Development of the cholinergic system in control … · Development of the cholinergic system in...

Developmental Brain Research, 47 (1989) 71-79 71 Elsevier

BRD 50902

Development of the cholinergic system in control and intra-uterine growth retarded rat brain

A. Represa, C. Chanez*, M.A. Flexor and Y. Ben-Ari INSERM U29, MaternitO de Port-Royal, Paris (France)

(Accepted 6 December 1988)

Key words: Intra-uterine growth retardation; Rat; Brain regional development; Choline acetyltransferase; Acetylcholinesterase; Muscarinic receptor

The activity of choline acetyltransferase (CHAT), acetylcholinesterase (ACHE), and muscarinic receptors was studied in control rats and in rats growth-retarded in utero because of reduction of the blood supply 5 days before birth. The different markers of the cholinergic system were estimated at P (postnatal day) 6, 9, 12, 15, 22 and 60 in cerebellum, hypothalamus, septum, striatum and CA1, CA3 and fascia dentata of the hippocampus. In control rats, there was a transient increase in ChAT activity in the septum during the second week of postnatal development. In the intrauterine growth retarded rats there was a marked delay in this developmental rise in CA1, CA3 at P6 and P9 and in the fascia dentata at P14 respectively. This delayed rise enzyme activity was associated with a significant reduction of muscarinic binding sites ([3H]QNB) in the hippocampus. AChE staining showed a similar development in both groups. Therefore, the undernutrition produced by a reduction of the blood supply 5 days before birth is associated with a delayed maturation of cholinergic functions.

INTRODUCTION

An early restriction of nutriments can have

considerable influence on somatic development. Undernutr i t ion during the prenatal period causes

growth disturbances, especially in tissues undergoing rapid cellular multiplication 9A°'37'39. The brain is

particularly vulnerable at this time; morphological

and biochemical maturat ion may be retarded and

lead to impaired functioning in the central nervous system 29'33"38. Undernutr i t ion induced by restriction

of the blood supply to the foetus by ligation of the uterine vessels five days before birth, has immediate and long lasting effects on rat development 31. Mod-

ifications in neurotransmitter metabolism have been

reported in intra-uterine growth retardation ( IUGR) rats 1'8'23. Moreover , in the cerebellum and hippo-

campus, D N A synthesis is delayed and the timing of cell acquisition altered in I U G R in relation to control rats 9.

The largest part of cholinergic neurons of medial

septal and diagonal band nuclei project to the hippocampus 3. Because the development of this system is essentially postnatal 16'19 we have studied

the effects of I U G R on the maturat ion of cholinergic

markers (acetylcholinesterase (ACHE) staining,

choline acetyltransferase (CHAT) activity levels and

muscarinic receptors) in the septal and hippocampal

regions. In intrauterine growth retarded rats the

development of ChAT activity and muscarinic bind-

ing sites were significantly delayed. Since the septal

innervation of hippoeampus has been implicated in memory 12'22'34 the present results could explain the

learning deficits found after perinatal anoxia is.

MATERIALS AND METHODS

Female rats of the Sherman strain were fed on a standard diet ad libitum. Gestational age was ascer- tained by allowing access to the male on one single

* Association Claude Bernard. Correspondence: A. Represa, INSERM U29, Maternit6 de Port-Royal, 123 Boulevard de Port-Royal, 75014 Paris, France.

0165-3806/89/$03.50 t~) 1989 Elsevier Science Publishers B.V. (Biomedical Division)

72

occasion. The uterine blood supply was restricted

according to the method of Wigglesworth 36. Briefly,

on the 17th day of gestation, a laparotomy was

performed under ether anesthesia, the uterine horns

were exposed and both the uterine artery and vein

were ligated at the lowest part of one horn. The

closer a fetus was to the ligation, the smaller its

weight; near the ligation some fetuses were even

resorbed completely. At birth, an animal was con-

sidered as being I U G R when its body weight was

reduced by at least 40% as compared to controls

from the same litter. Using this criterion, approxi-

mately 35% of the mothers undergoing laparotomy

had offspring showing IUGR. Eighty-seven % of

these animals were viable and showed no malfor-

mation. After birth, 4 control and 4 I U G R pups

were kept with lactating mothers until weaning. For

the whole period, all animals had free access to

standard chow and water. Preliminary experiments

indicated that there was no sex-related difference in

ChAT activity so both males and females were used

at random. The control offspring of laparotomized

mothers showed ChAT activities similar to those

found in offspring of control rats.

(-'hA T assay

At P6, 9, 12, 15, 22 and 60 days, the animals were

sacrificed between 10.00 and 12.00 h. The brains

were rapidly removed and placed on ice. The

cerebellum, hippocampus, hypothalamus, septum

and striatum were dissected. The tissues were

weighed and frozen at -80 °C for subsequent assay.

Under these conditions, the enzyme activity re-

mained unaltered for several months. In some cases,

each hippocampus of 14-day-old I U G R or control

rats was removed and cut with a razor blade into 8

slices. The CA1, CA3 and FD were dissected,

pooled and frozen for enzyme assays.

For measurements of ChAT the technique de-

scribed by Fonnum ~I was used. The assay mixture

contained in a final volume of 0.1 ml, 300 mM NaC1,

50 mM sodium phosphate buffer, pH 7,4, 0.5% v/v

Triton X-100, 8 mM choline chloride, 20 mM EDTA,

1 mM physostigmine and approximately 1 mg of

tissue homogenized in E D T A (10 raM). Reaction

tubes in triplicate were incubated in a shaking bath

for 30 rain at 37 °C. The reaction started by addition

of [14C]acetyl CoA (CEA, spec. act. 40-60 mCi/

mmol) to reach a final concentrat ion of 0.4 raM. In

TABLE I

Evolution of the weight o[ body, [orebrain, cerebellum, hippocampus, hypothalamus, septum and striatum

Each value expressed in g and mg wet weight _+ S.E.M. was the m e a n of 6-10 individual determinations in the IUGR (H) and control (C) groups.

Body Forebrain Cerebellum Hippocampus Hypothalamus Septum Striatum (g) (g) (rag) (rag) (rag) (rag) (rag)

C H C H C H C H C H C H C H

P6 15.5+ 10.1+ 0.481+ 0.41-+ 50.3-+ 33.3_+ 56.1_+ 49.8_+ 40.0_+ 43.6+ 17.6-+ 18.3+ 41.4-+ 0.8 0.9* 0.03 0.06* 3.6 2.9* 9.3 8.1 7.4 9.6 1.7 1.2 7.7

P9 20.0+ 11.8_+ 0.654+ 0.58+ 103.6+ 96.0_+ 73.4+ 72.3_ + 54.5± 58.6+ 32.6± 34.8+ 56.7_+ 0.3 0.2* 0.01 0.03 4.0 4.6 3.4 2.7 6.3 4.1 3.5 2.3 3.6

P12 29.1_+ 20.3_+ 0.850 + 0.77± 140.0_+ 115.0_+ 73.5+ 72.8_+ 41.6± 49.5± 29.3± 35.6+ 47.6+ 0.7 0.5* 0.02 0.01 7.1 8.9 4.2 6.3 4.3 6.3 3.5 4.9 3.9

P15 32.9_+ 22.0+ 0.980_+ 0.83_+ 173.9_+ 137.2_+ 100.0_+ 79.5_+ 50.1± 47.2-+ 33.9± 35.5_+ 61.0 + 0.8 0.6* 0.05 0.07* 5.9 7.4* 5.3 4.5 5.1 5.7 5.7 6.2 5.5

P22 53.1+ 32.1-+ 1.076_+ 0.90_+ 236.8+ 218.0_+ 135.4_+ 134.0_+ 96.6_+ 83.8_+ 55.8_+ 64.0_+ 96.0_+ 1.7 1.4" 0.06 0.05* 3.3 5.1 7.1 7.6 4.8 8.1 4.4 8,3 11.4

P60 242.0+ 141.5-+ 1.310-+ 1.16+ 289.0-+ 258.0_+ 140.0± 148.0_+ 99.2_+ 89.8± 58.0_+ 64.0__+ 111.2_+ 29.8 14.1" 0.09 0.05 11.0 13.0 3.6 8.0 8.9 6.5 4.2 6.7 9.7

40.2_+ 8.5

54.5 + 2.5

44.1___ 7.0

56.0_+ 8.3

101.0_+ 5.8

111.2+__ I0.8

*P < 0.05 when compared with values from control rats.

blank tubes, bidistilled water was added to the mixture in place of homogenate. After incubation for 30 min, reaction tubes were dropped into scintillation vials containing 4 ml sodium phosphate buffer, pH 7.4. A mixture of 2 ml acetonitrile, 10 mg Kalignost and 10 ml of scintillation liquid was added to each vial. After extraction of the [14C]ACh into the organic phase by gentle shaking for 1.2 min. The radioactivity content was measured in a liquid scintillation apparatus. Formation of radioactive acetylcholine was linear with incubation time and quantity of homogenate used. ChAT activity was expressed as nmol/mg/h. Protein was determined according to the method of Lowry et al. TM using BSA as standard. Differences between experimental and control data were considered to be significant with P values under 0.05 (ANOVA).

Autoradiographic experiments Controls and IUGR rats were sacrificed by de-

capitation at P6, 8, 12, 14, 21, 30 and 60 and the brains rapidly removed and frozen in isopentane at -50 °C. The procedure for autoradiographic study of muscarinic receptors was performed according to a previously described technique as with minor modifications. Briefly brain sections (20/zm) were prein- cubated for 30 min at 25 °C in 50 mM Tris-HCl to remove endogenous competitive ligands. Then they were incubated at room temperature in the same buffer containing 1 nM [3H]quinuclidinyl benzilate ([3H]QNB) (NEN, spec. act. = 46 Ci/mmol) for 2 h. Alternate sections were incubated in the presence of 100 ~M atropine sulphate to determine the non- specific binding. After rinsing and drying, the slices were put in an X-ray cassette and apposed to a 3H-sensitive film simultaneously with internal stan- dards (Amersham). Five IUGR and 5 controls rats were studied at each age. For each quantification at least 14 sections/animal were analyzed by a com- puter assisted image analyzer (Imstar).

Acetylcholinesterase staining procedure The distribution of acetylcholinesterase was stud-

ied by the procedures of Karnovsky and Roots 17 and Shute and Lewis 3°. The animals were perfused intracardially with a 4% buffered formaldehyde solution. Brains were cut (20/~m) with a cryostat and incubated with acetylthiocholine. Ethopropazine (30

73

/zM) was used to inhibit the non-specific cholinester- ase. Positive staining was eliminated when acetylthiocholine was omitted from the incubation mixture. At least 3 IUGR and 3 control rats were studied at each P4, 6, 8, 10, 12, 14 and 30.

120

100

T, eo

6 0

<• 40

2O

• cont ro l C e r e b e l l u m o iug r

6 9 12 15 22 610

P o s t n a t a l a g e

120

tO0

>,

>_. 80

"6 6C

,~ 4C

2 ( I

0

• control Hypotha lamus o iugr

Postnata l a g e

I

60

120

100

~ .o

' ~ 6O

~ 4 0

2 0

Str la tum

• control o iugt

/t~/"

, , t , 6 9 12 15 22 610

Postnata l age

Fig. 1. Developmental changes in ChAT activity in 3 brain regions: cerebellum, hypothalamus and striatum in IUGR and control rats. Each point was the mean + S.E.M. of 6-8 independent determinations of ChAT activity in nmol of ACh formed/rag protein/h. *P < 0.05 when compared with values of age-matched control rats.

74

RESULTS

L Effects of undernutrition on growth As noted previously 31, I U G R rats showed a

reduction of 40-50% in body weight at birth. The reduction was still as large at 60 days postnatal even though they were fed in the same way as control animals (Table I) and persisted into adulthood whatever the rearing conditions.

As shown, in Table I, the weight of the forebrain in I U G R rats was markedly less affected than was the body weight since only about a 15% reduction was found compared to control rats. Furthermore, postnatal development was associated with some recovery, since the brain weight of I U G R reached 90% of that of control rats. The cerebellum also was of significantly lower weight in I U G R than in control rats (30%). No significant reduction was observed in the other brain areas between I U G R and control rats during the period studied.

II. Developmental changes in ChAT activity in IUGR and control rats

In all brain structures with the exception of the

cerebel lum, there was a progressive rise in enzyme

activity during deve lopment as a function of age in

controls. As shown in Fig. 1, in the hypothalamus

and the s t r ia tum we found a progressive rise of

C h A T activity to reach adul t values during the three

weeks pos tna ta l age; the enzyme activity at weaning

reached 3.6- to 4.5-fold the values obta ined in the

youngest rats. In contrast , the ChAT activity de-

120 S e p t u r n

• con t ro l

100 o iugr

• ~ 8 0

g "~ 60

.1~ 40 0

20

I r I I I I I 6 9 12 15 22 28 60

P o s t n a t a l a g e

Fig. 2. Developmental changes in ChAT activity in the septum in IUGR and control rats. Each point was the mean + S.E.M. of 6 independent determinations of ChAT activity in nmol of ACh formed/mg protein/h. *P < 0.005 when compared with values of age-matched control rats.

creased in the cerebel lum (Fig. 1A) during postnatal

deve lopment and reached adult levels at PI 2. In the

septum (Fig. 2), the rise of ChAT activity w~s

typically bi-phasic with an initial peak at P9, fol-

lowed by a decrease at P12 and a fur ther rise to

reach adult values at P22. The bi-phasic curve was

consistently found in every control case (n = 6, f' 0.005). This initial peak of ChAT activity was

e l iminated in the I U G R rats, since the transient

increase at P9 was not present . The ChAT activity

levels in the hypotha lamus were significantly lower

in the I U G R rats at P22 (44.6 __ 6.9 nmol) than in

controls (26.1 _ 2.1 nmol for I U G R , P < 0.05). In

the cerebel lum, str iatum (Fig. 1) and total hippo-

campus (Fig. 3 top), no significant difference was

found between the two groups.

120

100

.-~ 8¢

<~

6C

ac 40 c)

2O

• con t ro l H i p p o c a m p u s o lug r

- - - - - - -T 6.

f 6 9 12 15 2=2

P o s t n a t a l

6'0' a g e

ChAT Activity -14 days-

50 45 40 35 30 25 20 15 10 5 0

CA1 CA3 FD

Fig. 3. Top: developmental changes in ChAT activity in total hippocampus in IUGR and control rats. Bottom: ChAT activity in CA1, CA3 and FD in IU(3R and control rats at 14 days. Each bar or point is the mean + S.E~M. of 6 independent determinations of ChAT activity in nmol of ACh/mg protein/h. *P < 0.05 when compared with values of control rats.

Since the hippocampus is composed of well defined subregions with different developmental time courses, we determined the ChAT levels in three specific regions of the hippocampus: CA1, CA3 and fascia dentata (FD) in 14 days IUGR and control rats. In control rats (Fig. 3 bottom), the ChAT activity in CA1 was not different from that in CA3 but the enzyme levels in FD were higher than in either CA1 or CA3 (P < 0.05). The IUGR group showed a similar regional distribution of ChAT activity, i.e. the highest levels were in FD. The levels were, however, significantly lower in each of the 3 regions in IUGR than in the control group. The regional analysis reveals therefore a difference between control and IUGR rats which was not found in total hippocampus.

CONTROL

75

IlL Developmental changes of [3H]QNB binding sites in the hippocampus of IUGR and control rats

In agreement with earlier studies 7'2° we found little QNB binding at P6 (Fig. 4 and Table II). The densities of these sites significantly increases by 70% (P < 0.001) between P6 and P8; thus, at P8 (Fig. 4) Ammon's horn showed already the laminar distribution of muscarinic receptors characteristic of adult hippocampus. The highest density of QNB binding sites at this age was found in the Ammon's horn, in particular in the stratum oriens of CA1 and CA3. A further increase in density in Ammon's horn was conspicuous until P14, when adult values were reached. In contrast in the dentate gyrus the maturation of muscarinic binding sites extended over the end of the 3rd week of postnatal development. At

IUGR

P6

P8

P30

Fig. 4. Autoradiographs depicting the maturation of muscarinic binding sites in the control and IUGR rat hippocampi. Note that at P8 the density of QNB binding sites in fascia dentata (FD) and CA3 is lower in IUGR than in control rats.

76

P21 the values of QNB binding sites were higher in

the denta te molecular layer than in A m m o n ' s horn.

In I U G R hippocampi (Fig. 4 and Table II) the

densi ty of QNB binding sites at P8 was significantly

lower in CA3 and denta te gyrus (but not in CA1)

when compared with control values. Nevertheless ,

the normal adult levels were reached as in control

group at P14 and P21 in the two zones respectively.

IV. Development o f acetylcholinesterase (ACHE)

staining in normal and I U G R rats

In keeping with ear l ier studies 16"19, posit ively

s ta ined fibers and neural e lements were se ldom seen

at P4-6 . There were no signs of laminat ion in the

rostral h ippocampus and AChE-pos i t ive e lements

were comple te ly absent from the caudal hippo-

campus. A few AChE-pos i t ive cells were seen in the

septum. The pat tern of A C h E staining in the control

h ippocampus was laminated at P8-10 with a dense

fiber stain in s t ra tum oriens of CA3 and CA1 (Fig.

5A). The hilar zone also had posit ively stained fibers

at P8; several A C h E neurons could be seen in the

TABLE II

Densities of specific [3H]QNB binding sites (fmol/mg tissue + S.D.) in three regions of the hippocarnpus of developing control (n = 5 at each age) and 1UGR (n = 5 at each age) rats

FD, fascia dentata.

CA1 CA3 FD Oriens Oriens Molecular

P6 Control 130 + 14 127 + 20 110 __+ 30 IUGR 108 + 20 116 + 24 122 +_ 27

P8 Control 482 + 16 432 + 44* 338 __+ 19" IUGR 437 + 20 280 _+ 14 128 + 13

P12 Control 529 + 13 424 + 46 336 _+ 57 IUGR 589 + 50 475 + 23 323 _+ 38

P14 Control 678 + 70 561 _+ 31 542 + 43 IUGR 676 + 45 534 + 60 538 + 76

P21 Control 697 + 30 557 + 46 647 + 58 IUGR 662 + 25 578 _+ 51 661 + 60

P30 Control 670 + 27 582 + 35 639 + 27 IUGR 660 + 21 583 + 61 574 + 38

P60 Control 625 _+ 22 556 + 45 648 + 29 IUGR 653 ___ 27 560 + 31 679 + 40

* Indicated P < 0.005.

polymorph-cel lu lar layer of fascia denta ta as well as

in the stratum radiatum and oriens of A m m o n ' s

horn. In the septum and diagonal band, because of

the intensity of fiber stain, it was difficult to visualize

AChE-pos i t ive neurons (see Fig. 5E). The adult

pat tern in the h ippocampus was reached al PI4 (Fig.

5C).

We found no clear-cut effect of I U G R on the t ime

course and distr ibution of A C h E staining. Thus

A C h E stained fibers appear in the h ippocampus at

the same t ime in the same fields in I U G R and

control rats. Figure 5B,F clearly shows that at P8

both h ippocampus and septum of I U G R animals

have a similar pat tern to that observed in control

cases at P8. Because A C h E staining is ra ther a

quali tat ive procedure we cannot exclude a quanti ta-

tive difference between I U G R and control rats in

A C h E contains.

DISCUSSION

The present observat ions are in agreement with

ear l ier studies on the deve lopmenta l changes in

cholinergic markers in the sep to-h ippocampal system

(see ref. 7, 20 for QNB, 16, 19 for A C h E and 16, 23,

28 for CHAT). However our study addi t ional ly

reveals that the deve lopment of ChAT activity

follows, in the septal region, a bi-phasic curve with

a transient rise between P6 and P9. It is unlikely that

these changes are due to dilution phenomenon

caused by growth since the weight of the septum did

not increase during the second postnata l week (Table

I). The transient ChAT decrease between P9 and

P12 is l ikely due to natural cel lular death or a

reduction in enzyme expression. I t is interest ing to

note that a similar t ransient over-expression of

t ransmit ter markers has been descr ibed during de-

ve lopment for G A B A 26, ChAT immunoreact iv i ty 4,

and for exci tatory amino acids binding sites 14'25"32. In

paral lel , the matura t ion of muscarinic binding sites

reveals that in CA1, CA3 the adult values were

reached at P14, whereas in the fascia den ta ta the

adult values were reached at P21. This is l ikely due

to the t ime course of granular cells matura t ion ,

which in contrast to the pyramida l neurons of C A 1 - C A 3 , essent ia l ly develops postnatal ly 2'6'27. Se-

vere prote in restr ict ion during gestat ion and lactation leads to impor tan t and long lasting diminut ion

A C O N T R O L IUGR

77

P8 P8

E ADULT ADULT

F

P8 P8 Fig. 5. Coronal sections through septum (E,F) and hippocampus (A-D) to compare the distribution of AChE stain at P8 and adult in control and IUGR rats.

78

of ChAT in different brain areas ~'232s. Compared to

this model, the mothers of the I U G R rats are fed

normally during gestation and lactation so that

indirect influence of nutritional or endocrine imbal-

ance can be excluded. Moreover, this model has clinical relevance since it corresponds to a situation encountered in gestational pathology 24.

Modifications in neurotransmitter metabolism

have been previously reported in I U G R rats ~. Thus,

the levels of serotonin were increased in the fore-

brain and the brainstem of the I U G R in relation to

pair-aged control rats until weaning. This rise of the

neurotransmitter was associated with a parallel

increase of its precursor, tryptophan. In the present study we show that cholinergic markers are also

affected. The most consistent effect of the procedure was found in the hypothalamus in which the ChAT

levels were lower in I U G R rats than in control rats

from P15 until after weaning. In addition, the ligation clearly altered the developmental sequence

of the septo-hippocampal cholinergic system; partic-

ularly in the septum, in which the ligation abolished the first peak of ChAT activity (between P6 and P9).

Our findings for the development of the ChAT

activity, were correlated with a late development of

the muscarinic receptors in I U G R hippocampus.

The density of receptors present in CA3 and FD at

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ACKNOWLEDGEMENTS

We are grateful to G. Charton and M, Kais for

technical assistance and to S. Guidasci for photo-

graphs.

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