Heterogeneity of muscarinic cholinergic receptors in the developing human fetal brain: regional distribution and characterization

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Publishers

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Heterogeneity of muscarinic cholinergic receptors in the developing human fetal brain: regional distribution and characterization Fulvia Gremoa, Marco Palombaa, Anna Maria Marchisio”, Costantino Marcellob, Maria Luisa Mulasa and Silvia Torelli” “Institute of Human

Anatomy and bZnstitute of Obstetrics and Gynecology, School of Medicine, Cagliari, Italy Accepted

for publication

6 January

1987

The ontogeny of muscarinic cholinergic receptors has been studied in different regions of the human fetal brain. For a comparison, the same study has been carried out on newborn and premature brain. Regarding on the areas examined (frontal cortex, cerebellum, hippocampus, thalamus and basal ganglia) either an increase or a decrease of receptor density during gestation was observed. Thus, the ontogeny of the receptors follows a different pattern in areas which differ in function, cholinergic innervation and embryological origin. However, in all the regions the affinity of the binding site for the ligand [3H]quinuclidinyl benzilate [3H]QNB was very similar to that reported for muscarinic receptors from adult mammalian brain. Data obtained from agonist binding (acetylcholine and carbachol) revealed the presence of a high (H)- and a low-affinity binding site (L) from 10 weeks of gestation. The selective antagonist pirenzepine (PZ) also distinguished two different muscarinic receptor subtypes, which however had higher affinity than that seen in adult brain. In conclusion, during ontogeny, the muscarinic acetylcholine receptor shares some but not all of the pharmacological properties shown in the adult brain. muscarinic cholinergic

Address for correspondence: Cagliari,

receptors;

Fulvia

fetal human brain; heterogeneity

Gremo

M.D.,

Institute

of Human

Anatomy.

Via

Porcell

2. 09100

Italy.

Abbreviations: H, high-affinity

binding site; L, low-affinity

binding site; PZ. pirenzepine;

lidinyl benzilate.

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in Ireland

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QNB,

quinuc-

166

Introduction* In the adult, muscarinic cholinergic synapses are involved in a variety of higher brain functions such as sleep, avoidance behaviour, learning, memory, mood and attention [7,16], as well as neuroendocrin regulation in the hypothalamus 1111and modulation of extrapyramidal control of movements 1181. Several authors have reported the presence of muscarinic receptors in specific areas of the adult human brain, such as cerebral cortex [10,12,32,39], amygdala [lo] and hippocampus [31]. However, few data are available on the ontogenesis of muscarinic receptors in the fetal human brain [1,4,35]. Moreover, there are no reports on their regional distribution and their characterization during gestation. Muscarinic receptors are known to be heterogeneous (for reviews see Refs. 2, 6 and 9) and different receptor subclasses presumably subserve different functions. In the fetus, information about the maturation of cholinergic circuits would increase, for example, our knowledge of the maturation of behavioural states (sleep and waking), fetal movements and control of heart rate, which have so far been studied only from a physiological point of view [8,30]. We have therefore examined the distribution of muscarinic cholinergic receptors in different brain regions, namely frontal cortex, basal ganglia, hippocampus, cerebellum and thalamus in fetuses from the 10th to the 32nd week of gestation. The findings have been compared with those obtained from a newborn infant.

Materials and Methods Collection of human fetal brains Human fetuses were obtained from either spontaneous or medically terminated pregnancies. In the latter case, the delivery was induced by intravenous administration of Prostin F, alpha (Upjohn). The fetuses were placed in ice postmortem, the brains dissected out and separated into frontal cortex, cerebellum, hippocampus, basal ganglia and thalamus. Tissues were stored at -70°C until assay. The age of the fetus was determined from the following criteria: (1) menstrual history of the mother; (2) clinical assessment by the obstetrician; (3) ultrasound analysis. If degenerations, hemorrhages and abnormalities were’observed, the samples were rejected. Dissection of cerebral areas Brain regions were separated on the basis of their morphological appearance, in agreement with Netter [29]. Since at lo-12 weeks the frontal cortex and the other areas are extremely immature, the whole cerebral hemispheres were used, after dissection of the midbrain and cerebellum. * Part of these data have been presented in abstract form in ref. 25.

167

Two premature infants (28 and 32 weeks of gestation) were also examined. The dissection of these was carried out 24 h after death, as well as in the single case of the newborn, which had died from general sepsis. Preparation of subcellular fractions

Tissues were homogenized 1:20 (wt./vol.) in 0.32 M sucrose +Tris (hydroxymethyl-aminomethane) 5 mM (pH 7.4) with five strokes in a Teflon-glass homogenizer. The homogenates were centrifuged at 1500 x g for 15 min. Pellets were discarded and the supernatants centrifuged at 40 000 x g for 20 min. After two washes the pellet was resuspended in sodium phosphate buffer (50 mM, pH 7.4) by sonication. Binding assay

Binding studies were performed according to the method of Sugiyama et al. [38] with slight modifications [26]. Kinetic experiments performed with human brain membranes (not shown in the text) demonstrated that at 0.8 nM [sH]QNB binding reached equilibrium by 45-60 min. Thus, samples were diluted to a final concentration of 0.2-0.4 mg prot./ml and incubated for 60 min. at 25°C with 8-12 different concentrations of [3H]QNB (New England Nuclear, spec. act., 33.1 Ci/mmol). The range of concentrations varied from 0.1 to 7 nM. The final volume was 1.0 ml. Specific binding was defined as the difference between total binding and binding in the presence of 50 nM unlabeled QNB. Non specific binding under these conditions was less than 5% of the total [3H]QNB binding. After incubation, samples were filtered through Whatman glass fiber filters (GF/C) under vacuum and rinsed twice with 5 ml of cold buffer. Filters were placed in counting vials with 10 ml of Instagel (Packard) and counted in a liquid scintillation counter (60% efficiency). Protein assay

Proteins were assayed by the method of Lowry et al. [231. Data analysis of competition curves

Competition studies were performed in the presence of 0.2 nM i3HlQNB with different concentrations of atropine (Sigma), pirenzepine (a gift from Boehringer), carbachol (Sigma), and acetylcholine (Sigma). When acetylcholine was used, each tube received 50 ~1 of eserine (Sigma) (1 x lO_” M), to inactivate endogenous acetylcholinesterase. Each competition curve was constructed with lo-12 points determined in duplicate. The results of two experiments were then averaged and the means subjected to data analysis, according to Munson and Rodbard [271. In brief, using an Apple II Europlus computer, data were analyzed by a nonlinear leastsquares curve-fitting technique using a generalized model for complex ligand receptor interaction to determine ligand dissociation constants for the binding sites. The data

168

were analyzed using a model for either one or two binding sites in the presence of competitors and the improvement in the goodness of fit on addition of a second class of receptor state was tested. One- or two-state fit is accepted only if there is a statisticaly significant improvement in the fit. According to Birdsall et al. [3,15] the values of agonist binding obtained by agonist-antagonist competition are representative of direct agonist binding.

Results Protein content

Protein content ranged between 1% and 2% of fresh tissue. It did not significantly change during development in the cerebral cortex and in the cerebellum. In diencephalon, basal ganglia and hippocampus a small increase (from 1% up to 2.5%) was measured. Binding data

Representative Scatchard plots of saturation curves are shown in Figs. 1 and 2. Apparent Kd value did not undergo significant changes during development of brain areas we considered (see also Tables I and II). On the other hand, the concentration of receptors changed with brain maturation, even if their relative distribution among the different areas was quite similar from the earliest ages to that of the adult. In the frontal cortex (Fig. 1 and Table I) a steady increase of muscarinic receptors was found between lo-12 weeks of gestation and the 21st week. Moreover the receptor concentration almost doubled after birth. In the thalamus (Table II), the concentration of receptors increased from 18-21 weeks to 28-32 weeks, but decreased after term. In the basal ganglia, the concentration of receptors tended to remain more constant. The hippocampus and the cerebellum showed a decrease, so that in the newborn cerebellum the concentration was extremely low (see also Fig. 2). Analysis of competition curves

Atropine was a potent displacer of [SHIQNB in all the regions at all the ages examined (not shown). Its ICso ranged between 2 X IO-“’ and 3 X 10-’ M, without showing significant differences with maturation. Moreover, computer analysis of the displacement curves never identified more than one binding site. On the other hand, both the agonists acetylcholine and carbachol and the selective antagonist pirenzepine bound to two binding sites. Agonist binding

As shown in Table III, at all the ages and in all the areas acetylcholine bound to a high-affinity (H) and to a low-affinity (L) binding site. In the basal ganglia, the

B/F 1900 If300 1700

*Newborn

1600

A Prmture

1500

ml9 - Heeks ?? lO-weeks

1400 1300 1200 1100 1000 900 800 700 600 500 400 300 200 100 0

Fig. I, Scatchard plot of sH-QNB binding in frontal cortex of human fetuses at different from a typical experiment. each point representing the mean of duplicates. The number shown to increase, whereas Kd values remains approximately constant. Ses also Table

TABLE

ages. Data are of receptors is 1.

I

[‘H]QNB

binding

in human

fetal cerebral

cortex during development

Age (weeks)

& (X IO-’ M)

R,,,

10-12” (4) IX-21 (6)

0.12 * 0.03 0.I.s + 0.01

72 * 7 I lo* IP 187zt6 3-W

28-32 (prem.) Newborn (2)

(3)

0.35 + 0. I7 0.19

Values are the mean of several experiments experiments. each run in duplicate. ;’ The whole cerebral

hemispheres

h P < 0.05

vs. ICI2

weeks.

c P < 0.02

I%2 I vs. nremature.

? S.E.

were used.

The numbers

in parenthesis

(fmolimg

represent

prot.)

the number

of

170

*21w&s ??Premtwe

??Naborn

0

20

40

60

80

100 120

140

160

180

200 220

240

260

280 300

Fig. 2. Scatchard plot of [3H]QNB binding in human fetal cerebellum, at different ages. Data are from a typical experiment, each point representing the mean of duplicates. The number of receptors strongly decreases with maturation. See also Table II.

affinity of both H and L remained basically unchanged, but the relative proportion of L was increased in the prematures and in the newborn. Both thalamus and cerebellum showed a similar pattern: the affinity and the relative proportion of H and L remained unchanged during gestation, but in the newborn the affinity of both was decreased. On the contrary, in the frontal cortex the affinity of H was increased, but not the affinity or the proportion of L. A striking change was found in the hippocampus: in the newborn the relative proportion of H and L was reversed in comparison to the earlier stages and the affinity of both was increased. Carbachol binding (Table IV) also revealed the presence of two binding sites. As already shown with acetylcholine, in the basal ganglia the relative proportion of H and L remained approximately the same. On the other hand, the thalamus showed a decrease in the percentage of H. Moreover, in the newborn hippocampus the affinity of H was significantly decreased in comparison with that at the 21st week. In the frontal cortex a decrease of H and an increase of L was observed. PZ binding

PZ, a compound with selective antimuscarinic activity, has been shown to distinguish different subclasses of muscarinic receptors in rat brain 1141.Apparently there

Kd (xlOP

H

6’)

6’) 6X 0.2 6X I.4

28 5.6

17.x

have been used. binding site; L, low-affinity

receptorswerecalculatedfromcomputeranalysisof

I4.X

a The whole cerebral hemispheres H, high-affinity -1 not determined;

31

6.6 4.2

71 32 1.7

4.x I .4

2.x

4.2 2.5

L ICS” (x10-5M)

45 10.3

4.5

37 4.5

%

site.

displacementcurves(see

32 14.2

72

67

H “h KS” (x10-‘M)

binding

L ICS” (xlW5M)

33 6.0

%

Cerebellum

X.0 I.1 3.7 4.7

Kd (X IOP M)

Basal ganglia

7.1 2.7 6.8 4.1

L ICSO (x10-‘M)

text for explanations).

6.5 9.1 1.3

H IC5” (x10-‘M)

56 17.0

52 52 44 46

%

1.3 6.3 5.2

L IC50 (x10-5M)

34 12.7

49 44 24

%

$G!imgprot.)

Basal ganglia

each run in duplicate.

44 26.0

48 48 55 54

H IC50 % (x10-’ M)

Thalamus

of experiments.

BIn.,\ (fmolimgprot.)

55 23.0

63 55 55

%

the number

Kd (xIOY”M)

Thalamus

regions

represent

cerebral

in parenthesis

&X,X (fmolimgprot.)

from different

IY.4

ICSO (xIW’M)

67.5

H

% Ic‘,, (x IO-’ M)

16.8 3.1

2.7

membranes

The numbers

Hippocampus

brain

-C S.E.

~lWVM)

Cerebellum

gestation

L

37 S 12.5

%

to fetal human

IO.‘)

I<‘50 (xIV’M)

lCsoandpercentagesof

Newhorn

(prcmat.)

I(!-138 IV 21 2X-32

(\*Wh\)

Frontal correx

of acetylcholine

Binding

A@e

III

experiments

M)

during

$%/mpprot.)

hrain regions

Hippocampus

in human

are the mean of several

binding

II

TABLE

Values

Newborn

(premat.)

3-32

Ix-21

(weeks)

Age

[‘H]QNB

TABLE

66

51 55 76

%

28.9 21.2 6.5 6.6

(x1(~-7~)

H G.0

V

10

(x

(~10~~

3.2

Cerebellum

60 -

- 69 2.4 70 2.9

H Go % (x10-‘M)

4x

0.6

Hippocampus

_

_.

- 46 3.4 37 8.4

% (xlO+M)

8.2

-

26

8.3

15 18.7 8X 22.7

-

14 42.0

25 95.0 12 3.0

-

46

11.8

40 59.3 42 1.9

H L Go Go (x10-9M) % (x10-‘M)

regions

2.0

0.7 2.8 -

L

28 11.2

- 65 1.4 40 6.6 - -

% (x10-JM)

Go

curves

54

59

5.3

25 10.2 31 12.1 30 1.3

41

11.0

75 is.0 63 49.9 70 13.1

82 14.5

71 8.5 70 13.3 70 6.1

IC50

67 50 74

18

29 30 30

% (x10-9M) % (x10-7M) %

H IC50

L

33 25.3 50 4.9 26 15.0

Basal ganglia

12 -

35 21.3 60 0.1 2.3

(see text for explanations).

10.0

3.0 60 16.4 58 0.3

% (x~O-~M) % (x10-‘M)

L Go

H IC50

Thalamus

Go

% (x10-5M) %

L

H IC50

Basal ganglia

% (x10-‘M)

--

curves (see text for explanations).

-

54 63

% (x10-‘M)

H Go

L

-

Thalamus

lC50

were calculated from computer analysis of displacement have been used. binding site; L, low-affinity binding site.

S2

S6 52 62 7.3 30 19.4

cerebral

Cerebellum

from different

H L % ICSO Go (x10-‘M)% M) (x10@‘M) %

IC5,, and percentages of receptors * The whole cerebral hemispheres -, not determined; H, high-affinity

Newborn

44 i.54 48 14.X 38 6.2 70 6.9

IO-’

L % IQ.,,

M)

12.7 11.6

40 26.8

31 30

L Go % (x10-5M)

to fetal human brain membranes

H lG0

Frontal cortex

19 42.5 21 3,s 28-32 6.4 (Premat.)

l&13-

Age (weeks)

Binding of pirenzepine

TABLE

-1 not determined;

76 65.9

57 -62 IO 6.0 7.5 3.7

H Go % (x10-‘M)

Hippocampus

of receptors were calculated from computer analysis of displacement hemispheres have been used. H, high-affinity binding site; L, low-affinity site.

B The who!e cerebral

ICSo and percentages

41.8 30.9 31.3 20.9

L Go (x10-“M)

24 21.5

43 38 30 25

%

Frontal cortex

(premat.) Newborn 14.3

10-13’ 19 21 28-32

(weeks)

Age

Binding of carbachol to fetal human brain membranes from different cerebral regions

TABLE IV

173

% 100 -

I -9

-9 log-Lc.

I

I

I

-9

-5

(Ml

Fig. 3. Competition curves of [3H]QNB (0.2 nM) by PZ in human frontal cortex of lO-12-week-old fetus (squares) and newborn (circles). Data are from a typical experiment, each point representing the mean of duplicates. Abscissa = concentration of the competing drug; ordinate = percentage of r3H]QNB binding inhibition. Computer analysis of the curves indicates the presence of Ivf, and Mz PZ binding sites in both. A decrease in affinity is shown in the newborn. See also Table V.

is no correlation between the subclasses defined by pirenzepine and those distinguished by agonists [3]. As shown in Table V, two subclasses, a high-affinity (M,) and low-affinity (M,), were present in all the areas at all the ages examined. The percentage of M, was slightly decreased in the basal ganglia of the newborn, compared to the fetuses. The thalamus showed important changes, after birth. In fact, the affinity of both M, and M, was only slightly decreased, but M, represented about 30% of the total population until term, after which they predominated. The opposite phenomenon was observed in the hippocampus, where a relative abundance of M, was detected in the fetuses and prematures. In the frontal cortex, a peculiar increase of MJ was detected in the premature, whereas during gestation a tendency for the affinity of both M, and M2 to increase was observed (Fig. 3j.

Discussion Muscarinic receptor binding has been considered an expression of cholinergic maturation in different systems [40]. Thus, the presence of muscarinic receptors at 10 weeks of gestation, with affinitjl very close to that in adult brain, could indicate

174

precocious maturation of both cholinergic systems and muscarinic receptors in the human brain. Aguilar and Lundt [l] have also concluded from their data that the ontogeny of muscarinic receptors in the human brain takes the form of a build-up of a receptor that has very similar kinetic and pharmacological properties to that of the adult receptor. Actually, literature reports show that during development very little differences in muscarinic receptor Kd value exists in rat cerebellum [37], in rabbit brain [42], in rat brain and in spiny mouse [33]. This is not necessarily a pattern common to all species. We have observed [26] in the retina of the chick embryo that the affinity of muscarinic receptors for the ligand significantly increased until the time of synaptogenesis, which occurs around the middle of the embryonic period. Moreover, Levy [2 l] reported that apparent Kd values were very similar in mouse above 8 days, but at the latter age, the muscarinic receptors’ affinity for [“HIQNB was lower. Thus, whether or not an immature stage of the receptor is present in the fetal human brain, 10 weeks of gestation seems to be an already late period for detecting it. Muscarinic receptor distribution and density change during gestation. This presumably reflects the major changes that the fetal brain undergoes before birth [20]. For example, at a relatively advanced stage of gestation, a significant decrease of muscarinic receptor density was observed in the cerebellum. This also occurred in rabbit [42]. In the adult cerebellum muscarinic binding was absent [lo]. Brooksbank et al. [4] also observed peak values in cholinergic parameters such as choline acetyltransferase activity and[3H]QNB binding in fetal cerebellum during gestation, which was followed by a marked fall to the adult level. They attributed it to the fact that archicerebellum develops early. Since it is relatively rich in cholinergic systems [ 171 muscarinic receptors become diluted out by the later expression of the phylogenetically newer, and predominantly non-cholinergic, cerebellar structures. On the other hand, development of cholinergic projections might be responsible for the increase of muscarinic receptors in the frontal cortex. Brooksbank et al. [4] and Ravikumar and Sastry 1351also reported a similar phenomenon in the human fetal cerebral cortex. However, it must be taken into consideration proliferation of glial cells [34], which have been shown to carry muscarinic receptors [5,28,36] and might contribute to a certain extent to the changes in receptor concentration. In the newborn hippocampus the concentration of receptors was much lower than in prematures. This is in accord with Nordberg and Winblad (311, who have also reported a decrease in this a;ea with age. Agonist binding studies showed that muscarinic receptors were heterogenous from an early stage. Both acetylcholine and carbachol bound to H and L. Both H and L were present in all the areas, as also detected in the human adult cerebral cortex 1321. However, in some areas, such as frontal cortex. hippocampus and basal ganglia, the relative proportion of H and L changed with maturity. A similar phenomenon was observed in rat brain [Is)]. In the chick embryo retina, the affinity of H and L for both agonists, as well as their relative proportion changed with age [26]. On the other hand, Nordberg and Winblad 1311observed no such changes of oxotremorine binding in the human hippocampus after birth. This finding could be due to the unsuitability of oxotremorine as an agonist for detecting differences with age or,

175

alternatively, these changes occur only during development. The hypothesis has been advanced that the low affinity binding sites represent the active form of the receptor [3,15]. Therefore an increase of L would constitute a functional maturation. This interpretation is supported by the general tendency towards a decrease in affinity during gestation. PZ distinguished two muscarinic receptor subtypes from the earliest ages in all the areas examined. Garvey et al. [12] also found two muscarinic binding sites in the adult frontal cortex. However the affinity in the adult was much lower than that measured in the fetus. On the other hand, with pH]PZ binding Lin et al. 1221found both M, and 4 receptors in adult human brain. They reported a rH]PZ K, = 5-10 nM, which is extremely close to that we observed with displacement experiments. Moreover, they measured in frontal cortex and putamen about 70% of M, subtype whereas cerebellum was the poorest. Thalamus had about 40% and hippocampus had 60%. We measured in prematures similar values for what frontal cortex, thalamus and basal ganglia were concerned. On the contrary, cerebellum showed in all the ages relatively high content of M, , closer to that reported for rat brain 1241. Hippocampus showed a percentage of M, higher than adult before term. Thus, during development muscarinic cholinergic receptor maturation and distribution follows different patterns in different areas. The fetal muscarinic receptor shares some but not all of the pharmacological properties of the adult receptor, as also suggested by Aguilar and Lunt [l] on the basis of their data on the entire brain.

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