Loss and reacquisition of hippocampal synapses after selective destruction of CA3–CA4 afferents with kainic acid

Brain Research, 191 (1980) 387-403

387

© Elsevier/North-Holland Biomedical Press

LOSS A N D REACQUISITION OF H I P P O C A M P A L SYNAPSES A F T E R SELECTIVE D E S T R U C T I O N OF CA3-CA4 A F F E R E N T S WITH K A I N I C ACID

J. VICTOR NADLER*, BRUCE W. PERRY, CHRISTINE GENTRY and CARL W. COTMAN Department of Psychobiology, University of California, Irvine, Calif. 92717 (U.S.A.)

(Accepted November 15th, 1979) Key words: hippocampus - - synaptic plasticity -- kainic acid -- brain lesions

SUMMARY Intraventricular injections of kainic acid were used to destroy the hippocampal CA3-CA4 cells bilaterally in rats, thus denervating the inner third of the molecular layer of the fascia dentata and stratum radiatum of area CA1. Electron microscopic studies showed that this lesion reduced the synaptic density of the CA1 stratum radiatum by an average of 86 ~ . The synaptic density of the inner third of the dorsal dentate molecular layer declined by two-thirds and the corresponding zone of the ventral dentate molecular layer by about half. Within 6-8 weeks the synaptic density of these laminae had been restored to the control value or nearly so. In the CA1 stratum radiatum about 72 ~ of the synaptic contacts destroyed by the lesion were replaced, the inner third of the ventral dentate molecular layer recovered 75 ~ of its lost synapses and the inner third of the dorsal dentate molecular layer apparently recovered virtually all of them. The newly formed synapses did not differ noticeably from those normally present. A kainic acid lesion reduced the synaptic density of the outer two-thirds of the dentate molecular layer by 30 ~ within 3-5 days, despite a virtual absence ofpresynaptic degeneration in that zone. This result implies a substantial disconnection of perforant path synapses. It did not appear to depend on the extent of denervation of the inner zone. The loss ofperforant path synapses was completely reversible. We suggest that the dentate granule cells shed a portion of their synapses in response to a substantial loss of neurons to which they project and regained them when their axons had formed new synaptic connections.

* To whom all correspondence should be addressed at present address: Department of Pharmacology, Duke University Medical Center, Durham, N.C. 27710, U.S.A.

388 INTRODUCTION Neurons of the hippocampal end blade are among the most sensitive in the human brain to destruction by such insults as anoxia, senile dementia and chronic temporal lobe epilepsy and status epilepticus4, 26. In seeking an animal model of this pathology, we discovered that intraventricular administration of the potent convulsant, kainic acid (KA), to rats preferentially destroys the homologues of these cells (CA3-CA4 pyramidal cells)2S, 29. Intraventricular KA does not directly damage fibers passing to or through the CA3-CA4 area nor does it destroy the dentate granule cells or the pyramidal cells of the h2 area (area CA2 and the immediately adjacent part of area CA3a). KA lesions may therefore provide information on the response of intact hippocampal tissue to loss of end blade neurons. Since these neurons have been shown in experimental animals to interconnect the various hippocampal regions 15,2~,3s, as well as to connect the two hippocampi to one another 13,18,2t,3v, their destruction must have devastating consequences for hippocampal function. One would like to know whether the synapses lost as a result of cell death are eventually replaced. Several instances of reinnervation or "reactive synaptogenesis" have been demonstrated in hippocampal regions after a mechanical or electrolytic lesion of particular afferents 6,v. Quantitative electron microscopic studies have shown that the great majority of the degenerated synaptic boutons are eventually replaced 12,24,~5. There is therefore every reason to think that such would be the case also in laminae denervated when CA3-CA4 cells are destroyed. We have presented light microscopic evidence that certain nearby afferents grow into the inner portion of the molecular layer of the fascia dentata after the CA4-derived innervation of this zone is destroyed bilaterally with KA ~0. In contrast, there was no evidence of growth into or proliferation in the denervated laminae of area CAl. The question arose whether reinnervation did, in fact, take place in area CA1. To investigate these issues, we quantitated the loss and reacquisition of synaptic contacts in the dentate molecular layer and stratum radiatum of area CA I after bilateral destruction of CA3-CA4 cells. Some of these results have been presented in preliminary form 31. MATERIALS AND METHODS Adult male Sprague-Dawley rats, 60-100 days of age, were used in this study. Electron microscopic data were obtained from 15 KA-treated rats, 2 animals injected with vehicle only and 5 untreated animals. Animals were injected intraventricularly with kainic acid as described by us previously 2s,29. Either 2.34 or 3.75 nmol of KA dissolved in l #1 of Elliott's artificial CSF 9 was injected over a period of 30 rain. This procedure was then repeated on the contralateral side. In the latter part of this study the dose was limited to 2.34 nmol into each lateral ventricle, since the higher dose killed about half the animals so treated but did not destroy any more CA3-CA4 neurons than the lower dose. Animals were killed at survival times ranging from 4 h to 55 days. Rats given artificial CSF alone were killed 3 days after treatment. Just before perfusion, 1 ml of

389 nitrite-heparin (12.8 mg/ml heparin, 10 mg sodium nitrite) was injected into the jugular vein under pentobarbital anesthesia. The animal was immediately perfused with a fixative containing 4 ~ (w/v) paraformaldehyde, 0.5 ~ (v/v) glutaraldehyde, 5 . 4 ~ (w/v) o-glucose and 0.1 m M sodium phosphate, pH 7.439. After an initial postfixation for 1-24 h in the perfusion medium, the hippocampal formation of one side was dissected out, and transverse slices of 150 # m thickness were cut with a Mcllwain tissue chopper (Brinkmann Inst., Westbury, N.Y.). Slices cut one-third the distance from the septal to the temporal end of the hippocampal formation were used for electron microscopy. Additional slices immediately adjacent to these were further cut into 20/zm-thick sections and stained with cresyl violet to determine the extent of the lesion. The extent of our bilateral KA lesions has been reported in detail elsewheree9,30. The transverse dorsal hippocampal slices were postfixed for 1 h in 1 ~ (w/v) osmium tetroxide in Caulfield's buffer 5 and stained with 0.5 ~ (w/v) uranyl acetate in Kellenberger buffer 19 for 1 h or overnight. Following dehydration in a graded series of ethanol solutions and propylene oxide, the slices were embedded flat in Epon-Araldite. Ultrathin sections (50-60 nm) were cut from these blocks through the entire length of area CA1 from alveus to hippocampal fissure midway between its junction with area CA2 and its medial border (i.e. area C A l b of Lorente de N6 e2) and through the length of both dorsal and ventral leaves of the fascia dentata from the upper part of the granule cell layer to the hippocampal fissure or pial surface midway between the ends and apex of the granule cell arch (Fig. 1). These sections were stained for 5 min with Reynolds' lead citrate a4, transferred to Formvar grids and examined and photographed in a Jeol model 100-C electron microscope.

Fig. 1. Coronal section through the dorsal hippocampal formation cut at the level used for electron microscopy 3 days after bilateral KA injection. Rectanglesenclose areas from which thin sections were cut. Hippocampal regions indicated are: CAI, CA3, CA4, h2 (area CA2 and immediately adjacent portion of area CA3a), fascia dentata dorsal leaf (FDD) and fascia dentata ventral leaf (FDV). Note destruction of CA3-CA4 neurons and accompanying gliosis. Cresyl violet stain. Scale bar = 1 mm.

390 Montages were constructed by taking rows of photographs perpendicular to the cell layer at a screen magnification of 10,000 ~. For purposes of synaptic analysis, the molecular layer was divided into thirds. The inner third (nearest the cell layer) corresponded to the zone of termination of CA4-derived afferents '~1 and the middle and outer thirds corresponded roughly to the zones of termination of mediaP 7,'~'~ and lateraP 6,35 perforant paths, respectively. Photomicrographs of area CAI were taken from the alvear surface to the hippocampal fissure, but only stratum radiatum was analyzed quantitatively. This lamina was defined as the zone which extended from the apical edge of the pyramidal cell bodies to the beginning of the temporo-ammonic fiber bundle. The border of this tract could easily be recognized on semi-thin sections cut from the same blocks and stained with thionin. In these montages every recognizable synapse was counted and classified as intact or degenerating and as containing mainly spheroidal or mainly flattened vesicles. Intact synapses were identified by the presence of synaptic vesicles in association with a synaptic junction. Degenerating synapses were identified as either an electron dense presynaptic profile associated with a synaptic junction (electron dense degeneration) or an abnormally lucent profile, whose synaptic vesicles were few and not in contact with the synaptic junction (electron lucent degeneration). The density of identified structures was calculated by averaging counts from the photomicrographs taken through each zone and dividing by the cross-sectional area sampled in each micrograph. An additional 33 rat brains prepared for light microscopy were used to determine the extent to which the denervated laminae atrophied. Of these, 26 had received bilateral intraventricular injections of KA and 7 had been operated and injected with artificial CSF alone. These animals were killed by transcardial perfusion with neutralized formalin-saline at the same survival times as those used for electron microscopy. After postfixation of the brain for 1-7 days, serial coronal sections of 20/zm thickness were cut through the entire hippocampal formation. Measurements of the relevant laminae were made on two sections stained with cresyl violet from each of two septotemporal levels (levels 1 and 2 of Nadler et al.27). Sections were projected onto white paper with use of a projecting microscope (× 45). Widths of the dentate molecular layer and stratum radiatum of area CA1 were measured normal to the cell layer at the same location from which sections were cut for electron microscopy. The upper boundary of stratum radiatum was taken as the beginning of the band of oligodendroglia that coincides with the myelinated temporo-ammonic fiber bundle. KA treatment did not affect the width of this band. RESULTS Normal ultrastructure The ultrastructure of the dentate molecular layer has been examined previously by Laatsch and Cowan 2o, Blackstad 2, Cotman et al. 8 and Matthews et al. 2a and stratum radiatum of area CA1 by Westrum and Blackstad 41. Our electron microscopic findings in untreated or sham-injected rats matched theirs so closely that a complete

391

Fig. 2. Stratum radiatum of area CA1 after bilateral KA administration. A: vehicle-injected rat. A number of S synapses are indicated, x 16,500. B: Presynaptic degeneration 1 day after KA treatment. Long arrow indicates an example of electron lucent degeneration. Broad arrows point to electron dense synaptic boutons, x 15,600. C: extensiveelectron dense presynaptic degeneration is present 3 days after KA administration. Two normal-appearing S synapses are indicated, x 17,400. D: Restoration of normal ultrastructure 55 days after KA administration, x 16,500. description need not be given here. In all laminae examined the great majority of synapses were formed by a small bouton, which contained spheroidal vesicles (S synapses), on a small dendritic spine (Figs. 2A, 3A, 4A). Virtually all of these contacts were of the asymmetric type (Gray type I). A minority of S synapses involved complex spines, which have been variously referred to as "calciform," "cup-" or "nose-like" or " U " - or "W"-shaped. In all control and K A treated rats, broad dendritic profiles dominated portions of the neuropil proximal to the cell bodies. Even here, the great majority of synapses were made on spines, which often could be seen to originate directly from the dendritic trunk. In the dentate molecular layer many of the synaptic bouton profiles proximal to the cell bodies appeared to be larger than any encountered in the more distal neuropil, but this apparent difference in terminal size

392 was not quantitated. Farther from the cell bodies, postsynaptic profiles in the dentate molecular layer were composed almost entirely of small dendritic branches and spines. In the distal portion of stratum radiatum, on the other hand, large dendritic profiles were still frequently encountered, although they were not so numerous as in the more proximal area. Glial profiles occupied relatively little area in these laminae. A small number of synapses in all laminae examined featured a bouton which contained predominantly flattened vesicles (F synapses) (Fig. 3B). These profiles were always larger than the average S synapse. The great majority were located on dendritic shafts or large spines, where they formed symmetric contacts (Gray type II). F synapses were rather rare, constituting on the average only 2.6 °~i of the total number of synapses in the dorsal dentate molecular layer, 1.8 ~'/,, of the total in the ventral dentate molecular layer and 0.9 to of the total in stratum radiatum of area CAl. In both dentate molecular layers, F synapses tended to cluster immediately above the granule cell bodies and at the border of the middle and outer thirds, but in material from any individual animal, they could appear at any depth of the layer. There was no obvious pattern to their distribution in the CA1 stratum radiatum. Synaptic contacts were more densely distributed in the ventral (or internal) dentate molecular layer than in the dorsal (or external) layer (Table I). On the dorsal leaf, the synaptic density tended to be highest in the middle third and on the ventral leaf, in the outer third. The presence of large dendritic profiles undoubtedly was at least partly responsible for the lower synaptic density of the inner zone. The synaptic density of CA1 stratum radiatum approximated that of the dorsal dentate molecular layer. Loss and reacquisition of synapses in CA1 stratum radiatum Destruction of CA3-CA4 neurons with KA denervated stratum oriens and stratum radiatum of area CA130. In addition, KA at the doses used here destroyed perhaps half the cell bodies in layer III of the entorhinal cortex and somewhat fewer in nucleus reuniens of the thalamus. These neurons provide much of the innervation of stratum lacunosum-molecularO4, 36. Except in one animal, whose CAI region was not ultrastructurally analyzed, the pyramidal cells of area CAlb remained intact by both light and electron microscopic criteria. Abundant terminal and fiber degeneration filled stratum radiatum and stratum oriens of area CA1 at survival times of I to 7 days. Sparser degeneration was present in stratum lacunosum-moleculare. No degenerating boutons were found apposed to the pyramidal cell bodies. Of these laminae, only stratum radiatum was analyzed in detail. The vast majority of the degenerating synaptic boutons atrophied somewhat and became electron dense (Fig. 2C). Only at 1-day survival did we detect evidence of socalled electron lucent degeneration (Fig. 2B). In these instances, about 10 ~ of the total degenerating population, the bouton appeared swollen and essentially empty of organelles, except for some light-to-dark flocculent material and perhaps a few raggedlooking synaptic vesicles. A number of other synaptic boutons exhibited some of these same features, but they were counted as intact as long as a number of synaptic vesicles were clustered at the presynaptic junction, since a few synapses of this ultrastructure

393 could occasionally be found in control material. Hence it is likely that some of the synaptic boutons that were classified as intact at 1-day survival were actually beginning to degenerate. We could identify no postsynaptic densities that were unapposed by an intact or degenerating bouton. The synaptic density of stratum radiatum appeared to have decreased somewhat already 4 h after KA administration, despite the absence of presynaptic degeneration (Table I). The synaptic density reached its nadir at about 14 ~ of normal in 3-7 days, and the incidence of presynaptic degeneration was also maximal at this time (Table II). During the first week after KA treatment, densities of intact and degenerating synaptic contacts combined remained essentially constant at the value attained 4 h after injection. In long-term survivors the synaptic density of stratum radiatum had returned to normal. In all cases a variable quantity of residual presynaptic degeneration remained after 6-8 weeks, including some electron dense synaptic boutons. Except for this, the neuropil appeared ultrastructurally normal. The apparently newly formed synapses exhibited the same features as those observed in control animals (Fig. 2D). Subtle differences might have escaped detection, however. There was no consistent change TABLE I Density o f intact synaptic contacts after bilateral K A lesion Values are expressed as means -4- S.E.M. for n = number of animals or as results from individual animals. Units are synapses per 100 sq./~m. Survival time

n

Dentate molecular layer - - dorsal leaf

Control 4h 1 day 3-5 days 6-8 weeks

Inner

Middle

Outer

33 ± 2 41, 38 26, 20 11 i 3 34 -4- 2

37 ± 2 32, 37 31, 28 25 -4- 3 38 -- 2

34 ± 2 33, 33 29, 34 24 -4- 1 36 -4- 1

4

5 5

Dentate molecular layer - - ventral leaf

Control 4h 1 day 3-5 days 6-8 weeks

lnner

Middle

Outer

42 ± 2 42, 49 30, 24 20 i 1 35 ± 1

44±3 46, 51 29, 28 30 i 2 39 4- 2

464-2 42, 48 36, 33 32 ± 2 42 -- 2

4

5 5

CA1 Stratum radiatum Control 4h 1 day 3-7 days 6-8 weeks

35±1 25, 30 14, 6 5±1 35±1

5

3 4

394

Fig. 3. Inner third of the dorsal dentate molecular layer after bilateral KA administration. A : vehicleinjected rat. A number of S synapses are indicated, z 17,400. B: F synapse forming a symmetric contacl and two S synapses forming asymmetric contacts in a control animal. ~ 25,000. C: presynaptic degeneration 5 days after KA treatment. Electron dense synaptic boutons are indicated by broad arrows. Other electron dense structures do not appear to be making synaptic contact. Note the absence of normal-appearing synapses and the presence of large dendritic profiles (D). t 6,500. D : restoration of normal ultrastructure 55 days after KA treatment. :~ 16,500. from n o r m a l at any time in the incidence of F synapses. Inspection of s t r a t u m oriens a n d s t r a t u m l a c u n o s u m - m o l e c u l a r e suggested that these laminae also had more or less recovered ultrastructurally by 6--8 weeks after treatment.

Loss and reacquisition of synapses in the dentate molecular layer I n t r a v e n t r i c u l a r a d m i n i s t r a t i o n of K A partially destroyed the CA4-derived (associational-commissural) afferents to the dentate molecular layer 3°. These projections terminate exclusively within the inner third of the molecular layer. Only perhaps 1-2 i~,~, of the stellate cells in layer I1 of the e n t o r h i n a l cortex were killed by the drug. Since the perforant path fibers arise p r e d o m i n a n t l y from these n e u r o n s ~6, this projection was

395

almost completely spared. We saw no evidence of degenerating or missing dentate granule cells in our material. No degenerating synaptic boutons could be recognized in the inner third of the molecular layer in control rats or in those which had survived only 4 h after KA administration, and at a survival time of 1 day only a few were present. Somewhat more extensive evidence of presynaptic degeneration appeared in 3-5 days. This took the form of electron dense boutons apposed to normal-appearing dendritic spines, which exhibited a thick postsynaptic density (Fig. 3C). In each animal killed at this survival time, the inner third of the dorsal molecular layer exhibited a greater density of degenerating synapses than the corresponding zone of the ventral molecular layer (Table II). Actually, only a minority of the electron dense profiles consisted of synaptic boutons. The greatest number of them could be recognized as boutons not associated with postsynaptic specializations (within the plane of section, at least) and preterminal fibers. In both molecular layers the degenerating elements were far more numerous in the most superficial part of the inner third and disappeared abruptly as one passed into the middle third of the layer. In contrast to the dentate molecular layer deprived of perforant path innervation 23, postsynaptic densities unapposed by a presynaptic element were exceedingly rare. Hence their incidence was not quantitated. We could identify only four degenerating synaptic contacts in the outer two-thirds of the molecular layer in all five rats combined (0.1 ~ of the total number of synapses). Other degenerating structures appeared there with even lesser frequency. This virtual absence T A B L E II Density o f intact and degenerating synaptic contacts after bilateral KA lesion Intact and degenerating synaptic contacts were identified as described in Materials and Methods. Values are expressed as means -4- S.E.M. for the number of animals given in Table I. Units are synapses per 100 sq./zm. Survival time

Intact

Degenerating

Total

Control

Inner third - - dorsal dentate molecular layer

4h 1 day 3-5 days 6-8 weeks

41,38 26, 20 11 i 3 34 -4- 2

0,0 < 1, < 1 3 4- 1 0

41,38 26, 20 14±2 34 -4- 2

33 4- 2

Inner third - - ventral dentate molecular layer

4 1 3--5 6-8

h day days weeks

42, 49 30, 24 20 -4- ! 35 -4- 1

0, 0 < 1, < 1 0.5 -4- 0.3 0

42, 49 30, 24 21 -4- 1 35 -4- 1

42 -4- 2

25,30 30,26 30±2 37 ± 2

354-1

CA1 Stratum radiatum

4 l 3-7 6-8

h day days weeks

25,30 14, 6 5 ± 1 35 ± 1

0,0 16,22 25 ± 3 1.8 ± 1.5

396

Fig. 4. Middle third of the dorsal dentate molecular layer in vehicle-injected rat (A) and rat that received bilateral injection of KA 5 days previously (B). Note in B the relative paucity of synapses and the unusual abundance of large dendritic profiles (D). Magnifications: A, × 17,400; B, 18,300.

397 of degeneration correlated well with the minimal destruction of perforant path fibers, which form the great bulk of synaptic contacts in this area. No residual degenerating synaptic boutons remained in 6-8 week survivors. At a 4-h survival time, the synaptic density of the inner third of the molecular layer appeared to have increased slightly (Table I), but because only two animals were killed at this time, this result might not have been representative. After this time, however, the synaptic density fell rapidly to about two-thirds of normal at 1-day survival and, at a 3-5 day survival period, to one-third of normal in the dorsal molecular layer and about one-half normal in the ventral molecular layer. Synapses having degenerating boutons accounted for only a small part of the difference between the synaptic densities of KA-treated and control rats (Table II). Surprisingly, despite the extreme rarity of presynaptic degeneration in the middle and outer thirds of the molecular layer, the synaptic density declined here too, by 30 ~ within 3-5 days (Table I). On the ventral leaf this response to KA treatment appeared to be fully developed even in 1 day. Rather than being associated with presynaptic degeneration, as in the inner third of the layer, the synaptic deficit appeared to correlate with a reduced incidence of small dendritic spines and a more frequent appearance of larger dendritic profiles. Only a small number of swollen dendritic profiles were seen, however, and they were only moderately hypertrophied. Occasionally, unusually large dendritic processes were seen to protrude from the shaft, presenting the appearance of enlarged spines (Fig. 4B). When examined 6-8 weeks after KA administration, the synaptic density of all zones of the dentate molecular layer had returned to normal or very nearly so (Table I). Except for an occasional remnant of electron dense material, the neuropil appeared ultrastructurally normal (Fig. 3D). The incidence o f F synapses was not altered by KA administration at any survival time. Although mossy fibers (granule cell axons) appear to invade the inner third of the molecular layer after destruction of CA4-derived fibers ~0, we found no clear evidence for the appearance of giant boutons, which characterize the mossy fiber projection 2,a.

TABLE III Widths o f denervated laminae after bilateral KA lesion Values are expressed as means :L S.E.M. for the number of animals given in parentheses. Widths in/~m. Survival time

Control 4h lday 3-5 days 33-105 days

Dentate molecular layer Dorsal

Ventral

257 261 246 251 243

274 4- 3 272±14 263 ~- 15 237 -+- 4* 246 ± 7*

-4- 8 (7) ± 3 (3) i 19 (3) ± 7 (8) ± 5 (10)

CA 1 Stratum radiatum

321 -4- 11 (7) 3 0 4 ± 10(3) 315 z~ 4 (3) 307 ± 9 (6) 243 ± 9 (5)*

* Significantly different from control at P < 0.005 (Student's t-test).

398 A trophy of denervated laminae In animals which had survived 33 days or longer after bilateral injections of KA, the dorsal dentate molecular layer had not atrophied significantly and the width of the ventral molecular layer had been reduced by only about 10 j"~ (Table III). Atrophy of the ventral molecular layer appeared to have reached its maximum in 3-5 days. Since degeneration products were found throughout the inner third of the ventral molecular layer at this time, the rather minor loss in dimension could not have been confined predominantly to the zone of degeneration. KA administration did not alter the width of stratum radiatum in area CA I for at least a week, but decreased it by 24 iI~iwithin 6-8 weeks.

DISCUSSION Reactive synaptogenesis in area CA1 Our results demonstrate that bilateral intraventricular administration of KA reduces the synaptic density of the CA1 stratum radiatum by 86 ~ within 3-7 days. This extensive denervation almost certainly resulted from the destruction of CA3-CA4 pyramidal cells bilaterally and the consequent degeneration of the Schaffer collateral and commissural afferents they provide, since no other known afferent to this lamina was damaged. Thus Schaffer collateral and commissural fibers evidently supply about 86 ~ of the synaptic contacts to this region of the apical dendrite. This density of innervation matches that provided by perforant path fibers in their terminal zone of the fascia dentata 28. Such figures should, however, be regarded as approximations only, since the size and shape of synapses and the ease with which they can be identified will affect the quantitation of these three-dimensional structures from two-dimensional micrographs. We found no evidence, however, that these factors differed much among the animals examined. This ultrastructural consistency and the close agreement among results from all the animals in each group suggest that our estimates of synaptic loss (and reacquisition) should not be far wrong. Synaptic degeneration proceeded very rapidly in area CAl. One day after KA treatment two forms of terminal degeneration could be clearly distinguished. These have been referred to as electron lucent and electron dense degeneration, according to whether the axoplasm becomes more or less electron dense than normal 1,1°,~1,~,4°. The electron lucent form was seen only at the l-day survival period. Its incidence might well have been underestimated somewhat, since we took a conservative approach to its identification. Nevertheless, even at the 1-day survival period, electron dense degeneration clearly predominated. We could not judge from the present material whether the electron lucent boutons eventually became electron dense or represented a separate form of presynaptic degeneration entirely. The pyramidal cell dendrites did not remain without synaptic contacts indefinitely, but regained a normal synaptic density. Taking into account a 24 ~ atrophy of stratum radiatum, the total number of synapses in this lamina was restored to 76 ~o of normal. Presumably, afferents to area CA1 that were left undamaged by KA administration formed additional synapses to replace those which had been lost. Light

399 microscopic studies have not yet revealed the identity of these reinnervating fibers, however30. Furthermore, F synapses, thought to be characteristic of those made by inhibitory afferents, were no more numerous in stratum radiatum of KA-treated rats than in stratum radiatum of controls. Some CA3-CA4 pyramidal cells in the temporal third of the hippocampal formation usually escape destruction by KA. Since these neurons have been shown to project a considerable distance rostrally3s, their axons could very well have contributed to the reinnervation process. Collaterals of the CA1 pyramidal cells themselves or of the undamaged pyramidal cells of the h2 area may also have participated. The synaptic changes obtained in the present study closely resemble those reported after nearly complete isolation of area CA1 from the CA3-CA4 area with a knife cut 12. In the latter study, 74 ~ of the synaptic contacts in stratum radiatum were disrupted and one can calculate from the data presented that 76 ~ of the lost synapses were replaced. However, the mechanical lesion interrupted not only the projections to area CA1 that originate from the CA3-CA4 area, but also septohippocampal and other projections that enter the CA1 area from the direction of the fimbria, and, in addition, at least some CA1 pyramidal cells were axotomized. The relatively specific chemical lesion of CA3-CA4 neurons employed in the present study avoided these complications. The similar results of the two studies strongly suggest that neither transection of afferents other than the Schaffer collateral-commissural fibers nor axotomy of the CA1 pyramidal cells greatly influenced the synaptic alterations in stratum radiatum that resulted from a hippocampal transection. On the other hand, the difference in lesion technique might well have affected the degree to which the apical dendritic zone atrophied. In the transection study little or no atrophy of this zone as a whole could be detected, whereas a bilateral KA lesion produced about a 24 ~ contraction of stratum radiatum and somewhat lesser shrinkage of the total apical dendritic zone. Previous studies of extensively deafferented regions of the CNS suggest that atrophy of the magnitude found in the present study is to be expectedz3,33. Perhaps the mechanical transection induced a swelling process that counteracted the tendency of deafferentation to cause atrophy of the tissue.

Reactive synaptogenesis hi the dentate molecular layer Bilateral intraventricular injection of KA destroyed most, but not all, of the neurons in area CA4 of the hippocampus. Hence a partial denervation of the inner third of the dentate molecular layer was expected and, indeed, was obtained. Rather few degenerating synaptic boutons were identified in this zone, however, although in 3-5-day survivors there were many electron dense presynaptic structures unassociated with a postsynaptic specialization. The densities of intact and degenerating synaptic contacts combined fell to about half the synaptic density of this zone in control rats. Previously, removal of perforant path 23 or commissura125 afferents to the dentate molecular layer was shown to produce a similar disparity in total synaptic density. In contrast, the combined densities of intact and degenerating synapses in stratum radiatum remained approximately constant between survival times of 4 h and 3-7 days at a value only slightly below normal. These observations suggest that degenerating synaptic boutons

400 are much more rapidly disconnected from granule cell dendrites than from pyramidal cell dendrites. The inner zone of the dentate molecular layer regained a normal or nearly normal synaptic density within 6 8 weeks. Since our quantitation showed an actual reduction in the density of postsynaptic sites (defined by the presence of a postsynaptic density) during the first few days after KA treatment, new synapses probably were created largely de novo, rather than by reoccupation of abandoned postsynaptic sites. Creation of new postsynaptic sites in the dentate molecular layer has been suggested to follow a perforant path lesion also 24. Thus the postsynaptic granule cell is an active participant in reactive synaptogenesis. Since the number of postsynaptic sites remained essentially constant in the CA1 stratum radiatum during the immediate postinjection period, previously existing sites might simply have been reoccupied by newly formed boutons in this region. More extensive studies are needed to evaluate this possibility. Perhaps the most intriguing effect of the lesion on the dentate molecular layer was the loss and reacquisition of 30 % of the synapses in the perforant path terminal zone (middle and outer thirds). This synaptic deficit developed in the virtual absence of recognizable presynaptic degeneration. The magnitude of the synaptic deficit and the great density of perforant path synapses suggested that most of the synapses that disappeared must have been those made by perforant path fibers. This loss of synaptic contacts was completely reversible. It probably did not arise from a direct action of KA, since the effect was not seen until hours after CA3-CA4 cells had already degenerated markedly29, al. It also appeared not to depend on loss ofinnervation from CA4 neurons, since the latter was much greater on the dorsal leaf than on the ventral leaf, yet KA reduced the synaptic density in the perforant path terminal zone to exactly the same extent on both leaves. The KA lesion destroyed the great majority of neurons (CA3-CA4 pyramidal cells) to which the mossy fibers project, and the number of mossy fiber boutons declined rapidly as their targets degeneratedaL One possible explanation for the synaptic deficit in the perforant path terminal zone is that the granule cells shed synapses as a result of the loss of trophic material normally accumulated by the mossy fiber boutons. This idea is consistent with the nearly equal loss of synapses by granule cells on both leaves of the fascia dentata and receives strong support from studies of axotomized neurons. Axotomy leads to the rapid disconnection of synapses on the injured cell in all systems that have been studied, but there is only scant evidence of presynaptic degeneration a~. All effects of axotomy are reversed once the axon regenerates and reforms its synaptic connections with its target cells. There is evidence to suggest that trophic material accumulated by the boutons normally acts to maintain synaptic connections, once it is conveyed by retrograde axonal transport to the cell body. If the dentate granule cells lost synapses after KA administration because they had been deprived of most of their postsynaptic targets, one would expect a similar synaptic loss to follow transection of the mossy fibers. Tarrant and Routtenberg have obtained just such a result (personal communication). The coincidence of the transection data with ours reinforces the view that the shedding of synapses from the

401 granule cell dendrite arose primarily from the disconnection and subsequent loss of mossy fiber boutons in areas CA3 and CA4. In contrast to axotomized peripheral neurons, the dentate granule cells could not have reacquired a normal synaptic complement as a result of axonal regeneration and reformation of the original contacts. However, the mossy fibers appear to form new boutons in the dentate molecular layer 3° and presumably also synaptic contacts. The formation of these and possibly other new mossy fiber connections could have permitted the granule cells to reacquire their normal density of innervation. One would expect the effects of mossy fiber disconnection to be distributed over the entire granule cell dendrite, not to be confined to the distal two-thirds. I f this idea is correct, much of the synaptic loss in the inner third of the molecular layer might have been attributable to this factor, rather than to presynaptic degeneration. Additionally, the CAI pyramidal cells lost some of their postsynaptic targets in the lateral septum and subiculum as a result of the K A lesion 29,3°. The number of target cells destroyed accounted for a much lesser percentage of the total than those lost by the dentate granule cells. Possibly, this partial deprivation of postsynaptic targets contributed to the small, but consistent, loss of synaptic contacts in stratum radiatum that appeared to precede presynaptic degeneration. In summary, our findings demonstrate that reactive synaptogenesis follows destruction of the hippocampal end blade in rats. The great majority of the synapses lost as a result of this lesion are eventually replaced. This observation raises the possibility of a similar response in man when this form of brain damage occurs. It remains to be determined whether restoration of the normal synaptic complement leads to recovery of hippocampal function. ACKNOWLEDGEMENTS We thank Dr. A. Routtenberg for permission to cite his unpublished work, Mr. D. L. Shelton for assistance with the perfusions and Mrs. V. Smith for typing the manuscript. This study was supported by N S F Grant BNS76-09973 and N I H Grants NS 08597, MS 19691 and A G 00538.

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