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Early septal lesion: Effect on the development of the cholinergic system in rat hippocampus
Brain Research, 185 (1980) 323-334 © Elsevier/North-HollandBiomedicalPress
323
EARLY SEPTAL LESION: EFFECT ON THE DEVELOPMENT OF THE CHOLINERGIC SYSTEM IN RAT HIPPOCAMPUS
JACOB BEN-BARAKand YADIN DUDAI* Department of Neurobiology, The Weizmann Institute of Science, Rehovot (lsrael)
(Accepted July 26th, 1979)
Key words: deafferentation-- hippocampus -- muscarinic receptors -- acetylcholinesterase
SUMMARY The development of the cholinergic system in the rat hippocampal formation was studied following lesion of the septal region at an age of 2-4 days postnatal (i.e. the lesion was performed prior to the establishment of the septohippocampal connections). The steep increase in acetylcholinesterase (ACHE) level, that under normal conditions takes place during the second and the third week postnatal, was not observed in early lesioned animals, and AChE level at maturity was about 30 ~ of control. AChE level of adult-lesioned animals was about 15 ~o of control, suggesting an age-dependent plasticity in response to the lesion. Early deafferentation did not seem to alter the pattern of development of muscarinic binding sites as measured by specific binding of [3H]quinuclidinyl benzilate ([3H]QNB). Total [aH]QNB bound per hippocampus of adult, early-lesioned animals was about 70~ of control, but this reduction could be accounted for by the atrophy observed in the hippocampal formation following early lesion. Binding of [aH]QNB per protein in early lesioned animals did not differ from normal. Thus the development and the level of muscarinic binding sites in the hippocampal formation do not seem to depend upon normal establishment of presynaptic contacts.
INTRODUCTION Deafferentation studies may shed light on the possible role ofpresynaptic signals in modulating the development and maintenance of postsynaptic elements, e.g. receptors for neurotransmitters. Denervation of vertebrate skeletal muscles leads to supersensitivity and redistribution of nicotinic cholinergic receptors3,1L Chemical destruction of central noradrenergic terminals led to an increase in density of fl* To whomcorrespondenceshould be addressed.
324 adrenergic receptors in rat cerebral cortex 30 and chemical lesions in the rat nigrostriatal system led to an increase of dopamine receptors 9 and of GABA receptors ~'~. Denervation did not affect muscarinic receptor level in rat sympathetic ganglia 7, and septal and fornix lesions in the adult rat did not seem to increase the level of cholinergic receptors in the hippocampal formation10, 3~. The effect of presynaptic lesions on the level of postsynaptic receptors seems thus to depend on the system studied. In the studies mentioned above, deafferentation was performed in a mature nervous system after the contacts between presynaptic and postsynaptic elements have been established. We set out to investigate the effect of destruction of incoming cholinergic axons in the immature brain, before they establish synaptic contacts with their target cells, on the subsequent development of postsynaptic cholinergic receptors. The rat hippocampal formation appears suitable for such a study, since it receives most of its cholinergic innervation via a single extrinsic afferent, the septohippocampal tract 19,2°,a2,3a and deafferentation is thus feasible. Indeed destruction of this tract in the adult deprives the hippocampal formation of about 80-90 ~ of its choline acetyltransferase (CAT) and acetylcholinesterase (ACHE) activitiesl°,la,2°, a2,35 (the latter enzyme is considered mainly of a presynaptic origin in the hippocampusa2). Septohippocampal axons start to invade the hippocampus at about 4 days postnatal, and proceed toward the temporal end of the region, until, about 7 days later, all parts of the hippocampal formation are innervated by cholinergic afferents 23. Major synaptogenetic events take place in the hippocampal formation between 11 and 25 days after birth 28. Lesions of the septum at the first days after birth are thus expected to prevent the formation of most of the cholinergic synapses in the hippocampal formation. In a previous study ~ we have reported that the rate of development ofmuscarinic receptors in the hippocampal formation, as revealed by specific binding of [aH]quinuclidinyl benzilate ([3H]QNB), is similar to the rate of development of the hippocampal presynaptic marker, acetylcholinesterase, and both processes may reflect the functional maturation of cholinergic synapses. It is thus pertinent to ask whether early septal lesions will affect the development and maintenance of the postsynaptic muscarinic receptors. The effect of such lesions on the hippocampal cholinergic system are described in the present study. MATERIALS AND METHODS
Animals. Wistar rats (from the Weizmann Institute Animal Breeding Center) were used. Septal lesions in 2--4-day-old rats were performed by inserting a fine tipped needle vertically through the bregma point on the skull at a depth of 2-3.5 mm and rotating it 1 mm in each direction. Histological examination revealed that such a procedure completely destroys the septum. Only animals in which post-mortem examination revealed complete loss of the septum but no apparent damage to the dorsal hippocampus were used. Septal lesions in adult rats were performed as described by Dudai and SegaP °.
325
Fig. 1. A: dissected hippocaxnpal formation of an adult, early septal-lesioned (sl) and of an adult control (c) rat. x 3.6. B and C: coronal sections ofhippocampal formations of a control (B) and an early septallesioned (C) adult rat, stained for AChE activity, x 37. D and E: coronal sections of hippocampal formations of a control (D) and an early septal-lesioned (E) adult rat, stained with haematoxylin-eosin. v, ventricle; f, fimbria. × 43. In each case the lesion was placed at day 4 postnatal.
326 At the appropriate age, rats were sacrificed, the brain immediately removed and the hippocampal formation (including the hippocampus, the dentate gyrus and the subiculum) dissected on ice. Tissue was homogenized (100 mg/ml) in ice-cold 0.32 M sucrose in a glass-Teflon homogenizer driven by a Heidolph motor at half-maximal speed. Assays. Muscarinic receptor binding level was determined by reacting aliquots of homogenates with the powerful muscarinic antagonist, [~H]quinuclidinyl benzilate ([ZH]QNB), as described by Ben-Barak and Dudai 2. For determination of [3H]QNB binding levels, incubation was carried out with 5 nM [3H]QNB. Specific [3H]QNBbinding was defined as total binding minus the binding in the presence of 10 -5 M atropine. Acetylcholinesterase activity was determined as described by Johnson and Russell 16, employing [3H]acetylcholine (ACh, 3.3 mM) as a substrate. Histochemical staining of cryostat-cut sections for AChE activity was performed as described by Mellgren and Srebro 24. Choline acetyltransferase activity was determined as described by Fonnum 13, employing [3H]acetyl CoA as a substrate. Protein was determined according to Lowry et al. 21, using BSA as a standard. Results of various assays are presented as mean ± S.E.M. for at least 3 determinations each. 5-20~o sucrose gradients with a 50~o sucrose cushion were performed as described by Dudai et al. 11. Chemicals. [3H]QNB (29.4 Ci/mmol) and [acetyl-3H]acetyl CoA (2.67 Ci/mmol) were from New England Nuclear (Boston, Mass.). [3H]Acetylcholine chloride (100-500 mCi/mmol) was from the Radiochemical Centre, Amersham. All other chemicals were of analytical grade. RESULTS Morphology o f the hippocampal formation following early septal lesions
Septal lesion at an age of 2-4 days postnatal led to a permanent loss of the fimbria (Fig. 1), and to a significant reduction in size (Fig. l) and weight (Table I, Fig. 2) of the hippocampal formation in the adult. The atrophy could not be accounted for TABLE I Fresh weight and AChE activity of the hippocampal formation of adult rats as afunction of the age at which the septal area was lesioned
Values are mean :L S.E.M. Age of lesion
Weight
AChE activity
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104 ± 98 ± 142 ± 138 ±
3 1 5 3
75 71 103 100
201 141 89 708
± 14 ~(: I I ± 8 ± 22
28 20 13 100
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Fig. 2. Fresh weight of hippocampal formations of early septal-lesioned(0----0) and of control (© (3) rats, as a function of age. The lesion was placed at day 4 postnatal. solely by loss of the fimbria since the weight of the fimbria was found to be only about 3 ~ of total hippocampal formation weight in adult animals. Weight loss was also not observed in cerebellum of operated animals, indicating the specificity of the effect. The protein content per wet weight of hippocampal homogenates of operated animals was (91 -+- 5) ~ of control. The weight reduction observed in adult hippocampi was evident only when the lesion was placed at days 4 or 13 postnatal but no significant weight loss was observed in animals operated at maturity (Table I). Cross-sections of the hippocampal formation of lesioned and control animals revealed a reduction in the width of dendritic layers. The most marked reduction (0.80 zk 0.05 of control) was observed in stratum oriens and stratum radiatum of area CA1, stratum oriens of area CA3 and the molecular layers of the dentate gyrus. We did not detect significant changes in the number of pyramidal and granular cells between operated and control animals.
Cholinergic enzymes activity in the hippocampalformation following early septal lesions Lesions of the septum or the fornix in the adult rat lead to a decrease of about 80-90 ~ in the level of AChE and CAT in the hippocampal formation10, ls,2°,zz,35. We observed a marked decrease in AChE level in the hippocampal formation following an early septal lesion, employing both histochemical (Fig. 1) and biochemical (Fig. 3) techniques. Enzyme activity, as revealed in stained sections, was very much reduced throughout the entire structure. The region most affected was the dentate whereas the highest residual activity was observed in CA3, area 31 and the subiculum. These observations confirmed the histochemical studies of Srebro and Mellgren zl. We have quantified, by biochemical measurements, the decrease in activity in the whole hippocampal formation during development. The effect of the early lesion was already detected 2 days after the operation (Fig. 3). A small increase in AChE level was detected in lesioned animals until the third week after birth, but enzyme activity increased very little, if at all, later on (Fig. 3). In contrast, a sharp increase in AChE
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Fig. 3. Development of acetylcholinesterase activity in the hippocampal formation of early septallesioned (O Q) and control (O O) rats as a function of age. The lesion was performed at day 4 postnatal. A: total activity per region. B: specific activity.
level took place in non-lesioned animals during the third week after birth and total activity continued to increase gradually until an age of about 7 weeks (Fig. 3, ref. 2). As seen in Table I, residual AChE activity in the adult was dependent upon the age at which the lesion was performed, with the lowest residual activity being found in adultoperated animals. We have examined some of the properties of the residual AChE activity observed in early-lesioned animals. Incubation with 10-6 M BW284C51 inhibited (90 ± 2) ~ of the residual activity vs (97 4- 2) ~o inhibition in control animals. Under these conditions BW284C51 is not expected to inhibit pseudocholinesterase 1. The residual AChE activity in early-lesioned animals was composed, as the AChE of non-lesioned animals, of two molecular forms, with apparent sedimentation coefficients of about 4S and 10S (Fig. 4). As was reported for the whole rat brain 2s, the 4S form was found to be the major form in the immature hippocampus, while the proportion of the 10S form increased during maturation (Fig. 4). However, although early septal lesion did not abolish either of these forms, the 10S form seemed to be more affected (Fig. 4), and the
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F i g 4. Sucrose gradient centrifugation ofaliquots of hippocampal formation homogenates from lesioned and non-lesioned animals. A: non-lesioned, age 2 days postnatal. B: non-lesioned, age 11 days postnatal. C: non-lesioned, adult. D: adult, following a septal-lesion at day 4 postnatal. The gradient was of 5-20% sucrose in 0.12 M NaCI, 0.1 )g Triton X-100, 0.05 M Tris'HCl, pH 7.4, and was layered on top of a 50 70 sucrose cushion in the same buffer. The arrows indicate the position of catalase (11.4S) which was run as a marker. The top of the gradient is on the right hand side.
ratio 10S:4S at maturity following an early lesion was 4:1 vs 9:1 in non-lesioned animals. CAT activity fell drastically following an early septal lesion, similar to ACHE. Thus activity in homogenates of lesioned animals was found to be 2.4 4- 0.3 #mol ACh synthesized/h/g tissue vs 5.6 4- 0.1 in a control, i.e. a reduction of about 60%. Again the fraction of activity observed following early lesion is higher than that previously reported for adult-lesioned animals TM.
Development of muscarinic receptors following early septal lesions Muscarinic receptol" level, as determined by specific binding of the powerful muscarinic antagonist, pH]QNB, increased in a similar developmental pattern both in control and in early-lesioned animals (Fig. 5). A sharp increase in total binding took place during the first 3 weeks after birth, a period which is characterized by massive synaptogenesis in normal animalsS, 26. Specific activity of pH]QNB-binding sites at maturity was essentially the same in lesioned and non-lesioned rats (Fig. 5B). Total binding per hippocampus was smaller in operated animals (Fig. 5A), but this reduction could be accounted for by the significant weight reduction and the slight decrease in protein content following early lesions (Table I, Fig. 2). No significant difference was detected in the apparent dissociation constant of QNB-binding sites from lesioned and control animals, as determined by Scatchard analysis of pH]QNB binding isotherms. A single class of binding sites was detected in both cases, and a K~:(0.20 :k 0.06) × 10-9 M was determined for early-lesioned animals vs (0.24 q0.08) x 10-9 M for control animals. We also did not detect a significant difference in the affinity of the muscarinic agonist, oxotremorine, for the receptor as determined by displacement of pH]QNB-binding at equilibrium. Thus the concentration of oxo-
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Fig. 5. Development of specific[aH]QNBbinding in the hippocampal formation of early septal-lesioned (0 O) and control (O ©) rats as a function of age. A: total activity per region. B: specific activity. The lesion was performed at day 4 postnatal. tremorine that displaced 50% of bound [3H]QNB (I50 value, in the presence of 5 nM [aH]QNB) was found to be (3.1 :]: 0.4)× 10-5 M in homogenates of early-lesioned animals vs (3.6 ± 0.2) × 10-5 M in homogenates of control animals. The Hill coefficient for oxotremorine binding was calculated to be 1.02 ~ 0.05 for early-lesioned animals vs 0.88 -~ 0.05 for controls (see Discussion). DISCUSSION The primary objective of our work was to study the development of postsynaptic muscarinic receptors in the rat hippocampal formation following early cholinergic deafferentation, and thus to determine whether normal establishment of presynaptic contacts is required for the development and maintenance of these receptors. Destruction of the septohippocampal tract in the adult deprives the hippocampal formation of 80-90% of CAT and AChE activities, both of which are considered indicators of presynaptic innervation in this systeml0,is,~0, 32,35. Our data show that destruction of the septum at the first postnatal days also causes a massive and permanent decrease in the level of hippocampal AChE and CAT. However, the response of the immature hippocampal formation to deafferentation differed in several aspects from the response of the mature hippocampus. Our data show that lesions of the septum at an age of 2-4 days postnatal, before its growing axons invade the hippocampal formation, or at day 13 postnatal, before most synapses are established, caused a significant weight loss of the region. The reduction in weight was accompanied by a reduction in the width of dendritic layers. Atrophy of a target organ due to deafferentation was observed in several studies of
331 brain and spinal nuclei (reviewed by Smith29). In addition, it was reported that early deafferentiated neurons may develop immature dendritic arborization2L Shrinkage of dendritic layers was also observed in the dentate by Zimmer et al. a~ following early entorhinal lesions. It is plausible to suggest that in the hippocampus, as in some other cases 29, it is the formation of synaptic contacts at an early age, rather than their maintenance, that is a major factor in maturation of dendritic morphology, since adult lesions did not seem to cause a significant atrophy. However, such an interpretation should be regarded with caution since the neonatal lesions, being coarser than the septal lesions performed at maturity, may have caused a greater interference with hippocampal afferents and efferents (for possible pathways see ref. 27). This possibility was not further investigated in the present study. The response of the hippocampal cholinergic system to a septal lesion, as measured by AChE level, seems to display features of age-dependent plasticity. Early lesions were followed by a smaller decrease in AChE level than later lesions. Our data are compatible with the histochemical observations of Srebro and Mellgrenal. Our biochemical studies show that the steep increase in AChE level, that under normal conditions takes place during the second and the third week postnatal TM, does not occur in early-lesioned animals. This developmental phase is characterized in normal animals by a marked increase in the level of the 10S AChE form. The 10S ACHE, which has been previously reported to be predominant in mature brain and to be more intimately associated with membranes than the 'immature', 4S form 2s, is also the major form in adult animals following an early lesion. However, the ratio 10S:4S in early-lesioned animals was smaller than in adult controls, and similar to that observed in 2-week-old animals. It thus again seems that the major effects of an early lesion become apparent especially at about 2-3 weeks after birth, when major synaptogenetic events occur in the normal hippocampal formation8,26. The residual AChE activity observed in the hippocampus following adult 1°,18, 24,3z,a5 or early septal lesions may be either of a cingulate origin, or of an endogenous origin, or both z4. The larger residual activity following an early lesion may represent sprouting of such non-septal fibers in the vacancies formed due to the lesion, as suggested by Mellgren and Srebro z4 and similar to the increase of cholinergic sprouting observed following entorhinal lesion2L The residual activity of cholinergic enzymes reflects the fact that a septal lesion does not completely deplete hippocampal cells of cholinergic innervation. This fact should be borne in mind while evaluating the effects of septal lesions on postsynaptic receptors. Nevertheless, even following an early lesion, the hippocampus becomes devoid of about 70 % of its cholinergic input, as estimated by AChE (and CAT) activities. Preventing such a massive cholinergic input does not seem to significantly affect the development and level of postsynaptic muscarinic receptors, as measured by specific binding of [aH]QNB. The difference in absolute QNB-binding per total hippocampus between early-lesioned and control animals could be accounted for by the lesion-induced alterations in weight and protein content, and specific activity of QNB-binding sites remains essentially unaltered after early lesion at all developmental phases. Harden et al. 14 reported that the time course of development of
332 postsynaptic fl-adrenergic receptors in rat cerebral cortex is also not regulated by presynaptic terminals. However, destruction of the latter did result in an increase in the number of fl-adrenergic receptors and in supersensitivity to catecholamines, in contrast with the lack of receptor-mediated supersensitivity in the hippocampal muscarinic system (see also refs. 4, 10 and 35). In our study we have determined the level of muscarinic receptors by measuring the binding of the powerful muscarinic antagonist, [aH]QNB. It has been reported that muscarinic receptors which display the same affinity for potent antagonists may in fact differ in their affinity for agonists5. The potency of agonists in displacing antagonists and the ratio of various classes of agonist-binding sites differ in various brain regions 5,6,17. Most of the muscarinic binding sites in the mature rat hippocampus were reported to be of a single type with a relatively low affinity for agonists6. Our results indicate no significant change in the 150 value of the agonist, oxotremorine, following early septal lesion. However, although the difference between the Hill coefficients was also not statistically significant (P > 0.05, two-sided t-test), we cannot as yet exclude the possibility of relatively small alterations in the proportions of different types of agonist-binding sites or in the mode of interaction of agonists with the receptor. Also, one cannot exclude the possibility that following early deafferentation, some changes occur in receptor distribution on cells or in some postreceptor events (e.g. cyclase activity15) which were not measured in our study. The observation by Bird and Aghajanian4 that following adult lesion pyramidal neurons do not display receptor-mediated hypersensitivity, as measured by the response to iontophoretically applied cholinergic ligands, renders the latter possibility not very likely. It is also not very likely that an increase in postsynaptic muscarinic receptors is masked by a concomitant decrease in presynaptic receptors, since possible binding of [3H]QNB to putative presynaptic receptors under the conditions employed is expected to be small33. Our data are compatible with lack of muscarinic supersensitivity following deafferentation of sympathetic ganglia7. In this respect muscarinic receptors studied to date seem to differ from peripheral nicotinic receptors 3,12 and from central dopaminergic 9, adrenergic14,3° and GABAergic3~ receptors. In conclusion, although under normal conditions development of postsynaptic muscarinic binding-sites in the hippocampal formation appears to proceed in parallel with maturation of synapses in the region 2, development of the postsynaptic receptor sites, as revealed by [aH]QNB-binding, does not seem to depend upon normal establishment of presynaptic contacts. ACKNOWLEDGEMENTS We thank Dr. M. Segal for valuable comments. Y.D. is incumbent of the Barecha Foundation Career Development Chair.
333 REFERENCES 1 Bayliss, B. J. and Todrick, A., The use of a selective acetylcholinesterase inhibitor in the estimation of pseudocholinesterase activity in rat brain, Biochem. J., 62 (1956) 62-67. 2 Ben-Barak, J. and Dudai, Y., Cholinergic binding sites in rat hippocampal formation: properties and ontogenesis, Brain Research, 166 (1979) 245-257. 3 Berg, D. K., Kelly, S. R., Sargent, P. B., Williamson,P. and Hall, Z., Binding of a-bungarotoxin to acetylcholine receptors in mammalian muscle, Proc. nat. Acad. Sci. (Wash.), 69 (1972) 147-151. 4 Bird, S. J. and Aghajanian, G. K., Denervation supersensitivity in the cholinergic septo-hippocampal pathway: a microiontophoretic study, Brain Research, 100 (1975) 355-370. 5 Birdsall, N. J. M., Burgen, A. S. V. and Hulme, E. C., The binding of agonists to brain muscarinic receptors, Molec. Pharmacol., 14 (1978) 723-736. 6 Birdsall, N. J. M., Burgen,A. S. V. and Hulme, E. C., Multiple classes ofmuscarinicreceptor binding sites in the brain, Proc. Vllth int. Cong. Pharmacol., Paris, 1978, in press. .~ 7 Burt, D. R., Muscarinic receptor binding in rat sympathetic ganglia is unaffected by denervation, Brain Research, 143 (1978) 573-579. 8 Cotman, C., Taylor, D. and Lynch, G., Ultrastructural changes in synapses in the dentate gyrus of the rat during development, Brain Research, 63 (1973) 205-213. 9 Creese, I., Burr, D. R. and Snyder, S. H., Dopamine receptor binding enhancement accompanies lesion-induced behavioral SUl:ersensitivity,Science, 197 (1977) 596-598. 10 Dudai, Y. and Segal, M., a-Bungarotoxin binding sites in rat hippocampus: localization in postsynaptic cells, Brain Research, 154 (1978) 167-171. 11 Dudai, Y., Silman, I., Kalderon, N. and Blumberg, S., Purification by affinity chromatography of acetylcholinesterase from electric organ tissue of the electric eel subsequent to tryptic treatment, Biochim. biophys. Acta ( Amst.) , 268 (1972) 138-157. 12 Fambrough,D. M.,Acetylcholinereceptors. Revised estimate ofextrajunctionalreceptor densityin denervated rat diaphragm, J. gen. Physiol., 64 (1974) 468-471. 13 Fonnum, F., A rapid radiochemical method for the determination of choline acetyltransferase, J. Neurochem., 24 (1975) 407-409. 14 Harden, T. K., Wolfe, B. B., Sporn, J. R., Poulos, B. K. and Molinoff, P. B., Effects of 6-hydroxydopamine on the development of the beta adrenergic receptor/adenylate cyclase system in rat cerebral cortex, J. Pharmacol. exp. Ther., 203 (1977) 132-143. 15 Heilbronn, E. and Bartfai, T., Muscarinic acetylcholine receptor, Progr. Neurobiol., 10 (1978) (in the press). 16 Johnson, C. D. and Russell, R. L., A rapid, simple radiometric assay for cholinesterase suitable for multiple determinations, Analyt. Biochem., 64 (1975) 229-238. 17 Kloog, Y.,Egozi, Y. and Sokolovsky, M.,Regionalheterogeneityofmuscarinicreceptors ofmouse brain, FEBS Lett., 97 (1979) 265-268. 18 Kuhar, M. J., Sethy, V. H., Roth, R. H. and Aghajanian, G. K., Choline: selective accumulation by central cholinergic neurons, J. Neurochem., 20 (1973) 581-593. 19 Lewis, P. K. and Shute, C. C. D., The cholinergic limbic system: projection of hippocampal formation, medial cortex nuclei of the ascending cholinergic reticular system and the subfornical organ and supraoptic crest, Brain, 90 (1967) 521-539. 20 Lewis, P. K., Shute, C. C. D. and Silver, A., Conformation from choline acetylase analysis of a massive cholinergic innervation to the rat hippocampus, J. Physiol. (Lond.), 191 (1967) 215-224. 21 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 22 Maeda, T., Tohyarna, M. and Shimizu, N., Modification of postnatal development of neocortex in rat brain with experimental deprivation of locus coeruleus, Brain Research, 70 (1974) 515-520. 23 Matthews, D. A., Nadler, J. V., Lynch, G. S. and Cotman, C. W., Development of cholinergic innervation in the hippocampal formation of the rat. I. Histochemical demonstration of acetyicholinesterase activity, Develop. Biol., 36 (1974) 130-141. 24 Mellgren, S. I. and Srebro, B., Changes in acetylcholinesterase and distribution of degenerating fibers in the hippocampal region after septal lesions in the rat, Brain Research, 52 (1973) 19-36. 25 Nadler, J. V., Cotman, C. W. and Lynch, G. S., Altered distribution of choline acetyltransferase and acetylcholinesterase activities in the developing rat dentate gyrus following entorhinal lesion, Brain Research, 63 (1973) 215-230. 26 Nadler, J. V., Matthews, D. A., Cotman, C. W. and Lynch, G. S., Development of cholinergic
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323
EARLY SEPTAL LESION: EFFECT ON THE DEVELOPMENT OF THE CHOLINERGIC SYSTEM IN RAT HIPPOCAMPUS
JACOB BEN-BARAKand YADIN DUDAI* Department of Neurobiology, The Weizmann Institute of Science, Rehovot (lsrael)
(Accepted July 26th, 1979)
Key words: deafferentation-- hippocampus -- muscarinic receptors -- acetylcholinesterase
SUMMARY The development of the cholinergic system in the rat hippocampal formation was studied following lesion of the septal region at an age of 2-4 days postnatal (i.e. the lesion was performed prior to the establishment of the septohippocampal connections). The steep increase in acetylcholinesterase (ACHE) level, that under normal conditions takes place during the second and the third week postnatal, was not observed in early lesioned animals, and AChE level at maturity was about 30 ~ of control. AChE level of adult-lesioned animals was about 15 ~o of control, suggesting an age-dependent plasticity in response to the lesion. Early deafferentation did not seem to alter the pattern of development of muscarinic binding sites as measured by specific binding of [3H]quinuclidinyl benzilate ([3H]QNB). Total [aH]QNB bound per hippocampus of adult, early-lesioned animals was about 70~ of control, but this reduction could be accounted for by the atrophy observed in the hippocampal formation following early lesion. Binding of [aH]QNB per protein in early lesioned animals did not differ from normal. Thus the development and the level of muscarinic binding sites in the hippocampal formation do not seem to depend upon normal establishment of presynaptic contacts.
INTRODUCTION Deafferentation studies may shed light on the possible role ofpresynaptic signals in modulating the development and maintenance of postsynaptic elements, e.g. receptors for neurotransmitters. Denervation of vertebrate skeletal muscles leads to supersensitivity and redistribution of nicotinic cholinergic receptors3,1L Chemical destruction of central noradrenergic terminals led to an increase in density of fl* To whomcorrespondenceshould be addressed.
324 adrenergic receptors in rat cerebral cortex 30 and chemical lesions in the rat nigrostriatal system led to an increase of dopamine receptors 9 and of GABA receptors ~'~. Denervation did not affect muscarinic receptor level in rat sympathetic ganglia 7, and septal and fornix lesions in the adult rat did not seem to increase the level of cholinergic receptors in the hippocampal formation10, 3~. The effect of presynaptic lesions on the level of postsynaptic receptors seems thus to depend on the system studied. In the studies mentioned above, deafferentation was performed in a mature nervous system after the contacts between presynaptic and postsynaptic elements have been established. We set out to investigate the effect of destruction of incoming cholinergic axons in the immature brain, before they establish synaptic contacts with their target cells, on the subsequent development of postsynaptic cholinergic receptors. The rat hippocampal formation appears suitable for such a study, since it receives most of its cholinergic innervation via a single extrinsic afferent, the septohippocampal tract 19,2°,a2,3a and deafferentation is thus feasible. Indeed destruction of this tract in the adult deprives the hippocampal formation of about 80-90 ~ of its choline acetyltransferase (CAT) and acetylcholinesterase (ACHE) activitiesl°,la,2°, a2,35 (the latter enzyme is considered mainly of a presynaptic origin in the hippocampusa2). Septohippocampal axons start to invade the hippocampus at about 4 days postnatal, and proceed toward the temporal end of the region, until, about 7 days later, all parts of the hippocampal formation are innervated by cholinergic afferents 23. Major synaptogenetic events take place in the hippocampal formation between 11 and 25 days after birth 28. Lesions of the septum at the first days after birth are thus expected to prevent the formation of most of the cholinergic synapses in the hippocampal formation. In a previous study ~ we have reported that the rate of development ofmuscarinic receptors in the hippocampal formation, as revealed by specific binding of [aH]quinuclidinyl benzilate ([3H]QNB), is similar to the rate of development of the hippocampal presynaptic marker, acetylcholinesterase, and both processes may reflect the functional maturation of cholinergic synapses. It is thus pertinent to ask whether early septal lesions will affect the development and maintenance of the postsynaptic muscarinic receptors. The effect of such lesions on the hippocampal cholinergic system are described in the present study. MATERIALS AND METHODS
Animals. Wistar rats (from the Weizmann Institute Animal Breeding Center) were used. Septal lesions in 2--4-day-old rats were performed by inserting a fine tipped needle vertically through the bregma point on the skull at a depth of 2-3.5 mm and rotating it 1 mm in each direction. Histological examination revealed that such a procedure completely destroys the septum. Only animals in which post-mortem examination revealed complete loss of the septum but no apparent damage to the dorsal hippocampus were used. Septal lesions in adult rats were performed as described by Dudai and SegaP °.
325
Fig. 1. A: dissected hippocaxnpal formation of an adult, early septal-lesioned (sl) and of an adult control (c) rat. x 3.6. B and C: coronal sections ofhippocampal formations of a control (B) and an early septallesioned (C) adult rat, stained for AChE activity, x 37. D and E: coronal sections of hippocampal formations of a control (D) and an early septal-lesioned (E) adult rat, stained with haematoxylin-eosin. v, ventricle; f, fimbria. × 43. In each case the lesion was placed at day 4 postnatal.
326 At the appropriate age, rats were sacrificed, the brain immediately removed and the hippocampal formation (including the hippocampus, the dentate gyrus and the subiculum) dissected on ice. Tissue was homogenized (100 mg/ml) in ice-cold 0.32 M sucrose in a glass-Teflon homogenizer driven by a Heidolph motor at half-maximal speed. Assays. Muscarinic receptor binding level was determined by reacting aliquots of homogenates with the powerful muscarinic antagonist, [~H]quinuclidinyl benzilate ([ZH]QNB), as described by Ben-Barak and Dudai 2. For determination of [3H]QNB binding levels, incubation was carried out with 5 nM [3H]QNB. Specific [3H]QNBbinding was defined as total binding minus the binding in the presence of 10 -5 M atropine. Acetylcholinesterase activity was determined as described by Johnson and Russell 16, employing [3H]acetylcholine (ACh, 3.3 mM) as a substrate. Histochemical staining of cryostat-cut sections for AChE activity was performed as described by Mellgren and Srebro 24. Choline acetyltransferase activity was determined as described by Fonnum 13, employing [3H]acetyl CoA as a substrate. Protein was determined according to Lowry et al. 21, using BSA as a standard. Results of various assays are presented as mean ± S.E.M. for at least 3 determinations each. 5-20~o sucrose gradients with a 50~o sucrose cushion were performed as described by Dudai et al. 11. Chemicals. [3H]QNB (29.4 Ci/mmol) and [acetyl-3H]acetyl CoA (2.67 Ci/mmol) were from New England Nuclear (Boston, Mass.). [3H]Acetylcholine chloride (100-500 mCi/mmol) was from the Radiochemical Centre, Amersham. All other chemicals were of analytical grade. RESULTS Morphology o f the hippocampal formation following early septal lesions
Septal lesion at an age of 2-4 days postnatal led to a permanent loss of the fimbria (Fig. 1), and to a significant reduction in size (Fig. l) and weight (Table I, Fig. 2) of the hippocampal formation in the adult. The atrophy could not be accounted for TABLE I Fresh weight and AChE activity of the hippocampal formation of adult rats as afunction of the age at which the septal area was lesioned
Values are mean :L S.E.M. Age of lesion
Weight
AChE activity
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201 141 89 708
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28 20 13 100
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Fig. 2. Fresh weight of hippocampal formations of early septal-lesioned(0----0) and of control (© (3) rats, as a function of age. The lesion was placed at day 4 postnatal. solely by loss of the fimbria since the weight of the fimbria was found to be only about 3 ~ of total hippocampal formation weight in adult animals. Weight loss was also not observed in cerebellum of operated animals, indicating the specificity of the effect. The protein content per wet weight of hippocampal homogenates of operated animals was (91 -+- 5) ~ of control. The weight reduction observed in adult hippocampi was evident only when the lesion was placed at days 4 or 13 postnatal but no significant weight loss was observed in animals operated at maturity (Table I). Cross-sections of the hippocampal formation of lesioned and control animals revealed a reduction in the width of dendritic layers. The most marked reduction (0.80 zk 0.05 of control) was observed in stratum oriens and stratum radiatum of area CA1, stratum oriens of area CA3 and the molecular layers of the dentate gyrus. We did not detect significant changes in the number of pyramidal and granular cells between operated and control animals.
Cholinergic enzymes activity in the hippocampalformation following early septal lesions Lesions of the septum or the fornix in the adult rat lead to a decrease of about 80-90 ~ in the level of AChE and CAT in the hippocampal formation10, ls,2°,zz,35. We observed a marked decrease in AChE level in the hippocampal formation following an early septal lesion, employing both histochemical (Fig. 1) and biochemical (Fig. 3) techniques. Enzyme activity, as revealed in stained sections, was very much reduced throughout the entire structure. The region most affected was the dentate whereas the highest residual activity was observed in CA3, area 31 and the subiculum. These observations confirmed the histochemical studies of Srebro and Mellgren zl. We have quantified, by biochemical measurements, the decrease in activity in the whole hippocampal formation during development. The effect of the early lesion was already detected 2 days after the operation (Fig. 3). A small increase in AChE level was detected in lesioned animals until the third week after birth, but enzyme activity increased very little, if at all, later on (Fig. 3). In contrast, a sharp increase in AChE
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level took place in non-lesioned animals during the third week after birth and total activity continued to increase gradually until an age of about 7 weeks (Fig. 3, ref. 2). As seen in Table I, residual AChE activity in the adult was dependent upon the age at which the lesion was performed, with the lowest residual activity being found in adultoperated animals. We have examined some of the properties of the residual AChE activity observed in early-lesioned animals. Incubation with 10-6 M BW284C51 inhibited (90 ± 2) ~ of the residual activity vs (97 4- 2) ~o inhibition in control animals. Under these conditions BW284C51 is not expected to inhibit pseudocholinesterase 1. The residual AChE activity in early-lesioned animals was composed, as the AChE of non-lesioned animals, of two molecular forms, with apparent sedimentation coefficients of about 4S and 10S (Fig. 4). As was reported for the whole rat brain 2s, the 4S form was found to be the major form in the immature hippocampus, while the proportion of the 10S form increased during maturation (Fig. 4). However, although early septal lesion did not abolish either of these forms, the 10S form seemed to be more affected (Fig. 4), and the
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F i g 4. Sucrose gradient centrifugation ofaliquots of hippocampal formation homogenates from lesioned and non-lesioned animals. A: non-lesioned, age 2 days postnatal. B: non-lesioned, age 11 days postnatal. C: non-lesioned, adult. D: adult, following a septal-lesion at day 4 postnatal. The gradient was of 5-20% sucrose in 0.12 M NaCI, 0.1 )g Triton X-100, 0.05 M Tris'HCl, pH 7.4, and was layered on top of a 50 70 sucrose cushion in the same buffer. The arrows indicate the position of catalase (11.4S) which was run as a marker. The top of the gradient is on the right hand side.
ratio 10S:4S at maturity following an early lesion was 4:1 vs 9:1 in non-lesioned animals. CAT activity fell drastically following an early septal lesion, similar to ACHE. Thus activity in homogenates of lesioned animals was found to be 2.4 4- 0.3 #mol ACh synthesized/h/g tissue vs 5.6 4- 0.1 in a control, i.e. a reduction of about 60%. Again the fraction of activity observed following early lesion is higher than that previously reported for adult-lesioned animals TM.
Development of muscarinic receptors following early septal lesions Muscarinic receptol" level, as determined by specific binding of the powerful muscarinic antagonist, pH]QNB, increased in a similar developmental pattern both in control and in early-lesioned animals (Fig. 5). A sharp increase in total binding took place during the first 3 weeks after birth, a period which is characterized by massive synaptogenesis in normal animalsS, 26. Specific activity of pH]QNB-binding sites at maturity was essentially the same in lesioned and non-lesioned rats (Fig. 5B). Total binding per hippocampus was smaller in operated animals (Fig. 5A), but this reduction could be accounted for by the significant weight reduction and the slight decrease in protein content following early lesions (Table I, Fig. 2). No significant difference was detected in the apparent dissociation constant of QNB-binding sites from lesioned and control animals, as determined by Scatchard analysis of pH]QNB binding isotherms. A single class of binding sites was detected in both cases, and a K~:(0.20 :k 0.06) × 10-9 M was determined for early-lesioned animals vs (0.24 q0.08) x 10-9 M for control animals. We also did not detect a significant difference in the affinity of the muscarinic agonist, oxotremorine, for the receptor as determined by displacement of pH]QNB-binding at equilibrium. Thus the concentration of oxo-
330
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Fig. 5. Development of specific[aH]QNBbinding in the hippocampal formation of early septal-lesioned (0 O) and control (O ©) rats as a function of age. A: total activity per region. B: specific activity. The lesion was performed at day 4 postnatal. tremorine that displaced 50% of bound [3H]QNB (I50 value, in the presence of 5 nM [aH]QNB) was found to be (3.1 :]: 0.4)× 10-5 M in homogenates of early-lesioned animals vs (3.6 ± 0.2) × 10-5 M in homogenates of control animals. The Hill coefficient for oxotremorine binding was calculated to be 1.02 ~ 0.05 for early-lesioned animals vs 0.88 -~ 0.05 for controls (see Discussion). DISCUSSION The primary objective of our work was to study the development of postsynaptic muscarinic receptors in the rat hippocampal formation following early cholinergic deafferentation, and thus to determine whether normal establishment of presynaptic contacts is required for the development and maintenance of these receptors. Destruction of the septohippocampal tract in the adult deprives the hippocampal formation of 80-90% of CAT and AChE activities, both of which are considered indicators of presynaptic innervation in this systeml0,is,~0, 32,35. Our data show that destruction of the septum at the first postnatal days also causes a massive and permanent decrease in the level of hippocampal AChE and CAT. However, the response of the immature hippocampal formation to deafferentation differed in several aspects from the response of the mature hippocampus. Our data show that lesions of the septum at an age of 2-4 days postnatal, before its growing axons invade the hippocampal formation, or at day 13 postnatal, before most synapses are established, caused a significant weight loss of the region. The reduction in weight was accompanied by a reduction in the width of dendritic layers. Atrophy of a target organ due to deafferentation was observed in several studies of
331 brain and spinal nuclei (reviewed by Smith29). In addition, it was reported that early deafferentiated neurons may develop immature dendritic arborization2L Shrinkage of dendritic layers was also observed in the dentate by Zimmer et al. a~ following early entorhinal lesions. It is plausible to suggest that in the hippocampus, as in some other cases 29, it is the formation of synaptic contacts at an early age, rather than their maintenance, that is a major factor in maturation of dendritic morphology, since adult lesions did not seem to cause a significant atrophy. However, such an interpretation should be regarded with caution since the neonatal lesions, being coarser than the septal lesions performed at maturity, may have caused a greater interference with hippocampal afferents and efferents (for possible pathways see ref. 27). This possibility was not further investigated in the present study. The response of the hippocampal cholinergic system to a septal lesion, as measured by AChE level, seems to display features of age-dependent plasticity. Early lesions were followed by a smaller decrease in AChE level than later lesions. Our data are compatible with the histochemical observations of Srebro and Mellgrenal. Our biochemical studies show that the steep increase in AChE level, that under normal conditions takes place during the second and the third week postnatal TM, does not occur in early-lesioned animals. This developmental phase is characterized in normal animals by a marked increase in the level of the 10S AChE form. The 10S ACHE, which has been previously reported to be predominant in mature brain and to be more intimately associated with membranes than the 'immature', 4S form 2s, is also the major form in adult animals following an early lesion. However, the ratio 10S:4S in early-lesioned animals was smaller than in adult controls, and similar to that observed in 2-week-old animals. It thus again seems that the major effects of an early lesion become apparent especially at about 2-3 weeks after birth, when major synaptogenetic events occur in the normal hippocampal formation8,26. The residual AChE activity observed in the hippocampus following adult 1°,18, 24,3z,a5 or early septal lesions may be either of a cingulate origin, or of an endogenous origin, or both z4. The larger residual activity following an early lesion may represent sprouting of such non-septal fibers in the vacancies formed due to the lesion, as suggested by Mellgren and Srebro z4 and similar to the increase of cholinergic sprouting observed following entorhinal lesion2L The residual activity of cholinergic enzymes reflects the fact that a septal lesion does not completely deplete hippocampal cells of cholinergic innervation. This fact should be borne in mind while evaluating the effects of septal lesions on postsynaptic receptors. Nevertheless, even following an early lesion, the hippocampus becomes devoid of about 70 % of its cholinergic input, as estimated by AChE (and CAT) activities. Preventing such a massive cholinergic input does not seem to significantly affect the development and level of postsynaptic muscarinic receptors, as measured by specific binding of [aH]QNB. The difference in absolute QNB-binding per total hippocampus between early-lesioned and control animals could be accounted for by the lesion-induced alterations in weight and protein content, and specific activity of QNB-binding sites remains essentially unaltered after early lesion at all developmental phases. Harden et al. 14 reported that the time course of development of
332 postsynaptic fl-adrenergic receptors in rat cerebral cortex is also not regulated by presynaptic terminals. However, destruction of the latter did result in an increase in the number of fl-adrenergic receptors and in supersensitivity to catecholamines, in contrast with the lack of receptor-mediated supersensitivity in the hippocampal muscarinic system (see also refs. 4, 10 and 35). In our study we have determined the level of muscarinic receptors by measuring the binding of the powerful muscarinic antagonist, [aH]QNB. It has been reported that muscarinic receptors which display the same affinity for potent antagonists may in fact differ in their affinity for agonists5. The potency of agonists in displacing antagonists and the ratio of various classes of agonist-binding sites differ in various brain regions 5,6,17. Most of the muscarinic binding sites in the mature rat hippocampus were reported to be of a single type with a relatively low affinity for agonists6. Our results indicate no significant change in the 150 value of the agonist, oxotremorine, following early septal lesion. However, although the difference between the Hill coefficients was also not statistically significant (P > 0.05, two-sided t-test), we cannot as yet exclude the possibility of relatively small alterations in the proportions of different types of agonist-binding sites or in the mode of interaction of agonists with the receptor. Also, one cannot exclude the possibility that following early deafferentation, some changes occur in receptor distribution on cells or in some postreceptor events (e.g. cyclase activity15) which were not measured in our study. The observation by Bird and Aghajanian4 that following adult lesion pyramidal neurons do not display receptor-mediated hypersensitivity, as measured by the response to iontophoretically applied cholinergic ligands, renders the latter possibility not very likely. It is also not very likely that an increase in postsynaptic muscarinic receptors is masked by a concomitant decrease in presynaptic receptors, since possible binding of [3H]QNB to putative presynaptic receptors under the conditions employed is expected to be small33. Our data are compatible with lack of muscarinic supersensitivity following deafferentation of sympathetic ganglia7. In this respect muscarinic receptors studied to date seem to differ from peripheral nicotinic receptors 3,12 and from central dopaminergic 9, adrenergic14,3° and GABAergic3~ receptors. In conclusion, although under normal conditions development of postsynaptic muscarinic binding-sites in the hippocampal formation appears to proceed in parallel with maturation of synapses in the region 2, development of the postsynaptic receptor sites, as revealed by [aH]QNB-binding, does not seem to depend upon normal establishment of presynaptic contacts. ACKNOWLEDGEMENTS We thank Dr. M. Segal for valuable comments. Y.D. is incumbent of the Barecha Foundation Career Development Chair.
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