Effects of aging on neuronal electrical membrane properties

Mechanisms of Ageing and Development, 44 (1988) 203--214

203

Elsevier Scientific Publishers Ireland Ltd.

EFFECTS OF AGING ON NEURONAL ELECTRICAL MEMBRANE PROPERTIES

BRIAN SCOTT', JAMES LEU and B. CINADER b °Surrey Place Centre, 2 Surrey Place, Toronto, Ontario MSS 2C2, Canada, and Dept. o f Zoology, University of Toronto, Toronto, and b Dept. o f Immunology, Medical Sciences Building, University of Toronto, Toronto, Ontario MSS lAB, (Canada) (Received June 8th, 1987) (Revision received January 25th, 1988)

SUMMARY

The electric membrane properties (EMP) of dorsal root ganglion (DR(;) neurons in cell cultures prepared from control mice (8--14 weeks) and old mice (90--92 weeks) were compared. The old neurons had a number of significant alterations in EMP compared to controls including decreased electrical excitability, increased action potential duration and more pronounced biphasicity of the repolarization phase. The old neurons also had larger action potential overshoot and afterhyperpolarization. The pattern of altered electric membrane properties was consistent with an age-induced shift from voltage-sensitive sodium channels to less excitable voltage-sensitive calcium channels and also a decrease in potassium permeability during the repolarizing phase of the action potential.

Key words: Senescence; Neurons; Dorsal root ganglion; Electric membrane properties; Aging; Neural cell culture INTRODUCTION

Despite the well known progressive declines in the function of the nervous system in senescence [9], relatively little effort has been made to investigate the effects of aging on the electric membrane properties (EMP) of individual neurons. This is most surprising in view of the important role of EMP in many critical functions of the nervous system including electrical excitability, conduction of the action potential (AP), secretion of hormones and neurotransmitters, and receptor-mediated signal transduction and electrotonic integration of post-synaptic potentials. In order to simplify discussion of previous electrophysiological studies of aging, definitions and abbreviations for the various EMP are enumerated in Table I. 0047.6374/88/$03.50 Printed and Published in Ireland

© 1988 Elsevier Scientific Publishers Ireland Ltd.

204 TABLE I DEFINITIONSAND ABBREVIATIONSOF EMP For methods of measuring or calculating see Methods.

Abbreviation Definition AP VM RI RM TICON CI CM VRH VTH DT RISE M or B-type FALLI FALL2 FAR OS AHP VK AREA IL IS

Action potential Resting membranepotential Cell input resistance Specificmembraneresistance Membrane time constant Cell input capacitance Specific membrane capacitance Threshold depolarization using 10 ms current pulse Threshold membranepotential using 10 ms current pulse Duration of AP Maximum rate of rise of AP Neurons could have either monphasicrepolarization (M-type) or biphasic repolarization (B-type) Rate of first phase of repolarization (B-typeonly) Rate of secondphase of repolarization (B-typeand M-type) Ratio of FALL 2 and FALLI Overshoot of AP Afterhyperpolarizationof AP A measure of potassium equilibrium potential Neuron surfacearea Threshold current (10 ms duration) Threshold current (0.1 ms duration)

A review of the literature indicated that research on aging and neuronal membrane electrophysiology has been relatively limited [3m6,20--23]. Frolkis et al. [5] have reported a study o f the effects of aging on the E M P o f identified neurons o f the pond snail (Lymnaea stagnalis). These mollusks consisted of an adult group (10--12 months) and a senescent group (22--24 months) and the following 12 variables were compared in the two groups: (1) VM; (2) IL; (3) RI; (4) frequency o f spontaneous APs; (5) A M P ; (6) DT; (7) OS; (8) RISE; (9) " b a c k slope" or rate of fall or repolarization of the A P ; (10) A H P ; (11) "synaptic activity frequency"; and (12) synaptic potential duration. Frolkis et al. found that only four o f the above 12 E M P changed significantly (P < 0.05) with age: IL increased 47W0; A P frequency decreased 45.2°70; back slope decreased 21.3070 and A H P decreased 45.9070. The decrease in F A L L 2 and A H P was interpreted by the authors as an age-induced decrease in the "rate o f K yield" and they concluded that "Further research into age-dependent shifts of the ion transport through a membrane and the state o f ion, in particular potassium, channels would explain the mechanism o f changes occurring with aging in the thresholds of neuronal

205 direct excitability, the period of action potential repolarization and trace hyperpolarization." Frolkis [4] has provided further support for an age-dependent decrease in potassium permeability (Px) in studies of rat spinal cord neurons. The spinal cord motor neurons had significantly increased DT which was largely due to decreases in the rate of repolarization again suggesting decreased PK" Similar to the observations of Frolkis et al. on aging neurons, Scott et al. [16] observed increased DT, decreased FALL2 and decreased A H P for Down syndrome neurons in cell culture and these authors also interpreted these altered EMP as reflecting a decreased Px" Since there is considerable evidence of premature aging in Down syndrome [18,19], the results obtained for the Down neurons could be considered added support for the occurrence of an age-dependent decrease in PK" Further evidence for a decrease in Px with aging has been obtained by Zs.-Nagy and colleagues in their attempts to validate the "membrane hypothesis" proposed by Zs.-Nagy to explain cellular aging [20--22]. According to this hypothesis, aging causes a reduction in membrane potassium permeability (PK)" The resulting increase in intracellular K disrupts basic intracellular processes, e.g. transcription causing the widespread cellular effects of aging. Zs.-Nagy and his colleagues have obtained evidence both for the decrease in PK in aged rat brain cortical neurons [7] and for the increased intracellular K in aged snail neurons [23]. Another investigation of the effects of aging on neurons has been carried out recently by Fukuda [6]. He conducted an extensive and sophisticated comparison of the morphological characteristics of young adult (4--8 weeks) and old (98--99 weeks) C57BL/6 male mice DRG maintained in monolayer culture for 7 days. He found some interesting differences including decreased neuron survival, and accelerated reduction in cell size for the old neurons. He also carried out a brief electrophysiological investigation but found no age difference in VM, RI, or CI, and RISE of both Na and Ca APs. However, this analysis was based on samples of 10--15 neurons. Previous work in our laboratory (for review, see Ref. 14) has indicated that much larger samples of approximately 100 neurons are required to detect subtle differences in EMP due to the masking effect of the variance of EMP even for control neurons. The purpose of the present study was to investigate the effect of age on the EMP of mammalian neurons and in particular to obtain evidence relevant to the hypothesis of reduced PK in old neurons. Voltage clamp techniques are the obvious method of choice to examine such permeability changes. However, DRG consist of a heterogeneous population of neuron types both morphologically and functionally [15] and each type probably has different ionic permeability characteristics. There is no doubt that the major part of the variance in EMP mentioned above is due to this heterogeneity. Voltage-clamp methodology, either intracellular or patch-clamp, requires considerable time and care for each neuron examined and it would be extremely difficult to obtain the requisite large sample of 100 neurons. Therefore in

206

the present study quantitative intracellular current-clamp techniques were used. These techniques were used previously to detect the effect of a variety of agents on EMP of neurons in cell culture [13--16] and here were used to investigate the effect of age on the EMP of mouse DRG neurons in cell culture. Even using this relatively simple and fast recording technique, it was found necessary to select experimental conditions to minimize unwanted variability. Previous studies had indicated that EMP changed with time in culture particularly during the first week in culture [14,15]. Therefore in the present study, approximately a 3-week culture duration was used to allow stabilizaton of EMP. Also preliminary experiments had indicated that to detect age-associated alterations of EMP it was necessary to use yoked experiments, i.e. simultaneous culturing of young and old neurons and to sample two mice for each condition in each experiment. Using this quantitative electrophysiological technique, a number of highly significant alterations in EMP were found for the old neurons. A possible explanation for these age-associated EMP changes was proposed in terms of an increased involvement of calcium channels in action potential generation and a decreased PK with aging. MATERIALS AND METHODS

Female mice of C57BL/6J strain purchased from Jackson Laboratory (Bar Harbout, ME) were used. The animals were aged in the animal facilities of the Medical Sciences Building of the University of Toronto. All animals were checked daily for disease and only healthy animals (i.e. without skin lesions or tumors) were selected for use. The techniques for both the cell culture and the electrophysiology have been described previously [13--16]. In each experiment approximately 45 ganglia were dissected from two backbones, softened by a 1-h incubation in 0.25°70 collagenase (CLSSOS, Cooper Biomedical, Malvern, PA), rinsed, and resuspended in culture medium. The latter consisted of CMRL-1415 (Connaught Medical Research Laboratories, Downsview, Canada) with penicillin and streptomycin present at 50 units/ ml and 50/ag/ml respectively and supplemented with 10°70 fetal calf serum (FCS, Gibco, Grand Island, NY). The softened ganglia were dissociated by gentle trituration and three drops (0.3 ml) of the cell suspension plated on each of six collagen-coated Aclar microculture vessels [1] placed in plastic petri dishes. Cultures were incubated at 35°C, maintained at pH 7.4 with approximately 50?0 CO 2 and fed three times weekly. In each experiment, the above procedure was carried out using two control mice (8--14 weeks) and two old mice (90--92 weeks). Cultures of control and old neurons were prepared simultaneously in order to have the two groups yoked with respect to experimental conditions including medium, collagenase, trituration, and culture conditions.

207 For the electrophysiological work, after 20 days in vitro (DIV), a culture was rinsed and mounted in a special recording chamber attached to an inverted phase contrast microscope. The culture was bathed in CMRL-1415 supplemented with 10V0 fetal calf serum without sodium bicarbonate and with pH adjusted to be 7.4 in air. Temperature was maintained at 36°C. Glass microelectrodes were filled with 3 M KCI and had initial resistances of 40-60 M o but were routinely broken to 30--50 M o on the collagen substrate. EMP were measured using an intracellular amplifier with bridge circuitry for impedance measurements (Model 9700, Dagan Corp., Minneapolis). Great care was taken to select only stable potentials (maximum permitted variations in VM was 5 mV over a 3-min period). Using procedures described previously [13--16], the EMP defined in Table I were determined. RM was calculated using RM = RI × AREA where AREA was calculated using the formula for either a sphere or a prolate spheroid depending on whether the axes were equal or different. DRG neurons were very suitable for this type of calculation because they were generally spherical or only slightly elliptical. Moreover they do not have dendrites and nearly always have a single small neurite whose effect on RM calculation is minimal. TICON was obtained from the approximation TICON = IS/IL [16]. CM was calculated from TICON = RM × CM. In previous studies, Frolkis [4,5] found a difference between the effects of aging on electrical excitability for mollusk and rat neurons; in the rat, IL decreased from 3.0 to 2.0 nA (P < 0.02) whereas in the mollusk IL had increased. With respect to measuring electrical excitability, it seems likely that comparisons of threshold depolarization (VRH = IL × RI) would have been more valid indications of changes in excitability since values of IL could be affected by changes in RI. It is for this reason that we have included VRH as well as IL as measures of electrical excitability in the present and previous investigations [16]. A third measure of electrical excitability was VTH which was set equal to VM minus VRH. VTH takes into consideration variations in VM for example due to differences in neuron types [15]. Since APs could have either monophasic or biphasic falling phases, both the rate of fall of the initial falling phase (FALL1) and that of the final falling phase (FALL2) were determined. The ratio of FALL2 and FALLI was computed (FAR). FAR resembles the variable MB used previously [15] in that it also is a measure of the degree of biphasicity. For APs with monophasic falling phase, FAR = 1 and for biphasics it is greater than one. Since many studies have shown that the maximum deflection reached during the AHP approaches VK, another variable calculated was VK = VM + AHP. However, VK was only a very rough indicator of VKchanges since it is also affected by PK changes. The measurements and the waveform of the AP were fed into an HP 9816 computer for automatic determination of certain AP variables (OS, AHP, DT, RISE, FALL1 and FALL2) and calculation of other computed variables (RM, T, CM,

2O8 TABLE II AGES AND NUMBER OF NEURONS EXAMINED ELECTROPHYSIOLOGICALLY IN YOKED DRG CELL CULTURES PREPARED FROM C57/6BL MICE

Expt. No

Controls Age (weeks)

1 2 3 4

Totals

8 12 13 14

Experimental No. neurons examined 27 30 24 22 103

Age (weeks)

90 92 90 92

No. neurons examined 35 29 25 22 111

VRH, AREA and FAR). It is to be noted that the measured and computed EMP were obtained individually for each neuron and these data were used to calculate group means. The entire procedure described above was repeated four times (Table II) and the data subjected to statistical analysis using the Statistical Package for the Social Sciences (SPSS/PC). RESULTS

The morphological development of adult mouse DRG neurons has been described in detail previously in several studies ([13], review Ref. 14). Although not the main focus of attention in this study, qualitative observation indicated no differences in ease of dissociation, neuron survival or morphological development in culture between the control and old cells. Initially, a two-way analysis of variance (ANOVA) was carried out for each EMP by condition (young or old) by experiment number (replication one through four). Although there were significant differences in EMP across replications, there were no significant interactions between condition and experiment number indicating that the effects of age were independent of experiment number and also of the minor age differences within each group. Therefore the data from the four replications were pooled and a one-way ANOVA of EMP by condition carried out. This analysis indicated a number of significant (P < 0.05) differences in EMP for the old neurons compared to the controls (Table III, Fig. 1). Three of the largest and statistically most significant changes were a 38.0070 increase in VRH, a 21.6% decrease in VTH and a 46.4°7o increase in IL. All three changes indicated a decrease in electrical excitability for the old neurons.

209 TABLE III COMPARISON OF THE EMP OF CONTROLS AND OLD DR(} NEURONS IN CELL CULTURE EMP"

Control ( N = 103) VM

RI RM TICON CI CM

VRH VTH DT RISE FALLI FALL2 OS AHP VK AREA FAR IL IS

A N O V A with covariate D T

A N O V A without covariates Old (AT = l i d

% change~

t~

58.7

59.1

+ 0.68

0.493

15.8 673 1.77 0.124 2.78 22.9 35.6 1.76 262 64.4 81.5 24.0 8.18 66.9 4384 1.45 1.66 27.0

14.3 649 1.66 0.126 2.71 31.6 27.9 2.33 269 44.7 69.0 29.3 9.75 68.8 4628 2.05 2.43 36.0

-9.49 - 3.57 -6.21 + 1.61 -2.52 + 38.0 - 21.6 + 32.4 + 2.67 - 30.6 - 15.3 +22.1 + 19.2 + 2.84 + 5.56 + 41.4 + 46.4 +33.3

0.038 0.462 0.111 0.805 0.616 0.000 0.000 0.000 0.312 0.000 0.003 0.000 0.005 0.001 0.018 0.000 0.000 0.000

Control Old ( N = 103) ( N = l i d 58.0

16.1 676 1.80 0.125 2.68 22.3 35.6 a 260 52.4 71.0 25.8 8.60 66.6 4339 1.79 1.55 27.4

59.8

14.3 651 1.53 0.125 2.34 31.6 25.4

% P change + 3.16

0.001

- 11.2 - 3.74 - 15.0 +0.00 - 12.3 +42.1 - 20.4

0.021 0.382 0.001 0.724 0.082 0.000 0.000

+ 4.23 + 6.66 + 7.79 +6.97 +8.96 + 3.89 + 7.58 + 0.00 + 55.4 +29.6

0.147 0.262 0.067 0.118 0.180 0.000 0.003 0.980 0.000 0.001

_

271 55.9 76.6 27.6 9.36 69.2 4668 1.79 2.41 35.6

•See Table I for definitions of different EMP. be/, is the difference between the controls and old EMP expressed as a % of the former. cp represents probability as determined from analysis of variance (ANOVA). dNot calculated because DT was used as a covariate in ANOVA.

As well there were large a n d significant changes in several other variables which indicated a shift to longer d u r a t i o n A P s with m o r e biphasic falling phases; D T was increased b y 3 2 . 4 % , F A R which is a m e a s u r e o f the degree o f biphasicity increased b y 4 1 . 4 % . OS a n d A H P b o t h increased b y a p p r o x i m a t e l y 2 0 % which is also consistent with a c h a n g e to m o r e biphasic types o f A P s [14]. There was a small b u t highly significant ( P = 0.001) increase in VK o f 2 . 8 4 % . A R E A was also increased significantly b u t this m u s t be interpreted with c a u t i o n since n e u r o n s were n o t s a m p l e d r a n d o m l y with respect to cell size; o n l y the largest n e u r o n s were selected in b o t h c o n d i t i o n s i n order to i m p r o v e electrical recordings. A linear regression analysis indicated t h a t for b o t h c o n t r o l a n d old n e u r o n s , V R H was significantly ( P < 0.01) linearly related to V M (Fig. 2), the correlation coefficients ( P e a r s o n ' s ) being 0.517 a n d 0.353, respectively. The two regression lines did n o t differ significantly i n slope b u t h a d highly significant ( P < 0.005) differences in y-intercepts s h o w i n g that the age-associated decrease i n electrical excitability was o f

210 IL

FALLI VTH FALL2 TICDN ( n . s . ) l

1

AREA

CM (n.s.) I

I VK I RISE [n.s.)

P~ (n.s.), VM (n.s.) I

-40

l

I

-30

i

i

-PO

i l ,

-~0

~

0

l

i

10

l

i

20

i

,

l

30

40

PERCENT CHANGE

Fig. 1. Age-associated percent changes in electrical m e m b r a n e properties (EMP). Horizontal bars represent the differences in m e a n E M P values between old a n d control neurons expressed as a percent of the m e a n values for the control neurons (for E M P abbreviations see Table I a n d for specific values see Table III, c o l u m n s 1--5). All differences were significant ( P < 0.05) except where marked n.s. Bar to right indicates an increase from control values and to the left a decrease.

4g.0

3g.0

i >~

29.0

19.0

I 56.3

I 56.8

I 61.3

I 63.8

I 66.3

VH Fig. 2. Threshold depolarization (VRH) vs. resting m e m b r a n e potential (VM). Note the parallel and linear relationship o f V R H to VM for both control ( ) a n d old (- - -) neurons. This indicates that there is an age-associated decrease in electrical excitability which is independent o f changes in VM.

211

constant magnitude for different VMs. In order words the observed differences in VRH were not just due to differences in the distribution of resting membrane potentials between young and old neurons, but rather represent an age-associated change in electrical excitability independent of any changes in VM. Because of the age-associated shift to longer duration APs, the data were also subjected to an ANOVA using DT as a covariate to determine if there were any effects of age independent of DT prolongation. As indicated in Table III, even with DT as a covariate, the three variables intimately related to electrical excitability (VRH, IL and VTH) still had significant alterations indicative of decreased sensitivity. Also regression analyses indicate that unlike the linear dependence of VRH on VM, VRH did not correlate with DT for either control or old neurons. Thus the decreased electrical excitability of the old neurons occurred independently of the age-associated increase in DT. The DT covariate analysis also indicated several other age-dependent changes; VM and VK increased (P < 0.001), and TICON decreased significantly (P < 0.001) due to non-significant decreases in RM and CM. DISCUSSION

This study has shown that the age of donor animals has a significant effect on the electrical properties of DRG neurons as determined after 3 weeks of cell culture. The largest and most significant changes were a decrease in electrical excitability and an increase in AP duration. The latter was due to decreased rate of repolarization of the AP and to increased amplitude of the OS and AHP. There are several difficulties in concluding that the observed EMP changes represent the effects of aging in situ. First, although the 8--14-week mice of the control group are sexually mature they may be immature with respect to other functions. Therefore there is the possibility that the observed EMP changes represent the effects of maturation rather than aging. Future studies should explore details of agerelated changes in EMP by including middle-aged mice. Another difficulty in interpreting the observed EMP changes arises out of the fact that DRG neurons do not constitute a homogenous population either morphologically or functionally [14,15]. Thus there is the possibility that with increasing age there is a differential drop-out of particular types which could have caused the observed EMP changes. Future work should include some way to control for this confounding factor. The question also arises whether the observed EMP changes are not just the result of a differential effect of culture conditions on the old vs. the control DRG cells. However there is evidence that the altered EMP observed here after culture do indeed reflect age-induced changes occurring in situ. Firstly, it is difficult to explain all of the EMP changes observed here on the basis of the results of a previous investigation of the effect of DIV on EMP [15]. This earlier study had indicated that

212

adult DRG APs could be divided into two types; a short duration M-type with monophasic falling phase and a longer duration B-type with biphasic falling phase. With increasing DIV there was a shift from M-type to B-type with concomitant alterations in a number of EMP. Since a similar shift to biphasicity was observed for the old neurons in the present study, one might suggest that the altered EMP of the old neurons were just due to an accelerated shift of the old neurons to B-type and consequently altered EMP. However, this suggestion is not plausible since not all of the altered EMP for the old neurons are consistent with those observed with increased DIV. For example, for both M and B-type, VRH decreased with DIV while in the present study one of the largest changes was increased VRH for the old neurons. Also for both types, RM which increased dramatically with DIV in the previous study did not show any significant change for the old neurons. The second reason for believing that the altered EMP observed here in vitro reflect age-induced EMP changes occurring in situ is that similar EMP changes have been observed in several other electrophysiological studies of old neurons in situ. Decreased electrical excitability, surprisingly similar to that observed in the present study, has also been reported for old snail neurons by Frolkis et al. [5] using in situ measurements. Although no significant increase in DT was observed by these investigators, the rate of repolarization was reduced by 21.5°70 which is in good agreement with the decreases of 30.6070 and 15.3070 for FALL1 and FALL2 observed in the present study. In a study of aged motor neurons in situ [4] Frolkis observed a significant increased in DT and mentioned that this increase was mainly due to a "decreasing rate of AP back slope drop". These in situ observations are also in agreement with the increased DT and decreased FALL 1 and FALL2 observed in the present study. Frolkis (see Ref. 3, page 274) had also reviewed in situ studies of aged muscle fibers in which increased DT and decreased electrical excitability have been reported. With respect to the increased amplitude of the A H P observed in the present study, it is most interesting that an increased duration of the Ca-dependent A H P was reported by Landfield and Pitier [10] in slice preparations of hippocampal neurons of aged rats. It is obvious from their Fig. 1 that the amplitude of the A H P was also increased as was observed in the present study for old mouse DRG neurons. Thus in view of the impossibility of explaining the observed EMP changes in terms of a DIV effect and in view of the similar results obtained by in vitro and in situ experiments, it seems reasonable to conclude that the altered EMP observed in the present study of old neurons in vitro reflect age-induced EMP alterations occurring in situ. The effects of age on EMP of DRG neurons in cell culture obtained here at 20 DIV are generally consistent with those obtained by Fukuda at 7 DIV [6]. Fukuda reported no significant effect of age on VM, RI, CI or maximum rate of rise of Na o r Ca spikes. Similarly in the present study, no significant alteration in VM, CI or RISE were detected (Table III). Although RI was increased significantly in the pre-

213 sent study, this did not result in a significant increase in RM. Unfortunately, Fukuda did not measure the various other EMP found to be affected by age in the present study such as VRH and DT. Although accurate interpretation of the age-dependent EMP changes observed here in terms of possible changes in underlying ionic mechanisms would require voltage clamp experiments, it is of interest to speculate on the most likely mechanisms. Two of the largest effects of age were to cause a decrease of electrical excitability and an increase in DT. Both of these effects are consistent with the hypothesis of an age-induced shift to Ca channels. Excitability would be decreased with a shift from Na to Ca voltage-sensitive channels since the latter generally require a greater depolarization for excitation than Na channels [8]. The prolongation of APs was due to a more biphasic type of AP with decreased FALL1 and FALL2. The 15.3e/0 decrease in FALL2 could be due to decreased Pz as suggested by Frolkis [4,5]. However, the decrease in FALL1 was twice as great and probably was due to an increased involvement of Ca channels since previous work has shown this phase of the AP to be Ca dependent [15]. Moreover as mentioned previously, adult DRG APs can be divided into monophasic M-type which are Na dependent and biphasic B-type which are both Na and Ca dependent. B-type also had increased OS, AHP and DT compared to M-type and all of these changes in EMP were also observed for the old neurons in the present study further supporting the idea of an age-associated shift to Ca channels. Since there is evidence of premature aging in Down syndrome [18,19], it is of interest to compare the results of the present study with those obtained for Down syndrome DRG neurons in cell culture [16]. In contrast to the old mouse neurons, the Down syndrome neurons had increased electrical excitability and reduced AHP compared to age-matched controls. However like the old neurons of the present study, the Down neurons has increased DTs and a switch to more biphasic falling phases suggesting an increased involvement of voltage-sensitive Ca channels. Also both the old neurons and the Down syndrome neurons had indications of decreased PK (e.g. decreased FALL2 in both, and decreased RM for the Down neurons). The results of the present study suggest that there is an increased involvement of voltage-sensitive Ca channels and to a lesser extent decreased PK in old neurons. This hypothesis is supported by a number of previous studies also suggesting an important role of Ca in neuron aging [10,11,17] and decreased Pz [3--5,7,12,20--22]. The investigation of the effects of aging on EMP deserves further study since it may shed light on the neurobiology of Down syndrome, neurobiology of aging, and the multicentric changes with age of the organism as a whole [23]. ACKNOWLEDGEMENTS We thank the Natural Sciences and Engineering Council of Canada, the Ontario Mental Health Foundation, the Hospital for Sick Children Foundation, the Con-

214 naught

Foundation,

the Canadian

Geriatric Research

Society and the Canadian

National Cancer Institute for providing funds for this research. We also thank Mrs. S. K o h a n d M s . U s h a P o n n a p p a n

for their assistance with the logistics of animal

procurement. REFERENCES 1

2

3 4 5

6

7

8 9 10 11 12

13 14 15 16 17

18 19 20 21 22 23

R.P. Bunge and P. Wood, Studies of the transplantation of spinal cord tissue in the rat. I. Development of a culture system for hemisections of embryonic spinal cord. Brain Res., 57 (1973) 261--276. B. Cinader, Immune senescence: Individual variation and multicentricity. In A.L. de Week (ed.), Lymphoid Cell Functions in Ageing: Topics in Ageing Research in Europe, Vol. 3, Eurage, Rijswijk, 1984, pp. 3--17. V.V. Frolkis, Aging and Life Prolonging Processes, Springer, Berlin, 1982. V.V. Frolkis (ed.), Physiology of cell aging. Interdiscip. Top. Gerontol., 18 (1984) 1--28. V.V. Frolkis, A.S. Stupina, O.A. Martinenko, S. Toth and A.I. Timchenko, Aging of neurons in the mollusc Lymnaea stagnalis: structure, function and sensitivity to neurotransmitters. Mech. Ageing Dee., 25 (1984) 91--102. J. Fukuda, Nerve cells of adult and aged mice grown in a monolayer culture: age-associated changes in morphological and physiological properties of dorsal root ganglion cells in vitro. Dee. Neurosci., 7 (1985) 374--394. M. Gyenes, G. Lustyik, V. Zs.-Nagy, F. Jeny and I. Zs.-Nagy, Age-dependent decrease of the passive Rb* and K* permeability of the nerve cell membrane in rat brain cortex as revealed by in vivo measurement of the Rb* discrimination ratio. Arch. GerontoL Geriatr., 3 (1984) 11--3 I. B. Hille, Ionic Channels o f Excitable Membranes, Sinauer Associates, Inc., Sunderland, Ma., 1984, p.80. D.C. Kimmel, Adulthood andAging, 2nd edn., Wiley, New York., 1980. P.W. Landfield and T.A. Pitier, Prolonged Ca dependent afterhyperpolarization in hippocampal neurons in aged rats. Science, 226 (1984) 1089-- 1091. S.W. Leslie, L.J. Chandler, E.M. Barr and R.P. Farrar, Reduced calcium uptake by rat brain mitochondria and synaptosomes in response to aging. Brain Res., 329 (1985) 177--183. G. Lustyik and V. Zs.-Nagy, Alterations of the intracellular water and ion concentration in brain and liver cells during aging and centrophenoxine treatment as revealed by energy dispersive X-ray microanalysis. Scanning Electron Microsc., I (1985) 323--337. B.S. Scott, Adult mouse dorsal root ganglia neurons in cell culture. J. Neurobiol., 8 (1977) 417-427. B.S. Scott, Adult neurons in cell culture: electrophysiological characterization and use in neurobiological research. Prog. Neurobiol., 19 187--211. B.S. Scott and B.A.V. Edwards, Electric membrane properties of adult mouse DRG neurons and the effect of culture duration. J. Neurobiol., 11 (1980) 291--301. B.S. Scott, T.L. Petit, L.E. Becker and B.A.V. Edwards, Abnormal electric membrane properties of Down's syndrome DRG neurons in cell culture. Dee. Brain Res., 2 (1982) 257. H. Selye and P. Prioreschi, Stress Theory of Aging. In N.W. Shock (ed.), Aging: Some Social and Biological Aspects, American Association for the Advancement of Science, Proceedings of a Symposium at Chicago, 1960. F.M. Sinnex and C.R. Merril, Alzheimer's disease, Down's syndrome and aging. Ann. N.Y. Acad. Sci., 396 (1982) 3--14. D. St. Clair and D. Blackwood, Premature senility in Down's syndrome. Lancet, 8445 (1985) 34. I. Zs.-Nagy, The biochemistry o f aging, Symposium at Santa Monica, California, Nov 30--Dec 2, 1977. I. Zs.-Nagy, A membrane hypothesis of aging. J. Theor. Biol., 75 (1978) 189--195. I. Zs.-Nagy, The role of membrane structure and function in cellular aging: a review. Mech. Ageing Dee., 9 (1979) 237--246. I. Zs.-Nagy, S. Toth and G. Lustyik, Verification of the membrane hypothesis of aging on the identified giant neurons of the snail Lymnaea stagnalis L. (Gastropoda, Pulmonata) by a combined application of intracellular electrophysiology and X-ray microanalysis. Arch. Gerontol. Geriatr., 4 (1985) 53--66.