Altered distribution of choline acetyltransferase and acetylcholinesterase activities in the developing rat dentate gyrus following entorhinal lesion

Brain Research, 63 (1973) 215-230

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© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

ALTERED DISTRIBUTION OF CHOLINE ACETYLTRANSFERASE AND ACETYLCHOLINESTERASE ACTIVITIES IN THE DEVELOPING RAT DENTATE GYRUS FOLLOWING ENTORHINAL LESION

J. VICTOR NADLER, CARL W. COTMAN AND G A R Y S. LYNCH

Department of Psychobiology, University of California at Irvine, Irvine, Calif. 92664 (U.S.A.) (Accepted May 28th, 1973)

SUMMARY

The entorhinal cortex of rats was removed unilaterally at the age of 11 days. At various times after the lesion was performed, the response of the cholinergic septohippocampal innervation of the ipsilateral dentate gyrus was assessed by quantitative enzyme histochemistry. Laminar analysis revealed that, within 5 days, the absolute and specific activities of choline acetyltransferase and acetylcholinesterase became significantly elevated in the outer part of the molecular layer on the operated side relative to the corresponding zone on the control side. These differences between the two sides were greatly reduced or abolished before the animals reached maturity. We propose that these acute neurochemical adjustments in the dentate gyrus reflected formation of an increased number of cholinergic synaptic terminals in partial compensation for lack of entorhinal input. We further suggest that the ability of these additional terminals to synthesize transmitter was eventually reduced, and thus they may have become functionally less effective. Additionally, the lesion produced a chronic, bilateral depletion of acetylcholinesterase activity. Between 32 and 80 days after the entorhinal cortex was removed, the specific activity of this enzyme was reduced, in all laminae, to 40 ~ of values in unoperated animals. Such neurochemical changes may play a role in recovery of the central nervous system from damage.

INTRODUCTION

A growing body of evidence suggests that, following certain lesions of the mammalian central nervous system (CNS), synaptic endings derived from intact fiber tracts in close proximity to the denervated region come to occupy some of the newly exposed membrane surface4,15,20,23,29,35,4z,47. In a few cases the capacity of the imma-

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ture CNS to undergo a similar type of synaptic reorganization has been examined ~;.~'. 45,48. The results of these studies have suggested a generally greater extent of morphologic alteration than that found in adult animals. These adjustments may be rela~ed to the attainment 15,2a,45 or prevention 42 of functional recovery tbllowing damage to the central nervous system. In this regard, the neurochemical consequences of lesions of the CNS should also be examined. Adjustments in enzyme activities related to neurotransmission could be among the most significant neurozhemical effects of brain lesions. In an effort to uncover such alterations, we have examined the activities of choline acetyltransferase (ChAc) and acetylcholinesterase (ACHE) in the dentate gyrus of the developing rat following unilateral lesion of the entorhinal cortex. The arrangement of the granule cells, the predominant neuronal type in the dentate gyrus, defines the overall organization of this region. The bodies of the granule cells are arranged in a closely-packed layer forming a hilus, their dendrites ramify in the molecular layer and their axons course through the hilus 25,44. Afferent fiber tracts relevant to the present study arise from cell bodies in the ipsilateral entorhinal cortex (temporo-ammonic tract; perforant path) and the septum (septo-hippocampal fibers). The temporo-ammonic tract terminates in the superficial (outer) two-thirds of the molecular layer ls,19. The septo-hippocampal fibers originate from cell bodies in the medial septal nucleus and nucleus of the diagonal band 1"~, enter the hippocampal formation from the fimbria and form synapses near the base of the granule cell dendrites 36 and immediately deep to the granule cell layer a6,4' in a discrete laminar fashion. Biochemically determined ChAc 14,s° and AChE 49 activities and ACHEdependent histochemical staining 2~,22,'~7,46,49-51 in the dentate gyrus are associated predominantly with the septo-hippocampal tract. The other extrinsic inputs are noncholinergic 5°, and there are few, if any, intrinsic cholinergic neurons 46. Thus changes in parameters of cholinergic transmission can be related directly to a single nerve pathway. Following unilateral removal of the entorhinal cortex of immature rats, an intense band of histochemically demonstrable AChE activity appears in the outer one-sixth to one-third of the molecular layer of the ipsilateral dentate gyrus ~. Only light AChE activity is normally demonstrable in this zone 21,sl. Electron microscopic studies reveal, in this same zone, an increased number of synaptic terminals which display AChE activity. Since the intense band of staining is dependent on the integrity of septo-hippocampal connections, the proposal has been made that a greater number of septo-hippocampal synaptic terminals are formed 9. In this report, we demonstrate that these changes are accompanied by large, time-dependent alterations in both the magnitude and distribution of ChAc and AChE activities. METHODS

Sprague-Dawley rats bred in our facilities were used in these studies. The day of birth was taken as day 0. The adult rats which were used were 105-137 days of age.

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Fig. 1. Camera lucida drawings of horizontal sections through the temporal half of the hippocampal region. The sections represent the most dorsal (a), intermediate (b) and most ventral (c) which were taken for dissection. The shaded area denotes the extent of typical lesions. White matter is indicated by solid black areas. Triangles (A), pyramidal cells; circles (©), granule cells. Abbreviations: DG, dentate gyrus; H, hippocampus; F, fimbria; LE, lateral part of the entorhinal cortex; ME, medial part of the entorhinal cortex; PS, parasubiculum. Arrows represent the boundaries of the last 3 subdivisions.

All animals received entorhinal lesions at the age of 11 days. Usually the lesions were performed on all the animals in a litter. Each rat was anesthetized individually with ethyl ether, and the skull was opened just anterior to the left transverse sinus. The entorhinal cortex and at least the posterolateral portion of the parasubiculum on that side were then removed by aspiration (Fig. 1). Sometimes the lesion encompassed adjacent regions as well, but this had no additional effect on either histochemical AChE staining or the enzyme activities which were studied. The animals were sacrificed by decapitation at various times after operation, and the brains were quickly removed and treated for quantitative histochemistry as previously described 38. Briefly, the brains were frozen rapidly and a number of horizontal sections of 100/~m thickness were cut in a cryostat maintained a t - - 1 8 ° to --22°C. The sections were then freeze-dried thoroughly. They were either dissected immediately after freeze-drying or stored at - - 7 0 °C for not longer than 2 weeks. At the same time, sections of 75 # m thickness, cut at similar dorsoventral levels, were stained for AChE activity 40 with an incubation period of 12-20 h. Promethazine at 1.8 × 10-4 M concentration was included in the incubation medium to inhibit non-specific cholinesterases52. The stained sections were used as a check on the proper placement of the lesion. Only freeze-dried sections taken f r o m dorsoventral levels at which a prominent band of staining could be seen along the superficial edge of the dentate gyrus on the operated side (Fig. 2) were dissected. This band of staining was unaffected by exposure to promethazine, but was abolished by inclusion of 5 × 10-6 M BW284C51, a specific inhibitor of AChE 3, in the incubation medium. Freeze-dried sections which had been cut transversely through the temporal half of the hippocampal region were selected for dissection. They were representative

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Fig. 2. Unfixed cryostat sections t h r o u g h the dentate gyrus stained for A C h E activity 19 days afte~~ unilateral removal o f the entorhinal cortex. T h e intense band o f staining (indicated by arrows) on the operated side (O) was never observed on the control side (C). Calibration bar = I ram.

of the area which lies about 1500-4000 # m ventral to the most dorsally lying granule cells, or about in the middle region of the temporal hippocampal formation. The operated and control sides of each section were separated, and the dentate gyrus from 8 to 12 sections was dissected into 5 layers (Fig. 3): outer (MO), middle (MM) and inner (MI) portions of the molecular layer, granule cell layer (G) and the superficial part of the hilus (H) immediately deep to the granule cell layer. The respective layers from each section were pooled, dispersed uniformly in 50/~1 of H20 and stored at --20 °C overnight prior to enzymatic analysis. ChAc and AChE activities were determined by sensitive radiochemical techniques as previously described 3s. Protein content was measured by the colorimetric method of Lowry et al. 26. To correct for considerable individual variation in enzyme activities of the control side, the statistical significance of differences between operated and control sides was determined with a t-test for inherently paired samples 11. Transections of the septo-hippocampal fibers were performed in a few cases to determine whether or not the changes in distribution of enzyme activities which followed removal of the entorhinal cortex were dependent on the presence of these connections. For this purpose, 27- or 28-day-old rats (16 or 17 days after entorhinal lesion) were positioned in a stereotaxic instrument under sodium pentobarbital anesthesia, and the skull was exposed. A rectangular piece of bone was removed just posterior to bregma, and then, under stereotaxic guidance, a bilateral cut was made

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Fig. 3. Demarcation of layers of the dentate gyrus on a section stained for AChE activity. In all cases, layers were dissected from both internal and external leaves. Note that the width of certain layers varied at different points in the dentate gyrus (the dissection followed the zones of AChE staining). Abbreviations as given in Methods. Arrows point to the hippocampal fissure. Magnification x 50.

in the coronal plane about 1.5 mm posterior to bregma to a depth of 5.5 mm from the surface of the dura. This procedure was judged sufficient to interrupt all the septohippocampal fibers on the basis of the nearly complete loss of histochemically demonstrable AChE activity from the hippocampal formation (Fig. 4). The animals were sacrificed at various times after the transection, and the brains were processed for quantitative histochemistry as described above. RESULTS

The specific activity of ChAc in layer MO on the operated side was more than twice that of the same layer on the control side 5-6 days after the entorhinal cortex was removed (Table I). There were no differences in any of the parameters which were measured between animals sacrificed at 5 or 6 days after operation. Therefore the data from all animals of these ages have been combined. The specific activity of ChAc in layer MO was also significantly higher on the operated side at 19 days survival, but by 32 days after operation the difference between the two sides was no longer significant. Nevertheless a difference in favor of the operated side was still obtained in 4 of the 5 animals examined at the latter age. When the animals were examined after reaching adulthood, no significant differences between operated and

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Fig. 4. Unfixed cryostat sections through the hippocampal region stained for AChE activiiy 23 days after bilateral transection of the septo-hippocampal fibers and 39 days after unilateral removal of the entorhinal cortex. Transections were performed at the level of the pre-commissural fornix. Most of the normal staining in the dentate gyrus is absent from operated (O) and control (C) sides, as well as the intense band of staining induced by the entorhinal lesion. The residual staining in the molecular layers ofhippocampus regio superior and subiculum is of unknown origin:'~LCalibration bar 1 mm.

control sides were detected in the specific activity of ChAc. Although the specific activity of ChAc in the molecular layer of the operated side tended to be slightly greater than that of unoperated animals, the activity on the control side was distributed normally, i.e., the highest specific activity of ChAc was consistently found in layer G, and it was lowest in the molecular layer 38. The specific activity of ChAc in the dentate gyrus as a whole was only slightly greater on the operated side at 5-6 days survival, because the relatively high specific activity in layer MO was consistently offset by relatively low specific activities in layers G and H, the regions in which septo-hippocampal fibers normally terminate. Although the specific activity of ChAc on the operated side at 19 days survival tended to be lower than control in layers other than MO, this effect could not consistently be attributed to a difference in any particular layer. These differences in specific enzyme activity between operated and control sides were attributable mainly to differences in enzyme activity rather than in protein content. Comparison in terms of absolute enzyme activity (total activity in a 1.2 mm length of the temporal half of the dentate gyrus) revealed that removal of the entorhinal cortex altered the laminar distribution of ChAc activity in the dentate gyrus, but did not alter the total amount of activity. Layer MO was relatively enriched at the

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TABLE I SPECIFIC AND ABSOLUTE ACTIVITIES OF C h A c IN DENTATE GYRUS AFTER REMOVAL OF ENTORHINAL CORTEX

Values are expressed as means :k S.E.M. for 5 animals (from at least 3 different litters) studied at each age. See Methods and Fig. 3 for identification of layers. Activity of the whole dentate gyrus is the sum of activities in all layers. This quantity was divided by the total protein in all layers to obtain specific activity. O, operated side; C, control side. Layer

Days survival 5-6

Specific activity (/~mole ACh synthesized/ 30 min/g protein)

MO

O 11.2 C 15.0 MM O 0.6 C 7.6 MI O 6.8 C 11.0 G O 6.7 C 8.8 H O 6.4 C 9.3 Whole O 8.2 dentate C 7.8 gyrus

Absolute activity (/~mole ACh synthesized/ 30 min/12 × 100#m sections)

MO

O C MM O C MI O C G O C H O C Whole O dentate C gyrus

± 0.9*** d- 0.9 i 2.2 ± 0.5 ± 3.5 ± 3.4 ± 0.6** ± 1.0 ± 0.6* ± 1.2 ± 0.5* ± 0.6

0.085 ± 0.050 ± 0.079 ± 0.066 ± 0.011 ± 0.021 ± 0.109 ± 0.126 ~ 0.039 ± 0.059 ± 0.323 ± 0.321 ±

0.008*** 0.010 0.011 0.011 0.005 0.003 0.023* 0.019 0.005* 0.009 0.032 0.020

19

32

18.3 :k 2.5** 10.9 ± 1.2 12.8 ± 2.3 12.2 ± 1.8 10.3 ± 4.4 8.4 ± 1.7 13.6 ± 2.2 14.1 ± 1.2 10.7 ± 2.7 12.4 ± 2.3 13.4 ± 1.8 12.1 ± 1.4

14.5 ± 10.4 :k 13.0 ± 11.5 ± 8.7 ± 7.7 ± 14.8 ± 11.6 ± 10.2 ± 7.6 ± 12.9 ± 10.5 ±

0.32 ± 0.22 ± 0.22 ± 0.32 ± 0.07 ± 0.08 ± 0.29 ± 0.38 ± 0.11 ± 0.16 ± 1.02 ± 1.16 ±

0.48 ± 0.15 0.40 ± 0.09 0.38 ± 0.12 0.48 ± 0.12 0.15 ± 0.05 0.15 ± 0.04 0.60 ± 0.23 0.46 ± 0.14 0.23 ± 0.10 0.14 zk 0.06 1.8 ± 0.5 1.6 ± 0.4

0.06* 0.03 0.05 0.04 0.03 0.01 0.07 0.03 0.03 0.02 0.18 0.05

80 ±

2.8 1.2 2.9 1.7 2.3 1.7 3.2 1.8 2.3 2.2 2.0 1.4

16.9 ± 14.9 ± 14.0414.0 ± 16.7 ± 15.0± 16.0± 16.4 ± 12.1 ± 15.5 ± 14.9 ± 15.1 ±

3.3 2.0 1.2 1.4 2.9 2.2 1.5 1.4 1.9 1.9 1.5 1.2

0.46 ± 0.68 ± 0.52 ± 0.94 ± 0.15 ± 0.23 ± 0.78 ± 1.00 ± 0.19 ± 0.33 ± 2.1 ± 3.2 ±

0.09 0.13 0.06 0.19 0.04 0.06 0.10 0.21 0.03 0.06 0.2 0.6

Operated side differed from control at * P < 0,05; ** P < 0.025; *** P < 0.005 (paired t-test).

expense o f o t h e r layers. A f t e r this effect c o u l d no longer be detected, there was no further net gain o f C h A c activity on the o p e r a t e d side. H o w e v e r , a d d i t i o n o f e n z y m e activity o n the c o n t r o l side c o n t i n u e d normallyS< In contrast to that o f C h A c , the specific activity o f A C h E in layer M O r e m a i n e d m o r e than twice as great on the o p e r a t e d side relative to c o n t r o l until at least 32 days after o p e r a t i o n (Table II). This difference was no longer significant in animals which had reached a d u l t h o o d , a l t h o u g h the specific activity on the o p e r a t e d side o f 4 o f the 5 animals studied at m a t u r i t y still exceeded that on the c o n t r o l side. U n t i l at least 32 days survival, the specific activity o f A C h E was also significantly greater o n the o p e r a t e d side in layer M M , a l t h o u g h the difference was less than in layer M O . It also b e c a m e insignificant in a du l t animals. Th e specific activities o f layers

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TABLE I1 SPECIFICAND ABSOLUTEACTIVITIESOF AChE IN DENTATE(;YRUSAFTERREMOVALOF ENTORHINAL('ORTEX See Table I for details. Layer

Days survival 5-0

Specific activity (/~mole ACh hydrolyzed/ 30 min/g protein)

MO

O C MM O C O MI C O G C O H C O Whole dentate C gyrus O C O C O C O G C O H C O Whole dentate C gyrus

Absolute activity MO (/zmole ACh hydrolyzed/ MM 30 rain/12 × 100/~m sections) M1

540 240 610 400 460 600 510 470 510 720 540 450

± 60*** ± 50 -- 60* ± 110 :k 130 ~200 ± 90 ± 80 _L 60 ~: 140 ~ 60*** ~ 70

4.2 ± 2.4 t. 4.8 ± 4.0 ± 0.7 ± 1.2 ~: 8.4_~ 7.0± 3.4 ±_ 4.5 ± 22 :L 19 i

0.7** 0.6 0.5 1.4 0.2 0.3 2.2 1.4 0.8 0.8 3** 3

19

32

80 .-

1510 %_ 140"** 7 0 0 ± 60 1090 ± 60* 890 ± 40 1170 ± 90 8 2 0 ± 70 1300 :k 140 1200 :k 110 1110±140 1080 ± 110 1270 ± 80** 960 t: 40

1670 ± 160"** 6305:120 1420 + 140"** 780 ~:: 130 940 ~_ 140 740zk 100 •390 5:170 1260 ~_ 190 980:3 140 1120 ± 240 1360 ± 120"** 910 £ 150

610 ± II0 4 6 0 L 60 610 ± 110 600 ~: 50 510 ± 100 4 3 0 ± 100 890 ± 170 980 ± 100 5 9 0 ± 80 660 :~: 60 710 -~: 110 680:~ 40

31 i 16 ± 18 2: 24 ± 10 ± 9± 28 5 33 ± 12 ~ 16 ± 99 ~ 98 ±

8* 4 3 3 3 3 5 5 3 3 18 14

55 j:: 22 ± 41 ± 31 _~ 16 ~ 14 k: 52± 48 ± 22 ± 18 :~ 186 ± 133 !::

17'* 6 9* 5 4 2 14 11 6 3 33* 20

19 + 6 21 ~: 4 24 ~ 5 45 ~ 14 5~ 2 8 _L 2 42~ 7 57 " 9 9~ 1 14 2: 3 100 :.t: 14 145 ± 29

Operated side differed from control at * P < 0.05; ** P < 0.025; *** P < 0.005 (paired t-test).

G a n d H w e r e e a c h v e r y n e a r l y e q u a l o n o p e r a t e d a n d c o n t r o l sides at all times. A s a result, the specific a c t i v i t y o f A C h E in the d e n t a t e g y r u s as a w h o l e was signifi c a n t l y h i g h e r o n the o p e r a t e d side f o r at least 32 days a f t e r the e n t o r h i n a l c o r t e x was r e m o v e d . T h e r e l a t i v e l y h i g h A C h E a c t i v i t y o f l a y e r M O o n the o p e r a t e d side was due m a i n l y to e n r i c h m e n t in e n z y m e a c t i v i t y a n d n o t to l o w e r p r o t e i n c o n t e n t . H o w e v e r , the differences b e t w e e n o p e r a t e d a n d c o n t r o l sides in the specific a c t i v i t y o f layer M M c o u l d n o t be c l e a r l y a t t r i b u t e d to differences in a b s o l u t e activity. E x c e p t f o r o n e a n i m a l at 19 d a y s s u r v i v a l in w h i c h the d e n t a t e g y r u s o n the o p e r a t e d side was severely d e p l e t e d o f p r o t e i n , the g r e a t e r A C h E a c t i v i t y o f l a y e r M O o n the o p e r a t e d side was a l w a y s reflected in a small, b u t significant, difference in a c t i v i t y o f the d e n t a t e gyrus as a whole. T h e e n r i c h m e n t o f A C h E a c t i v i t y in layer M O was n o t offset by l o w e r

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TABLE III PROTEIN CONTENT OF DENTATE GYRUS AFTER REMOVAL OF ENTORHINAL CORTEX

Protein content of the whole dentate gyrus is the sum of protein in all layers. The units are pg/12 × 100 pm sections. See Table I for other details. Layer

MO

O C MM O C MI O C G O C H O C Whole dentate O gyrus C

Days survival 54

19

32

80 q-

7.6 q- 0.7 10.4A_2.2 8.14-1.0 8.74.1.6 1.74,0.3 2.3±0.6 15.6:kl.8 14.3 4. 1.1 6.24-0.8 6.3±0.6 39 4, 3 42 -4- 4

20.5 _% 5.7 21.5± 4.1 16.9i 2.1 27.1± 2.7 8.14. 2.3 10.5~: 2.4 20.9± 2.3 27.4 5 : 2 . 0 11.3± 2.0 14.7:k 2.7 78 ± 13 101 ~ 12

31 4- 7 384- 7 28 ± 5 40 4- 5 184. 4 194. 3 38! 8 38 -- 6 224- 4 17± 2 137 ± 20 152 4, 17

30 4- 7 46±10 38 ± 3 72 4- 17 94, 2 164, 3 49± 6 63 4- 14 174, 2 24± 6 143 4, 15 220 4, 47

than normal activity in other layers. It thus represented a gain in the total amount of enzyme activity. Between 32 days after operation and maturity, the specific activity of AChE declined by more than one-fourth on the control side and by almost one-half on the operated side. Comparable effects were observed in all layers. The dentate gyrus on the operated side actually lost AChE activity during this period, and no net addition took place on the control side. In the dentate gyrus of unoperated animals, absolute and specific activities of AChE increased steadily throughout postnatal developmental The changes in protein content during maturation of the dentate gyrus (Table III) displayed a pattern similar to that of changes in the absolute activity of ChAc. The differences between the two sides could be resolved into two components. A generalized depletion of protein in the dentate gyrus as a whole was observed on the operated side of most animals 19 days after operation and later. When adjustment was made for the differences between the two sides in the protein content of layers MI, G and H, a difference of 17 ~ remained in layers MO and MM, the regions in which afferent fibers from the ipsilateral entorhinal cortex terminate. This difference probably resulted directly from lack of temporo-ammonic elements on the operated side. The reasons for the more generalized difference in protein content are not evident at present, but they may not necessarily be related to loss of temporo-ammonic innervation. Bilateral transection of the septo-hippocampal fibers reduced the specific activities of ChAc and AChE in all layers to very low levels (Table IV). Thus the altered distributions of these enzymes which followed removal of the entorhinal cortex were dependent on the integrity of septo-hippocampal connections. The time course of disappearance differed for the two enzymes, AChE activity being reduced at a much

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T A B L E IV S P E C I F I C A C T I V I T I E S OF

ChAc

AND AChE

A N D T R A N S E C T I O N OF S E P T O - H I P P O C A M P A L

IN D E N T A T E ( i Y R U S A F T E R R E M O V A l , Ol ~ ENTOP, H I N A L ( ' O R I I X P R O J E C T I O ] ~,

The entorhinal lesion was performed at 11 days of age and the transection at 27 28 days of age. Data are from one animal at each time of survival. See Table I for other details.

Enzyme

Layer

Dayssurvivalaftertransect~n 3

7

23

ChAc

MO

O C MM O C MI O C G O C H O C Whole dentate O gyrus C

9.6 6.6 8.2 5.4 6.2 6.6 6.9 6.2 5.8 6.8 7.4 6.1

11.2 7.6 7.6 7.4 12.2 7.7 5.0 6.9 7.3 9.1 7.8 7.6

1.6 2.0 1.2 2.4 2.4 1.6 2.4 1.9 0.9 2.0 1.7 2.0

AChE

MO

360 310 310 220 370 310 320 210 460 360 350 260

110 ~z 50 ~:~ 40 80 < 100 < 100 40 130 50 80 ~ 50 ~ 80

100 200 130 160 120 800 140 130 270 200 150 200

O C MM O C MI O C G O C H O C Whole dentate O gyrus C

faster rate. In adult rats with no prior lesion, transection of the septo-hippocampal tract reduces ChAc activity in the dentate gyrus to less than 20 ~ of normal within 7 days 5°. In our animals, on the other hand, more than 50 ~ was retained for at least 7 days after transection. This discrepancy may possibly reflect a difference between normal rats and those with entorhinal lesions or a difference between developing animals and adults. DISCUSSION

Our quantitative histochemical approach has revealed significant enzymatic adjustments within the cholinergic septo-hippocampal innervation of the dentate gyrus in response to entorhinal lesion. First, removal of the ipsilateral entorhinal cortex at the age of 11 days initially increased the absolute and specific activities of ChAc and AChE in the outer part of the molecular layer, when compared with the

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corresponding zone on the control side. These differences eventually became insignificant. Second, the specific activity of AChE declined in all layers between 32 days after operation and maturity, without a concomitant decrease in ChAc activity. Both of these alterations could have facilitated cholinergic transmission in the region of the molecular layer normally occupied by temporo-ammonic synaptic terminals. In analyzing the basis for the acute alterations in enzyme activity, the developmental state of the dentate gyrus and its cholinergic innervation at the time when the entorhinal cortex was removed is of major importance. At 11 days of age, less than one-fourth of the granule cells have differentiated1 and only 5-10~ of the mature number of synaptic terminals in the molecular layer are present 1°. Hence, at the time of operation, we were dealing with a region of the CNS in the very early stages of development. The cholinergic innervation of the temporal part of the dentate gyrus is also quite immature at this age, as judged by the low activity of ChAc3s. The specific activity of this enzyme is only about one-third of adult values at 11 days, and it is uniformly distributed in all layers. It abruptly reaches mature values around 16-17 days, suggesting that the major part of the cholinergic innervation attains maturity at this age. The ipsilateral entorhinal cortex is a very significant source of synaptic input to the granule cells of the dentate gyrus~,7,1s,19,z4,ag,43. Unpublished studies in our laboratory based on observation of terminal degeneration with the electron microscope have suggested that more than 50 ~ of the synaptic endings in the outer two-thirds of the molecular layer are derived from temporo-ammonic fibers, but relatively few can have formed by the age of 11 days. Hence removal of the entorhinal cortex at this age should have prevented establishment of the normal synaptic input to a large percentage of the dendritic surface. Sharp increases in ChAc activity have been shown to accompany development of cholinergic synaptic terminalsS,S,la. On this basis, a number of hypotheses can be formulated to explain the relatively high activities of ChAc and AChE in the outer part of the molecular layer following entorhinal lesion: (1) the altered distribution results from pathological damage unrelated to removal of the entorhinal cortex; (2) the effects are attributable to removal of the entorhinai cortex, but they are confined to the granule cells, processes of polymorphic neurons which stain positively for AChE activity21,a4,46,5~, or terminals of extrinsic afferents other than the septo-hippocampal fibers; (3) the increases in enzyme activity reflect an induction of additional enzyme in the few septo-hippocampal synaptic terminals which may normally be present in the outer part of the molecular layer; (4) the loss of synaptic input from the entorhinal cortex increases the rate of formation of septo-hippocampal synaptic input to this zone, but does not alter the final laminar distribution of these synapses; (5) this partial denervation of the granule cell dendrites triggers development of a greater than normal number of septo-hippocampal synaptic terminals in the region of the superficial dendritic branches, and these additional terminals cease to function after a short period of time. Preliminary studies established that the altered distributions of ChAc and AChE activities which followed the lesion were almost certainly attributable to removal

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of the entorhinal cortex rather than to operative trauma. When sections were taken at dorsoventral levels from which little or no entorhinal cortical tissue had been removed, no band of histochemica[ AChE staining could be seen in the outer part of the molecular layer, and there was no difference in the distributions of enzyme activity between operated and control sides. For this reason, only sections in which no residual entorhinal cortex was present were dissected in the studies reported here. However, operative trauma apparently did influence our results in another way. The developmental increases in ChAc and AChE activities and in protein content, which normally occur between l 1 and 16-17 days of age, were reduced by the operative procedure, i.e., the values of these parameters on the control side 5-6 days after operation were consistently lower than those of unoperated animals of the same age ~s. This effect was not observable 19 days after operation. The simple loss of mechanical support from the entorhinal cortex was probably irrelevant also, since we have observed the same effects on AChE staining at the septal end of the dentate gyrus 9, which is far removed from the entorhinal cortex. The changes in enzyme activity can be localized to the septo-hippocampal input on several grounds. The histochemical staining and activities of ChAc and AChE in the molecular layer were abolished by transection of these fibers. In unpublished experiments, the lesion-induced band of staining was not abolished by lesion of other afferents to the hippocampal formation. Finally, electron microscopic studies have shown that the increased AChE staining in the outer part of the molecular layer was present in synaptic terminal membranes and preterminal axons, never within dendrites, cell bodies or other structures 9. The possibility that increased amounts of enzyme were synthesized in the few septo-hippocampal elements which may be present normally in the outer part of the molecular layer cannot be evaluated definitively until these connections can be traced by a method independent of enzyme activity (see also ref. 9). The remaining explanations postulate some type of altered terminal distribution, similar to changes in the commissural and crossed temporo-ammonic innervations which take place under the same circumstances 2s,48. Since the ipsilateral entorhinal cortex was removed prior to the time when the major portion of the septo-hippocampal fibers are likely to form mature synapses as, the growth of these axons toward available postsynaptic sites may have been altered. The argument that an increased rate of formation of septo-hippocampal synaptic terminals accounts for the differences between operated and control sides predicts that the final state of cholinergic innervation should be normal. Indeed there were no significant differences in the specific activities of either ChAc or AChE between the operated and control sides of adult animals. However, electron microscopic investigations have revealed that a significantly greater number of synaptic terminals which stain for AChE activity persist indefinitely in the outer one-third of the molecular layer on the operated side 9. This finding must be interpreted with some caution since the histochemical reaction does not always correlate with biochemical data. For example, sections from operated adult rat brains studied by light microscopic histochemistry exhibited AChE-dependent staining similar in pattern and intensity to that of sections from brains of animals

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examined 19 or 32 days after operation despite large differences in biochemical activity. On balance though, the available evidence tends to support the hypothesis of a permanent change in the distribution of septo-hippocampal synaptic terminals. We tentatively propose that an increased number of cholinergic synaptic terminals is established in the molecular layer as partial compensation for lack of entorhinal input. In some of these terminals, the content of enzymes related to transmission evidently decreases with time. This in turn might lead to eventual failure of synaptic function, but not necessarily to loss of the terminals themselves. Although the evidence for this conclusion is only suggestive at present, it serves as a reasonable working hypothesis which is testable by available anatomical and electrophysiological techniques. Although the hypothesis that anomalous synaptic endings of transient function form in response to denervation has not previously been put forward to explain lesioninduced alterations in the CNS, it derives considerable support from studies of the peripheral nervous system, in which similar phenomena have been reported to occur. In these studies, intact axons have been shown to form morphologically normal and functionally effective synaptic terminals with denervated postsynaptic87 or postjunctionala°-z3 membrane by collateral sprouting. These new connections were functionally suppressed following reinnervation by the 'correct' nerve17,30,3z, but in one case at least31 they did not degenerate. This last finding is especially relevant to our argument, since we have observed that the number of AChE-staining synaptic terminals in the outer part of the molecular layer on the operated side continues to exceed the number in that zone on the control side for at least 200 days after operation 9. In the absence of anatomical and electrophysiological data these provocative findings cannot be applied to our study in a straightforward manner. For one thing we do not know whether septo-hippocampal synaptic terminals normally contact the granule cells or other neuronal types. However, acetylcholine evidently can increase the spontaneous activity of granule cells by a direct action~3, and thus one would expect cholinergic terminals on the granule cells to be effective in initiating or facilitating excitation. Hence the anomalous synaptic terminals we have postulated could be functionally effective. Also, since the ipsilateral entorhinal cortex was removed in our study, re-establishment of the correct innervation could not have suppressed the function of these terminals. But fibers which originate in the contralateral entorhinal cortex innervate the dentate gyrus in response to this lesion48, and the granule cells may possibly be unable to distinguish these connections from the normal temporoammonic innervation. The long-term response of the dentate gyrus to lack of ipsilateral entorhinal innervation included a severe, bilateral decline of AChE activity. The specific activity of this enzyme in adult animals was only about 40 ~ of normal 38. This effect probably could not have been due to loss of cholinergic innervation, since the specific activity of ChAc in adult animals was normal. Comparison of animals with and without lesions shows that this bilateral loss of AChE activity was clearly abnormal and thus related in some way to removal of the entorhinal cortex. An indirect mechanism is indicated, since the entorhinal cortex does not normally project to the contralateral dentate

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g y r u s 6,4s. In a n y case, this m a s s i v e r e d u c t i o n in A C h E a c t i v i t y w o u l d be e x p e c t e d to facilitate c h o l i n e r g i c t r a n s m i s s i o n in the d e n t a t e gyrus. ACKNOWLEDGEMENTS T h i s s t u d y was s u p p o r t e d by N I M H G r a n t G B 3 5 3 1 5 X to G . S . L .

Grant MH

19691 to C . W . C . a n d N S F

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