The development of the cholinergic system in rat hippocampus following postnatal X-irradiation

Brain Research, 226 (1981) 171-186

171

Elsevier/North-Holland Biomedical Press

T H E D E V E L O P M E N T OF T H E C H O L I N E R G I C SYSTEM I N R A T HIPPOCAMPUS FOLLOWING POSTNATAL X-IRRADIATION

JACOB BEN-BARAK Department of Neurobiology, The Weizmann Institute of Science, Rehovot (Israel)

(Accepted April 30th, 1981) Key words: hippocampus - - X-irradiation - - muscarinic receptors - - acetylcholinesterase - - choline

acetyltransferase

SUMMARY Postnatal X-irradiation of the rat hippocampus results in a marked reduction in the number of the postnatally developing granular neurons in the dentate gyrus and also caused a marked increase in the specific activity of acetylcholinesterase (ACHE) and choline acetyltransferase (CAT) and a slight but consistent increase in the activity per whole hippocampus of ACHE. The effect of irradiation on the granular neurons and on the cholinergic enzymes was found to be dose and age dependent. Drastic increase in specific enzymatic activities is also observed in the irradiated cerebellum whose granular neurons differentiate postnatally and to a lesser extent in the cerebral cortex in which cell formation is accomplished prior to birth. Staining for AChE activity ievealed enhanced staining in the molecular layer and the hilar zone of the irradiated dentate gyrus, and in the striatum lucidum of area CA3 which corresponds to the projection area of the mossy fibers. Enhanced staining in area CA1 and subiculum was noticed especially in the supra- and infrapyramidal layers. Biochemical analysis demonstrated that AChE and C A T activities were 140-180 ~o higher in the subareas of the irradiated vs non-irradiated hippocampus. The development and distribution of the postsynaptic muscarinic receptors in the irradiated hippocampus by [aH]quinuclidinyl benzilate (QNB) -binding were also studied. It was found that the elimination of the postnatally formed neurons does not appear to change the developmental pattern of the [aH]QNB-binding sites but reduced receptor level to about 75 ~ of control to adulthood. Measurements of the [aH]QNBbinding in the subareas within the hippocampus revealed marked reduction in the specific [3H]QNB-binding in the molecular layer of the dentate gyrus but not in other subareas. However, the reduction in [aH]QNB-binding sites in the dentate is not as drastic as the reduction in the number of granular neurons. It is suggested that 0006-8993/81/0000-0000/$02.75 © Elsevier/North-Holland Biomedical Press

172 muscarinic sites may be located on early formed neurons, non-cholinergic afferents, or glial elements in this area.

INTRODUCTION The hippocampal formation of the rat contains two major types of neurons which are located in two well-defined zones: the pyramidal cells that reside in areas CAI, CA2 and CA3, and the granular cells which constitute the major neuronal population in the dentate gyrus "~5.The two neuronal types differ also in their dates of origin : pyramidal neurons accomplish their final division prior to birth, whereas about 85 ~ of the granular cells are formed in the first postnatal weeks 4,6,27. The vulnerability of dividing cells to X-irradiation makes it possible to selectively prevent the differentiation of the granular neurons in the dentate gyrus by neonatal X-irradiation, thus sparing the already differentiated pyramidal neurons in the othe~ zones of the hippocampus 4,1°,H. The X-irradiated hippocampus is therefore a suitable preparation for the study of the development of afferents projecting into an area which is devoid of most of its target neurons2~,32,a1,4L In previous publications 13,14 we described the development of the cholinergic, muscarinic and nicotinic binding sites and the cholinergic enzymes acetylcholinesterase (ACHE) and choline acetyltransferase (CAT). These enzymes are derived primarily from the septum 14,3'%34,4s,49 and innervate all parts of the hippocampus 38. Septal neurons accomplish their final mitotic differentiation prior to birth 9 thus rendering the cholinergic input to the hippocampus unperturbed by neonatal irradiation. This study examines changes in quantity and distribution of the cholinergic enzymes and receptors induced by the elimination of the granular neurons in the dentate gyrus. MATERIALS AND METHODS

Animals and irradiation Wistar rats were obtained from the Weizmann Institute Breeding Center. Irradiation was performed by a Picker Vanguard X-ray apparatus at 250 kV at a distance of 50 cm and a dose rate of 50 rads/min (R). The absorbed dose was measured and confirmed by Thermoluminescent Dosimetry (T.L.D.). Rats were divided into 5 groups: Group 1. Newborns irradiated at 200 R on postnatal days 1, 2 and 3, followed by 150 R on days 5, 7, 9, 12 and 15. This dosage is termed 'full dose'. Group 2. Newborns irradiated at the same ages by half the above dose ('half dose'). Group 3. Newborns irradiated at 'full dose' on postnatal days 10, 11, 12, 14, 16, 18, 21 and 24. Group 4. Mature males irradiated at 'full dose' on postnatal days 75-77, 79, 81, 83, 85 and 88 under Nembutal anesthesia (45 mg/kg).

173

Group 5. Control rats of the same litters which were held in the animal center. Irradiation was limited by lead shielding of the right side of the head only. At the appropriate ages rats were sacrificed by cervical dislocation, the brain immediately removed and the hippocampal formation (including the hippocampus, the dentate gyrus and the subicular system) dissected on ice. Tissue was homogenized, unless otherwise specified, in sucrose 0.32 M (1:10 w/v) in a glass-Teflon homogenizer. Slices of the hippocampus were performed by a Mcllwain tissue chopper. Fornix lesions were performed as described by Dudai and Sega123. Enzymes and receptor assays Serial cryostat-cut sections were stained for AChE activity as described by Mellgren and Srebro 40 and stained with cresyl violet for cell counts. Muscarinic receptor binding was determined by reacting aliquots of homogenates with the powerful muscarinic antagonist, [3H]quinuclidinyl benzilate (QNB) as previously published 13. For determination of [aH]QNB-binding levels, incubation was carried out at saturating concentration (5 nM). Specific binding was defined as total binding minus the binding in the presence of 10-5 M atropine. AChE activity was determined as described by Johnson and Russell 28, employing [3H]acetylcholine (ACh, 3.3 nM) as a substrate. Choline acetyltransferase activity was determined as described by Fonnum 24 employing [3H]acetyl-CoA as a substrate at suboptimal substrate concentration of 126 mM. Protein was measured according to Lowry et al. ~6, using BSA as a standard. The two-tailed t-test was used to determine statistical significance. Chemicals [SH]QNB (29.4 Ci/mmol) and [3H-acetyl]CoA (2.67 Ci/mmol) were form New England Nuclear (Boston, MA). [ZH]acetylcholine chloride (100-500 mCi/mmol) was from Radiochemical Centre, Amersham. All other chemicals were of analytical grade. RESULTS

The results presented below were obtained from animals whose right hemispheres were exposed to the ionizing irradiation whereas the left hemispheres were shielded by lead plates. This procedure completely abolished mortality, and provided intrinsic controls for the various parameters measured in this study both in homogenates and in brain slices.

Weight and morphology Neonatal irradiation reduced hippocampal weight throughout development in a dose-dependent fashion (Fig. 1). The effect persisted for up to 5 months, the oldest age group assayed. Cell staining of slices from mature hippocampi demonstrates a loss of about 85 ~ of the granular neurons in the treated dentate gyrus. This effect is in agreement with previous studies11,12,2L The extent of neural loss depends upon the age of the irradiated animal and the dosage of irradiation (Fig. 2). Thus, exposing 10-day-

174 old neonates to the irradiation procedure affected less hippocampal weight and caused a reduction of only about 60 ~ in the number of granular neurons. No changes in hippocampal weight and granular cell number were observed when animals past the proliferative period of the granular neurons were irradiated. Lowering the irradiation dose also produced milder effects on hippocampal weight (Fig. 1) and granular neuronal loss (Fig. 2). The irradiation procedure utilized in this study enabled us to accurately compare cell numbers in the treated and non-treated hippocampi in the same slice. In addition, a small but consistent diminution in the number of pyramidal neurons in areas CA 1 was also observed. Comparison of cell counts in the right (exposed) and the left (shielded) dorsal hippocampus in the same slices taken from 3 animals revealed a reduction of 16 4- 2 ~ (P < 0.01) in the number of pyramidal neurons in area CA1. The width of the stratum pyramidale was smaller and an unusally large number of cells appeared in the stratum oriens. It should be stressed that the irradiation causes volumetric reduction of the hippocampus 12, thus the total number of the pyramidal cells in the whole structure may even be lower than 84 ~. This effect may result from species differences, or alternatively from indirect transsynaptic degeneration due to the paucity of mossy fibers. This point was not elaborated further in this study.

Activity of cholinergic enzymes Most (up to 85 ~ ) of the cholinergic innervation of the hippocampus is derived from the septal area z3,s4,48'49. It was demonstrated that septal cholinergic innervation as detected by the activity of the presynaptic markers AChE and CAT takes place in the first few weeks after birth ls,14,zS. Septal neurons, in contrast to the granular neurons in the hippocampus, accomplish their formation before birth 9. Indeed, no noticeable difference was found in this study between cell counts in the right (exposed) and left (shielded) medial septal nuclei of the adult irradiated rats. Measurement of i

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Fig. 1. Fresh weights of hippocampi taken from the shielded side ( O - - O ) , hippocampus irradiated at 'half dose' ( A - - A ) , hippocampus irradiated at 'full dose' starting at the age of the 10th postnatal day ( A - - ~ ) , hippocampus irradiated at 'full dose' starting at the 1st postnatal day (C.--©). Values are weight of hippocampus multiplied by 2.

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Fig. 2. Cresyl violet staining of coronal sections of dorsal hippocampus of a 75-day-old rat irradiated at 'full dose' (A, shielded side; B, exposed side taken from the same slice) and 'half dose' (C, shielded side; D, exposed side taken from the same slice) from the 1st postnatal day. DG, dentate gyrus; hi, hilus of the dentate gyrus; gr, granular layer of the dentate gyrus; tool, molecular layer of the dentate gyrus; SUB, subiculum. Bar equals 1 mm. A C h E activity shows that in all phases o f development the activity of the enzyme in the whole irradiated hippocampus was slightly but consistently higher than in the contralateral unexposed area. The experiment was repeated 3 times with comparable results. Fig. 3 shows a typical developmental pattern of enzyme activity in one such set o f experiments. C A T activity at 23 days o f age is not significantly different in the whole

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Fig. 3. Development of acetylcholinesterase activity in whole hippocampus irradiated at 'full dose' from the first postnatal day (O--O) and the shielded hippocampi of the same animals ( 0 - - 0 ) .

treated hippocampus than in the shielded side (Table I). At adulthood, however, the total activity of the two enzymes in the treated hippocampus does not significantly differ from control. Specific activities on the other hand remained higher (Fig. 4 and Table I). I n order to establish that the increase in the level of the presynaptic markers is not due to elevation in enzyme synthesis a n d / o r t r a n s p o r t to the target area, but to the reduced n u m b e r of target neurons, two experiments were performed: I n the first, m e a s u r e m e n t of A C h E activity in h i p p o c a m p i taken from animals irradiated at adulthood, where no observable change in weight a n d cell n u m b e r was found, did not reveal a n y difference in enzyme activity. The adult skull bone did n o t reduce the dose of irradiation, thus eliminating the possibility that the lack of effect of the irradiation in adults is due to diminished effective dose in the brain. In a second set of experiments we measured the level of A C h E a n d C A T in TABLE I Effect o f irradiation on CA T activity in the hippocampus, cerebellum and cortex

For the determination of CAT activity homogenates (1 : 10 w/v in sucrose 0.32 M) were prepared from the right (exposed) and the left (shielded) brain areas of 3 (23-day-old) rats and 4 adults (4.5 months old). Values are mean ± S.E.M. Numbers in parentheses are per cents of shielded side. At 23 days Shielded area

Hippocampus

Cerebellum Cortex

* P < 0.05. ** P < 0.01.

nmol/mg tissue/h

5.0 ± (100) nmol/hippocampus/h 438 ± (100) nmol/mg tissue/h 0.9 ± (lO0) nmol/mg tissue/h 4.3 ± (100)

0.8 104 0.26 0.25

Adult Exposed area Shielded area

Exposed area

6.8 ± (136) 473 ± (108) 2.1 ± (233) 5.0 ± (116)

9.2 £ (124) 840 ~ (98) 1.8 ~ (300) 6.1 ~ (115)

1.2" 85 0.7** 0.25

7.4 ± 0.1 (100) 853 -~- 96 (100) 0.6 i 0.06 (I 00) 5.3 ~ 0.55 (100)

0.6** 54 0.15"* 0.6

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Fig. 4. Development of acetylcholinesterase. A: hippocampus. B: cerebellum. C: cerebral cortex. Irradiations were delivered at 'full dose' to 1-day-old pups ((D--G), to 10-day-old pups ( x . . . x), at 'half dose' to l-day-old pups ( A - - A ) , shielded hippocampus of the 'full dose' irradiated pups ( O - - Q ) and hippocampus of control animals of the same litters left in the breeding c e n t e r + . . . + ) .

irradiated cerebellum and cerebral cortex. In the cerebellum, the granular neurons, which constitute a major neuron population and serve as the main target for the cholinergic mossy fibers 5, are formed during the first postnatal weeksZ, a, whereas cortical neurons complete their differentiation before birthT,26,2L It was expected that if elimination of target neurons is the cause of the elevated activity of presynaptic enzymes then it would be manifested in the agranular cerebellum and to a lesser degree in the cortex. The results shown in Fig. 4 and in Table I confirm this assumption. The effect of irradiation on the level of AChE (Fig. 4) and CAT (Table I) are more

Fig. 5. Acetylcholinesterase staining in shielded (A) and irradiated (B) hippocampus of 75-day-old rats irradiated at 'full dose' from the first postnatal day. Photomicrographs are from the same slice. DG, dentate gyrus. Bar equals 1 ram.

178 TABLE 1I

Effect o f irradiation on AChE and CA T activities in subarea o f the h ippocampus Slices of 400/~m thickness from irradiated and contralateral non-irradiated hippocampi of 7 adult rats were cut and kept in ice-cold 0.32 M sucrose. Dissections were performed on ice by a fine-tipped razor blade under binocular inspection. Pooled subareas from several slices of each hippocampus were weighed and homogenized in 0.32 M sucrose (1:40 w/v) and assayed for enzymatic activity as described in Materials and Methods. Subareas were dissected, as shown in Fig. 2A. Whole dentate gyrus (excluding the hilus), areas CA3 (including the CA4 area) and CAI (including area CA2 and subiculum) were collected from 4 rats and the molecular layers of the dentate gyrus were collected from 3 animals. Values are mean ± S.E.M, numbers in parentheses are per cents of shielded sides. Values for AChE are in nmol/mg tissue/min and for CAT in nmol/mg tissue/h.

AChE

Whole dentate gyrus CA3 CA1 Molecular layer of dentate gyrus

CAT

Shielded subarea

Exposed subarea

Shielded subarea

Exposed area

2,84 ± (100) 4.37 ± (100) 3.84 ± (100) 4.4 ± (100)

4.42 :j__0.5** (156) 7.13 :k 1.4"* (163) 5.47 A 1.2 (142) 7.2 £ 0.7** (164)

4.92 ± (100) 6.48 ± (100) 7.10 ± (100) 3.2 ± (100)

7.75 ± (158) 11.3 ± (174) 9.92 ± (140) 5.8 ± (181)

0.5 0.5 0.8 1.3

1.9 2.8 3.5 0.03

2.5 2.9* 1.2 0.9**

* P < 0.05. ** P < 0.01.

pronounced in the cerebellum than in the hippocampus, whereas the level of the enzymes in the cortex is relatively unaltered. Histochemical staining for AChE activity (Fig. 5) demonstrated that the intensified enzyme activity is confined to discrete zones within the hippocampus. Thus, TABLE Ill

[ 3HI QNB-binding and cholinergic enzymatic activities in intact and fornix-lesioned irradiated dentate gyrus Whole dentate gyrus of adult rats were dissected from ice-cold 400 p m thick slices taken from 4 irradiated hippocampi and 7 fornix-lesioned hippocampi. The deafferentation was performed 10 days prior to sacrifice. Dentate gyrus from several slices of each hippocampus were pooled, weighted, and homogenized 1:40 (w/v) in sucrose 0.32 M and assayed immediately for [aH]QNB-binding and enzymatic activities.Values are mean ± S.E.M. Numbers in parentheses are per cents of exposed dentate gyrus.

Exposed dentate gyrus Fornix-lesioned dentate gyrus

[ 3HI QNB-binding (pmol/mg tissue)

AChE

CA I

(nmol/mg tissue/rain)

( nmol/ mg tissue~h)

0.103 ± 0.005 (100)

4.15 i 0.48 (100)

7.7 ± 0.41 (100)

0.102 ± 0.014 (99)

0.28 ± 0.09 (6.7)

0.56 ± 0.16 (7.3)

179 higher esterase staining is found in the hilar zone and the proximal fourth of the molecular layer in the dentate gyIus. In addition, a clear band of staining appears in the stratum lucidum of area CA3 and a diffuse heavier staining is observed at supraand subpyramidal layers and the dendritic fields of area CA1. In order to obtain quantitative estimates of the distribution of the cholinergic enzymes, defined areas in the irradiated and shielded hippocampi were dissected from 400 # m thick slices of four adult animals and molecular layers of three animals. The results presented in Table II demonstrate parallel increase in CAT and AChE levels in the molecular layer of the dentate gyrus, in the whole dentate gyrus, in area CA3 and a smaller but still considerable increase in area CA1. That the septal area is the source of the increase in the cholinergic enzyme activity is demonstrated by the observation that severing the septo-hippocampal pathway by fornix lesions reduces esterase staining and activity, and CAT activity almost completely (Table III). The increased AChE level was apparently not due to enhancement of pseudo cholinesterase activity since incubation with 10 -6 M BW 284C51, a specific inhibitor of acetylcholinesterase, inhibited (95 :t: 2 ) ~ enzyme activity in three treated dentate gyri, a value which is similar to the inhibition obtained in control hippocampus ~4.

Development and distribution of muscarinic receptors The muscarinic receptors in the hippocampus develop largerly during the second postnatal week and reach mature level at the age of about 7 weeks 13. The postnatal drastic increase in the quantity of the receptors may be due to the accumulation of receptors on the prenatally formed neurons and also to differentiation of new cells, especially the granular neurons in the dentate gyrus. The irradiation technique which abolishes a major portion of postnatal neuronal acquisition makes it possible to separate between the two contributions and furthermore, to study their distribution within discrete areas in the agranular hippocampus. 14

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Fig. 6. Development of 13H]QNB-binding in whole hippocampi following postnatal X-irradiation. Neonates were irradiated at 'full dose' from the 1st postnatal day. The irradiated (©--©) and the shielded ( 0 - - 0 ) hippocampi from the same animals were assayed for [3H]QNB-binding at the appropriate ages.

180 TABLE IV {3tI] QNB-binding to subareas of irradiated and non-irradiated hippocampus

13H]QNB-binding assays were performed on the same homogenates as were used for enzymatic activity determination. For details see Table III. Values are mean ± per cent.

Whole hippocampus Whole dentate gyrus CA3 CAI Molecular layer

shielded exposed shielded exposed shielded exposed shielded exposed shielded exposed

I~g protein/ mg wet weight

[aH]QNBbinding (pmol/ mg protein)

[3H]QNBbinding(pmol/ mg wet weight)

101 ._k_5.5 110 ± 7.6 73 ± 19 62 ± 6 107 ± 21 107 ± 18 96 ± 20 101 ± 17 80 ± 12 84 ~: 14

1.22 ± 1.20 ± 1.90 ± 1.90 ± 1.12 ± 1.18 ± 1.35 ± 1.39 ± 2.25 ± 1.19 ±

0.12 0.13 0.14 0.12 0.12 0.13 0.13 0.14 0.18 0.10

0.14 0.18 0.3 0.6 0.1 0.1 0.2 0.05 0.03** 0.01

3_ 0.03 ± 0.02 ± 0.02 ± 0.02 ± 0.02 ± 0.03 ± 0.02 ± 0.02 ± 0.03* ± 0.03

* P < 0.05. ** P < 0.01.

Fig. 6 shows the d e v e l o p m e n t a l p a t t e r n o f the Q N B - b i n d i n g sites in the i r r a d i a t e d h i p p o c a m p u s is essentially similar to the d e v e l o p m e n t in the c o n t r a l a t e r a l , n o n - i r r a d i a t e d h i p p o c a m p u s , b u t the total content o f the receptors is a b o u t 25 ~ lower with no recovery observed at maturity. The specific b i n d i n g to i r r a d i a t e d h i p p o c a m p u s m e a s u r e d at a d u l t h o o d does not differ m a r k e d l y f r o m control. The d i s t r i b u t i o n o f the muscarinic receptors following i r r a d i a t i o n was studied within discrete areas o f the h i p p o c a m p u s (Table IV). It can be seen that in the m o l e c u l a r layer o f the dentate gyrus (which contains mostly dendrites o f the g r a n u l a r cells a n d afferents), but n o t in o t h e r subareas, the elimination o f the g r a n u l a r cells m a r k e d l y reduced the specific [3H]QNB-binding. However, the effects o f i r r a d i a t i o n on the muscarinic levels in the whole dentate gyrus and in the m o l e c u l a r layer are unexpectedly mild c o m p a r e d to the d r a m a t i c effect on the n u m b e r o f g r a n u l a r cells. One possible e x p l a n a t i o n for this is that p a r t o f the muscarinic receptors are located on the cholinergic presynaptic terminals which, as can be j u d g e d from the activity o f the presynaptic markers, are m o r e a b u n d a n t in the whole i r r a d i a t e d dentate gyrus than in the control. Indeed the existence o f such receptors was suggested by the ability o f [3H]QNB at relatively high c o n c e n t r a t i o n s to m o d u l a t e acetylcholine release in rat h i p p o c a m p u s 49. To date, a t t e m p t s to detect muscarinic a u t o r e c e p t o r s by direct b i n d i n g assays have yielded negative results (e.g. refs. 14,51). In c o n t r a s t to these studies, which e m p l o y e d the whole h i p p o c a m p u s to reveal the existence o f presynaptic receptors, the i r r a d i a t e d dentate seems a m o r e suitable p r e p a r a t i o n because o f the m a r k e d increase in the ratio between presynaptic a n d p o s t s y n a p t i c elements. In o r d e r to check this possibility the connections between the septum and the i r r a d i a t e d h i p p o c a m p u s were severed by fornix lesions in 7 animals a n d [3H]QNB-

181 binding at the deafferented irradiated dentate gyrus areas was compared to irradiated, non-operated dentate gyrus. The results presented in Table III show that fornix lesions decreased AChE and CAT activities to less than 10 ~. However, the failure to detect any reduction in the [3H]QNB-binding sites in the dentate gyrus deprived of most of its cholinergic input indicates that autoreceptors are either absent, or constitute a minute fraction of receptors in this area. DISCUSSION The results presented in this study demonstrate that irradiation of newborn hippocampus caused marked changes in the pre- and postsynaptic elements of its cholinergic system. Several explanations could account for the altered distribution of the cholinergic innervation within the hippocampus. The first explanation is that irradiation caused a change in the number of cholinergic fibers invading the area during development. This possibility cannot be ruled out completely, but it appears unlikely since septal neurons accomplish their differentiation prior to birth 9 and are not expected to be affected by postnatal irradiation. A second explanation for the observed effects of irradiation on the cholinergic enzymes is that the irradiation interferes with the metabolism and/or the transport of the enzymes, thus leading to enhanced activity in pre-existing cholinergic terminals. This possibility is also not very likely since the activity of the two enzymes increased in parallel at all ages and in all areas of the hippocampus. It would be difficult to assume that irradiation affected the synthetic or transport mechanisms of the two enzymes similarly. Moreover, it was shown 50 that the rate of synthesis of AChE is not altered following irradiation of the cerebellum. It is also not plausible to assume that irradiation would affect cholinergic neurons innervating the cerebellum and hippocampus, but not those terminating at the cortex. The results support, however, the contention that the primary cause for the altered distribution and the dose and age dependent higher specific activities of the cholinergic enzymes is the reduced number of target neurons in the hippocampus. In agreement with Valcana and Timiras 50, the elevated specific activities of CAT and AChE at maturity are conceivably due to the relative radioresistance of the cholinergic system compared to the non-cholinergic target neurons. Measurements of AChE levels during development indicated that there is an increase not only in the specific activity of the eznyme, but also in its total content in the hippocampus. Total content of CAT in the developing and mature hippocampus, on the other hand, did not significantly differ from control. Nadler et al. 42 also found enhanced AChE staining and CAT activity following entorhinal removal which did not persist in adulthood. They also observed differential response of the two cholinergic enzymes following the insult. It appears that some adaptive changes may take place during maturation that regulate enzymatic levels separately. Therefore, the transient elevations in the total content of AChE may not signal a total change in the amount of innervation, but may conceivably result from elevated enzyme activity in existing terminals and/or alterations in AChE levels occurring on postsynaptic elements in development but not in

182 adulthood. In support of the latter possibility are the observations that Purkinje cells in the cerebellum stain for AChE only during a specific developmental phase. At later phases and at maturity esterase activity is abolished 5 but may reappear following denervation 30. The major changes in the cholinergic innervation in the irradiated hippocampus are secondary to the reduced neuronal population, and reflect the reorganization of the cholinergic input following the irradiation-induced alterations in hippocampal internal structure. Staining for esterase activity revealed enhanced staining in discrete zones within the structure. Thus, a sharp band of staining is seen in the proximal fourth of the molecular layer and in the hilar zone of the dentate area, at the stratum lucidum of area CA3, and at the supra- and infrapyramidal layers in area CA 1 and the subicular system. That the septum is the origin of the altered cholinergic activity is evident from the drastic reduction in enzymatic level and virtual abolishment of esterase staining following fornix lesion. It is well documented that septal cholinergic fibers are capable of invading vacated synaptic territories and forming synaptic contacts in the molecular layer of the dentate gyrus, following removal of the normal afferents in immature 20 as well as mature 21,37 animals. The causes of these plastic changes, and in particular, the factors that initiate the sprouting of the septal fibers are as yet not clear 21. One possible explanation of the lesion-induced sprouting is that afferents release retardants which under normal conditions prevent invasion of afferents into their territory2L Removal of the adjacent fibers will create permissive conditions for sprouting into the vacated territory. Alternatively, one can conceive that degenerating fibers secrete some factor(s) thereby signalling neighboring fibers to initiate sprouting. While the present results cannot exclude such possibilities they indicate that reorganization of septal (as well as monoaminergic 41 and entorhina125,47) fibers, can take place in a neuronal target which is devoid of degenerating tissue. One of the factors that appears to participate in the reorganization of the septal fibers following X-irradiation is the occupation by septal fibers of dendritic territories normally occupied by mossy fibers. The enhanced staining in the hilar zone of CA4 and the sharp band of activity in the molecular layer of CA3 terminating on the border between CA3 and CA2, corresponds to the mossy fiber projections~5,sL Abnormal projections in this area were also noted for monoaminergic 41 and perforant path fibers 25. The observed expansion of septal projections into the mossy fiber layer suggests that the lamination of the septal and mossy fibers in areas CA4 and CA3 in the normally developing hippocampus cannot be accounted for solely on the basis of temporal competition between the two afferents since both systems arise gradually and simultaneously during the first weeks after birth16,~s,sL Thus the boutons of the mossy fibers attain adult size but still increase in number at postnatal day 15 (Stirling and Bliss, quoted in ref. 52). Ingrowing cholinergic fibers also develop from birth at the septal edge until by day 1 1 all parts of the hippocampus exhibit esterase activity 38. If competition were the only factor one would expect the two afferent systems to comingle and not to generate two segregated layers upon the common dendritic tree. It is plausible to assume then, that two major factors may play a role in the development of the two afferent systems. First, discrete areas on the postsynaptic dendritic area

183 display preference for a specific type of innervation 21. The absence of one type creates free target areas that can be occupied by the less preferred fibers. Second, laminar distribution can be induced at the presynaptic level by the capability of two afferent systems to retard each other 21. The existence of such mechanisms is suggested by the observation that pathways in the X-irradiated dentate gyrus 47 and in the 'reeler' mouse 22 retain their lamination in spite of the abnormal location of postsynaptic elements. Laminar distribution is also retained by septal fibers in the irladiated supragranular zone of the molecular layer in the dentate (Fig. 5). The increased esterase and CAT activities (164 and 181 ~ respectively) do not seem to be the result of hyperinnervation on the remaining granular dendrites since Lewis and Cotman 32 were unable to detect changes in the density of synapse in the molecular layer of the irradiated dentate gyrus. It appears that shrinkage of the molecular layer and/of failure to establish successful contacts may induce sprouting in this area and in remote areas of the fiber system. Thus, the intensified esterase staining and the 4 0 ~ increase in CAT and AChE activities in area CA1 do not seem to be accounted for solely by the slight loss of pyramidal cells since no significant change in the width of the dendritic fields in the supra- and infrapyramidal layer was noticed. The elevated enzymatic levels in area CA I and the subicular system, and probably also in the molecular layer of the dentate gyrus, may reflect compensatory sprouting as a result of the failure to form connections in the drastically diminished target field in the irradiated dentate. Indeed, sprouting of noradrenergic fibers 44 and retinal projections a5 is observed following removal of a part of the projection area of these fibers. Also, sprouting of motor fibers is evident when nmscle is rendered inactive 17,a3, axons are transected z9 or axonal flow is blocked by colchicine 1,19,21. The muscarinic receptors in the hippocampus were shown to reside on postsynaptic cells 51. Their development in this area is characterized by sharp quantity increases during the second postnatal week 13. Fig. 4 shows that in the X-irradiated hippocampus in which contribution to the quantity of muscarinic receptors by postnatally developing neurons is eliminated, the developmental pattern is essentially reserved, although the total number of the [3H]QNB-binding sites in this area is reduced by about 25 ~ and does not recover at adulthood. This reduction is probably the result of drastic diminution in the number of granular neurons in the dentate and the slight reduction in the number of pyramidal cells. It was shown earlier lz that muscarinic receptors are present in the hippocampus at embryonic day 16, long before septal innervation takes place. Moreover, their development is not dependent upon cholinergic innervation 14. We have also recently shown that the distribution of the muscarinic receptors on the dendritic and cell body layers in the CA1 area is not altered following deafferentation at infancy or adulthood (unpublished data). This evidence together with the present observation that the postnatal sharp increase in muscarinic receptor levels occurs in the prenatally folmed cells indicate that the synthetic machinery of the muscarinic receptors in hippocampal cells retains a marked degree of autonomy with respect to quantity, timing of appearance and distribution on the cell layers. Measurements of the distribution of muscarinic sites in the hippocampus

184 revealed that the specific b i n d i n g o f [aH]QNB in the whole i r r a d i a t e d d e n t a t e a r e a is not significantly different f r o m c o n t r o l area. The only difference observed was the reduction in the n u m b e r o f g r a n u l a r neurons. This relatively high muscarinic b i n d i n g in the i r r a d i a t e d dentate was not due to p r e s y n a p t i c receptors on cholinergic fibers which s p r o u t in this area since fornix lesions failed to alter [ a H ] Q N B - b i n d i n g sited in this area. There still r e m a i n 3 possibilities to a c c o u n t for the differential effects o f i r r a d i a t i o n on n e u r o n a l n u m b e r a n d muscarinic sites: (a) The r e m a i n i n g g r a n u l a r n e u r o n s synthesize m o r e receptors. (b) M o s t o f the muscarinic receptors reside on p r e n a t a l l y f o r m e d ( a n d therefore radioresistant) p o l y m o r p h i c cells a n d i n t e r n e u r o n s in the hilus a n d in the m o l e c u l a r layer 27. (c) A m a j o r fraction o f the muscarinic receptors reside also on other types o f afferents or on glia cells which, unlike neurons, are m o r e c a p a b l e o f recovery 8. It remains to be established on the electron m i c r o s c o p y level what the exact l o c a t i o n o f the muscarinic receptor is on the cells c o n t a i n i n g them or whether they can be f o u n d on p r e s y n a p t i c terminals as well. A l t h o u g h g r a n u l a r cell loss (and consequently the loss o f the mossy fiber system) is the m o s t p r o n o u n c e d consequence o f p o s t n a t a l irradiation, the neural reorganization o f the cholinergic system as presented in this study, a n d o f o t h e r afferents to the area 25,41,49 has to be considered in the analysis o f the b e h a v i o r a l changes occurring following i r r a d i a t i o n to the h i p p o c a m p u s 19',1s. ACKNOWLEDGEMENTS This w o r k was s u p p o r t e d by grants f r o m the I s r a e l - U n i t e d States Binational Science F o u n d a t i o n , Jerusalem, a n d f r o m the Israel Center for Psychobiology, The Charles Smith F a m i l y F o u n d a t i o n , Jerusalem, to Dr. Y a d i n Dudai.

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