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Developmental differences in post-lesion axonal growth in the hippocampus
Brain Research, 59 (•973) 155-168
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© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
D E V E L O P M E N T A L D I F F E R E N C E S IN POST-LESION A X O N A L G R O W T H IN T H E HIPPOCAMPUS
G A R Y LYNCH, B R E N T STANFIELD AND CARL W. C O T M A N
Department of Psychobiology, University of California, lrvine, Calif. 92664 (U.S.A.) (Accepted February 22nd, 1973)
SUMMARY
The effects of~esions of the entorhinal cortex on the distribution of the commissural projections to dentate gyrus and hippocampus were studied with the FinkHeimer techniques in immature and mature rats. Early entorhinal lesions caused a hyperdevelopment of the commissural system in both areas while lesions in adults produced similar but quantitatively smaller effects. Two types of post-lesion growth were observed: (1) an increase of the density of innervation in areas normally occupied by commissural fibers, and (2) a movement of commissural fibers into dendritic areas in which they are not found in normal rats.
INTRODUCTION
It has long been suspected that immature brains possess greater capacity for change (or 'plasticity') than do those of an adult. One of the most striking examples of this is the common observation that recovery from brain damage is often far more complete in young animals than it is in adults. Recent experiments have greatly increased our understanding of the morphological adjustments which take place after discrete brain lesions and in doing so have made it feasible to ask what factors might impart greater plasticity to the immature nervous system. The evidence is now quite good that partial deafferentation of a brain structure causes in some, but not all la, cases a massive growth of the afferents remaining to that structure 4,6,9,11,12,15,~6,1s,~°,21. Three types of post-lesion growth have been reported: (1) an increase in the density of terminals to a region normally innervated11,15,18; (2) a 'spread' of axons into dendritic areas that would not normally be innervated12; and (3) a rerouting of an afferent into brain structures that normally would not receive projections from that afferente0,22.
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The degree to which these various phenomena are influenced by developmental status is not known. Different studies have used either neonatal or adult organisms but to date there have been only a few direct comparisons15, ~°. The experiments reported in this paper were directed at this problem. In previous papers we reported that entorhinal lesions in rats produce dramatic changes in the remaining afferent projections to the dentate gyrus. The dentate gyrus was chosen for study because in the normal rat its afferents are precisely laminated as shown in Fig. 1. Note that projections from the entorhinal cortex terminate in the outer molecular layer while projections from the contralateral CA3 field end in the inner molecular layer. The inner molecular layer also receives an associational projection from the ipsilateral CA3 fields TM. Recent work 17 has shown that fibers from the medial septal nucleus innervate a thin zone immediately below the commissural terminals among the most superficial granule cells. Finally there are acetylcholinesterase (ACHE) containing fibers and terminals in two layers of the molecular layer of the dentate gyrus4,S, 2a and the evidence is quite good that these originate in the septum17,23. Entorhinal lesions profoundly alter the precise organization of the afferents remaining to the molecular layer. Stated briefly, light microscopic histochemical studies showed that the AChE system increased in staining intensity in the molecular layer of the dentate gyrus (the area in which the entorhinal projections terminate) in
OUTER nuJ >-
AChE
MIDDLE
, rr ..J l.g i
IO INNER
AL/ACHE
.,di
~ GRANULAR LAYER
~EPTAL AChE
..dl ~i
Fig. 1. Schematic representation of the distribution of afferents to a granule cell of the dentate gyrus.
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both young and old rats but in the former case this growth was restricted to the outermost quarter of the molecular layer 4 while in the adult it occupied the outer half 11. Electron microscopic analysis revealed that the great majority of the enzyme was bound to small diameter axons and terminals in normal and lesioned rats and established that these elements were confined to the outermost portions of the molecular layer. Furthermore there was a tremendous increase in terminals that stained positively in animals with neonatal lesions 4. Since all AChE in the molecular layer disappears in both lesioned and unlesioned rats after lesions of the medial septal nucleus, it was concluded that entorhinal lesions cause a growth of an AChE containing septal system and that this growth was vastly different in young and mature rats4,1L These are not the only changes which take place in the dentate gyrus after entorhinal lesions. The commissural projection to the dentate from the contralateral hippocampus has been shown to undergo a massive hyperdevelopment after neonatal entorhinal lesions 12. Neurophysiological work has provided strong evidence that the commissural axons which invade the outer molecular layer after neonatal entorhinal lesion establish functional synaptic connections 14. In this report we characterize the post-entorhinal lesion growth of the commissural system more fully and report developmental differences in the effect.
METHODS
Entorhinal lesions were performed by aspiration in 11-day-old rats (n -----6) who were returned to their mothers and weaned at 21 days. The animals were allowed a minimum of 80 post-surgical recovery days before being subjected to a second lesion to remove the commissural system. Two types of lesions were used for this purpose: (l) a complete aspiration lesion of the entire dorsal hippocampus (including fimbria) contralateral to the original entorhinal lesion, or (2) a small electrolytic lesion of the ventral hippocampal commissure. Lesions of the entorhinal cortex in 5 adult rats (at least 100 days old) were performed using electrolytic techniques. Small lesions were placed at 3 dorsoventral placements on 2 separate electrode 'drops' in an attempt to equate the size of the adult lesions with those of the neonates. The entorhinal lesions in both the neonates and the adults were successful in destroying both the medial and lateral entorhinal areas and the most posterior part of the parasubiculum. Following recovery periods of at least 150 days, the dorsal hippocampus contralateral to the original entorhinal lesion was removed by aspiration. Both hippocampal and commissural lesions were placed in adult rats (n ---- 7) with no prior entorhinal damage. These animals served as controls for the normal location of the commissural system. Following the hippocampal or commissural lesion the 3 groups of rats were processed for the Fink-Heimer (1967) method. Five days after the lesion the animals were sacrificed, perfused with 10 ~ formalin and their brains removed and post-fixed for at least 7 days after which they were placed for 3 days in 30 ~ sucrose. The brains
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were sectioned at 40 #m; most brains were cut horizontally but a few were sectioned coronally. The interoperative time period of 80 days is more than adequate for the complete disappearance of all degeneration produced by neonatal entorhinal lesions as shown by other workers 24 and verified by unpublished data from our laboratory. Therefore all degeneration seen after the second (adult commissural) lesion was caused by the destruction of commissural fibers and terminals. The problem is more complicated when both lesions are performed on adult rats. Some degeneration from adult entorhinal lesions is still evident after the longest interoperative time periods used in this study (Lynch et al., unpublished) and this presents a problem in deciding which degeneration is due specifically to the second (commissural) lesion. However in the dentate gyrus of the dorsal hippocampal formation the long-term degeneration products from the first lesion are large and scattered evenly throughout the outer molecular layer of the dentate and are easily distinguished from the dense fine grained degeneration found immediately above the granule cells seen after the secondary commissural lesion. That is, the commissural degeneration forms a dense band which has a sharp boundary and this degeneration is distinguishable from that caused by the initial lesion. The commissural projection to the dentate thins out (that is, there are fewer terminals) along the septo-temporal axis of the hippocampal formation and in more ventral levels it was much more difficult to distinguish the degeneration caused by the second (commissural) lesion from those products remaining from the original entorhinal lesion. For this reason we will place more emphasis on the results obtained in the more dorsal aspects of the dentate gyrus (that part of the dentate closer to the septum) in the adult rats. RESULTS
Methods of analysis Because of its simplicity and rigid orderliness the hippocampus is an ideal model system on any given 2-dimensional section but when it is necessary to consider the structure in 3 dimensions (as it is in this study) a number of problems arise. Because the hippocampus twists in several directions as it proceeds from the septum towards the amygdala it is impossible to collect more than a few sections at right angles to its axis with any of the 3 standard sectioning angles (horizontally, coronally or sagitally). As a consequence, when any of these 3 sectioning angles are used the vast majority of sections are not at right angles to the axis of the hippocampus and the resulting distortion varies greatly depending on the locus of a particular section. Such distortions complicate rapid quantitative comparisons of the size of particular components or layers in different levels of the hippocampus within an animal to say nothing of comparisons between different animals. As an aid in analyzing our material we have selected 8 horizontal sections through the hippocampus and 2 measurement points within each section. The 8 levels and 2 measurement points were selected both for ease of identification and because
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V Ill IV v Vl
VII viii IX x
m
Fig. 2. Drawing of the hippocampus as it appears in a section taken approximately in the plane of its septo-temporal axis. The layer of dentate granule cells is represented by the row of circles while the pyramidal cell layer is shown as a row of triangles. The Roman numerals illustrate the dorsoventral location of the levels selected for analysis of growth of the commissural system after entorhinal lesions. Levels I and II are not indicated because they do not intersect the hippocampus this far laterally.
they introduce minimal distortion. Fig. 2 illustrates the levels and their approximate positions on a section taken parallel to the septo-temporal axis of the hippocampus (in a plane roughly midway between the coronal and sagittal planes). With this type of approach it was possible to perform a rapid and reasonably complete analysis of the changes in the distribution of the commissural system after entorhinal lesions; equally important it allowed valid comparisons between animals. An additional problem in quantifying changes in the commissural system was caused by tissue shrinkage. Early in this study it became obvious that the sizable lesions needed to destroy the commissural system and entorhinal cortex were producing varying amounts of tissue shrinkage not only in the hippocampus but throughout the brain. The shrinkage problem was not restricted to the dentate gyrus, where most of the quantification was done, but was general to the entire hippocampal formation and cerebral cortex. In an attempt to provide a ~omplete picture of the thickness of the commissural band, the data are presented both as absolute size and as a percentage of the entire molecular layer. This latter value to a degree corrects for the amount of shrinkage if it can be assumed that shrinkage is equal through all sublevels within the molecular layer.
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The normal distribution o f the commissural system to the dentate gyrus A l l o f the c o n t r o l rats which received only h i p p o c a m p a l a b l a t i o n or t r a n s e c t i o n o f the ventral h i p p o c a m p a l c o m m i s s u r e showed the same p a t t e r n o f d e g e n e r a t i o n in the dentate. T h e only differences were caused by i n c o m p l e t e lesions o f the ventral h i p p o c a m p a l c o m m i s s u r a l which caused d e g e n e r a t i o n in some cases restricted to the d o r s a l end o f the h i p p o c a m p u s or d e g e n e r a t i o n restricted to the p y r a m i d a l cell fields w i t h o u t involving the d e n t a t e gyrus. O u r o b s e r v a t i o n s are in a g r e e m e n t with earlier descriptions o f the c o m m i s s u r a l p r o j e c t i o n s to the d e n t a t e gyrus 1,2,7,17,19. T w o gradients o f density o f i n n e r v a t i o n can be distinguished. A l o n g the s e p t o - t e m p o r a l axis the p r o j e c t i o n to the dentate gyrus thins o u t so t h a t r o s t r a l d e n t a t e regions are m u c h m o r e densely i n n e r v a t e d t h a n the c a u d a l areas. F u r t h e r m o r e , a g r a d i e n t o f density was also evident a r o u n d the 'V' o f the d e n t a t e gyrus with the n u m b e r o f terminals decreasing going f r o m the tip o f the exterior wing (the r o w o f granule cells facing the t h a l a m u s ) t o w a r d s the tip o f the int e r i o r wing.
I
Fig. 3. Photomicrographs of the degeneration products in the dentate gyrus after a complete lesion of the contralateral hippocampus. Panel A is from a normal rat, panel B is from one which received a lesion of the entorhinal cortex 150 days earlier (as an adult), while panel C is from an animal which received an entorhinal lesion at 11 days of age. All photographs were taken from comparable points in the dentate at level III (see Fig. 2). The dotted line emphasizes the hippocampal fissure and the arrows denote the upper boundary of degeneration caused by the commissural lesion. (× 400.)
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Despite these changes in density of innervation, the commissural system always occupied the same zone of the molecular layer (Figs. 3-5), namely a region extending to about 90 # m above the granule cell bodies. A space was located between the granule cells and the commissural terminals and the size of this varied roughly with the density of innervation. Earlier work has shown this commissural free zone to be innervated by septal afferentslL The distribution o f the dentate commissural system in rats with neonatal or adult entorhinal lesions Entorhinal lesions in 11-day-old rats caused a remarkable change in dentate commissural system. At rostro-dorsal levels (I-III) the commissural projections expanded to fill the entire molecular layer, essentially occupying the areas normally innervated by entorhinal projections. This result was obtained in 4 rats with neonatal lesions and successful secondary commissural or contralateral hippocampal lesions. In the lower levels of the hippocampal formation the spread of the commissural system in the dentate gyrus was not as great and did not completely occupy the outer molecular layer. These findings are summarized in Figs. 3-5 and Table I; note
Fig. 4. Same as Fig. 3 except that all micrographs were taken at level V (see Fig. 2); also the dotted line emphasizes the exterior surface of the dentate gyrus rather than the hippocampal fissure.
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eta[.
I
b
C
Fig. 5. Reconstructions of the degeneration (dots) seen in the hippocampus in a normal rat (panel a), a rat which had received entorhinal lesions 150 days prior to the commissural lesion (panel b), and one which had received an entorhinal lesion at 11 days of age and the commissural lesion as an adult (panel c). The broken lines in panels b and c represent the limits of the entorhinal lesions. The dorsoventral levels shown in this figure are level II (upper panels) and level IV (lower panels).
t h a t the g r o w t h effect was obvious b o t h in terms o f a b s o l u t e a n d percentage growth. E n t o r h i n a l lesions in a d u l t rats also caused the c o m m i s s u r a l system in the dentate gyrus to e x p a n d b u t the effect was n o t so d r a m a t i c as t h a t seen after e n t o r h i n a l lesions in the l 1-day-old rat. A s m e n t i o n e d above, we are m o r e confident o f the results o b t a i n e d in the m o r e d o r s a l (that is, closer to the septum) aspects o f the h i p p o c a m p a l f o r m a t i o n since the d e g e n e r a t i o n there was very dense a n d easily d i s c r i m i n a t e d f r o m the d e g e n e r a t i o n p r o d u c t s left f r o m the original (entorhinal) lesion. I n the d o r s a l h i p p o c a m p u s it can be seen t h a t the dentate c o m m i s s u r a l system e x p a n d e d nearly 50 # m to occupy a b o u t 50 ~ o f the t o t a l m o l e c u l a r layer. Similar increases were f o u n d in o t h e r levels o f d e n t a t e b u t precise quantification was m o r e difficult because the
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TABLE I W I D T H OF THE DEGENERATION BAND IN THE MOLECULAR LAYER OF THE DENTATE GYRUS AFTER DESTRUCTION OF THE COMMISSURAL SYSTEM IN RATS W I T H PRIOR ENTORHINAL LESIONS
The upper table gives the width of band in microns while the lower table presents the data as the percentage of the molecular layer occupied by the degeneration products produced by the second (commissural system) lesion. The numbers in parentheses refer to the number of rats analyzed at each level while the asterisks denote data statistically different (by the Mann-Whitney U test; P < 0.05) from control data; the terms 'll-day lesion' and 'adult lesion' indicate the age at which the first (i.e. entorhinal) lesion was performed. Dorsoventral level
Median width of commissural degeneration band in dentate gyrus (lzm) 11-day lesion
Adult l e s i o n
Control
II III IV V VI VII VIII
*200 (3) "188 (4) 128 (3) "111 (3) "118 (2) 100 (2) 130 (1)
"157 (5) "137 (5) "131 (5) 112 (4) "118 (5) *110 (5) 133 (3)
112 (7) 93 (7) 110 (5) 89 (6) 87 (6) 74 (5) 72 (3)
Dorsoventral level
Median percentage of molecular layer of dentate gyrus occupied by commissural degeneration band
II III IV V VI VII VIII
11-day lesion
Adult l e s i o n
Control
"71.8 (3) "81.6 (4) *63.4 (3) *52.5 (3) "51.9 (2) *47.0 (2) 44.0 (1)
"51.3 (5) *47.8 (5) *48.5 (5) *43.8 (4) *39.4 (5) *40.8 (5) "40.1 (3)
32.7 (7) 29.2 (7) 31.4 (5) 27.0 (6) 25.8 (6) 24.8 (5) 24.1 (3)
c o m m i s s u r a l d e g e n e r a t i o n was sparser, t h e r e b y m a k i n g the b o r d e r o f the c o m m i s s u r a l system less distinct. Despite this p r o b l e m , it was evident t h a t the c o m m i s s u r a l system h a d increased in the d e n t a t e gyrus o f lower h i p p o c a m p a l f o r m a t i o n levels a n d a p p a r ently to the same degree as it h a d in m o r e d o r s a l sections. This is in c o n t r a s t to the g r o w t h seen after n e o n a t a l lesions in which the s p r e a d o f the c o m m i s s u r a l system was m u c h greater in the d o r s a l levels. The normal distribution o f the commissural projections to the pyramidal cell fields The c o m m i s s u r a l p r o j e c t i o n to the p y r a m i d a l cell fields has been described by several authorsl,2,17,lL U n l i k e the c o m m i s s u r a l p r o j e c t i o n s to the dentate, those to the p y r a m i d a l cells d o n o t have an obvious s e p t o - t e m p o r a l g r a d i e n t as d e m o n s t r a t e d in Fig. 5.
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The distribution o f the commissural system projections to the pyramidal cell fields in rats with lesions o f the entorhinal cortex as neonates or adults
A massive growth of the commissural system takes place in the stratum moleculare of the hippocampus after neonatal entorhinal lesions (Fig. 5). This takes place in both regio superior (CAI) and regio inferior (CA2-4). In CA1 the growth is particularly evident in the more rostral levels of the hippocampus where the degeneration extends along the entire length of the hippocampal fissure (Fig. 5). At more caudal (temporal) levels, the outer molecular layer also contains dense degeneration (in contrast to normal rats) but this is restricted to one-third of the zone above the fissure facing the free end of the interior wing of the dentate ( C A l c of Lorente de N61°). These results were obtained in 6 rats with neonatal entorhinal lesions and secondary commissural or hippocampal lesions performed when the animals were adults. For two reasons we conclude that the dense degeneration seen in the stratum moleculare of CA1 is due to sprouting of the commissural system as opposed to growth of the septo-hippocampal system or the crossed temporo-ammonic tract. First, it could not be explained by possible growth of the crossed temporo-ammonic system since it was obtained in 2 rats with lesions of the ventral commissure and in these involvement of the dorsal psalterium was impossible. Secondly, the effect was obtained in 4 rats with contralateral hippocampal ablations that did not involve the septo-hippocampal system. There was a strong indication of commissural growth into the stratum moleculare of the regio inferior after lesions of the entorhinal cortex in 11-day-old rats (Table II) but, unlike the situation in the dentate gyrus, it did not completely fill this field at any septo-temporal level. Table II presents a summary of the thickness of the commissural degeneration band in CA2 at a point 100 # m from the end of the projection of the mossy fibers at level II. It can be seen from this table that the median width of the degeneration band and the percentage of the apical dendritic fields that it occupies were greater in the lesioned animals but due to the relatively small number of rats involved and considerable variance in the data these results are only marginally significant by the Mann-Whitney test. It was not possible to arrive at a definitive answer about commissural expansion into the stratum moleculare of CA1 in adult rats. Degeneration in this zone was clearly not as dense as that in the stratum radiatum of CA1 but subtle changes could not be evaluated because of the residual degeneration products from the original entorhinal lesions. TABLE I1 Median width (/~m) and ~ of apical dendritic fields occupied by commissural degeneration band in regio inferior approximately 150/~m from the end of the mossy fiber band at dorsoventral level II. ll-day lesion (n ~ 5)
Adult lesion (n = 5)
Control (n ~ 7)
466 #m 78.2 ~
427 #m 71.7 ~
366 #m 65.2
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There did appear to be an increase in the width of the commissural band in CA2 after adult lesions, at least in the dorsal hippocampus (Table II). These results, however, were not so consistent as those obtained in the dentate and were only of borderline statistical significance. DISCUSSION
From both qualitative and quantitative analysis of these results two conclusions are obvious. First, the commissural system undergoes growth after entorhinal lesions in both immature and mature rats and secondly, this growth is greater in the younger animals. Two types of changes in the commissural projection were seen after entorhinal lesions. The first type of post-lesion growth observed was what we have referred to as 'spreading'. Spreading occurs when a synaptic population expands and occupies areas of a dendritic field of a particular cell in which it would not normally be found. The second type of growth has been observed by several authors and will be referred to here as 'sprouting'. This effect takes place when an afferent system proliferates and increases its density ofinnervation in a dendritic area that it normally occupies. In the dentate gyrus, the commissural system spreads into new territory in the outer molecular layer, an area in which commissural terminals are never seen in the normal rat1,2, lz. This type of growth in which axons invade new territory on a particular cell can be distinguished from that which occurred in the stratum moleculare of the pyramidal cell field CA1. The few terminals normally found in that region undergo extensive proliferation and become as dense as the normal innervation of stratum radiatum of CA1. Whether sprouting and spreading of afferent projections are qualitatively different phenomena or are simply different degrees of a single effect is unknown. Solution of this question will have to await development of techniques for manipulating postlesion axonal and synaptic growth. From the results of this study and those of earlier experiments, it is apparent that the effects of entorhinal lesions on the distribution of septal and commissural projections to the hippocampal formation are age dependent. This observation provides a possible morphological explanation for the greater 'plasticity' in immature brains observed in neurobehavioral studies using lesion techniques. There are several hypotheses which could account for the greater post-lesion axonal growth in immature animals. In discussing some of these we will restrict ourselves to the post-lesion growth of commissural system described in this paper. (1) It is possible that the cell bodies which give rise to the commissural terminals have greater growth potential or are more responsive to the denervation changes going on in contralateral dentate gyrus in the younger animals. It is widely believed that retrograde changes are more pronounced in neonatal neurons than in adults a; and it is conceivable that this difference could be involved in the greater commissural growth after 11-day entorhinal lesions. (2) The glia are more numerous in adult brains than in neonatal ones and it is
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possible that the upward movement of the commissural terminals is retarded in the adults by post-lesion growth and proliferation of these elements. (3) The degenerating elements produced by the entorhinal lesion in adults persist for months and possibly years (Lynch et al., unpublished). In the neonate the lesion causes much less degeneration and what is produced disappears very rapidly. Therefore, it is possible that the outward movement of the commissural system is blocked by degenerating fragments of the original entorhinal innervation in the adult but not in the neonate. (4) The status of the dendritic fields of the granule cells is radically different in the neonatal and adult rat and this could be responsible for the differences in commissural growth. The dentate gyrus is undergoing tremendous growth in the 11-day-old rat while it is, of course, relatively stable in the adult. At 11 days, most of the granule cells have not yet migrated into their final positions to say nothing of differentiating dendritic trees and spines (see Crain et aL 5, for a description of the molecular layer at 11 days of age). Removing the entorhinal cortex at this age, then, means that the stilldeveloping commissural system is given access to dendritic space that has never been occupied by the temporo-ammonic system. In the adult, the commissural afferents must invade territory that once was innervated by the entorhinal terminals. The growth into the molecular layer of the hippocampal fields is a different matter. The pyramidal cells are present before birth and presumably are considerably more mature than the later appearing granule cells. Despite this more advanced condition, the sprouting of commissural terminals was still much greater after neonatal entorhinal lesions. This does not of course rule out the possibility that the immature developmental state of the dendritic territory to be invaded is the reason for the greater growth in immature animals but it does indicate that this effect is not unique to newly formed nerve cells. More definitive statements may become possible when studies on the ontogeny of the dentate gyrus and hippocampus combined with longitudinal studies on sprouting in these areas are completed. (5) A final possibility is that competition goes on between the remaining dentate afferents for the space vacated by the entorhinal projection and that the commissural fibers are less successful in this in the adult animals than in the neonates. We have previously reported evidence that the acetylcholinesterase containing septal projection undergoes massive growth after entorhinal lesions and the nature of this growth is also dependent upon maturational stage< 11. In the neonates, the growth is restricted to the outermost levels of the molecular layer while in the adult it occupies about 50 ~ of the layer. This, of course, is the reverse of the growth of the commissural system in which the greatest spread of terminals is seen in the neonatal animals. This reciprocity of effects between septal and commissural systems suggests that the final disposition of these systems after entorhinal lesions may be dependent upon an interaction between them. It is pertinent to note at this point that the above arguments are most applicable to the rostral (septal) segments of the hippocampus. In the caudal (temporal) regions of the hippocampal arch the growth of the commissural system is only slightly greater in the neonates than in the adults. Since it is apparent that the developmental condi-
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tions are relatively u n i f o r m t h r o u g h o u t the structure, it is necessary to consider w h a t other c o n d i t i o n s c o u l d p r o d u c e this restricted g r o w t h in the c a u d a l h i p p o c a m p u s . One attractive hypothesis is suggested by the o b s e r v a t i o n t h a t the c o m m i s s u r a l p r o j e c t i o n b e c o m e s progressively less dense a l o n g the s e p t o - t e m p o r a l axis (as well as a r o u n d the ' V ' o f the dentate). A s described above, G o t t l i e b a n d C o w a n 7 have r e p o r t e d d a t a i n d i c a t i n g t h a t the relative density o f the d e n t a t e c o m m i s s u r a l p r o j e c t i o n is the result o f a d e v e l o p m e n t a l c o m p e t i t i o n between t h a t system a n d the recently described ipsilateral associational systemV, 24. Therefore, it is possible t h a t the failure o f the d e n t a t e c o m m i s s u r a l p r o j e c t i o n to show greater g r o w t h in the c a u d a l h i p p o c a m p u s is due to the m o r e successful g r o w t h o f the a s s o c i a t i o n a l system in these h i p p o c a m p a l areas. T h a t is, j u s t as the associational p r o j e c t i o n a p p e a r s to o c c u p y p r o p o r t i o n a l l y greater dendritic space in c a u d a l h i p p o c a m p u s d u r i n g n o r m a l development, so it m a y be able to c o m p e t e m o r e successfully t h a n the c o m m i s s u r a l p r o j e c t i o n for the areas left v a c a n t by the d e s t r u c t i o n o f the e n t o r h i n a l cortex. ACKNOWLEDGEMENTS
This research was s u p p o r t e d by N I H G r a n t s M H 19793-01, M H 19691 and N S F G r a n t G B 16973.
REFERENCES 1 ALKSNE,J. F., BLACKSTAD,T. W., WALBERG,F., AND WHITE, L. E., JR., Electron microscopy of axon degeneration: A valuable tool in experimental neuroanatomy, Ergebn. Anat. Entwickl.Gesch., 39 (1966) 1-31. 2 BLACKSTAD,T. W., Commissural connections of the hippocampal region in the rat, with special reference to their mode of termination, J. camp. NeuraL, 105 (1956) 417-537. 3 BLEIER,R., Retrograde transynaptic cellular degeneration in mammillary and ventral tegmental nuclei following limbic decortication in rabbits of various ages, Brain Research, 15 (1969) 365-393. 4 COTMAN,C. W., MATTHEWS,n . A., TAYLOR,D., AND LYNCH, G., Plasticity in a brain cholinergic system: Developmental differences in post lesion adjustments, Submitted for publication. 5 CRAIN, B., COTMAN, C. W., TAYLOR,D., AND LYNCH, G., A quantitative electron microscopic study of synaptogenesis in the dentate gyrus of the rat, Brain Research, 63 (1973) in press. 6 GOODMAN,D. C., AND HOREL,J. A., Sprouting of optic tract projections in the brainstem of the rat, J. camp. Neural., 127 (1966) 71-88. 7 GOTTLIEB,D., AND COWAN, W., Evidence for a temporal factor in the occupation of available synaptic sites during development of the dentate gyrus, Brain Research, 41 (1972) 452-456. 8 LEWIS,P. R., SHUTE, C. C. n., AND SILVER,A., Confirmation from choline acetylase analyses of a massive cholinergic innervation to the rat hippocampus, d. Physiol. (Land.), 191 (1967) 215-224. 9 LIU, C. W., AND SCOTT, D., JR., Regeneration in the dorsal spino-cerebellar tract of the cat, J. camp. Neural., 109 (1958) 153-167. 10 LORENTEDE NO, R., Studies on the structure of the cerebral cortex. II. Continuation of the study of the ammonic system, J. PsychaL Neural. (Lpz.), 46 (1934) 113-177. 11 LYNCH, G. S., MATTHEWS,D., MOSKO, S., PARKS, T., AND COTMAN, C. W., Induced acetylcholinesterase rich layer in rat dentate gyrus following entorhinal lesions, Brain Research, 42 (1972) 311-318. 12 LYNCH, G. S., MOSKO, S., PARKS, T., AND COTMAN, C. W., Hyperdevelopment of the dentate gyrus commissural system after entorhinal lesions in immature rats, Brain Research, 50 (1973) 174-178.
13 LYNCH, G. S., PARKS,T., STANFIELD,B., AND COTMAN,C. W., Effects of commissural lesions on
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the distribution of acetylcho!inesterase and the temporo-ammonic tract in the hippocampus, Submitted. LYNCH, G. S., DEADWYLER, S., AND COTMAN, C. W., Post-lesion axonal growth produces functional contacts, Science, in press. LUND, R. D., AND LUND, J. S., Synaptic adjustment after deafferentation of the superior colliculus of the rat, Science, 171 (1971) 804-807. MOORE, R. Y., BJ6RKLUND, A., AND STENEVI, U., Plastic changes in the adrenergic innervation of the rat septal area in response to denervation, Brain Research, 33 (1971) 13-35. Mos~o, S., LYNCH, G. S., AND COTMAN, C. W., Comparison of acetylcholinesterase distribution in the dentate gyrus with the localization of septal and commissural projections, J. comp. Neurol., in press. RAISMAN,G., Neuronal plasticity in the septal nuclei of the adult rat, Brain Research, 14 (1969) 25-48. RAISMAN, G., COWAN, W, M., AND POWELL, T. P. S., The extrinsic afferent, commissural and associational fibers of the hippocampus, Brain, 88 (1965) 963-996. SCrtNE[DER, G. E., Mechanisms of functional recovery following lesions of visual cortex or superior colliculus in neonate and adult hampsters, Brain Behav. Evol., 3 (1970) 295-323. STENEVI,U., BJ~3RKLUND,A., AND MOORE,R. Y., Growth of intact central adrenergic axons in the denervated lateral geniculate body, Exp. Neurol., 35 (1972) 290-299. STEWARD, O., COTMAN, C. W., AND LYNCH, G., Re-establishment of electrophysiologically functional entorhinal cortical input to the dentate gyrus deafferented by ipsilateral entorhinal lesions: innervation by the contralateral entorhinal cortex, Exp. Brain Res., in press. STORM-MATH1SEN,J., Quantitative histochemistry of acetylcholinesterase in rat hippocampal region correlated to histochemical stain, J. Neuroehem., 17 (1970) 739-750. ZIMMER, J., lpsilateral afferents to the commissural zone of the fascia dentata, demonstrated in decommissurated rats by silver impregnation, J. comp. Neurol., 142 (1971) 393-416.
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© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
D E V E L O P M E N T A L D I F F E R E N C E S IN POST-LESION A X O N A L G R O W T H IN T H E HIPPOCAMPUS
G A R Y LYNCH, B R E N T STANFIELD AND CARL W. C O T M A N
Department of Psychobiology, University of California, lrvine, Calif. 92664 (U.S.A.) (Accepted February 22nd, 1973)
SUMMARY
The effects of~esions of the entorhinal cortex on the distribution of the commissural projections to dentate gyrus and hippocampus were studied with the FinkHeimer techniques in immature and mature rats. Early entorhinal lesions caused a hyperdevelopment of the commissural system in both areas while lesions in adults produced similar but quantitatively smaller effects. Two types of post-lesion growth were observed: (1) an increase of the density of innervation in areas normally occupied by commissural fibers, and (2) a movement of commissural fibers into dendritic areas in which they are not found in normal rats.
INTRODUCTION
It has long been suspected that immature brains possess greater capacity for change (or 'plasticity') than do those of an adult. One of the most striking examples of this is the common observation that recovery from brain damage is often far more complete in young animals than it is in adults. Recent experiments have greatly increased our understanding of the morphological adjustments which take place after discrete brain lesions and in doing so have made it feasible to ask what factors might impart greater plasticity to the immature nervous system. The evidence is now quite good that partial deafferentation of a brain structure causes in some, but not all la, cases a massive growth of the afferents remaining to that structure 4,6,9,11,12,15,~6,1s,~°,21. Three types of post-lesion growth have been reported: (1) an increase in the density of terminals to a region normally innervated11,15,18; (2) a 'spread' of axons into dendritic areas that would not normally be innervated12; and (3) a rerouting of an afferent into brain structures that normally would not receive projections from that afferente0,22.
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The degree to which these various phenomena are influenced by developmental status is not known. Different studies have used either neonatal or adult organisms but to date there have been only a few direct comparisons15, ~°. The experiments reported in this paper were directed at this problem. In previous papers we reported that entorhinal lesions in rats produce dramatic changes in the remaining afferent projections to the dentate gyrus. The dentate gyrus was chosen for study because in the normal rat its afferents are precisely laminated as shown in Fig. 1. Note that projections from the entorhinal cortex terminate in the outer molecular layer while projections from the contralateral CA3 field end in the inner molecular layer. The inner molecular layer also receives an associational projection from the ipsilateral CA3 fields TM. Recent work 17 has shown that fibers from the medial septal nucleus innervate a thin zone immediately below the commissural terminals among the most superficial granule cells. Finally there are acetylcholinesterase (ACHE) containing fibers and terminals in two layers of the molecular layer of the dentate gyrus4,S, 2a and the evidence is quite good that these originate in the septum17,23. Entorhinal lesions profoundly alter the precise organization of the afferents remaining to the molecular layer. Stated briefly, light microscopic histochemical studies showed that the AChE system increased in staining intensity in the molecular layer of the dentate gyrus (the area in which the entorhinal projections terminate) in
OUTER nuJ >-
AChE
MIDDLE
, rr ..J l.g i
IO INNER
AL/ACHE
.,di
~ GRANULAR LAYER
~EPTAL AChE
..dl ~i
Fig. 1. Schematic representation of the distribution of afferents to a granule cell of the dentate gyrus.
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both young and old rats but in the former case this growth was restricted to the outermost quarter of the molecular layer 4 while in the adult it occupied the outer half 11. Electron microscopic analysis revealed that the great majority of the enzyme was bound to small diameter axons and terminals in normal and lesioned rats and established that these elements were confined to the outermost portions of the molecular layer. Furthermore there was a tremendous increase in terminals that stained positively in animals with neonatal lesions 4. Since all AChE in the molecular layer disappears in both lesioned and unlesioned rats after lesions of the medial septal nucleus, it was concluded that entorhinal lesions cause a growth of an AChE containing septal system and that this growth was vastly different in young and mature rats4,1L These are not the only changes which take place in the dentate gyrus after entorhinal lesions. The commissural projection to the dentate from the contralateral hippocampus has been shown to undergo a massive hyperdevelopment after neonatal entorhinal lesions 12. Neurophysiological work has provided strong evidence that the commissural axons which invade the outer molecular layer after neonatal entorhinal lesion establish functional synaptic connections 14. In this report we characterize the post-entorhinal lesion growth of the commissural system more fully and report developmental differences in the effect.
METHODS
Entorhinal lesions were performed by aspiration in 11-day-old rats (n -----6) who were returned to their mothers and weaned at 21 days. The animals were allowed a minimum of 80 post-surgical recovery days before being subjected to a second lesion to remove the commissural system. Two types of lesions were used for this purpose: (l) a complete aspiration lesion of the entire dorsal hippocampus (including fimbria) contralateral to the original entorhinal lesion, or (2) a small electrolytic lesion of the ventral hippocampal commissure. Lesions of the entorhinal cortex in 5 adult rats (at least 100 days old) were performed using electrolytic techniques. Small lesions were placed at 3 dorsoventral placements on 2 separate electrode 'drops' in an attempt to equate the size of the adult lesions with those of the neonates. The entorhinal lesions in both the neonates and the adults were successful in destroying both the medial and lateral entorhinal areas and the most posterior part of the parasubiculum. Following recovery periods of at least 150 days, the dorsal hippocampus contralateral to the original entorhinal lesion was removed by aspiration. Both hippocampal and commissural lesions were placed in adult rats (n ---- 7) with no prior entorhinal damage. These animals served as controls for the normal location of the commissural system. Following the hippocampal or commissural lesion the 3 groups of rats were processed for the Fink-Heimer (1967) method. Five days after the lesion the animals were sacrificed, perfused with 10 ~ formalin and their brains removed and post-fixed for at least 7 days after which they were placed for 3 days in 30 ~ sucrose. The brains
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were sectioned at 40 #m; most brains were cut horizontally but a few were sectioned coronally. The interoperative time period of 80 days is more than adequate for the complete disappearance of all degeneration produced by neonatal entorhinal lesions as shown by other workers 24 and verified by unpublished data from our laboratory. Therefore all degeneration seen after the second (adult commissural) lesion was caused by the destruction of commissural fibers and terminals. The problem is more complicated when both lesions are performed on adult rats. Some degeneration from adult entorhinal lesions is still evident after the longest interoperative time periods used in this study (Lynch et al., unpublished) and this presents a problem in deciding which degeneration is due specifically to the second (commissural) lesion. However in the dentate gyrus of the dorsal hippocampal formation the long-term degeneration products from the first lesion are large and scattered evenly throughout the outer molecular layer of the dentate and are easily distinguished from the dense fine grained degeneration found immediately above the granule cells seen after the secondary commissural lesion. That is, the commissural degeneration forms a dense band which has a sharp boundary and this degeneration is distinguishable from that caused by the initial lesion. The commissural projection to the dentate thins out (that is, there are fewer terminals) along the septo-temporal axis of the hippocampal formation and in more ventral levels it was much more difficult to distinguish the degeneration caused by the second (commissural) lesion from those products remaining from the original entorhinal lesion. For this reason we will place more emphasis on the results obtained in the more dorsal aspects of the dentate gyrus (that part of the dentate closer to the septum) in the adult rats. RESULTS
Methods of analysis Because of its simplicity and rigid orderliness the hippocampus is an ideal model system on any given 2-dimensional section but when it is necessary to consider the structure in 3 dimensions (as it is in this study) a number of problems arise. Because the hippocampus twists in several directions as it proceeds from the septum towards the amygdala it is impossible to collect more than a few sections at right angles to its axis with any of the 3 standard sectioning angles (horizontally, coronally or sagitally). As a consequence, when any of these 3 sectioning angles are used the vast majority of sections are not at right angles to the axis of the hippocampus and the resulting distortion varies greatly depending on the locus of a particular section. Such distortions complicate rapid quantitative comparisons of the size of particular components or layers in different levels of the hippocampus within an animal to say nothing of comparisons between different animals. As an aid in analyzing our material we have selected 8 horizontal sections through the hippocampus and 2 measurement points within each section. The 8 levels and 2 measurement points were selected both for ease of identification and because
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V Ill IV v Vl
VII viii IX x
m
Fig. 2. Drawing of the hippocampus as it appears in a section taken approximately in the plane of its septo-temporal axis. The layer of dentate granule cells is represented by the row of circles while the pyramidal cell layer is shown as a row of triangles. The Roman numerals illustrate the dorsoventral location of the levels selected for analysis of growth of the commissural system after entorhinal lesions. Levels I and II are not indicated because they do not intersect the hippocampus this far laterally.
they introduce minimal distortion. Fig. 2 illustrates the levels and their approximate positions on a section taken parallel to the septo-temporal axis of the hippocampus (in a plane roughly midway between the coronal and sagittal planes). With this type of approach it was possible to perform a rapid and reasonably complete analysis of the changes in the distribution of the commissural system after entorhinal lesions; equally important it allowed valid comparisons between animals. An additional problem in quantifying changes in the commissural system was caused by tissue shrinkage. Early in this study it became obvious that the sizable lesions needed to destroy the commissural system and entorhinal cortex were producing varying amounts of tissue shrinkage not only in the hippocampus but throughout the brain. The shrinkage problem was not restricted to the dentate gyrus, where most of the quantification was done, but was general to the entire hippocampal formation and cerebral cortex. In an attempt to provide a ~omplete picture of the thickness of the commissural band, the data are presented both as absolute size and as a percentage of the entire molecular layer. This latter value to a degree corrects for the amount of shrinkage if it can be assumed that shrinkage is equal through all sublevels within the molecular layer.
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The normal distribution o f the commissural system to the dentate gyrus A l l o f the c o n t r o l rats which received only h i p p o c a m p a l a b l a t i o n or t r a n s e c t i o n o f the ventral h i p p o c a m p a l c o m m i s s u r e showed the same p a t t e r n o f d e g e n e r a t i o n in the dentate. T h e only differences were caused by i n c o m p l e t e lesions o f the ventral h i p p o c a m p a l c o m m i s s u r a l which caused d e g e n e r a t i o n in some cases restricted to the d o r s a l end o f the h i p p o c a m p u s or d e g e n e r a t i o n restricted to the p y r a m i d a l cell fields w i t h o u t involving the d e n t a t e gyrus. O u r o b s e r v a t i o n s are in a g r e e m e n t with earlier descriptions o f the c o m m i s s u r a l p r o j e c t i o n s to the d e n t a t e gyrus 1,2,7,17,19. T w o gradients o f density o f i n n e r v a t i o n can be distinguished. A l o n g the s e p t o - t e m p o r a l axis the p r o j e c t i o n to the dentate gyrus thins o u t so t h a t r o s t r a l d e n t a t e regions are m u c h m o r e densely i n n e r v a t e d t h a n the c a u d a l areas. F u r t h e r m o r e , a g r a d i e n t o f density was also evident a r o u n d the 'V' o f the d e n t a t e gyrus with the n u m b e r o f terminals decreasing going f r o m the tip o f the exterior wing (the r o w o f granule cells facing the t h a l a m u s ) t o w a r d s the tip o f the int e r i o r wing.
I
Fig. 3. Photomicrographs of the degeneration products in the dentate gyrus after a complete lesion of the contralateral hippocampus. Panel A is from a normal rat, panel B is from one which received a lesion of the entorhinal cortex 150 days earlier (as an adult), while panel C is from an animal which received an entorhinal lesion at 11 days of age. All photographs were taken from comparable points in the dentate at level III (see Fig. 2). The dotted line emphasizes the hippocampal fissure and the arrows denote the upper boundary of degeneration caused by the commissural lesion. (× 400.)
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Despite these changes in density of innervation, the commissural system always occupied the same zone of the molecular layer (Figs. 3-5), namely a region extending to about 90 # m above the granule cell bodies. A space was located between the granule cells and the commissural terminals and the size of this varied roughly with the density of innervation. Earlier work has shown this commissural free zone to be innervated by septal afferentslL The distribution o f the dentate commissural system in rats with neonatal or adult entorhinal lesions Entorhinal lesions in 11-day-old rats caused a remarkable change in dentate commissural system. At rostro-dorsal levels (I-III) the commissural projections expanded to fill the entire molecular layer, essentially occupying the areas normally innervated by entorhinal projections. This result was obtained in 4 rats with neonatal lesions and successful secondary commissural or contralateral hippocampal lesions. In the lower levels of the hippocampal formation the spread of the commissural system in the dentate gyrus was not as great and did not completely occupy the outer molecular layer. These findings are summarized in Figs. 3-5 and Table I; note
Fig. 4. Same as Fig. 3 except that all micrographs were taken at level V (see Fig. 2); also the dotted line emphasizes the exterior surface of the dentate gyrus rather than the hippocampal fissure.
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eta[.
I
b
C
Fig. 5. Reconstructions of the degeneration (dots) seen in the hippocampus in a normal rat (panel a), a rat which had received entorhinal lesions 150 days prior to the commissural lesion (panel b), and one which had received an entorhinal lesion at 11 days of age and the commissural lesion as an adult (panel c). The broken lines in panels b and c represent the limits of the entorhinal lesions. The dorsoventral levels shown in this figure are level II (upper panels) and level IV (lower panels).
t h a t the g r o w t h effect was obvious b o t h in terms o f a b s o l u t e a n d percentage growth. E n t o r h i n a l lesions in a d u l t rats also caused the c o m m i s s u r a l system in the dentate gyrus to e x p a n d b u t the effect was n o t so d r a m a t i c as t h a t seen after e n t o r h i n a l lesions in the l 1-day-old rat. A s m e n t i o n e d above, we are m o r e confident o f the results o b t a i n e d in the m o r e d o r s a l (that is, closer to the septum) aspects o f the h i p p o c a m p a l f o r m a t i o n since the d e g e n e r a t i o n there was very dense a n d easily d i s c r i m i n a t e d f r o m the d e g e n e r a t i o n p r o d u c t s left f r o m the original (entorhinal) lesion. I n the d o r s a l h i p p o c a m p u s it can be seen t h a t the dentate c o m m i s s u r a l system e x p a n d e d nearly 50 # m to occupy a b o u t 50 ~ o f the t o t a l m o l e c u l a r layer. Similar increases were f o u n d in o t h e r levels o f d e n t a t e b u t precise quantification was m o r e difficult because the
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TABLE I W I D T H OF THE DEGENERATION BAND IN THE MOLECULAR LAYER OF THE DENTATE GYRUS AFTER DESTRUCTION OF THE COMMISSURAL SYSTEM IN RATS W I T H PRIOR ENTORHINAL LESIONS
The upper table gives the width of band in microns while the lower table presents the data as the percentage of the molecular layer occupied by the degeneration products produced by the second (commissural system) lesion. The numbers in parentheses refer to the number of rats analyzed at each level while the asterisks denote data statistically different (by the Mann-Whitney U test; P < 0.05) from control data; the terms 'll-day lesion' and 'adult lesion' indicate the age at which the first (i.e. entorhinal) lesion was performed. Dorsoventral level
Median width of commissural degeneration band in dentate gyrus (lzm) 11-day lesion
Adult l e s i o n
Control
II III IV V VI VII VIII
*200 (3) "188 (4) 128 (3) "111 (3) "118 (2) 100 (2) 130 (1)
"157 (5) "137 (5) "131 (5) 112 (4) "118 (5) *110 (5) 133 (3)
112 (7) 93 (7) 110 (5) 89 (6) 87 (6) 74 (5) 72 (3)
Dorsoventral level
Median percentage of molecular layer of dentate gyrus occupied by commissural degeneration band
II III IV V VI VII VIII
11-day lesion
Adult l e s i o n
Control
"71.8 (3) "81.6 (4) *63.4 (3) *52.5 (3) "51.9 (2) *47.0 (2) 44.0 (1)
"51.3 (5) *47.8 (5) *48.5 (5) *43.8 (4) *39.4 (5) *40.8 (5) "40.1 (3)
32.7 (7) 29.2 (7) 31.4 (5) 27.0 (6) 25.8 (6) 24.8 (5) 24.1 (3)
c o m m i s s u r a l d e g e n e r a t i o n was sparser, t h e r e b y m a k i n g the b o r d e r o f the c o m m i s s u r a l system less distinct. Despite this p r o b l e m , it was evident t h a t the c o m m i s s u r a l system h a d increased in the d e n t a t e gyrus o f lower h i p p o c a m p a l f o r m a t i o n levels a n d a p p a r ently to the same degree as it h a d in m o r e d o r s a l sections. This is in c o n t r a s t to the g r o w t h seen after n e o n a t a l lesions in which the s p r e a d o f the c o m m i s s u r a l system was m u c h greater in the d o r s a l levels. The normal distribution o f the commissural projections to the pyramidal cell fields The c o m m i s s u r a l p r o j e c t i o n to the p y r a m i d a l cell fields has been described by several authorsl,2,17,lL U n l i k e the c o m m i s s u r a l p r o j e c t i o n s to the dentate, those to the p y r a m i d a l cells d o n o t have an obvious s e p t o - t e m p o r a l g r a d i e n t as d e m o n s t r a t e d in Fig. 5.
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The distribution o f the commissural system projections to the pyramidal cell fields in rats with lesions o f the entorhinal cortex as neonates or adults
A massive growth of the commissural system takes place in the stratum moleculare of the hippocampus after neonatal entorhinal lesions (Fig. 5). This takes place in both regio superior (CAI) and regio inferior (CA2-4). In CA1 the growth is particularly evident in the more rostral levels of the hippocampus where the degeneration extends along the entire length of the hippocampal fissure (Fig. 5). At more caudal (temporal) levels, the outer molecular layer also contains dense degeneration (in contrast to normal rats) but this is restricted to one-third of the zone above the fissure facing the free end of the interior wing of the dentate ( C A l c of Lorente de N61°). These results were obtained in 6 rats with neonatal entorhinal lesions and secondary commissural or hippocampal lesions performed when the animals were adults. For two reasons we conclude that the dense degeneration seen in the stratum moleculare of CA1 is due to sprouting of the commissural system as opposed to growth of the septo-hippocampal system or the crossed temporo-ammonic tract. First, it could not be explained by possible growth of the crossed temporo-ammonic system since it was obtained in 2 rats with lesions of the ventral commissure and in these involvement of the dorsal psalterium was impossible. Secondly, the effect was obtained in 4 rats with contralateral hippocampal ablations that did not involve the septo-hippocampal system. There was a strong indication of commissural growth into the stratum moleculare of the regio inferior after lesions of the entorhinal cortex in 11-day-old rats (Table II) but, unlike the situation in the dentate gyrus, it did not completely fill this field at any septo-temporal level. Table II presents a summary of the thickness of the commissural degeneration band in CA2 at a point 100 # m from the end of the projection of the mossy fibers at level II. It can be seen from this table that the median width of the degeneration band and the percentage of the apical dendritic fields that it occupies were greater in the lesioned animals but due to the relatively small number of rats involved and considerable variance in the data these results are only marginally significant by the Mann-Whitney test. It was not possible to arrive at a definitive answer about commissural expansion into the stratum moleculare of CA1 in adult rats. Degeneration in this zone was clearly not as dense as that in the stratum radiatum of CA1 but subtle changes could not be evaluated because of the residual degeneration products from the original entorhinal lesions. TABLE I1 Median width (/~m) and ~ of apical dendritic fields occupied by commissural degeneration band in regio inferior approximately 150/~m from the end of the mossy fiber band at dorsoventral level II. ll-day lesion (n ~ 5)
Adult lesion (n = 5)
Control (n ~ 7)
466 #m 78.2 ~
427 #m 71.7 ~
366 #m 65.2
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There did appear to be an increase in the width of the commissural band in CA2 after adult lesions, at least in the dorsal hippocampus (Table II). These results, however, were not so consistent as those obtained in the dentate and were only of borderline statistical significance. DISCUSSION
From both qualitative and quantitative analysis of these results two conclusions are obvious. First, the commissural system undergoes growth after entorhinal lesions in both immature and mature rats and secondly, this growth is greater in the younger animals. Two types of changes in the commissural projection were seen after entorhinal lesions. The first type of post-lesion growth observed was what we have referred to as 'spreading'. Spreading occurs when a synaptic population expands and occupies areas of a dendritic field of a particular cell in which it would not normally be found. The second type of growth has been observed by several authors and will be referred to here as 'sprouting'. This effect takes place when an afferent system proliferates and increases its density ofinnervation in a dendritic area that it normally occupies. In the dentate gyrus, the commissural system spreads into new territory in the outer molecular layer, an area in which commissural terminals are never seen in the normal rat1,2, lz. This type of growth in which axons invade new territory on a particular cell can be distinguished from that which occurred in the stratum moleculare of the pyramidal cell field CA1. The few terminals normally found in that region undergo extensive proliferation and become as dense as the normal innervation of stratum radiatum of CA1. Whether sprouting and spreading of afferent projections are qualitatively different phenomena or are simply different degrees of a single effect is unknown. Solution of this question will have to await development of techniques for manipulating postlesion axonal and synaptic growth. From the results of this study and those of earlier experiments, it is apparent that the effects of entorhinal lesions on the distribution of septal and commissural projections to the hippocampal formation are age dependent. This observation provides a possible morphological explanation for the greater 'plasticity' in immature brains observed in neurobehavioral studies using lesion techniques. There are several hypotheses which could account for the greater post-lesion axonal growth in immature animals. In discussing some of these we will restrict ourselves to the post-lesion growth of commissural system described in this paper. (1) It is possible that the cell bodies which give rise to the commissural terminals have greater growth potential or are more responsive to the denervation changes going on in contralateral dentate gyrus in the younger animals. It is widely believed that retrograde changes are more pronounced in neonatal neurons than in adults a; and it is conceivable that this difference could be involved in the greater commissural growth after 11-day entorhinal lesions. (2) The glia are more numerous in adult brains than in neonatal ones and it is
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possible that the upward movement of the commissural terminals is retarded in the adults by post-lesion growth and proliferation of these elements. (3) The degenerating elements produced by the entorhinal lesion in adults persist for months and possibly years (Lynch et al., unpublished). In the neonate the lesion causes much less degeneration and what is produced disappears very rapidly. Therefore, it is possible that the outward movement of the commissural system is blocked by degenerating fragments of the original entorhinal innervation in the adult but not in the neonate. (4) The status of the dendritic fields of the granule cells is radically different in the neonatal and adult rat and this could be responsible for the differences in commissural growth. The dentate gyrus is undergoing tremendous growth in the 11-day-old rat while it is, of course, relatively stable in the adult. At 11 days, most of the granule cells have not yet migrated into their final positions to say nothing of differentiating dendritic trees and spines (see Crain et aL 5, for a description of the molecular layer at 11 days of age). Removing the entorhinal cortex at this age, then, means that the stilldeveloping commissural system is given access to dendritic space that has never been occupied by the temporo-ammonic system. In the adult, the commissural afferents must invade territory that once was innervated by the entorhinal terminals. The growth into the molecular layer of the hippocampal fields is a different matter. The pyramidal cells are present before birth and presumably are considerably more mature than the later appearing granule cells. Despite this more advanced condition, the sprouting of commissural terminals was still much greater after neonatal entorhinal lesions. This does not of course rule out the possibility that the immature developmental state of the dendritic territory to be invaded is the reason for the greater growth in immature animals but it does indicate that this effect is not unique to newly formed nerve cells. More definitive statements may become possible when studies on the ontogeny of the dentate gyrus and hippocampus combined with longitudinal studies on sprouting in these areas are completed. (5) A final possibility is that competition goes on between the remaining dentate afferents for the space vacated by the entorhinal projection and that the commissural fibers are less successful in this in the adult animals than in the neonates. We have previously reported evidence that the acetylcholinesterase containing septal projection undergoes massive growth after entorhinal lesions and the nature of this growth is also dependent upon maturational stage< 11. In the neonates, the growth is restricted to the outermost levels of the molecular layer while in the adult it occupies about 50 ~ of the layer. This, of course, is the reverse of the growth of the commissural system in which the greatest spread of terminals is seen in the neonatal animals. This reciprocity of effects between septal and commissural systems suggests that the final disposition of these systems after entorhinal lesions may be dependent upon an interaction between them. It is pertinent to note at this point that the above arguments are most applicable to the rostral (septal) segments of the hippocampus. In the caudal (temporal) regions of the hippocampal arch the growth of the commissural system is only slightly greater in the neonates than in the adults. Since it is apparent that the developmental condi-
POST-LESION AXONAL GROWTH IN THE HIPPOCAMPUS
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tions are relatively u n i f o r m t h r o u g h o u t the structure, it is necessary to consider w h a t other c o n d i t i o n s c o u l d p r o d u c e this restricted g r o w t h in the c a u d a l h i p p o c a m p u s . One attractive hypothesis is suggested by the o b s e r v a t i o n t h a t the c o m m i s s u r a l p r o j e c t i o n b e c o m e s progressively less dense a l o n g the s e p t o - t e m p o r a l axis (as well as a r o u n d the ' V ' o f the dentate). A s described above, G o t t l i e b a n d C o w a n 7 have r e p o r t e d d a t a i n d i c a t i n g t h a t the relative density o f the d e n t a t e c o m m i s s u r a l p r o j e c t i o n is the result o f a d e v e l o p m e n t a l c o m p e t i t i o n between t h a t system a n d the recently described ipsilateral associational systemV, 24. Therefore, it is possible t h a t the failure o f the d e n t a t e c o m m i s s u r a l p r o j e c t i o n to show greater g r o w t h in the c a u d a l h i p p o c a m p u s is due to the m o r e successful g r o w t h o f the a s s o c i a t i o n a l system in these h i p p o c a m p a l areas. T h a t is, j u s t as the associational p r o j e c t i o n a p p e a r s to o c c u p y p r o p o r t i o n a l l y greater dendritic space in c a u d a l h i p p o c a m p u s d u r i n g n o r m a l development, so it m a y be able to c o m p e t e m o r e successfully t h a n the c o m m i s s u r a l p r o j e c t i o n for the areas left v a c a n t by the d e s t r u c t i o n o f the e n t o r h i n a l cortex. ACKNOWLEDGEMENTS
This research was s u p p o r t e d by N I H G r a n t s M H 19793-01, M H 19691 and N S F G r a n t G B 16973.
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