Muscarinic cholinergic receptors in the hippocampus of aged rats: influence of choline alphoscerate treatment

ELSEVIER

Mechanisms of Ageing and Development 76 (1994) 49-64

Muscarinic cholinergic receptors in the hippocampus of aged rats: influence of choline alphoscerate treatment Francesco Amenta *a, Aiping Liu a, Yong-Chun Zeng a, Damiano Zaccheo b aSezione di Anatomia Umana, Istituto di Farmacologia, Universith di Camerino, Via M. Scalzino, 5, 62032 Camerino, Italy blstituto di Anatomia Umana, Universith di Genova, Genova, Italy

Received 24 March 1994; revision received 4 June 1994; accepted 16 June 1994

Abstract

The present study was designed to investigate age-dependent changes of muscarinic M] and M2 cholinergic receptors in the rat hippocampus using radioreceptor, assay and autoradiographic techniques with [3H]pirenzepine and [3H]AF-DX 116 as ligands. The analysis was performed on 2-, 12- and 27-month-old male Wistar rats, considered young, adult and old, respectively. Moreover, the influence of a 6-month treatment with choline alphoscerate on the density and pattern of M~ and M2 ch01inergic receptors was assessed. Choline alphoscerate (L-a-glyceryl phosphorylcholine) is a precursor in the biosynthesis of several brain phospholipids which increases the availability of acetylcholine in various tissues. Muscarinic M~ cholinergic receptors were significantly decreased with increasing age whereas M 2 cholinergic receptors did not show changes. Choline alphoscerate treatment countered, in part, the loss of muscarinic M~ receptor sites in old rats. Light microscope autoradiography revealed a loss of silver grains developed after exposure of sections of hippocampus to [3H]pirenzepine in the stratum oriens of CA] and CA 3 fields in rats of 12 and 27 months in comparison with young animals. Choline alphoscerate restored, in part, the decrease of silver grains noted in old rats. Quantitative analysis of the density of silver grains developed in the cell body of pyramidal neurons of CA~ and CA3 fields processed for the demonstration of muscarinic Mt receptor sites revealed a decrease of these grains in rats of 27 months in comparison with younger cohorts. These findings suggest that the reduction in muscarinic M I sites noticeable between 2- and 12-month rats is probably dependent on the loss of nerve cells * Corresponding author. 0047-6374/94/$07.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved SSDI 0047-6374(94)0 ! 478-5

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F. Amenta et al./ Mech. Ageing Dev. 76 (1994) 49-64

and/or terminals in these hippocampal fields rather than to a reduction of their density per neuron. Treatment with choline alphoscerate increased the expression of muscarinic Micholinergic receptors within the cell body of pyramidal neurons of CA~ and C A 3 fields compared to age-matched control old rats. Consistent with radioreceptor assay data, no changes in the density of muscarinic M2 cholinergic receptors in the animal groups examined were demonstrated by light microscope autoradiography. The possible pharmacological relevance of the increased expression of muscarinic M l cholinergic receptors elicited by choline alphoscerate in the hippocampus of aged rats is discussed.

Keywords: Muscarinic M~ receptors; Muscarinic M2 receptors; Hippocampus; Radioligand binding; Autoradiography; Aging

1. Introduction

The observation that the administration of the muscarinic receptor antagonist scopolamine to healthy young subjects induced a cognitive impairment resembling that found in senile dementia [1] and the subsequent demonstration of a remarkable decrease in choline acetyltransferase activity in the cerebral cortex and hippocampus in Alzheimer's disease (AD) [2,3] contributed to the development of the cholinergic hypothesis of geriatric memory dysfunction [4]. Although the cholinergic system is not the only neurotransmitter system affected in age-related impairment of memory [5], changes in the cholinergic function are strongly implicated in the pathogenesis of learning and memory alterations occurring in the elderly [4,6,7]. Central muscarinic receptors are involved in learning and memory [8]. Cholinergic transduction through muscarinic receptors enhances sensory processing in the cerebral cortex and neuronal activity in the hippocampus [9,10]. Moreover, administration of muscarinic receptors antagonists affects performance of memory tasks in a manner consistent with involvement of cortical and hippocampal muscarinic receptors in memory function [ 11,12]. Age-related changes in the status of muscarinic cholinergic receptors have been reported in both animal and human central nervous systems [13]. In the cerebral cortex and hippocampus of aged rats, large, modest or no decreases in muscarinic binding sites have been described [17-21]. The discepancies in the literature are probably due in part to the different strains, age groups and experimental approaches used for assaying muscarinic receptors. Furthermore, the majority of studies on the aging of cortical or hippocampal muscarinic cholinergic receptors did not characterize the influence of senescence on the subtypes of these sites or have characterized the subtypes of muscarinic cholinergic receptors by using non-selective radioligands in the presence of agonists or antagonists for muscarinic M1 and M2 sites [14-20]. Only more recent investigations have analyzed age-dependent changes in muscarinic M1 and M2 cholinergic receptors using selective ligands for these subtypes of receptors and Long Evans or Fisher 344 rats [21,22].

F. Amenta et al./Mech. Ageing Dev. 76 (1994) 49-64

51

The present study was designed to investigate the influence of aging on muscarinic MI and M2 cholinergic receptors labelled using the selective antagonists [3H]pirenzepine and [3H]AF-DX 116 in the hippocampus of Wistar rats. Moreover, we have assessed whether treatment with choline alphoscerate (L-ot-glycerylphosphorylcholine) is able to counter the age-dependent changes in muscarinic M1 and M2 cholinergic receptors in the hippocampus. Choline alphoscerate is a precursor in the biosynthesis of several brain phospholipids and increases the bioavailability of acetylcholine in the neurous tissue [23]. Choline alphoscerate has been recently shown to counter the age-related microanatomical changes in the rat hippocampus [24] and has been proposed for the treatment of age-related cerebral impairment

[251. 2. Materials and methods 2.1. Animals and tissue preparation

Male Wistar rats (l-month-old) were purchased from Charles River Italy (Calco, Italy) and maintained in temperature controlled animal quarters with water and food ad libitum, for 26 months. Animals were sacrificed at 2 (n = 8) (considered to be young) and 12 (n = 8) (considered to be adult) months of age. Senescent rats of 21 months of age were divided into two groups of 10 animals each. Ten rats were used as controls and did not receive any treatment. The other 10 rats were treated for 6 months with choline alphoscerate dissolved in drinking water at a concentration which allowed for an average daily dose of 100 mg/kg. Animals in the four groups (young, adult, old (27-month-old) and old treated with choline alphoscerate) were weighed, anaesthetized with diethyl ether and decapitated. The brain was removed and divided into the two hemispheres. The right hippocampus was excised and used for the preparation of membranes for muscarinic receptor assay. In the left hemisphere, the hippocampal region was dissected out and embedded in a cryoprotectant medium (OCT, Ames, USA). OCT blocks were frozen in a dry ice/acetone mixture and used for the autoradiographic demonstration of muscarinic receptors. 2.2. Muscarinic receptor assay

The right hippocampi were homogenized in Krebs buffer containing the following substances (in mM): NaCI, 120; MgSO4.7H20, 1.2; KH2PO4, 1.2; NaHCO3, 25; CaCI2, 2.5; KCI, 4.7; glucose, 5.6 (pH, 7.4). Homogenates were centrifuged for 10 min at 49 000 x g and the supernatant was discarded. The pellets were washed twice by resuspending in fresh buffer and recentrifuging. The final membrane pellets were resuspended in buffer. Aliquots of membrane preparations (200-300 mg protein) were then used for muscarinic receptor assay. For labelling muscarinic M1 sites, tissues were incubated with increasing concentrations of [3H]pirenzepine (1-50 nM) for 60 min at 22°C. For labelling muscarinic M 2 sites, tissues were incubated with increasing concentrations of [3H]AF-DX 116 (1-60 nM) for 60 min at 4°C. Non-specific binding was defined by incubating membranes with radioligands in the presence of 1 #M atropine. At the end of incubation,

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F. Amenta et aL / Mech. Ageing Dev. 76 (1994) 49-64

bound radioligands were separated from free ones by rapid filtration using GF-B Whatman glass fibre filtres. Filters were washed four times with 4 ml of ice-cold Tris-HCl buffer (50 mM). Filters were then transferred into scintillation vials which were counted with a Beckman liquid scintillation spectrometer at an efficiency of 40%. Specific binding values were determined by subtracting non-specific from total binding. In a series of preliminary experiments, the pharmacological specificity of [3Hlpirenzepine or of [3H]AF-DX 116 binding for muscarinic Mt and M2 cholinergic receptors was assessed. Protein content was measured by the method of Lowry and co-workers [26], using bovine serum albumin as a standard.

2.3. Light microscope autoradiography OCT blocks were put in a microtome cryostat chamber and equilibrated at -20°C. Parasagittal sections of the hippocampal formation 8 #m thick were cut serially and mounted onto gelatine-coated microscope slides. Sections were air dried and incubated at 25°C in the same buffer above described for receptor assay with a 5-nM [3H]pirenzepine or with a 10-nM [3H]AF-DX 116 concentration to label muscarinic MI and M2 cholinergic receptors, respectively. Incubation with the two radioligands was done for 60 min at 22°C for [3H]pirenzepine and for 60 min at 37°C for [3H]AF-DX 116. Non-specific binding was defined by incubating sections with the radioligands plus 1 ~M atropine. At the end of incubation sections were washed in ice-cold incubation buffer and air dried. Sections were processed for light microscope autoradiography by attaching Ilford K5 nuclear emulsion- (diluted 1:1 with distilled water) coated coverslips to the slides. After 4-6 weeks of exposure in darkness, the autoradiographs were developed in Kodak D-19 and fixed in Agefix. Sections were then stained with toluidine blue or cresyl violet and examined under a Zeiss Axiophot microscope. The density of silver grains developed within the stratum oriens of the fields CA and CA 3 of the hippocampus was assessed by counting the number of grains developed in a 100-ttm 2 area of the stratum oriens and within the nerve cell bodies of pyramidal neurons. Analysis was made at a final magnification of x 400 for measurements of the stratum oriens and of ×600 for counting silver grains developed within the cell body of pyramidal neurons. Silver grain analysis was done on five consecutive sections per animal by two researchers independently without indication of the animal group analyzed. The 100-/zm2 area of the stratum oriens was delimited with a Videoplan (Kontron Zeiss, FRG) image analyzer connected via a TV camera with the microscope. For counts of silver grains in the body of pyramidal neurons, 10 nerve cell bodies per section in the CA1 field (i.e. 50 neurons per animal) and six nerve cell bodies per section in the CA 3 field (i.e. 30 neurons per animal) were examined.

2.4. Data analysis Binding data were derived from the saturation experiments for [3H]pirenzepine and [3H]AF-DX 116. Values for the dissociation constant (Ko) and for the maximum density of binding sites (Bmax) w e r e obtained by computerized analysis using the programme Ligand [27] with the modification introduced by McPherson [28].

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F. Amenta et aL / Mech. Ageing Dev. 76 (1994) 49-64

Results in the text are expressed as means ± S.E.M. of the number of animals indicated. Statistical differences among the four animal groups examined were assessed by analysis of variance (ANOVA), followed by the Duncan's multiple range test or the Newman-Keuls test as post hoc tests. 2.5. C h e m i c a l s

[3H]pirenzepine (specific activity, 72 Ci/mmol) and [3H]AF-DX 116 [112 [[2[(diethylamino)methyl]- l-piperidinyllacetyll5,11-dihydro-6H- yrido[2,3b][l,4]benzodiazepin-6-1] (specific activity, 73 Ci/mmol) were purchased from New England Nuclear (Boston, MA). Other chemicals were obtained from Sigma Chemical Co. (St. Louis, MO). 3. R ~ [3Hl]Pirenzepine and [3H]AF-DX 116 were specifically bound to membranes of the rat hippocampus. The binding was time, temperature (data not shown) and concentration-dependent (Figs. 1,2) belonging to a single class of high affinity sites

fmol/mg protein 1000

8OO

6O0

400

2OO

0

0

10

I

I

I

I

20

30

40

50

[ 3H]-Pirenzepine (nM) Fig. 1. Saturation curve of [3H]pirenzepinebinding to membranes in the hippocampus of rats of 2 months (young). Membranes were incubated with increasing concentrations of [3Hlpirenzepinealone (total binding: O) or plus !/~M atropine to definenon-specificbinding (ll). Specificbinding values (El) were obtained by subtracting non-specificfrom total binding. The points are mean values ± S.E.M. of 3-5 triplicate determinations.

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F. Arnenta et al. / Mech. Ageing Dev. 76 (1994) 49-64

fmol/mg protein 35O, 300 I

250 200 150 100

50 0 0

I

I

I

1

I

I

10

20

30

40

50

60

[3H]-AF-DX 116 (nM) Fig. 2. Saturation curve of [3H]AF-DX 116 binding to membranes in the hippocampus of rats of 2 months (young). Membranes were incubated with increasing concentrations of [3H]AF-DX 116 alone (total binding: O) or plus 1 #M atropine to define non-specific binding (11). Specific binding values (El) were obtained by subtracting non-specific from total binding. The points are mean values ± S.E.M. of 3~5 triplicate determinations.

(data not shown). Using a [3H]pirenzepine c o n c e n t r a t i o n o f 5 n M a n d a concentration o f [ 3 H ] A F - D X 116 o f 10 n M , a p p r o x i m a t e l y 75% o f [3H]pirenzepine (Fig. 1) a n d 55% o f [ 3 H ] A F - D X 116 (Fig. 2) were b o u n d specifically. Since these concentrations caused the d e v e l o p m e n t o f the highest specific/non-specific binding ratios, they were used in subsequent b i n d i n g a n d a u t o r a d i o g r a p h y experiments. The pharmacological specificity o f [3H]pirenzepine a n d o f [ 3 H ] A F - D X 116 binding is consistent with the labelling o f muscarinic MI a n d M2 cholinergic receptors, respectively (data n o t shown). The K d a n d Bmax values for [3H]pirenzepine binding in the four animal groups examined are s u m m a r i z e d in T a b l e 1. A s can be seen, the K d did n o t change,

Fig. 3. Autoradiographic localization of [3H]pirenzepine binding sites in the CA I field in the hippocampus of rats of 2 months (young). Sections were incubated with a 5 nM concentration of [3H]pirenzepine alone (A) or plus I #M atropine (C) to define non-specific binding. A and C are dark-field pictures; B is a bright-field picture of the micrograph A stained with toluidine blue to verify microanatomical details. P, pyramidal neurons layer; o, stratum oriens. The highest accumulation of silver grains corresponding to [3H]pirenzepine binding sites occurs in the stratum oriens of the hippocampus. Magnification: x 85.

F. Amenta et al./Mech. Ageing Dev. 76 (1994) 49-64

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F. Amenta et a l . / Mech. Ageing Dev. 76 (1994) 49-64

Table 1 Influence of aging and of choline alphoscerate treatment on muscarinic M~ and M2 receptor binding in membranes of rat hippocampus Group

Young Adult Old Old-treated

N

8 8 8 8

[3H]Pirenzepine

[3H]AF-DX 116

Kd (nM)

B~ (fmol/mg protein)

Kd (nM)

Bmax (fmol/mg protein)

15.4 4- 0.04 15.8 4- 0.07 15.6 4- 0.05 15.7 4- 0.06

515 453 325 385

19.2 ± 0.04 19.8 4- 0.06 19.9 4- 0.05 19.4 ± 0.07

175 4- 9.3 186 4- 7.5 195 4- 8.2 184 4- 6.6

4- 22 4- 17" 4- 15"* 4- 12+

Values are means 4- S.E.M; Old-treated: Choline alphoscerate-treated old rats. *P < 0.05 vs. young. **P < 0.01 vs. young or adult. +P < 0.05 vs. old.

whereas the Bmax values progressively decreased with age. The density o f muscarinic M l sites in the h i p p o c a m p u s o f a d u l t rats was reduced by a b o u t 12% (P < 0.05) vs. y o u n g animals. The loss in the h i p p o c a m p u s o f old animals averaged 37% ( P < 0.01) vs. y o u n g a n d 25% ( P < 0.01) vs. a d u l t rats (Table 1). T r e a t m e n t with choline alphoscerate increased the density o f muscarinic M1 cholinergic receptors by a b o u t 18% ( P < 0.05) in treated vs. u n t r e a t e d old rats (Table 1). The K d a n d Bmax values for [ 3 H ] A F - D X 116 binding in the four animal groups examined are summarized in Table 1. As can be seen, neither the K a n o r the Bmax changed with age or after choline alphoscerate treatment. Sections o f the h i p p o c a m p u s i n c u b a t e d with [3H]pirenzepine or with [3HIAFD X 116 developed silver grains within the s t r a t u m oriens and the p y r a m i d a l n e u r o n layer o f the C A I a n d C A 3 fields (Figs. 3,4). The density o f silver grains was higher in sections i n c u b a t e d with [3H]pirenzepine than in those exposed to [ 3 H ] A F - D X 116 (Figs. 3,4). Q u a n t i t a t i v e analysis o f the density o f silver grains in the stratum oriens o f the C A l a n d C A 3 fields in the h i p p o c a m p u s i n c u b a t e d with [3H]pirenzepine revealed the highest density o f silver grains in y o u n g rats followed in descending o r d e r by a d u l t a n d old animals (Fig. 5). T r e a t m e n t with choline alphoscerate restored the loss o f silver grains in the s t r a t u m oriens o f the C A I and C A 3 fields in the h i p p o c a m p u s in old rats (Fig. 5). The density o f silver grains developed in the stratum oriens o f the C A 1 a n d C A 3 fields o f sections incubated with [ 3 H ] A F - D X 116 to label muscarinic M 2 receptors did n o t change in the four animal groups examined (Fig. 6). Fig. 4. Autoradiographic localization of [3H]AF-DX 116 binding sites in the CA3 field in the hippocampus of rats of 2 months (young). Sections were incubated with a 7.5 nM concentration of [3H]AF-DX 116 alone (A) or plus 1 ~tM atropine (C) to define non-specific binding. A and C are dark-field pictures; B is a bright-field picture of the micrograph A stained with toluidine blue to verify microanatomical details. P, pyramidal neurons layer. Magnification: x 65.

E Amenta et al./Mech. Ageing Dev. 76 (1994) 49-64

57

58

F. Amenta et al./ Mech. Ageing Dev. 76 (1994) 49-64

Silver graina/lO0 ,urn 2

800

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400

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Fig. 5. Quantitative analysis of the density of silver grains developed in the stratum oriens of the CA I and CA 3 fields in the hippocampus of young (A), adult (B), old (C) and choline alphoscerate-treated old (D) rats. Sections were exposed to a concentration of 5 nM [3H]-pirenzepine alone (total binding, B) or plus 1/~M atropine to define non-specific binding (1"1). Specific binding (Irl) was obtained by subtracting non-specific from total binding. Values express the number of silver grains developed in a 100-tzm 2 area of the stratum oriens -u S.E.M. and were calculated as described in section 2. Materials and methods. *P < 0.001 vs. total. **P < 0.05 vs. specific binding of young. +P < 0.01 vs. specific binding of young or adult. ++P < 0.05 vs. specific binding of old.

Analysis of silver grain density developed within the cell body of pyramidal neurons of the CA 1 and CA 3 fields in the hippocampus in sections incubated with [3H]pirenzepine did not reveal significant differences in the number of silver grains per nerve cell body between young and adult rats (Figs. 7,8). A significant decrease in the density of silver grains per nerve cell body was noticeable between old and young or adult rats (Figs. 7,8). This loss was countered in part by treatment with choline alphoscerate (Figs. 7,8). Similarly, as already described for the density of silver grains in the stratum oriens of the CAt and CA 3 fields, no differences in the number of [3H]AF-DX 116 were seen within the cell body of the pyramidal neurons of the two fields in the different animal groups examined (data not shown). 4. Discussion

Many of the actions of acetylcholine in the central nervous system are mediated through the activation of muscarinic cholinergic receptors. It is known that these

59

F. Amenta et al./ Mech. Ageing Dev. 76 (1994) 49-64

5oo-

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Fig. 6. Quantitative analysis of the density of silver grains developed in the stratum oriens of the CA~ and CA3 fields in the hippocampusof young (A), adult (B), old (C) and choline alphoscerate-treatedold (D) rats. Sections were exposed to a concentration of 7.5 nM 13H]AF-DX 116 alone (total binding, ~) or plus 1 #M atropine to define non-specificbinding (El). Specificbinding (Irl) was obtained by subtracting non-specificfrom total bindig. Values express the number of silver grains developed in a 100-/zm2 area of the stratum oriens ± S.E.M. and werecalculatedas described in section 2. Materialsand methods. *P < 0.001 vs. total.

receptors in the frontal cortex and in the hippocampus are involved in cognitive processes such as learning and memory [4,29]. Muscarinic cholinergic receptors have been classified formerly into two main subtypes, namely muscarinic M 1 and M 2 receptors based on different affinities for various ligands. Muscarinic Ml receptors have a high affinity for pirenzepine, whereas M2 receptors show a low affinity for pirenzipine and a high affinity for A F - D X 116 [30,311. The application of molecular biology to musearinic receptor research has allowed the identification of five distinct musearinic receptor genes (m], m2, m3, m4, ms), the properties of which cannot adequately account for the characteristics of receptor sites established with receptor binding techniques [32-34]. As mentioned in the introduction, the findings on the age-dependent changes of cortical and hippocampal muscarinic cholinergic receptors are conflicting, although the majority of investigators agree on the occurrence of age-related loss of these receptor sites [13-21]. Unfortunately, the majority of studies centered on aging of muscarinic cholinergic receptors in the hippocampus did not analyze the sensitivity to aging of the different subtypes of muscarinic receptors. Studies on the status of the subtypes of muscarinic cholinergic receptors in aged rats reported no changes of

60

F. Amenta et al./Mech. Ageing Dev, 76 (1994) 49-64

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F. Amenta et al. / Mech. Ageing Dev. 76 (1994) 49-64

Silver grains/neuron 70 60

4-

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Fig. 8. Quantitative analysis of the density of silver grains developed within the cell body of pyramidal neurons of the CA] field in the hippocampus of young (A), adult (B), old (C) and choline alphosceratetreated old rats (D). Sections were exposed to a 5 nM concentration of [3H]pirenzepine alone (total binding, D) or plus l #M atropine to defin.e non-specific binding (D). Specific binding (I~1)was obtained by subtracting non-specific from total binding. Values express the number of silver grains per neuron 4- S.E.M. and were calculated as described in section 2. Materials and methods. *P < 0.001 vs. total. **P < 0.01 vs. specific binding of young or adult. +P < 0.05 vs. specific binding of old.

m u s c a r i n i c M] a n d M2 receptors in the h i p p o c a m p u s o f W i s t a r rats [19] a n d loss o f m u s c a r i n i c M E receptors w i t h o u t c h a n g e s o f m u s c a r i n i c MI receptors in the hipp o c a m p u s o f L o n g E v a n s rats [21]. F u r t h e r m o r e , n o a g e - d e p e n d e n t c h a n g e s in m u s c a r i n i c a c e t y l c h o l i n e r e c e p t o r s u b t y p e m e s s e n g e r r i b o n u c l e i c acid ( m R N A ) were detected in the h i p p o c a m p u s o f old W i s t a r rats [35]. O u r stud.y is o n e o f the first reports a d d r e s s i n g the p r o b l e m o f the age-related c h a n g e s o f m u s c a r i n i c M 1 a n d M 2 receptor s u b t y p e s in W i s t a r rats u s i n g selective ligands for the two p o p u l a t i o n s o f receptors. F r o m o u r data, it a p p e a r s that, whereas the density o f h i p p o c a m p a l

Fig. 7. Bright-field autoradiographs showing the accumulation of silver grains within the cell body of pyramidal neurons (P) of the CA l field in the hippocampus of young (A), adult (B), old (C) and choline alphoscerate-treated old rats (D). Sections were incubated with a 5 nM concentration of [3H]pirenzepine to label muscarinic M I receptors. The accumulation of silver grains is similar in the cell body of pyramidal neurons of young and adult rats and decreases in old rats. Treatment with choline alphoscerate restores in part the loss of silver grains occurring in pyramidal neurons of old rats. O, Stratum oriens. Magnification: x 250.

62

F. Amenta et al./ Mech. Ageing Dev. 76 (1994) 49-64

muscarinic M1 receptors is reduced with age, the population of muscarinic M 2 receptors is unaffected by aging. The use of a non-selective muscarinic receptor ligand and of a different technical approach is probably the reason for the inconsistency of our findings on muscarinic M l sites with those of Biegon and coworkers [19]. Strain differences are the probable explanation for the disagreement with the data of Aruajo and collaborators [21]. Pharmacological manipulation of cerebrocortical and hippocampal muscarinic receptors has been proposed as a possile treatment for Alzheimer's disease and related disorders. On the basis of the data currently available about the role of muscarinic cholinergic receptors in the cerebral cortex and hippocampus, several investigators have suggested that stimulation of muscarinic M t receptors and/or antagonism of muscarinic M2 receptors may represent a therapeutic principle for the treatment of senile dementia of the Alzheimer's type and related disorders associated with cholinergic hypofunction. This hypothesis is supported by the observation that stimulation of cortical and hippocampal muscarinic M1 receptor sites increases phosphoinositide turnover and mimics the actions of acetylcholine, whereas inhibition of muscarinic M2 receptors inhibits the release of acetylcholine from cortical and hippocampal terminals [36-38]. Although on the basis of our findings, we are unable to hypothesize through which mechanism choline alphoscerate increases the density of muscarinic M1 cholinergic receptors in the hippocampus of aged rats, the above observation shows that the compound counters the expression of a phenomenon occurring with aging. Furthermore, choline alphoscerate seems to increase the reserve of muscarinic Ml receptors, which represent a receptor subtype without reserve [39,40]. The functional significance of this observation, if any, should be clarified in future studies. The analysis of the density of silver grains developed in [3H]pirenzepine and [3H]AF-DX 116 autoradiographs in the stratum oriens of the hippocampus is consistent with the results obtained in biochemical assays. However, the evaluation of the density of M l sites within the cell body of pyramidal neurons of the CA1 and CA 3 fields revealed that these sites are unchanged between young and adult subjects and decreased in old rats. These findings which are different from binding studies showing a progressive loss of muscarinic M~ receptors with aging suggest that the decrease of these sites noticeable between young and adult subjects' is dependent on the loss of nerve cells and/or terminals occurring in the hippocampal fields examined between young and adult age [24,41]. On the other hand, a progressive agedependent loss of pyramidal neurons of the CA1 and CA 3 fields in the hippocampus has been demonstrated in male Wistar rats [24]. In contrast, hippocampal pyramidal neurons in old rats show a specific loss of muscarinic M l receptors located within their cell bodies. Hence, the decrease of this subtype of muscarinic cholinergic receptors in old age probably depends in part on the loss of the cellular population expressing muscarinic Ml cholinergic receptors and in part on the decreased receptor expression of single neurons. Receptors located in the cell body of pyramidal neurons in the hippocampus of aged rats are sensitive to choline alphoscerate treatment. This suggests that the compound restores in part the expression of muscarinic Ml cholinergic receptors by pyramidal neurons in the CA1 and CA 3 fields in the hippocampus of aged rats.

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