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The effect of acute and chronic centrophenoxine treatment on the synaptic plasticity of old rats
Arch. Gerontol. Geriatr., 1 (1982)365-373
365
Elsevier Biomedical Press
The effect of acute and chronic centrophenoxine treatment on the synaptic plasticity of old rats C. B e r t o n i - F r e d d a r i ], C. G i u l i a n d C. Pieri Center of Cytology, Gerontological Research Department of lNRCA (VILEG, Italian Section), Via Birarelli 8, 60100 Ancona, Italy (Received 24 June 1982; accepted in revised form 25 October 1982)
Summary The cerebellar glomerulus was studied by electron microscopic morphometry in female Wistar rats. Age-dependent alterations have been revealed from 3 to 28 ruth of age, and the effect of centrophenoxine (CPH) was analyzed in two different patterns of administration. First, 27-mth-old rats were treated daily for 6 wk (acute treatment), and second, 18-mth-old rats were treated 3 times per week for 5 months (chronic treatment). The dose was 100 mg C P H / k g body weight, injected intraperitoneally. The surface density (Sv), the numerical density ( N v ) and the average length (L,) of the synaptic junctions were calculated from data obtained on ethanol-phosphotungstic acid stained ultrathin sections. An age-dependent reduction of S v and N v of the synaptic contact zones was found, and the i. increased in the oldest animals. CPH-treatment resulted in a marked increase of Sv in both types of application, whereas the other two parameters behaved differently in the two groups. The chronic treatment resulted in a significant slowing down of the decrease of N v, whereas L, remained invariate. On the contrary, the acute treatment increased L but did not alter significantly N v. The results and the differences between the treatment types are discussed in terms of synaptic plasticity and are interpreted as different manifestations of the same reactive synaptogenetic process. cerebellar glomerulus; synaptic plasticity; age-dependence of synaptic parameters; centrophenoxine
Introduction Cellular aging can be defined as a gradual deterioration of the organization of cellular biological mechanisms, resulting in functional impairments and finally the death of the cell. This definition implies that the plasma membrane being deeply involved in the maintenance of cellular homeostasis, may play an important role in the aging process (Zs.-Nagy, 1978, 1979; Sun and Sun, 1979). This is particularly valid for the neurons that are postmitotic cells and forced to synthesize 'spare parts' ] To whom correspondence should be addressed. 0167-4943/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press
366 of their membranes, in order to assure a proper substitution of the constituents damaged during the normal metabolic processes. The synaptic junctions are highly differentiated areas of the neuronal membranes and one can assume that they are very exigent for a proper renewal of their functional components. As a matter of fact, the synaptic membranes need a continuous supply of specific macromolecules synthesized by the nerve cell soma, in order to exchange the worn out components. In addition, the synapses of the central nervous system are able to modify their morpho-functional features by remodelling both their distribution and size, as has been shown in well defined areas of the brain (Sotelo and Privat, 1978; Cotman and Sheff, 1979; Purves and Lichtman, 1980; Bertoni-Freddari et al., 1982). The recently introduced term 'synaptic plasticity' refers to such a dynamic metabolic state of synapses. The structural, morphological and physiological changes of the synapses taking place even in the fully developed nervous systems have been explained by the concept of synaptic plasticity. Aging also influences the plasticity of the synapses. In our previous work we described that some morphometric parameters, namely the surface- and numerical density (S v and N v, respectively), as well as the average and total length per unit area (L and LA, respectively) of synapses undergo significant changes in the cerebellar glomeruli of old animals (Bertoni-Freddari and Giuli, 1980). Centrophenoxine (p-chlorphenoxy acetic acid dimethylamino ethylester) (CPH) is used in human therapy as a so-called 'neuroenergeticum' to cure patients suffering from different kinds of brain injuries. CPH has been shown to improve the learning capacity of old animals (Nandy, 1978) and a considerable series of aging parameters of the nerve cells and myocardium (Zs.-Nagy et al., 1979). Moreover, it has also been observed that after an acute administration of CPH, the old rats show a partial recovery of the morphometric parameters mentioned above: S v, L A and f~ returned to younger values, whereas N v remained unchanged in the cerebetlar glomeruli of old rats (Giuli et al., 1980). Since according to our previous data the numerical density of synapses begins to decrease from the age of 18 mth of rats in the cerebellar glomeruli (Bertoni-Freddari and Giuli, 1980), we decided to treat rats of this age with CPH, in order to reveal whether the decrease of the number of synapses can be slowed down by an early pharmacological intervention. A second aim of this experiment was to compare the effect of acute and chronic administration of CPH on the synaptic plasticity. Materials and methods
Female Wistar rats of our own breed were used in 2 experimental groups according to the type of CPH-treatment. Group 1. This group received acute C P H treatment as follows: Five rats of 27 mth of age were treated with a daily dose of 100 m g / k g body weight of C P H (Helfergin ®, Promonta, Hamburg) in the form of an intraperitoneal injection for 6 wk. Group 2. This group of 5 rats of 18 mth of age was treated with C P H chronically. The drug was injected intraperitoneally (the same dose as in the previous group) 3 t i m e s / w k for 5 mth.
367
The respective control groups of identical sizes received an identical volume of physiological saline solution for identical periods of time. The method of tissue sampling, fixation, embedding and the staining with ethanol-phosphotungstic acid (E-PTA) was carried out exactly as it has been applied and described in our previous papers (Bertoni-Freddari and Giuli, 1980; Giuli et al., 1980; Bertoni-Freddari et al., 1982). Electron microscopic morphometric analysis was carried out on the cerebellar glomeruli in order to reveal alterations of the main synaptic parameters. Twenty-five micrographs were taken per animal from 5 randomly chosen tissue blocks, i.e., the total number of micrographs per group was 125. The following morphometric parameters were measured: the surface density (S v), the numerical density (N v) and the average length (f~) of the synaptic contact zones. The total length of synaptic contacts can be derived from these basic parameters. The values obtained have been corrected for the Holmes' effect. Statistical comparisons were carried out by using Student's t-test. For all other details of the morphometric analysis see our previous papers (Bertoni-Freddari and Giuli, 1980; Giuli et al., 1980; Bertoni-Freddari et al., 1982).
Results
Using the preferential staining method of Bloom and Aghajanian (1968) applying E-PTA, the paramembranous densities of the synaptic junctions are highlighted
Fig. 1. Eighteen-month-old rat cerebellar glomerulus stained with E - P T A preferential staining for the synaptic paramembranous densities. A 1 cm quadratic lattice was printed on the area of the photo to calculate the morphometric parameters. Arrow: synapse with dotted pre-synaptic density. Black circles: intersection points. For further details see the text. x 30,000.
368 140.
130 0 C, ql-
120
110
100
90
3
11
18
23
28
age in months
Fig. 2. Surface density of the synaptic contact zones (Sv). Each point represents the mean + SEM of 125 E.M. photographs of rat cerebellar glomeruli. • controls; • chronically CPH-treated rats; • acutely CPH-treated animals.
rather well. As can be seen in Fig. 1, synapses are easily recognizable as parallel lines of high electron density due to the pre- and postsynaptic thickenings. In some cases, however, the presynaptic density appears as a dotted line (see the arrow). The quadratic lattice printed on the micrograph represents the test system used for counting the intersection points of the lines with the synapses (e.g. as indicated by the circles on Fig. 1). This count of intersection allows us to calculate S v. The morphometric parameters were calculated individually for each animal first. Since, however, there were no significant differences between the individual values within the same group, the data were pooled together within the groups. The present paper describes the group averages of Sv, Nv and L,. In order to compare the effect of CPH on the age-dependent alterations of the morphornetric parameters, we present here data regarding 3-, 11- and 18-mth-old TABLE ] Average length of synaptic contact zones (L,) during aging. Mean + SEM of 125 cerebellar glomeruli per age group. Age (mth)
L (/~m)
3 11 18 23 28
0.2290 + 0.0055 0.2331 +0.0029 0.2040 + 0.0035 0.2201 -t- 0.0043 0.2197 -I-0.0059
Statistical significance: (3-18) P < 0.0001; (11-18) P < 0.0001; (18-23) P < 0.001; (18-28) P < 0.005.
369
2.2. Z 2.1 >-
2.0 1.9 18 1.7 1.6 1.5 1.4. 13.
TT
TT
3
'11
T 18
~lH~ 23
T ~T 28
AGE
IN M O N T H S
Fig. 3. Numerical density of synapses ( N v). Mean + SEM [] controls; [] chronically CPH-treated rats; [] acutely CPH-treated rats.
non-treated rats. These data were taken partly from our previous papers (BertoniFreddari and Giuli, 1980; Giuli et al., 1980); however, the number of rats per age group was increased from 3 to 5 during the present experiments. Figure 2 summarizes the surface density of synapses in the cerebellar glomerulus. This parameter displays rather high values in the young ages; nevertheless, there is a progressive decrease from the age of 11 mth. It is evident from the data given in Fig. 2 that CPH-treatment results in a significant increase of S v in both types of treatment. The acute treatment of old rats increased by about 22%, whereas the chronic treatment between 18 and 23 mth of age improved this parameter to a lesser extent (9%).
TABLE II Average length of synaptic contact zones ( i , ) in control and CPH-treated rats. M e a n + S E M of 125 cerebellar glomeruli per age group. Age (mth)
Control
CPH treated
23 28
0.2201 + 0.0043 0.2197 + 0.0059
0.2280 5:0.0036 a 0.2404 + 0.0038 b,c
a Chronically CPH-treated animals. b Statistically significant difference of CPH-treated rats from their controls: P < 0.001. c Acutely CPH-treated rats.
370 The average length of the synaptic contact zones observed in different untreated age groups is given in Table I. This parameter does not show any age-dependent alteration except the age of 18 mth when it is significantly lower than in the other age-groups. The effects of CPH on this parameter are summarized in Table II. The acute treatment of old rats resulted in a quite considerable increase of this parameter, whereas the chronic treatment between the ages of 18 and 23 ruth did not alter this parameter significantly. The numerical density of synapses in the cerebellar glomerulus increases up to the age of 18 mth (Fig. 3), and declines rather rapidly by the age of 28 mth. The differences between 18 and 23 as well as 23 and 28 mth of age are highly significant. The effect of chronic CPH-treatment is also reported in Fig. 3: the N v of synapses proved to be significantly higher in the chronically treated 23-mth-old rats than in the untreated control group. On the other hand, the acute treatment of old rats could not influence significantly this parameter (Fig. 3).
Discussion
Our data show that the synaptic plasticity is influenced by the age of the animals. The young rats display less frequent and large synaptic contacts resulting in a large total contact area. In the adult age group the same size of total contact area is maintained; however, important differences occur as well. Namely, the synaptic contacts of the adults are more frequent and smaller in size. During the aging after 18 mth the synapses tend to be larger again; however, their number decreases considerably, i.e., the total contact area is reduced. It is quite clear from our data that CPH is able to influence the age-dependent alterations of the synapses studied. The possible explanation of this effect may be derived from several observations, as follows. (1) It is known that CPH is hydrolized in the organism and it is believed that dimethylamino-ethanol (DMAE) enters the choline and acetylcholine synthesis (Hochschild, 1973; London and Coyle, 1978). Although this possibility has been discussed and contradicted by others (Hochschild, 1973a; Haubrich et al., 1975a; Zaniser et al., 1977; Jope and Jenden, 1979), it seems to us an important fact that free choline availability is increased after DMAE administration (Haubrich et al., 1975, 1981; Ceder et al., 1978). Since only a very small increase of choline level is sufficient to saturate the acetylcholine synthesis, and the excess of choline is used for phospholipid production (Haubrich et al., 1975a; Freeman and Jenden, 1976; Ansell and Spanner, 1977; Haubrich and Chippendale, 1977; Bartus et al., 1980), CPH may also be able to increase the synthesis of acetylcholine and phospholipids. Both of these processe s are involved in the synaptic functions as well as in the synaptic plasticity. On the other hand, it has also been shown that the choline-phospholipid synthesis is reduced during aging (Brunetti et al., 1979), and a number of papers report on reduced synaptic density and impaired functions of the cholinergic system (Huttenlocher, 1979; Bartus et al., 1980; Uemura, 1980; Gibson et al., 1981). (2) It has also been shown that DMAE becomes phosphorylated in the brain
371
tissue and persists for rather a long time in the neuronal membranes in the form of phosphatidyl-DMAE (Miyazaki et al., 1976). Since this compound differs from the common phospholipids (like lecithin) only in the possession of one methyl group less in its hydrophilic part, and it has been shown that DMAE is a very efficient free-radical scavenger (Zs.-Nagy and Nagy, 1980), it is quite reasonable to assume that this compound may render the nerve cell membrane more fluid and at the same time, may protect the membrane proteins against free-radical damage. This possible interpretation fits with the membrane hypothesis of aging (Zs.-Nagy, 1978; 1979). It should be stressed that the possibilities listed above for the CPH-effect are not contradictory to each other. Both kinds of effects may take place parallel with each other, and both of them can positively influence the synaptic plasticity. Comparing the results of the two types of CPH treatment, it seems to be important that in both cases we observed a tendency to increase or maintain the synaptic contact area size. Since it has been shown that malnutrition results in a decrease of number of synapses and an increase of the contact area of the existing junctions (Chen and Hillman, 1980; Hillman and Chen, 1981)just in the cerebellar cortex, one can assume that the increased individual synaptic size may represent a kind of compensation for the reduced number of synapses even during aging. Such kinds of compensation, however, cannot be considered very efficient. Namely, it has been demonstrated that although some electric parameters of the synapses of the aged animals improved (Barnes and MacMaughton, 1980) as compared to the young animals, the total performance of the neuronal circuitry of the old animals cannot reach that of the young ones (Smith, 1979). Therefore, one can conclude that it is better to prevent the age-dependent decline of the synaptic N v by an early introduced CPH treatment than to try to improve the functional capacity of the old animals by an acute CPH-dosage. Both types of CPH treatment resulted in changes of the synaptic parameters which can be considered as homotypic synaptogenetic effects, i.e. the functional possibilities of the existing synapses were improved. These effects of CPH are in agreement with the observations of many other authors studying various aspects of CPH effect (Oeriu et al., 1973; Riga and Riga, 1974; Nandy, 1978). We are of the opinion that the CPH effect represents primarily a protective-stabilizing influence on the plasma membrane of the nerve cells: it has been shown that CPH is able to reverse the age-dependent increase of the intracellular potassium concentration (Pied et al., 1977; Zs.-Nagy et al., 1979). Furthermore, CPH proved to be able to delay the damaging effect of E-avitaminosis on the brain cells of rats (BertoniFreddari et al., 1981). If the general metabolic activity including the rate of protein synthesis is improved by this effect of CPH (Zs.-Nagy, 1978; 1979), it is quite obvious to expect that the availability of all the components being necessary for the synaptic remodelling and provided by the nerve cell soma through the continuous axonal flow (Droz, 1973; Marko and Cuenod, 1973; Krygier-Brevart,. 1974) will be improved. Further studies are in progress in our laboratory regarding the interrelationship between the axonal transport processes and the CPH treatment.
372
References Ansell, B.G. and Spanner, S. (1977): Functional metabolism of brain phospholipids. Int. Rev. Neurobiol., 20, 1-29. Barnes, C.A. and McNaughton, B.L. (1980): Physiological compensation for loss of afferent synapses in rat hippocampal granule cells during senescence. J. Physiol., 309, 473-485. Bartus, R.T., Dean, R.L., Goas, J.A. and Lippa, A.S. (1980): Age-related changes in passive avoidance retention: modulation with dietary choline. Science, 209, 301-303. Bertoni-Freddari, C. and Giuli, C. (1980): A quantitative morphometric study of synapses of rat cerebellar glomeruli during aging. Mech. Age. Dev., 12, 127-136. Bertoni-Freddari, C., Giuli, C., Lustyik, Gy. and Zs.-Nagy, I. (1981): In vivo effects of vitamin E-deficiency on the intracellular monovalent electrolyte concentrations in brain and liver of rat. An energy dispersive X-ray microanalytic study. Mech. Ageing Dev., 16, 169-180. Bertoni-Freddari, C., Giuli, C. and Pieri, C. (1982): The effect of vitamin E-deficiency on the synapses of cerebellar glomeruli of young rats. Mech. Ageing Dev., submitted for publication. Bloom, F.E. and Aghajanian, G.K. (1968): Fine structural and cytochemical analysis of the staining of synaptic junctions with PTA. J. Ultrastruct. Res., 22, 361-375. Brunetti, M., Gaiti, A. and Porcellati, G. (1979): Synthesis of phosphatidylcholine and phosphatidylethanolamine at different ages in the rat brain in vitro. Lipids, 14, 925-931. Ceder, G., Dahlberg, L. and Schuberth, J. (1978): Effects of 2-dimethylaminoethanol (Deanol) on the metabolism of choline in plasma. J. Neurochem., 30, 1293-1296. Chen, S. and Hillman, D.E. (1980): Giant spines and enlarged synapses induced in Purkinje cells by malnutrition. Brain Res., 187, 487-493. Cotman, C.W. and Sheff, S.W. (1979): Synaptic growth in aged animals. In: Physiology and Cell Biology of Aging, pp. 109-120. Editors: A. Cherkin, N. Kharasch, F.L. Scott, C.E. Finch, T. Makinodan and B.L. Strehler. Raven Press, New York. Droz, B. (1973): Renewal of synaptic proteins. Brain Res., 62, 383-394. Freeman, J.J. and Jenden, D.J. (1976): The source of choline for acetylcholine synthesis in.brain. Life Sci., 19, 949-962. Gibson, G.E., Peterson, C. and Jenden, D.J. (1981): Brain acetylcholine synthesis declines with senescence. Science, 213, 674-676. Giuli, C., Bertoni-Freddari, C. and Pieri, C. (1980): Morphometric studies on synapses of cerebellar glomerulus: the effect of Centrophenoxine treatment in old rats. Mech. Ageing Dev., 14, 265-271. Haubrich, D.R., Wang, P.F.L. and Wedeking, P.W. (1975): Distribution of intravenously administered choline methyl-3H and synthesis in vivo of acetylcholine in various tissues of guinea pigs. J. Pharmacol. Exp. Ther., 193, 246-255. Haubrich, D.R., Wang, P.F., Clody, D.E. and Wedeking, P.W. (1975a): Increase in rat brain acetylcholine induced by choline or Deanol. Life Sci., 17, 975-980. Haubrich, D.R. and Chippendale, T.J. (1977): Regulation of acetylcholine synthesis in the nervous system. Life Sci., 20, 1465-1478. Haubrich, D.R., Gerber, N.H. and Pflueger, A.B. (1981): Deanol affects choline metabolism in peripheral tissues of mice. J. Neurochem., 37, 476-482. HiUman, D.E. and Chen, S. (1981): Vulnerability of cerebellar development in malnutrition. II. Intrinsic determination of total synaptic area on Purkinje cell spines. Neuroscience, 7, 1263-1275. Hochschild, R. (1973): Effect of dimethylaminoethanol on the life span of senile male A / J mice. Exp. Gerontol., 8, 185-191. Hochschild, R. (1973a): Effect of dimethylaminoethyl p-chlorophenoxyacetate on the life span of male Swiss Webster albino mice. Exp. Gerontol., 8, 177-183. Huttenlocher, P.R. (1979): Synaptic density in human frontal cortex. Developmental changes and effects of aging. Brain Res., 163, 195-205. Jope, R.S. and Jenden, D.J. (1979): Dimethylaminoethanol (Deanol) metabolism in rat brain and its effect on acetylcholine synthesis. J. Pharmacol. Exp. Ther., 211,472-479. Krygier-Brevart, V., Weiss, D.G., Mehl, E., Schubert, P. and Kreutzberg, G.W. (1974): Maintenance of synaptic membrane by the fast axonal flow. Brain Res., 77, 97-110.
373 London, E.D. and Coyle, J.T. (1978): Pharmacological augmentation of acetylcholine.levels in kainate lesioned rat striatum. Biochem. Pharmacol., 27, 2962-2965. Marko, P. and Cuenod, M. (1973): Contribution of the nerve cell body to renewal of axonal and synaptic glycoproteins in the pigeon visual system. Brain Res., 62, 419-423. Miyazaki, H., Nambu, K., Minaki, Y., Hashimoto, M. and Nakamura, K. (1976): Comparative studies on the metabolism of beta-dimethylamino ethanol in the mouse brain and liver following administration of beta-dimethylamino ethanol and its p-chlorophenoxyacetate, Meclofenoxate. Chem. Pharm. Bull., 24, 763-769. Nandy, K. (1978): Centrophenoxine: effects on aging mammalian brain. J. Am. Gerontol. Soc., XXXVI, 2, 74-81. Oeriu, S., Winter, D., Dobre, V. and Bruhis, S. (1973): Glucose transport across blood brain as related to age and Meclofenoxate treatment. J. Pharmacol., 4, 497-503. Pieri, C., Zs.-Nagy, I., Zs.-Nagy, V., Giuli, C. and Bertoni-Freddari, C. (1977): Energy dispersive X-ray microanalysis of the electrolytes in biological bulk specimen. II. Age-dependent alterations in the monovalent ion contents of cell nucleus and cytoplasm in rat liver and brain cells. J. Ultrastruct. Res., 59, 320-331. Purves, D. and Lichtman, J.V. (1980): Elimination of synapses in the developing nervous system. Science, 210, 153-157. Riga, S. and Riga, D. (1974): Effects of Centrophenoxine on the lipofuscin pigments in the nervous system of old rats. Brain Res., 72, 265-275. Smith, D.O. (1979): Reduced capabilities of synaptic transmission in aged rats. Exp. Neurol., 66, 650-666. Sotelo, C. and Privat, A. (1978): Synaptic remodelling of the cerebellar circuitry in mutant mice and experimental cerebellar malformations. Study "in vivo" and "in vitro". Acta Neuropathol., 43, 19-34. Sun, A.Y. and Sun, G.Y. (1979): Neurochemical aspects of the mebrane hypothesis of aging. Interdiscip. Top. Gerontol., 15, 34-53. Uemura, E. (1980): Age-related changes in prefrontal cortex of Macaca Mulatta: synaptic density. Exp. Neurol., 69, 164-172. Zaniser, N.R., Chou, D. and Hanin, I. (1977): Is 2-dimethylaminoethanol (Deanol) indeed a precursor of brain acetylcholine? A gas chromatographic evaluation. J. Pharmacol. Exp. Ther., 200, 545-559. Zs.-Nagy, I. (1978): A membrane hypothesis of aging. J. Theor. Biol., 75, 189-195. Zs.-Nagy, I. (1979): The role of membrane structure and function in cellular aging. A review. Mech. Ageing Dev., 9, 237-246. Zs.-Nagy, I. and Nagy, K. (1980): On the role of cross linking of cellular proteins in aging. Mech. Ageing Dev., 14, 245-251. Zs.-Nagy, I., Pieri, C., Giuli, C. and Del Moro, M. (1979): Effects of Centrophenoxine on the monovalent electrolyte contents of the large brain cortical cells of old rats. Gerontology, 25, 94-102.
365
Elsevier Biomedical Press
The effect of acute and chronic centrophenoxine treatment on the synaptic plasticity of old rats C. B e r t o n i - F r e d d a r i ], C. G i u l i a n d C. Pieri Center of Cytology, Gerontological Research Department of lNRCA (VILEG, Italian Section), Via Birarelli 8, 60100 Ancona, Italy (Received 24 June 1982; accepted in revised form 25 October 1982)
Summary The cerebellar glomerulus was studied by electron microscopic morphometry in female Wistar rats. Age-dependent alterations have been revealed from 3 to 28 ruth of age, and the effect of centrophenoxine (CPH) was analyzed in two different patterns of administration. First, 27-mth-old rats were treated daily for 6 wk (acute treatment), and second, 18-mth-old rats were treated 3 times per week for 5 months (chronic treatment). The dose was 100 mg C P H / k g body weight, injected intraperitoneally. The surface density (Sv), the numerical density ( N v ) and the average length (L,) of the synaptic junctions were calculated from data obtained on ethanol-phosphotungstic acid stained ultrathin sections. An age-dependent reduction of S v and N v of the synaptic contact zones was found, and the i. increased in the oldest animals. CPH-treatment resulted in a marked increase of Sv in both types of application, whereas the other two parameters behaved differently in the two groups. The chronic treatment resulted in a significant slowing down of the decrease of N v, whereas L, remained invariate. On the contrary, the acute treatment increased L but did not alter significantly N v. The results and the differences between the treatment types are discussed in terms of synaptic plasticity and are interpreted as different manifestations of the same reactive synaptogenetic process. cerebellar glomerulus; synaptic plasticity; age-dependence of synaptic parameters; centrophenoxine
Introduction Cellular aging can be defined as a gradual deterioration of the organization of cellular biological mechanisms, resulting in functional impairments and finally the death of the cell. This definition implies that the plasma membrane being deeply involved in the maintenance of cellular homeostasis, may play an important role in the aging process (Zs.-Nagy, 1978, 1979; Sun and Sun, 1979). This is particularly valid for the neurons that are postmitotic cells and forced to synthesize 'spare parts' ] To whom correspondence should be addressed. 0167-4943/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press
366 of their membranes, in order to assure a proper substitution of the constituents damaged during the normal metabolic processes. The synaptic junctions are highly differentiated areas of the neuronal membranes and one can assume that they are very exigent for a proper renewal of their functional components. As a matter of fact, the synaptic membranes need a continuous supply of specific macromolecules synthesized by the nerve cell soma, in order to exchange the worn out components. In addition, the synapses of the central nervous system are able to modify their morpho-functional features by remodelling both their distribution and size, as has been shown in well defined areas of the brain (Sotelo and Privat, 1978; Cotman and Sheff, 1979; Purves and Lichtman, 1980; Bertoni-Freddari et al., 1982). The recently introduced term 'synaptic plasticity' refers to such a dynamic metabolic state of synapses. The structural, morphological and physiological changes of the synapses taking place even in the fully developed nervous systems have been explained by the concept of synaptic plasticity. Aging also influences the plasticity of the synapses. In our previous work we described that some morphometric parameters, namely the surface- and numerical density (S v and N v, respectively), as well as the average and total length per unit area (L and LA, respectively) of synapses undergo significant changes in the cerebellar glomeruli of old animals (Bertoni-Freddari and Giuli, 1980). Centrophenoxine (p-chlorphenoxy acetic acid dimethylamino ethylester) (CPH) is used in human therapy as a so-called 'neuroenergeticum' to cure patients suffering from different kinds of brain injuries. CPH has been shown to improve the learning capacity of old animals (Nandy, 1978) and a considerable series of aging parameters of the nerve cells and myocardium (Zs.-Nagy et al., 1979). Moreover, it has also been observed that after an acute administration of CPH, the old rats show a partial recovery of the morphometric parameters mentioned above: S v, L A and f~ returned to younger values, whereas N v remained unchanged in the cerebetlar glomeruli of old rats (Giuli et al., 1980). Since according to our previous data the numerical density of synapses begins to decrease from the age of 18 mth of rats in the cerebellar glomeruli (Bertoni-Freddari and Giuli, 1980), we decided to treat rats of this age with CPH, in order to reveal whether the decrease of the number of synapses can be slowed down by an early pharmacological intervention. A second aim of this experiment was to compare the effect of acute and chronic administration of CPH on the synaptic plasticity. Materials and methods
Female Wistar rats of our own breed were used in 2 experimental groups according to the type of CPH-treatment. Group 1. This group received acute C P H treatment as follows: Five rats of 27 mth of age were treated with a daily dose of 100 m g / k g body weight of C P H (Helfergin ®, Promonta, Hamburg) in the form of an intraperitoneal injection for 6 wk. Group 2. This group of 5 rats of 18 mth of age was treated with C P H chronically. The drug was injected intraperitoneally (the same dose as in the previous group) 3 t i m e s / w k for 5 mth.
367
The respective control groups of identical sizes received an identical volume of physiological saline solution for identical periods of time. The method of tissue sampling, fixation, embedding and the staining with ethanol-phosphotungstic acid (E-PTA) was carried out exactly as it has been applied and described in our previous papers (Bertoni-Freddari and Giuli, 1980; Giuli et al., 1980; Bertoni-Freddari et al., 1982). Electron microscopic morphometric analysis was carried out on the cerebellar glomeruli in order to reveal alterations of the main synaptic parameters. Twenty-five micrographs were taken per animal from 5 randomly chosen tissue blocks, i.e., the total number of micrographs per group was 125. The following morphometric parameters were measured: the surface density (S v), the numerical density (N v) and the average length (f~) of the synaptic contact zones. The total length of synaptic contacts can be derived from these basic parameters. The values obtained have been corrected for the Holmes' effect. Statistical comparisons were carried out by using Student's t-test. For all other details of the morphometric analysis see our previous papers (Bertoni-Freddari and Giuli, 1980; Giuli et al., 1980; Bertoni-Freddari et al., 1982).
Results
Using the preferential staining method of Bloom and Aghajanian (1968) applying E-PTA, the paramembranous densities of the synaptic junctions are highlighted
Fig. 1. Eighteen-month-old rat cerebellar glomerulus stained with E - P T A preferential staining for the synaptic paramembranous densities. A 1 cm quadratic lattice was printed on the area of the photo to calculate the morphometric parameters. Arrow: synapse with dotted pre-synaptic density. Black circles: intersection points. For further details see the text. x 30,000.
368 140.
130 0 C, ql-
120
110
100
90
3
11
18
23
28
age in months
Fig. 2. Surface density of the synaptic contact zones (Sv). Each point represents the mean + SEM of 125 E.M. photographs of rat cerebellar glomeruli. • controls; • chronically CPH-treated rats; • acutely CPH-treated animals.
rather well. As can be seen in Fig. 1, synapses are easily recognizable as parallel lines of high electron density due to the pre- and postsynaptic thickenings. In some cases, however, the presynaptic density appears as a dotted line (see the arrow). The quadratic lattice printed on the micrograph represents the test system used for counting the intersection points of the lines with the synapses (e.g. as indicated by the circles on Fig. 1). This count of intersection allows us to calculate S v. The morphometric parameters were calculated individually for each animal first. Since, however, there were no significant differences between the individual values within the same group, the data were pooled together within the groups. The present paper describes the group averages of Sv, Nv and L,. In order to compare the effect of CPH on the age-dependent alterations of the morphornetric parameters, we present here data regarding 3-, 11- and 18-mth-old TABLE ] Average length of synaptic contact zones (L,) during aging. Mean + SEM of 125 cerebellar glomeruli per age group. Age (mth)
L (/~m)
3 11 18 23 28
0.2290 + 0.0055 0.2331 +0.0029 0.2040 + 0.0035 0.2201 -t- 0.0043 0.2197 -I-0.0059
Statistical significance: (3-18) P < 0.0001; (11-18) P < 0.0001; (18-23) P < 0.001; (18-28) P < 0.005.
369
2.2. Z 2.1 >-
2.0 1.9 18 1.7 1.6 1.5 1.4. 13.
TT
TT
3
'11
T 18
~lH~ 23
T ~T 28
AGE
IN M O N T H S
Fig. 3. Numerical density of synapses ( N v). Mean + SEM [] controls; [] chronically CPH-treated rats; [] acutely CPH-treated rats.
non-treated rats. These data were taken partly from our previous papers (BertoniFreddari and Giuli, 1980; Giuli et al., 1980); however, the number of rats per age group was increased from 3 to 5 during the present experiments. Figure 2 summarizes the surface density of synapses in the cerebellar glomerulus. This parameter displays rather high values in the young ages; nevertheless, there is a progressive decrease from the age of 11 mth. It is evident from the data given in Fig. 2 that CPH-treatment results in a significant increase of S v in both types of treatment. The acute treatment of old rats increased by about 22%, whereas the chronic treatment between 18 and 23 mth of age improved this parameter to a lesser extent (9%).
TABLE II Average length of synaptic contact zones ( i , ) in control and CPH-treated rats. M e a n + S E M of 125 cerebellar glomeruli per age group. Age (mth)
Control
CPH treated
23 28
0.2201 + 0.0043 0.2197 + 0.0059
0.2280 5:0.0036 a 0.2404 + 0.0038 b,c
a Chronically CPH-treated animals. b Statistically significant difference of CPH-treated rats from their controls: P < 0.001. c Acutely CPH-treated rats.
370 The average length of the synaptic contact zones observed in different untreated age groups is given in Table I. This parameter does not show any age-dependent alteration except the age of 18 mth when it is significantly lower than in the other age-groups. The effects of CPH on this parameter are summarized in Table II. The acute treatment of old rats resulted in a quite considerable increase of this parameter, whereas the chronic treatment between the ages of 18 and 23 ruth did not alter this parameter significantly. The numerical density of synapses in the cerebellar glomerulus increases up to the age of 18 mth (Fig. 3), and declines rather rapidly by the age of 28 mth. The differences between 18 and 23 as well as 23 and 28 mth of age are highly significant. The effect of chronic CPH-treatment is also reported in Fig. 3: the N v of synapses proved to be significantly higher in the chronically treated 23-mth-old rats than in the untreated control group. On the other hand, the acute treatment of old rats could not influence significantly this parameter (Fig. 3).
Discussion
Our data show that the synaptic plasticity is influenced by the age of the animals. The young rats display less frequent and large synaptic contacts resulting in a large total contact area. In the adult age group the same size of total contact area is maintained; however, important differences occur as well. Namely, the synaptic contacts of the adults are more frequent and smaller in size. During the aging after 18 mth the synapses tend to be larger again; however, their number decreases considerably, i.e., the total contact area is reduced. It is quite clear from our data that CPH is able to influence the age-dependent alterations of the synapses studied. The possible explanation of this effect may be derived from several observations, as follows. (1) It is known that CPH is hydrolized in the organism and it is believed that dimethylamino-ethanol (DMAE) enters the choline and acetylcholine synthesis (Hochschild, 1973; London and Coyle, 1978). Although this possibility has been discussed and contradicted by others (Hochschild, 1973a; Haubrich et al., 1975a; Zaniser et al., 1977; Jope and Jenden, 1979), it seems to us an important fact that free choline availability is increased after DMAE administration (Haubrich et al., 1975, 1981; Ceder et al., 1978). Since only a very small increase of choline level is sufficient to saturate the acetylcholine synthesis, and the excess of choline is used for phospholipid production (Haubrich et al., 1975a; Freeman and Jenden, 1976; Ansell and Spanner, 1977; Haubrich and Chippendale, 1977; Bartus et al., 1980), CPH may also be able to increase the synthesis of acetylcholine and phospholipids. Both of these processe s are involved in the synaptic functions as well as in the synaptic plasticity. On the other hand, it has also been shown that the choline-phospholipid synthesis is reduced during aging (Brunetti et al., 1979), and a number of papers report on reduced synaptic density and impaired functions of the cholinergic system (Huttenlocher, 1979; Bartus et al., 1980; Uemura, 1980; Gibson et al., 1981). (2) It has also been shown that DMAE becomes phosphorylated in the brain
371
tissue and persists for rather a long time in the neuronal membranes in the form of phosphatidyl-DMAE (Miyazaki et al., 1976). Since this compound differs from the common phospholipids (like lecithin) only in the possession of one methyl group less in its hydrophilic part, and it has been shown that DMAE is a very efficient free-radical scavenger (Zs.-Nagy and Nagy, 1980), it is quite reasonable to assume that this compound may render the nerve cell membrane more fluid and at the same time, may protect the membrane proteins against free-radical damage. This possible interpretation fits with the membrane hypothesis of aging (Zs.-Nagy, 1978; 1979). It should be stressed that the possibilities listed above for the CPH-effect are not contradictory to each other. Both kinds of effects may take place parallel with each other, and both of them can positively influence the synaptic plasticity. Comparing the results of the two types of CPH treatment, it seems to be important that in both cases we observed a tendency to increase or maintain the synaptic contact area size. Since it has been shown that malnutrition results in a decrease of number of synapses and an increase of the contact area of the existing junctions (Chen and Hillman, 1980; Hillman and Chen, 1981)just in the cerebellar cortex, one can assume that the increased individual synaptic size may represent a kind of compensation for the reduced number of synapses even during aging. Such kinds of compensation, however, cannot be considered very efficient. Namely, it has been demonstrated that although some electric parameters of the synapses of the aged animals improved (Barnes and MacMaughton, 1980) as compared to the young animals, the total performance of the neuronal circuitry of the old animals cannot reach that of the young ones (Smith, 1979). Therefore, one can conclude that it is better to prevent the age-dependent decline of the synaptic N v by an early introduced CPH treatment than to try to improve the functional capacity of the old animals by an acute CPH-dosage. Both types of CPH treatment resulted in changes of the synaptic parameters which can be considered as homotypic synaptogenetic effects, i.e. the functional possibilities of the existing synapses were improved. These effects of CPH are in agreement with the observations of many other authors studying various aspects of CPH effect (Oeriu et al., 1973; Riga and Riga, 1974; Nandy, 1978). We are of the opinion that the CPH effect represents primarily a protective-stabilizing influence on the plasma membrane of the nerve cells: it has been shown that CPH is able to reverse the age-dependent increase of the intracellular potassium concentration (Pied et al., 1977; Zs.-Nagy et al., 1979). Furthermore, CPH proved to be able to delay the damaging effect of E-avitaminosis on the brain cells of rats (BertoniFreddari et al., 1981). If the general metabolic activity including the rate of protein synthesis is improved by this effect of CPH (Zs.-Nagy, 1978; 1979), it is quite obvious to expect that the availability of all the components being necessary for the synaptic remodelling and provided by the nerve cell soma through the continuous axonal flow (Droz, 1973; Marko and Cuenod, 1973; Krygier-Brevart,. 1974) will be improved. Further studies are in progress in our laboratory regarding the interrelationship between the axonal transport processes and the CPH treatment.
372
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