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Induced acetylcholinesterase-rich layer in rat dentate gyrus following entorhinal lesions
BRAIN RESEARCH
311
INDUCED ACETYLCHOLINESTERASE-RICH LAYER IN RAT DENTATE GYRUS FOLLOWING ENTORHINAL LESIONS
GARY LYNCH, DEE ANN MATTHEWS, SARAH MOSKO, THOMAS PARKS AND CARL COTMAN
Department of Psychobiology, University of California, Irvine, Calif. 92664 (U.S.A.) (Accepted January 20th, 1972)
INTRODUCTION
In recent years, it has become increasingly clear that the adult central nervous system has the capacity to undergo structural changes in response to mild or drastic influences~,e,ll,ls,15,17,2L The manner in which the central nervous system responds to such situations is important, not only in determining the nature of processes associated with the learning and storage of information, but also in establishing functional adjustments associated with discrete brain lesions. Following brain lesions, remarkable changes seem to occur in intact nerve fibers adjacent to degenerating terminals. These changes appear to play a role in the reorganization of the brain. For example, several studies using Nauta stain techniques have shown changes in the distribution and quantity of degenerating terminals within a structure, if at some earlier time other afferent systems had been removede, 11. More recent degeneration studies, using the electron microscope, have also suggested the possibility of 'sprouting' by remaining intact axons following lesions2,1L While these studies have provided initial insights into the adjustments which occur after damage and the capacity of brain to undergo changes, the existing approaches do not readily lend themselves to an analysis of the time course of the changes, the specificity of the change to particular cellular populations, or the biochemical nature of the associated processes. Moreover, present approaches have not permitted the testing of the functional significance of these adjustments. An ideal system should be a relatively ordered projection of nerve fibers with a well defined structural organization and known electrophysiology so that functional changes can be assessed. Likewise, the transmitter of various neuronal elements should be known. The hippocampal formation, while not meeting all requirements, has some important advantages. Its structure is relatively simple and has been extensively studied3,4,s,14,16,19,21; electrophysiological knowledge is detailed; and the transmitter of the septal pathway, one of the major inputs, is believed to be acetylcholine 1°. In view of the advantages afforded by the hippocampus, we are currently engaged in a project studying the morphological, biochemical, and electrophysiological responses of the hippocampus Brain Research, 42 (1972) 311-318
312
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to removal of its various extrinsic afferents. In this paper, we report adjustments in the dentate gyrus of the rat following discrete brain lesions. Specifically, we demonstrate histochemically the induction of an intense layer of acetylcholinesterase activity (ACHE) in the dentate gyrus after lesions of the entorhinal cortex. The highly ordered organization of the dentate gyrus of the hippocampus is particularly appropriate for studying alterations in adult brain following brain damage. It is a rigidly layered structure, whose major extrinsic inputs arise in the septum and the entorhinal and subicular cortices a,14,16. The cortical fibers project topographically to the outer two-thirds of the apical dendrites of the dentate granule cells, while the septo-hippocampal pathway seems to terminate basal to the granule cells and on the inner one-third of the apical dendrites. Electron microscopic evidencO 9 and subcellular fractionation studies 5 indicate that the septal terminals and fibers contain high concentrations of ACHE. Removing septal input causes the disappearance of essentially all AChE activity, suggesting that septal fibers and terminals are the predominant locus of hippocampal AChE 10. Other studies, using light microscope histochemistry, show that AChE is discretely layered in relation to the granule cells 1s,~1. Two distinct bands are seen: one just below the granular cell bodies in the hilus of the dentate gyrus and another immediately above them, occupying approximately the inner one-fourth of the molecular layer. The supragranular zone is sandwiched between the granular cells and the zone of termination of the commissural afferents4. The latter zone and the AChE zone occupy together roughly the inner one-third of the molecular layer. Microchemical analyses have confirmed the general organizational features of the cholinergic system seen histochemically within the hippocampal formationS, ~0. Our own preliminary studies, using a modified Fink-Heimer technique, confirm that the distribution of AChE coincides with the degeneration pattern following a septal lesionlL The uniqueness of AChE in septal terminals provides a tag to examine the reaction of septal fibers and terminals to brain damage. We hypothesized that if the entorhinal cortex was lesioned, causing entorhinal afferents to degenerate, septal afferents might spread to open sites in the apical dendrites previously occupied by entorhinal terminals. If this is the case, it should be possible to very accurately resolve changes in the locus and density of the septal input by changes in the AChE staining pattern within the dentate gyrus. Accordingly, intense staining might appear in the outer two-thirds of the molecular layer, the normal locus of the entorhinal terminals. METHODS
Adult Sprague-Dawley rats of both sexes were used in these experiments. Unilateral electrolytic lesions of the entorhinal cortex or septum were performed with the aid of a stereotaxic apparatus, using a monopolar stainless steel electrode. Lesion placement was verified on sections stained with cresyl violet. At various times, as indicated for the particular experiment, the rats were anesthetized and perfused via the heart with 10~o formalin. The brains were postfixed in the cold ( + 4 °C) for 2 h, frozen sections were cut at 50/~m, and the sections then processed for staining Brain Research,
42 (1972) 311-318
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Fig. 1. ChE stain in rat hippocampus 40 days after unilateral entorhinal lesion. On the lesioned (L) side, an intense band of ChE is evident in the dentate gyrus (arrows) which is not present on the control side (C).
of cholinesterases by a modification of the Koelle procedure 7, as described by Landmesser 9. The incubation medium consisted of 52 m M sodium acetate buffer pH 5.3, 20 m M glycine, 6.5 m M copper sulfate and 5 m M acetylthiocholine. Incubations were carried out for 1.5-2.5 h at room temperature, at which time they were developed in a 1 ~ ammonium sulfide solution. Some sections were counterstained with cresyl violet. Except for the initial experiments, promethazine at 5 • 10-5 M was used to suppress non-specific cholinesterases. RESULTS After a unilateral lesion in the entorhinal cortex, an intense band of cholinesterase (ChE) appears in the outer parts of the molecular layer of the dentate gyrus, an area which normally gives a very slight reaction (Fig. 1). The increased staining intensity is restricted to the outer two-thirds of the molecular layer. The pattern on the control side agrees in all respects with earlier reports on ChE distribution in the hippocampus. On the lesioned side, there is a clearly visible intense band in the distal molecular layer of the dentate gyrus in addition to the normal intensity staining adjacent to the granular layer. Fig. 2C shows the two sides of the dentate gyrus at a higher magnification and emphasizes that this newly developed band is situated in the region of the molecular layer normally occupied by the entorhinal fiber terminals. Brain Research, 42 (1972) 311-318
314
G. L Y N C H
et at.
Fig. 2. Dentate gyrus stained for AChE at various times following entorhinal lesions. Control sides are on the right. Small arrows denote the new band. A, 15 days after entorhinal lesion. B, 30 days postoperative. C, 40 days postoperative. In C promethazine was eliminated. Symbols: M, molecular layer; C, zone of commissural afferents in inner one-third of M; S, normal, supragranular band of AChE staining; G, granule cell bodies; large arrows, obliterated hippocampus fissure.
Brain Research, 42 (1972) 311-318
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While some controversy exists regarding a restricted termination of fibers from entorhinal cortex with the molecular layera, 14,16, our studies using anterograde degeneration methods have shown that entorhinal lesions of the type used in the study produce terminal degeneration from the commissural layer to the hippocampal fissure. Similar histochemical changes were obtained in every animal (n : 20) in which histologically verified entorhinal lesions were made. By using various well-documented inhibitors of ChE, we determined that the cholinesterase staining which develops in molecular layer represents acetylcholinesterase activity. The cholinesterase staining is blocked by a highly specific inhibitor of acetylcholinesterase BW284C51 (ref. 1) (2 • l0 -6 M), but is not blocked by inhibitors of non-specific cholinesterases. Neither promethazine at 5. l0 -~ M, which inhibits 90% of non-specific ChE in rat brain or iso-OMPA (5 • 10-4 M) 1 had any noticeable effect on the band. When butyrylthiocholine is used as substrate in place of acetylthiocholine, the induced band is not seen. In order to determine the time course for the development of the band of AChE, histochemical analysis for AChE was carried out at 5, 15, 30, 40 and 60 days after the entorhinal lesions in adult rats. At 5 clays postoperative, there is no evidence of the band. At 15 days, AChE staining is just detectably enhanced. At 30 days there is a strong staining in the outer parts of the molecular layer, and no further change is noticed after 40 or 60 days. Fig. 2 shows the same region of the molecular layer at various times alter the lesion. These findings show that removal of the entorhinal
Fig. 3. Absence of AChE staining in hippocampus following a septal lesion 30 days after an entorhinal cortex lesion. Staining was done 5 days after the septal lesion.
Brain Research, 42 (1972) 311-318
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input to the hippocampus in adult rats results in the progressive development of a layer of AChE in the dentate gyrus. Our AChE histochemical results obtained after unilateral entorhinal lesions suggest an alteration in the composition or localization of septal terminals and fibers. In order to demonstrate the hypothesized dependence of the induced AChE band on the septum, an electrolytic lesion was made in the medial septal nucleus 30 days after the initial entorhinal lesion. Five days after this secondary lesion, the rats were sacrificed. Essentially all AChE in the hippocampal formation disappeared; the septal lesion eliminated both the normally present AChE bands and the newly developed one as shown in Fig. 3. The disappearance of hippocampal AChE 5 days after septal lesions is in agreement with previous studies on normal animals18,el. Our results conclusively and dramatically demonstrate that, like the normal bands, the induced band of AChE depends on the integrity of septo-hippocampal fibers. The possibility that the increase in AChE could be due to a degeneration process can be ruled out for the following reasons: (1) The band does not appear to be associated with vascular or glial elements present in the region. It is unaffected by inhibitors of non-specific cholinesterases which normally block glial and vascular cholinesterase. (2) The band takes approximately 15-30 days to develop, which is longer than expected for most degeneration processes. (3) A primary or secondary lesion in the septum, the origin of the supposed cholinergic afferents to the dentate gyrus, also results in the disappearance of the band of increased AChE seen following entorhinal lesions.
DISCUSSION
Several mechanisms can be proposed which might account for the observed increase in AChE staining. These include sprouting of axonal elements, with the possible formation of new functional synapses, selective enzyme induction in presynaptic terminals and the development of intense AChE activity on the granule cell dendrites. We feel the most plausible explanation of our results is that the loss of entorhinal endings triggers a massive growth of AChE containing septalfibers and terminals. Alternate explanations cannot be excluded, such as the induction of enzyme in intrinsic hippocampal elements. Whatever the structural locus of ACHE, its activity is completely dependent on the integrity of the septo-hippocampal fibers. Further work will be required to determine if these changes involve the formation of new synapses, but this study provides strong evidence that neural alterations can be induced in adult brain. The induction of a rich AChE band in the dentate gyrus illustrates a remarkable response of the adult central neurons to undergo specific changes. The stimulus for AChE change may be the opening of synaptie sites vacated by terminals from area entorhinalis. Once entorhinal terminals vacate, septal terminals sprout. Yet if the increase in AChE activity in fascia dentata is due to destruction of the afferents from area entorhinalis, one would also expect changes in the hippoBrain Research, 42 (1972) 311-318
LESION INDUCED ACETYLCHOLINESTERASE
317
campus which receives entorhinal afferents as well. At present, we cannot rule out some intensification of AChE in regio superior, but certainly the AChE zone in this region does not spread and is not dramatically more intense. Currently we are exploring various possibilities, but as yet we do not have a unique explanation for lack of marked AChE intensification in regio superior. It is not known whether changes after the lesion are of functional significance, but the hippocampal system is particularly favorable for answering this question and for further studying the response of the central nervous system to injuries. The change observed after entorhinal lesions is unilateral and confined to a discrete layer. This system should prove favorable for electrophysiological as well as anatomical and biochemical studies on the exact processes involved in the undergoing change. Our results have potentially profound implications for studies using lesions to examine behavior. They raise the question of whether the consequence of a lesion is not only to remove tissue elements, but also to catalyze a reorganization of other elements in the neuronal network. It is possible that post-lesion adjustments, such as recorded here, are instrumental in determining the nature of the behavioral changes which have been recorded after hippocampal lesions. SUMMARY Unilateral lesions were placed in the entorhinal cortex of rats and the hippocampal formation examined histochemically for the localization o f acetylcholinesterase. On the lesioned side, an intense band of acetylcholinesterase developed in the molecular layer of the dentate gyrus exactly where the entorhinal fibers normally terminate. This layer develops over a period of 30-40 days and is eliminated by septal lesions, which cause the disappearance of the normal bands ofacetylcholinesterase. The suggestion is made that the sprouting of acetylcholinesterase containing septal terminals occurs under these conditions. ACKNOWLEDGEMENTS This research was supported in part by Grant GB 16973 to G. L. from National Science Foundation, and by Grant M H 19691 to C. C. from the National Institute of Health.
REFERENCES 1 BAYLISS,B. J., ANDTODRICK,A., The use of a selectiveacetylcholinesterase inhibitor in the estimation of pseudocholinesterase activity in rat brain, Biochem. J., 62 (1956) 62-67.
2 BERNSTEIN,J. J., ANDBERNSTEIN,i . E., Axonal regeneration and formation of synapses proximal to the site of lesion followinghemisection of the rat spinal cord, Exp. Neurol., 30 (1971) 336-351. 3 BLACKSTAO,T. W., On the termination of some afferents to the hippocampus and fascia dentata, Acta anat. (Basel), 35 (1958) 202-214. 4 BLACKSTAD,T. W., Commissural connections of the hippocampal region in the rat, with special reference to their mode of termination, J. comp. Neurol., 105 (1956) 417-537. Brain Research, 42 (1972) 311-318
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5 FONNUM, F., Topographical and subcellular localization of choline acetyltransferase in rat hippocampal region, J. Neurochem., 17 (1970) 1029-1037. 6 GOODMAN,D. C., AND HOREL,J. A., Sprouting of optic tract projections in the brain stem of the rat, J. comp. Neurol., 127 (1966) 71-88. 7 KOELLE,G. B., AND FRIEDENWALD,J. S., A histochemical method for localizing cholinesterase activity, Proc. Soc. exp. Biol. ( N. Y.), 70 (1949) 617-622. 8 LAATSCH,R. H., ANDCOWAN,W. M., Electron microscopic studies of the dentate gyrus of the rat, J. comp. Neurol., 128 (1966) 359-396. 9 LANDMESSER,L., Properties of the frog sartorius muscle re-innervated by pre-ganglionic autonomic nerve fibers, UCLA, Ph.D. Thesis (1969). 10 LEW~S,P. R., SHUTE,C. C. D., AND SILVER,A., Confirmation from choline acetylase analyses of a massive cholinergic innervation to the rat hippocampus, J. Physiol. (Lond.), 191 (1967) 215-224. 11 LIu, C. W., AND CHAMRERS,W. W., Intraspinal sprouting of dorsal root axons, Arch. Neurol. Psychiat. (Chic.), 79 (1958) 46-61. 12 LYNCH,G., AND MOSKO,S., Septo-hippocampal projections studied by two techniques, in preparation. 13 MOORE,R. Y., BJ6RKLUND,A., AND STENEVt,U., Plastic changes in the adrenergic innervation of the rat septal area in response to denervation, Brain Research, 33 (1971) 13-35. 14 NAFSTAD,P. H. J., An electron microscope study of the termination of the perforant path fibers in the hippocampus and the fascia dentata, Z. Zellforsch., 76 (1967) 532-542. 15 RAISMAN,G., Neural plasticity in the septal nuclei of the adult rat, Brain Research, 14 (1969) 25-48. 16 RAISMAN,G., COWAN, W. M., AND POWELL, T. P. S., The extrinsic afferent, commissural and association fibres of the hippocampus, Brain, 88 (1965) 963-996. 17 ROSENZWEIG,M. R., BENNETT,E. L., DIAMOND,M. C., Wu, S., SLAGLE,R. W., AND SAFFRAN,E., Influences on environmental complexity and visual stimulation on development of occipital cortex in rat, Brain Research, 14 (1969) 427-445. 18 SHUTE,C. C. D., AND LEWIS,P. R., The use of cholinesterase techniques combined with operative procedures to follow nervous pathways in the brain, Bibl. anat. (Basel), 2 (1961) 34-49. 19 SHUTE, C. C. D., AND LEWIS, P. R., Electron microscopy of cholinergic terminals and acetylcholinesterase-containing neurones in the hippocampal formation of the rat, Z. Zellforsch., 69 (1966) 334-343. 20 STORM-MATHISEN,J., Quantitative histochemistry of acetylcholinesterase in rat hippocampal region correlated to histochemical staining, J. Neurochem., 17 (1970) 739-750. 21 STORM-MATHISEN, J., AND BLACKSTAD, T. W., Cholinesterase in the hippocampal region, Acta anat. (Basel), 56 0964) 216-253. 22 YANAGIHARA,T., AND HYDEN, H., Protein synthesis in various regions of rat hippocampus during learning, Exp. Neurol., 31 (1971) 151-162.
Brain Research, 42 (1972) 311-318
311
INDUCED ACETYLCHOLINESTERASE-RICH LAYER IN RAT DENTATE GYRUS FOLLOWING ENTORHINAL LESIONS
GARY LYNCH, DEE ANN MATTHEWS, SARAH MOSKO, THOMAS PARKS AND CARL COTMAN
Department of Psychobiology, University of California, Irvine, Calif. 92664 (U.S.A.) (Accepted January 20th, 1972)
INTRODUCTION
In recent years, it has become increasingly clear that the adult central nervous system has the capacity to undergo structural changes in response to mild or drastic influences~,e,ll,ls,15,17,2L The manner in which the central nervous system responds to such situations is important, not only in determining the nature of processes associated with the learning and storage of information, but also in establishing functional adjustments associated with discrete brain lesions. Following brain lesions, remarkable changes seem to occur in intact nerve fibers adjacent to degenerating terminals. These changes appear to play a role in the reorganization of the brain. For example, several studies using Nauta stain techniques have shown changes in the distribution and quantity of degenerating terminals within a structure, if at some earlier time other afferent systems had been removede, 11. More recent degeneration studies, using the electron microscope, have also suggested the possibility of 'sprouting' by remaining intact axons following lesions2,1L While these studies have provided initial insights into the adjustments which occur after damage and the capacity of brain to undergo changes, the existing approaches do not readily lend themselves to an analysis of the time course of the changes, the specificity of the change to particular cellular populations, or the biochemical nature of the associated processes. Moreover, present approaches have not permitted the testing of the functional significance of these adjustments. An ideal system should be a relatively ordered projection of nerve fibers with a well defined structural organization and known electrophysiology so that functional changes can be assessed. Likewise, the transmitter of various neuronal elements should be known. The hippocampal formation, while not meeting all requirements, has some important advantages. Its structure is relatively simple and has been extensively studied3,4,s,14,16,19,21; electrophysiological knowledge is detailed; and the transmitter of the septal pathway, one of the major inputs, is believed to be acetylcholine 1°. In view of the advantages afforded by the hippocampus, we are currently engaged in a project studying the morphological, biochemical, and electrophysiological responses of the hippocampus Brain Research, 42 (1972) 311-318
312
G. LYNCHet a/.
to removal of its various extrinsic afferents. In this paper, we report adjustments in the dentate gyrus of the rat following discrete brain lesions. Specifically, we demonstrate histochemically the induction of an intense layer of acetylcholinesterase activity (ACHE) in the dentate gyrus after lesions of the entorhinal cortex. The highly ordered organization of the dentate gyrus of the hippocampus is particularly appropriate for studying alterations in adult brain following brain damage. It is a rigidly layered structure, whose major extrinsic inputs arise in the septum and the entorhinal and subicular cortices a,14,16. The cortical fibers project topographically to the outer two-thirds of the apical dendrites of the dentate granule cells, while the septo-hippocampal pathway seems to terminate basal to the granule cells and on the inner one-third of the apical dendrites. Electron microscopic evidencO 9 and subcellular fractionation studies 5 indicate that the septal terminals and fibers contain high concentrations of ACHE. Removing septal input causes the disappearance of essentially all AChE activity, suggesting that septal fibers and terminals are the predominant locus of hippocampal AChE 10. Other studies, using light microscope histochemistry, show that AChE is discretely layered in relation to the granule cells 1s,~1. Two distinct bands are seen: one just below the granular cell bodies in the hilus of the dentate gyrus and another immediately above them, occupying approximately the inner one-fourth of the molecular layer. The supragranular zone is sandwiched between the granular cells and the zone of termination of the commissural afferents4. The latter zone and the AChE zone occupy together roughly the inner one-third of the molecular layer. Microchemical analyses have confirmed the general organizational features of the cholinergic system seen histochemically within the hippocampal formationS, ~0. Our own preliminary studies, using a modified Fink-Heimer technique, confirm that the distribution of AChE coincides with the degeneration pattern following a septal lesionlL The uniqueness of AChE in septal terminals provides a tag to examine the reaction of septal fibers and terminals to brain damage. We hypothesized that if the entorhinal cortex was lesioned, causing entorhinal afferents to degenerate, septal afferents might spread to open sites in the apical dendrites previously occupied by entorhinal terminals. If this is the case, it should be possible to very accurately resolve changes in the locus and density of the septal input by changes in the AChE staining pattern within the dentate gyrus. Accordingly, intense staining might appear in the outer two-thirds of the molecular layer, the normal locus of the entorhinal terminals. METHODS
Adult Sprague-Dawley rats of both sexes were used in these experiments. Unilateral electrolytic lesions of the entorhinal cortex or septum were performed with the aid of a stereotaxic apparatus, using a monopolar stainless steel electrode. Lesion placement was verified on sections stained with cresyl violet. At various times, as indicated for the particular experiment, the rats were anesthetized and perfused via the heart with 10~o formalin. The brains were postfixed in the cold ( + 4 °C) for 2 h, frozen sections were cut at 50/~m, and the sections then processed for staining Brain Research,
42 (1972) 311-318
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Fig. 1. ChE stain in rat hippocampus 40 days after unilateral entorhinal lesion. On the lesioned (L) side, an intense band of ChE is evident in the dentate gyrus (arrows) which is not present on the control side (C).
of cholinesterases by a modification of the Koelle procedure 7, as described by Landmesser 9. The incubation medium consisted of 52 m M sodium acetate buffer pH 5.3, 20 m M glycine, 6.5 m M copper sulfate and 5 m M acetylthiocholine. Incubations were carried out for 1.5-2.5 h at room temperature, at which time they were developed in a 1 ~ ammonium sulfide solution. Some sections were counterstained with cresyl violet. Except for the initial experiments, promethazine at 5 • 10-5 M was used to suppress non-specific cholinesterases. RESULTS After a unilateral lesion in the entorhinal cortex, an intense band of cholinesterase (ChE) appears in the outer parts of the molecular layer of the dentate gyrus, an area which normally gives a very slight reaction (Fig. 1). The increased staining intensity is restricted to the outer two-thirds of the molecular layer. The pattern on the control side agrees in all respects with earlier reports on ChE distribution in the hippocampus. On the lesioned side, there is a clearly visible intense band in the distal molecular layer of the dentate gyrus in addition to the normal intensity staining adjacent to the granular layer. Fig. 2C shows the two sides of the dentate gyrus at a higher magnification and emphasizes that this newly developed band is situated in the region of the molecular layer normally occupied by the entorhinal fiber terminals. Brain Research, 42 (1972) 311-318
314
G. L Y N C H
et at.
Fig. 2. Dentate gyrus stained for AChE at various times following entorhinal lesions. Control sides are on the right. Small arrows denote the new band. A, 15 days after entorhinal lesion. B, 30 days postoperative. C, 40 days postoperative. In C promethazine was eliminated. Symbols: M, molecular layer; C, zone of commissural afferents in inner one-third of M; S, normal, supragranular band of AChE staining; G, granule cell bodies; large arrows, obliterated hippocampus fissure.
Brain Research, 42 (1972) 311-318
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While some controversy exists regarding a restricted termination of fibers from entorhinal cortex with the molecular layera, 14,16, our studies using anterograde degeneration methods have shown that entorhinal lesions of the type used in the study produce terminal degeneration from the commissural layer to the hippocampal fissure. Similar histochemical changes were obtained in every animal (n : 20) in which histologically verified entorhinal lesions were made. By using various well-documented inhibitors of ChE, we determined that the cholinesterase staining which develops in molecular layer represents acetylcholinesterase activity. The cholinesterase staining is blocked by a highly specific inhibitor of acetylcholinesterase BW284C51 (ref. 1) (2 • l0 -6 M), but is not blocked by inhibitors of non-specific cholinesterases. Neither promethazine at 5. l0 -~ M, which inhibits 90% of non-specific ChE in rat brain or iso-OMPA (5 • 10-4 M) 1 had any noticeable effect on the band. When butyrylthiocholine is used as substrate in place of acetylthiocholine, the induced band is not seen. In order to determine the time course for the development of the band of AChE, histochemical analysis for AChE was carried out at 5, 15, 30, 40 and 60 days after the entorhinal lesions in adult rats. At 5 clays postoperative, there is no evidence of the band. At 15 days, AChE staining is just detectably enhanced. At 30 days there is a strong staining in the outer parts of the molecular layer, and no further change is noticed after 40 or 60 days. Fig. 2 shows the same region of the molecular layer at various times alter the lesion. These findings show that removal of the entorhinal
Fig. 3. Absence of AChE staining in hippocampus following a septal lesion 30 days after an entorhinal cortex lesion. Staining was done 5 days after the septal lesion.
Brain Research, 42 (1972) 311-318
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input to the hippocampus in adult rats results in the progressive development of a layer of AChE in the dentate gyrus. Our AChE histochemical results obtained after unilateral entorhinal lesions suggest an alteration in the composition or localization of septal terminals and fibers. In order to demonstrate the hypothesized dependence of the induced AChE band on the septum, an electrolytic lesion was made in the medial septal nucleus 30 days after the initial entorhinal lesion. Five days after this secondary lesion, the rats were sacrificed. Essentially all AChE in the hippocampal formation disappeared; the septal lesion eliminated both the normally present AChE bands and the newly developed one as shown in Fig. 3. The disappearance of hippocampal AChE 5 days after septal lesions is in agreement with previous studies on normal animals18,el. Our results conclusively and dramatically demonstrate that, like the normal bands, the induced band of AChE depends on the integrity of septo-hippocampal fibers. The possibility that the increase in AChE could be due to a degeneration process can be ruled out for the following reasons: (1) The band does not appear to be associated with vascular or glial elements present in the region. It is unaffected by inhibitors of non-specific cholinesterases which normally block glial and vascular cholinesterase. (2) The band takes approximately 15-30 days to develop, which is longer than expected for most degeneration processes. (3) A primary or secondary lesion in the septum, the origin of the supposed cholinergic afferents to the dentate gyrus, also results in the disappearance of the band of increased AChE seen following entorhinal lesions.
DISCUSSION
Several mechanisms can be proposed which might account for the observed increase in AChE staining. These include sprouting of axonal elements, with the possible formation of new functional synapses, selective enzyme induction in presynaptic terminals and the development of intense AChE activity on the granule cell dendrites. We feel the most plausible explanation of our results is that the loss of entorhinal endings triggers a massive growth of AChE containing septalfibers and terminals. Alternate explanations cannot be excluded, such as the induction of enzyme in intrinsic hippocampal elements. Whatever the structural locus of ACHE, its activity is completely dependent on the integrity of the septo-hippocampal fibers. Further work will be required to determine if these changes involve the formation of new synapses, but this study provides strong evidence that neural alterations can be induced in adult brain. The induction of a rich AChE band in the dentate gyrus illustrates a remarkable response of the adult central neurons to undergo specific changes. The stimulus for AChE change may be the opening of synaptie sites vacated by terminals from area entorhinalis. Once entorhinal terminals vacate, septal terminals sprout. Yet if the increase in AChE activity in fascia dentata is due to destruction of the afferents from area entorhinalis, one would also expect changes in the hippoBrain Research, 42 (1972) 311-318
LESION INDUCED ACETYLCHOLINESTERASE
317
campus which receives entorhinal afferents as well. At present, we cannot rule out some intensification of AChE in regio superior, but certainly the AChE zone in this region does not spread and is not dramatically more intense. Currently we are exploring various possibilities, but as yet we do not have a unique explanation for lack of marked AChE intensification in regio superior. It is not known whether changes after the lesion are of functional significance, but the hippocampal system is particularly favorable for answering this question and for further studying the response of the central nervous system to injuries. The change observed after entorhinal lesions is unilateral and confined to a discrete layer. This system should prove favorable for electrophysiological as well as anatomical and biochemical studies on the exact processes involved in the undergoing change. Our results have potentially profound implications for studies using lesions to examine behavior. They raise the question of whether the consequence of a lesion is not only to remove tissue elements, but also to catalyze a reorganization of other elements in the neuronal network. It is possible that post-lesion adjustments, such as recorded here, are instrumental in determining the nature of the behavioral changes which have been recorded after hippocampal lesions. SUMMARY Unilateral lesions were placed in the entorhinal cortex of rats and the hippocampal formation examined histochemically for the localization o f acetylcholinesterase. On the lesioned side, an intense band of acetylcholinesterase developed in the molecular layer of the dentate gyrus exactly where the entorhinal fibers normally terminate. This layer develops over a period of 30-40 days and is eliminated by septal lesions, which cause the disappearance of the normal bands ofacetylcholinesterase. The suggestion is made that the sprouting of acetylcholinesterase containing septal terminals occurs under these conditions. ACKNOWLEDGEMENTS This research was supported in part by Grant GB 16973 to G. L. from National Science Foundation, and by Grant M H 19691 to C. C. from the National Institute of Health.
REFERENCES 1 BAYLISS,B. J., ANDTODRICK,A., The use of a selectiveacetylcholinesterase inhibitor in the estimation of pseudocholinesterase activity in rat brain, Biochem. J., 62 (1956) 62-67.
2 BERNSTEIN,J. J., ANDBERNSTEIN,i . E., Axonal regeneration and formation of synapses proximal to the site of lesion followinghemisection of the rat spinal cord, Exp. Neurol., 30 (1971) 336-351. 3 BLACKSTAO,T. W., On the termination of some afferents to the hippocampus and fascia dentata, Acta anat. (Basel), 35 (1958) 202-214. 4 BLACKSTAD,T. W., Commissural connections of the hippocampal region in the rat, with special reference to their mode of termination, J. comp. Neurol., 105 (1956) 417-537. Brain Research, 42 (1972) 311-318
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