04 3.03

45

55

6.11 6.25 5.57 = 5.97±0.27

63 66 58 62~3

37 34 42 37 ± 3

9.3

46

54

2.03 ± 1.00 11

ventral leaf ll-la ll-lb 11-2 dorsal leaf 11-2

25

25-1a 25-1b 25-2

26.2 28.5 27.5 =27.4 ~:0.7

37 19 20 25 ± 7

63 81 80 74 ~:7

Adult

A-la A-lb A-2

27.7 31.5 25.7 =28.2 ~:2.0

14 12 10 12~ 1.3

86 88 90 88 ~ 1.3

* Data are reported as mean with average deviation, a and b samples are taken from the ventral leaf of the same animal approximately 200-400/~m apart.

metric in older animals (Table I). In the ventral leaf of 4- or 11-day-old animals approximately 6 0 ~ of the contacts are symmetric. This class decreases and in the adult molecular layer only approximately 1 0 ~ of total contacts are symmetric. A concomitant increase in asymmetric contacts is seen. At any age no dramatic differences were observed in either the total number or the proportion of asymmetric and symmetric synapses among different thirds of the molecular layer although the inner one-third always contained fewer synapses (Table ii). At 11 days of age, as well as at 4 days, the dorsal leaf contains a higher proportion of asymmetric synapses which are characteristic of older animals (Table I). The proportion of space occupied by presynaptic 'bags' (including PSVs) rose more slowly than expected (Fig. 5a) since the presynaptic terminals of the two younger age groups were on the average much larger than those of the older animals. The mean cross-sectional area of presynaptic profiles, as determined graphically, decreased by one-half between 11 and 25 days and then rose again slightly in the adult (Fig. 5b). Size differences did not appear to be correlated with other morphological characteris-

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o c~ Z

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20

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Adult

DAYS AFTER BIRTH

Fig. 4. Increase in total number of synapses in ventral leaf of the anterior dentate gyrus during development.

TABLE II SYNAPTIC CHANGES IN THIRDS OF THE VENTRAL LEAF OF THE MOLECULAR LAYER OF THE DENTATE GYRUS DURING DEVELOPMENT

Age

Layer

% Total

% Asymmetric

% Symmetric

4

Outer Middle Inner

28 38 34

11

Outer Middle Inner

35 q- 4 36 4- 8 27 4- 8

36 4- 8 41 4- 5 34 4- 7

63 4- 8 58 4- 5 64 4- 4

25

Outer Middle Inner

36 4- 2 34 4- 2 29 4- 1

75 4- 6 76 ! 3 76 4- 5

24 4- 6 24 4- 10 24 4- 4

Adult

Outer Middle Inner

38 4- 5 33 4- 3 29 4- 1

86 4- 2 89 ± 2 89 4- 4

13 4- 2 11 4- 2 10 4- 3

tics, and the number of synaptic vesicles was approximately proportionate to the cross-sectional area. DISCUSSION

During the first weeks after birth, dentate granule cells are still multiplying rapidly, migrating to their final positions, and sending out dendritic processes z, while afferent fiber systems to the molecular layer are also arriving. The small variation between animals and within the same animal at any age in our data is a strong testament for the precision and uniformity by which the ventral leaf of the molecular layer normally develops. Although the volume of the anterior portion of the dentate gyrus increases at least 25 times between 4 days after birth and maturity, the rate of synaptogenesis exceeds both overall growth and the rate of granule cell formation. The 100-fold

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DAYS AFTER BIRTH

Fig. 5. Changes in the area of the molecular layer of the dentate gyrus occupied by presynaptic profiles (upper) and the average cross-sectional area of presynaptic profiles during development (lower).

increase in synapse density in the ventral leaf is more dramatic than the changes observed in the rat in the motor cerebral cortex 4, but is approximately comparable to that observed in the cerebellum ~s. Both the dentate gyrus and cerebellum are remarkably immature at 4 days after birth. Between 4 days and 25 days synapses form at an explosive rate. At 25 days synaptic densities reach adult values in the dentate gyrus. Other brain regions studied to date, such as the parietal cortex 1, cerebellum and probably the motor cortex, mature at about 25 days. Although the data is sparse, it appears that synaptogenesis in the dentate gyrus is completed at approximately the same time as in other brain regions; but because initially the dentate gyrus is immature and small, growth in this region must proceed at an accelerated rate to catch up with other brain regions. Because of the dramatic volume change which occurs in the dentate gyrus during development, the rate of increase in the total number of synapses is the most dramatic so far measured. In the ventral leaf, the number of synapses approximately doubles every day between 4--11 days of age. Based on estimates from available data 2, synaptogenesis during the periods 4-11 and 11-25 days probably occurs more rapidly than the addition of new granule cells. However, this conclusion must be regarded as preliminary until further data on granule cell development along the septo-temporal axis is available. The incomplete development of the neonatal brain may explain in part the remarkable responses to entorhinal cortical lesions that we have observed in immature animals. Less than 5 ~ of the total synapses in the ventral leaf and less than 1 0 ~ in the dorsal leaf have formed by 11 days. The establishment of all but a small portion of

SYNAPTOGENESISIN DENTATEGYRUS

203

a major afferent system is, therefore, prevented by lesion of the entorhinal cortex at this time. Unfilled synaptic sites are available on many or all of the granule cells that have been formed throughout the entire dendritic tree and will become available on new granule cells not yet formed. In this open system, an altered organizational pattern emerges following an entorhinal lesion. Commissural terminals torm synaptic contacts that extend beyond the usual inner one-third of the dendritic tree towards the outer surface of the dentate gyrus 13 and above the enlarged commissural zone additional acetylcholinesterasecontaining synapses are formed 7. Lesions in older animals result in progressively less dramatic organizational changes since normal synaptic and granule cell development have proceeded farther before disruption occurs. The transitional period, when the adult lesion-induced effects prevail over those characteristics of developing animals, occurs at about 25 days at which time the system is complete. Synaptic densities have reached adult values and granule cell development is essentially complete by that age. ACKNOWLEDGEMENTS Supported by Research Grants NSF GB 35315X and N I M H M H 19793 to G.L. and N I H NS 08597 and N I M H 19691 to C.C. We are grateful to Mrs. P. Lemestre for secretarial assistance.

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