Transient increase in the number of cholinergic neurons in the developing rat dentate gyrus

Neuroscwnce Letters, 101 (1989) 23-28

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Elsewer Scientific Pubhshers Ireland Ltd NSL 06112

Transient increase in the number of cholinergic neurons in the developing rat dentate gyrus Shinobu Kanaya-Ida* and Y. Ben Ari INSERM U 29 H6pital de Port-Royal, ParLs (France)

(Recewed 8 August 1988, Revised versmn received 13 February 1989, Accepted 13 February 1989) Key words

Chohne acetyltransferase, Immunocytochemistry; Hlppocampus, Dentate gyrus, Development

Using a monoclonal antibody against chohne acetyltransferase (CHAT), we have exanuned the &stnbutlon of chohnerglc neurons in the rat dentate gyrus dunng development ChAT-positive neurons were occasionally detected m the hilus on postnatal day 2 (P2) There was a transient abrupt increase in the number and density of ChAT-poslave neurons between P 15 and P20 and then a decline to the adult level with few ChAT-trnmunoreactlve neurons. A few ChAT-posttlve varicose fibers and ptmctae were first seen at P5 They increased m number and density untd P20 when they reached the adult level and distribution These observations suggest the occurrence of a transient expression of cholinergic markers m the hlppocampus

The distribution of chohnergic neurons during development has been studied by means of the acetylcholinesterase (ACHE) stain. ACHE, however, may be present m b o t h chollnergic a n d n o n - c h o h n e r g i c n e u r o n s [7]. C h o l i n e a c e t y l t r a n s f e r a s e (CHAT), the synthesizing e n z y m e for acetylcholine, is presently a c c e p t e d as the best m a r k e r o f cholinergic n e u r o n s [13]. I n the p r e s e n t s t u d y we have used a m o n o c l o n a l a n t i b o d y a g a i n s t C h A T a n d i m m u n o c y t o c h e m l c a l staining to s t u d y the d i s t r i b u t i o n o f CHATpositive n e u r o n s in the d e n t a t e gyrus o f the r a t d u r i n g d e v e l o p m e n t . S h e r m a n rats were used in this study. A t least 3 b r a i n s were e x a m i n e d o n each o f the following p o s t n a t a l d a y s (P): P1, P2, P5, P15, P20, P28 a n d adult. All a n i m a l s were killed by p e r f u s i o n u n d e r s o d i u m p e n t o b a r b i t o n e anesthesia (20 m g / k g , 1.p.) T h e y were p e r f u s e d t h r o u g h the h e a r t with ice-cold n o r m a l saline f o l l o w e d b y a fixatwe c o n t a m m g 3% p a r a f o r m a l d e h y d e a n d 0.01% g l u t a r a l d e h y d e in 0.05 M p h o s p h a t e buffer ( p H 7.4, 100 ml/100 g), then they were perfused with the s a m e s o l u t i o n w i t h o u t g l u t a r a l d e h y d e (50 ml/100 g) a n d finally perfused with a s o l u t i o n c o n t a i n i n g 10% sucrose m 0.1 M p h o s p h a t e buffer (25 ml/100 g). T h e b r a i n s were r e m o v e d a n d i m m e r s e d *Present address Department of Pedlatncs, Osaka University Medical School, 1-1-50, Fukushima Fukushlma-ku, Osaka, Japan. Correspondence Y_ Ben-An, INSERM U29, H6pital de Port-Royal, 123 Bd de Port Royal, 75014 Pans,

France

25 TABLE I N U M B E R S O F C h A T - P O S I T I V E N E U R O N S IN T H E H I L U S O F T H E D O R S A L D E N T A T E G Y R U S . S H O W I N G A T R A N S I E N T . S I G N I F I C A N T I N C R E A S E 1N T H E N U M B E R O F CHATPOSITIVE N E U R O N S D U R I N G D E V E L O P M E N T . IN P A R T I C U L A R B E T W E E N P15 A N D P20

Age

No of animals

N o of sections

N o of C h A T posmve neurons

PI P2 P5 PI5 P20 P28 Adult

4 3 3 3 5 3 4

12 9 9 9 15 9 12

0 0 3 3 + 0 50 / 1 3 3 + 1 11~* 1_55~0 521, 17 I _+8 381, 4_66--+3 87~, 1_60_+ 1.45"

*P < 0 01 non-parametric. M a n n - W l t h n e y . U-test

in 0.1 M phosphate buffer containing 30% sucrose for ! or 2 days. Frontal secttons (30/zm) were cut on a cryostat. Immunocytochemistry was performed with a monoclonal antibody against ChAT from rat-mouse hybridoma (Type I, Boehrlnger Mannhetm, F R.G.) [4]. The peroxldase-antiperoxidase (PAP) technique was applied. The secttons were rinsed m icecold phosphate-buffered saline (PBS) 3 times for l0 min and incubated for 2 h at room temperature with rabbit anti-rat IgG (Miles Scienttfic, U.S.A.) diluted 1:250 with PBS containing 1% normal rabbit serum. The sections were agam rinsed in PBS 3 times for l0 min and incubated for 4 days at 4°C with 1:20 anti ChAT in PBS. After rinsing they were incubated for 2 h at room temperature with rat PAP complex (Miles Scientific) diluted 1:100 with PBS containing 1% normal rabbit serum. After rinsmg twice with PBS and once with Tris buffer (0.05 M, pH 7.6), the sections were treated for 10 min with a 0.05% solution of 3,3'-diaminobenzidine (DAB) in Tris buffer followed by the same solution of DAB containing 0.01% hydrogen peroxidase for 5 or l0 min. In control experiments, i.e. when the sections were treated in the same way but with rat serum instead of the primary antibody, immunoreactive neurons or fibers were not observed. Furthermore, sections containing the septum and diagonal band regions immunostained together with hippoeampal sections revealed the typical staining pattern which has been reported in earlier studies [2, 5, 6]. Immunolabeled tissue sections were then mounted onto glass slides, air-dried, dehydrated wtth graded alcohols and xylene, and then covered with glass coverslips and Eukitt. The remaming sections were stained with Cresyl violet for identification of the brain regions. Fig 1 Photographs of ChAT-positive neurons, varicose fibers and punctae at different ages in dentate gyrus A at P5 C h A T - p o s m v e neurons are seen m the hdus Fme varicose fibers and punctae are also labeled ( x 150) B_ at p20_ There is a remarkable increase in the number of C h A T - p o s m v e neurons and lmmunoreactlvlty of varicose fibers and punctae ( x 75) C adult only 2 positive neurons are seen ( x 75)

26 A t P2, C h A T - p o s i t i v e n e u r o n s were o c c a s i o n a l l y o b s e r v e d m the htlus but fiber~ were n o t clearly detected A t P5, as few C h A T - p o s i t i v e n e u r o n s were observed m the hllus and very fine varicose fibers a n d p u n c t a e were for the first ttme seen in this area ( F i g 1A) A n increase in the n u m b e r o f C h A T - p o s i t i v e n e u r o n s a n d l m m u n o r e a c t l w t y o f varicose fibers a n d p u n c t a e was o b s e r v e d at P20 (Ftgs 1B, 2) In the adult, in k e e p m g with earlier studtes [5, 8, 14], only a few C h A T - l m m u n o r e a c t l v e neurons were seen, b u t varicose fibers a n d p u n c t a e were o b s e r v e d in all layers o f this region, m p a r t i c u l a r m the s u p r a g r a n u l a r layer (Fig. IC). A t all ages, C h A T - p o s l t w e n e u r o n s were o b s e r v e d m a m l y in the htlus o f the d e n t a t e gyrus a l t h o u g h they were o c c a s i o n a l l y seen m the m o l e c u l a r a n d g r a n u l a r layers. T a b l e I shows q u a n t l t a t w e values o b t a i n e d by c o u n t i n g n u m b e r s o f C h A T - p o s l t t v e n e u r o n s f r o m c a m e r a luctda d r a w m g s in the hilus at the d o r s a l level. T h e n u m b e r o f C h A T - p o s l t w e n e u r o n s was significantly higher at P20 than at earlier ages ( P < 0_01, M a n n W h i t n e y ) ; furthermore, there was a significant decrease in the n u m b e r o f C h A T - p o s i t i v e n e u r o n s from P20 to P28 ( P < 0 01 M a n n - W h i t n e y ) Fig. 2 shows typical C h A T - p o s i t i v e neurons at P20 These n e u r o n s were m a i n l y fuslform or m u l t t p o l a r , a n d the labeled c y t o p l a s m s u r r o u n d e d u n s t a i n e d nuclei and e x t e n d e d into d e n d r i t l c - h k e processes which often o r i e n t e d parallel to the granule cell layer. The p r m c l p a l results o f the p r e s e n t s t u d y are that C h A T - p o s i t w e n e u r o n s are first

Fig 2 Photograph of ChAT-positive neurons, varicose fibers and punctae at P20 with high magmficatlon (×400) ChAT-positwe neurons are fuslform or multlpolar and the labeled cytoplasm surrounds unstained nuclei and extends into dendrltlc-hke processes which are often oriented parallel to the granule cell layer

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seen between P2 and P5 m the dentate gyrus and that there is a significant increase in number at P20 and then a decline. At P20, ChAT-positive neurons are found mainly m the hllus and occasionally in the granular and molecular layers. This localization of ChAT-posittve neurons is comparable to acetylcholinesterase staining during development [9]. It is of interest to note that ChAT-positive neurons are reminiscent of Amaral's multlpolar or fusiform cells [1] and that their distribution is comparable to that of somatostatln-reactive cells [12, 15]. ChAT-positive varicose fibers and punctae were found at P5 and increased m density until P20 when they reached the adult level and distribution, with ChAT-positive fibers and punctae m all layers of dentate gyrus in agreement wtth Frotscher and Leranth [5]. The observed low number of ChAT-positive neurons in adult rat is in general agreement with earlier studies [5, 8, 14]. Our results suggests that ChAT is transiently expressed in neurons in the developmg rat dentate gyrus between P15 and P28. The role of the transient increase in the number of ChAT-positive neurons is unknown. An increased production of ChAT in the medial septum and diagonal band between one and two weeks after birth, previously described both with tmmunocytochemical [2] and biochemical methods [11], may precede the transtent expression of ChAT in the htppocampus around the 3rd week of postnatal hfe. Thts corresponds to a phase with maximal axonal growth and arborlzatlon in thts region [3]. A slmdar transient expression of neurotransmltter markers has been observed in the developing neocortex [10]. These transient phenomena appear to revolve both early and late forming neurons as well as afferent and efferent projections. We are grateful to Dr. A. Represa for his contnbutton and criticism. 1 Amaral, D G , A Golgl study of cell types m the hllar region of the hlppocampus in the rat, J. Comp Neurol, 182 (1978) 851-914 2 Armstrong, D M , Bruce, G , Hersh, L B and Gage, F_H, Development of chohnergic neurons m the septal/dlagonal band complex of the rat, Dev_ Brain Res., 36 (1987) 149-256 3 Cram, B, Cotman, C_, Taylor, D and Lynch, G , A quantltatwe electron microscopic study ofsynaptogenesls m the dentate gyrus of the rat, Brain Res, 63 (1973) 195-204_ 4 Eckensteln, F, and Thoenen, H , Production of specific antisera and monoclonal antibodies to chohneacetyltransferase Characterization and use for identification of chollnergic neurons, EMBO J , 1 (1982) 363-368 5 Frotscher, M and Leranth, C , The chohnerglc innervatlon of the rat fascia dentata: identification of target structures on granule cells by combimng chohne acetyltransferase immunocytochemlstry and Golgl impregnation, J Comp Neurol, 243 (1986) 58-70. 6 Houser, C R , Crawford, G D., Barber, R P , Salvaterra, P.M. and Vaughn, J N , Organization and morphological characteristics of chohnergic neurons an lmmunocytochermcal study with a monoclonal antibody to chohne acetyltransferase, Brain Res., 226 (1983) 97-119_ 7 Levy, A I , Rye, D_B, Warner, B_H, Mufson, E_J and Mesulam, M - M , Chohne acetyltransferaseimmunoreactwe neurons intnnslc to rodent cortex and distinctions from acetylcholmesterase-posmve neurons, Neurosclence, 13 (1984) 341-353 8 Mattews, D A , Salvaterra, P M , Crawford, G.T, Houser, C.R and Vaughn, J N_, An lmmunocytochemical study of chohne acetyltransferase containing neurons and axon terminals in normal and partrolly deafferented hippocampal formation, Brain Res., 402 (1987) 30--43

28 9 MJlner, T A , Loy, R and Amaral, D G , An anatomical study of the development of the septo-hlppocampal projection in the rat, De,, Brain Res, 8 (t983) 34-371 10 Parnavelas, J G and Cavanagh. M E , Transient expression of neurotransmltters m the developing neocorten, Trends Neuroscl, 11 (1988) 92 93 11 Represa, A . Chanez, C , Flexor, M A and Ben Arl, Y , Effects of mtra uterine growth retardation on the development of the chohnerglc system, Brain Res, m press 12 Slovlter, R S and Ndaver, G , Immunocytochemlcal IocahzatLon ofGABA-, cholecystokmm-, vasoact~ve intestinal polypeptlde- and somatostatm-hke immunoreactlvlty m the area dentata and hlppocampus of the rat, J Comp Neurol, 256 (1987) 42~o0 13 Warner, B H , Levey, A_I, Mufson, E J and Mesulam, M - M , Chohnerglc system m mammahan brain identified with antibodies against chohne acetyltransferase, Neurochem Int, 6 (1984) 163 182 14 Warner, B H , Levey, A I, Rye, D B, Mesulam. M -M and Mufson, E J , Chollnerglc and non-choltnerglc septohlppocampal pathways, Neuroscl Lett_, 54 (1985) 45 52 15 Z~mmer, J , Laurberg, S and Sunde, N , Neuroanatomlcal aspects of normal and transplanted hlppocampal tissue In W SeJfert (Ed), NeurobJology of the Hlppocampus, Academic Press, New York, 1983, pp 39 64