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Aging enhances the calcium sensitivity of central neurons of the mouse as an adaptive response to reduced free intracellular calcium
Neuroscience Letters, 152 (1993) 181-184
181
© 1993 ElsevierScientificPublishers Ireland Ltd. All rights reserved0304-3940193/$06.00 NSL 09411
Aging enhances the calcium sensitivity of central neurons of the mouse as an adaptive response to reduced free intracellular calcium H e n r i k e H a r t m a n n , A n n e E c k e r t a n d W a l t e r E. Miiller Department of Psychopharmacology. Central Institute of Mental Health, Mannheim ( FRG)
(Received 30 October 1992; Revised version received 16 December 1992; Accepted 11 January 1993) Key words." Dissociatedneuron; Brain aging; Depolarization;Intracellular calcium; Inositolphosphate
Age-relatedchanges in CaZ+-homeostasishave been investigatedin mechanicallydissociated neurons from young and aged mice. In aged animals, basal intracellular calcium ([Ca2+]i) was significantlyreduced and depolarization (KC1)-inducedrise in [CaZ*]~was lower, probably as a result of increased activationof Ca2+-dependentmechanisms terminating Ca 2+ influx.Additionally,depolarization-inducedinositolphosphate(IP) accumulation in aged animals was found to be significantlyincreased. Both findingssuggest that Ca2+-dependentintracellularprocessesbecomemore sensitive to Ca> in aged animals due to decreasedCa> availability.
Intracellular free calcium ([Ca2+]i) represents the most important intracellular messenger for many signaltransduction pathways of central neurons. Changes of Ca2+-homeostasis have been suggested to play a crucial role for brain aging in general and for age-related deficits of learning, memory and other cognitive functions particularly [4, 20]. Although this hypothesis is quite attractive in many ways, its general acceptance is hampered by conflicting observations which do not allow to draw a final conclusion if [Ca>]j availability in central neurons is decreased or increased in aging [14, 20]. This controversy might originate from the fact that in different studies various tissue preparations have been used and investigations have concentrated on only one of many cellular processes which actually regulate [Ca2+]~. Accordingly, reduced Ca 2÷ influx into synaptosomes [4], but enhanced inward Ca 2+ currents [14] and enhanced calcium-dependent afterhyperpolarization in hippocampal slices [15] are examples for these divergent findings. In an attempt to find a comprehensive answer on age-related changes in neuronal Ca2+-homeostasis, we compared baseline [Ca2+]i and depolarization-induced rise of [Ca2+]i in mechanically dissociated neurons from young and aged mice. Dissociated mouse brain cells were prepared from young (3 months) and aged (22 months) femal N M R I mice (Interfauna, Tuttlingen, F R G ) essentially following Correspondence.. H. Hartmann, Department of Psychopharmacology, Central Institute of Mental Health, 6800 Mannheim, FRG.
the method of Stoll et al. [23]. The protein content of the preparation of young and aged animals was not different and averaged between 60-100/.tg/ml. For measurements of [Ca2+]~, the cell preparation was resuspended in 2 ml Hank's balanced salt solution (HBSS) containing 1 mmol/1 CaCI2 and 1 mmol/1 MgSO4, and incubated with fura-2 AM (Molecular Probes, USA) (10/1moll1) for 45 min in a shaking water bath (37°C). After dye-loading, cells were washed twice and resuspended in 14 ml HBSS (see above). Aliquots of 2 ml were taken and kept at 37°C. For studies on time dependence, depolarization was started by adding 50/11 KC1 (20 mmol/1) and cells kept at 37°C for the according time period. At the end of the appropriate incubation time (15, 30, 45, 60 min), each sample was washed immediate before measurement. Fluorescence was measured with a SLM Aminco 4800 spectrofluorometer, where samples were kept at 37°C under magnetic stirring. After equilibration to get the basal [Ca2+]i KC1 (50 pl) was added. [Ca2+]i was calculated according to Grynkiewiecz et al. [8] (Rma x 0.2% S D S , Rmin EGTA 6 mmol/1, TRIS 30 mmol/1). Inositolphosphate accumulation was determined as described previously by Stoll et al. [23] with slight modifications [9] using a stimulation period of 60 min. Statistical comparison was performed by ANOVA (SAS package) and by t-test as post hoc analysis. The preparation of mechanically dissociated neurons [11] has been increasingly used in the last years to study a variety of functions of central neurons including receptor regulation [3, 22], phosphoinositide hydrolysis [23,
182
1200
O
Et -
900
.
~- =°:
!~"
1
~ Young Aged, T,
:v- i
lC=~]., [Ca=*I., lmmoi 'I
3.1mmol/l
600
! I
O 300
Basal
20
60
80
K*(mmol/I)
Fig. 1. Basal and potassium-induced [Ca:~]~in mechanically dissociated neurons from young (3 months, open bars) and aged (23 months, hatched bars) female NMRI mice. Values represent the maximal initial response (see Fig. 2) and are means + S.D. In = 6 7), each representing an individual animal (ANOVA P<0.001, young vs. aged). Inset: [Ca2+]i([Ca2+]iminus basal [Ca2+]i)in mechanically dissociated neurons from young and aged mice (see above) alter depolarization with K +(20 mmol/l). Extracellular calcium concentration was 1 mmol/I or 0.1 mmoV1, respectively. Values represent means _+S.D. (n = 6 7), each representing an individual animal, ("P < 0.01. *"P < 0.001, t-test).
24] and accumulation o f c A M P and c G M P [1,21]. Moreover, a very recent report has clearly demonstrated the suitability o f this preparation to study [Ca2+]~ [25]. The ATP content o f this preparation has been shown to be lower than in intact brain, but substantially higher than ATP levels f o u n d in synaptosomes. Furthermore, the respiration rate o f dissociated brain cells was found to be in the range o f that f o u n d in cultured neurons, indicating a g o o d preservation o f metabolic activity. In agreement with findings using the same tissue preparation but rat brain [25], [Ca2+]~ o f adult mice averages at about 350+41 nmol/1 (n = 13). Very interestingly, baseline [Ca2+]i was significantly (P < 0.001 ) lower in dissociated neurons o f aged animals (265 + 4 4 nmol/1) (n = 13). The significant difference in baseline [Ca2+]~ was not altered by incubating cells for up to 60 min, since there was only a small increase (about 10%) in baseline [Ca2+]~ during that time in y o u n g and aged neurons. Depolarization o f cells with KCI elevated [Ca2+]~ significantly more in neurons f r o m y o u n g than from aged animals. The increase in [Ca2+]i was dependent on the potassium concentration (Fig. 1). Furthermore, KC1 (20 mmol/1)-induced rise in [Ca2+]i was dependent on the extracellular calcium concentration for neurons of y o u n g and aged animals in a similar fashion (Fig. 1, inset), indicating that different mobilization o f Ca 2+ f r o m intracel-
lular pools might not be the explanation for the a g e - d i f ference given in Fig. 1. Fig. 2 shows the time course o f KC1 (20 mmol/l)-induced rise in [Ca2+]i. The KCl-induced increase in [Ca2+]1 was already seen after a few seconds and remained oil el high level over 60 min. Reduced [Ca2~]i levels in aged animals were seen t h r o u g h o u t the same time period (Fig. 2). However, 15 min after the initial maximal response [Ca2+]~ was significantly reduced in the y o u n g g r o u p (from 613 +_ 65 nmol/1 to 511 _+ 34 nmol/l, P < 0.01), but not in the aged g r o u p (from 385 + 44 nmol/1 to 357 _+ 30 nmol/1, P > 0.05) (Fig. 2). A possible explanation is olfered by the findings o f Michaelis et al. [18] indicating that the affinity o f the Na+/Ca 2+ exchanger is rather decreased than increased in the aging brain. The sustained high [Ca2+]i levels in our preparation even 60 rain after depolarization contrast to observations in rat brain synaptosomes where the potassium-induced rise of [Ca2'], was rapidly reversed to baseline levels, even when K ' was still present [5]. In our experiments, K+-induced elevations of [Ca2+]i remained constant t"oi at least 60 min, indicating the formation o f a new stable equilibrium between increased Ca TM influx, increased inactivation o f calcium channels and stimulation of Ca 2+ extrusion mechanisms. A specific impairment o f the energy metabolism in the aged neurons would presumedly result in a reduced gradient between [Ca2+]~x and [Ca2+]i. This has not been found in the present study. Therefore, the ability of the aged neuron to keep the [Ca2~}~ response lower when c o m p a r e d with neurons of y o u n g animals rather suggests that Ca2~-dependent mechanisms terminating Ca > influx might be activated by lower Ca 2~ concentra-
800
600 O
Et-~-
400
o 200
0
I
L
I
I
I
0
15
30
45
60
time
(rnin}
Fig. 2. Time course of[Ca2+],upon depolarization with KC1 (20 mmol/I) in dissociated neurons from young (open circles) and aged (closed circles) mice. Values represent means -+ S.D. (n = 7), each representing an individual animal (ANOVA P < 0.001, young vs. aged).
183
0.20
0.15
~ ~
0.t0
~0.,0
0.10
~: 0.05
0
10
20
60
80
K*(mmol/I) Fig. 3. Potassium-induced [3H]InsP~ accumulation in dissociated neurons from young (open bars) and aged (hatched bars) mice. Values represent means + S.E.M. (n = 8), each representing an individual animal (ANOVA P < 0.05, young vs. aged). Inset: relationship between KCl-induced IP response (Fig. 3) and increase in [Ca2+], (Fig. 1) in dissociated neurons from young (open circles) and aged (closed circles) female NMRI mice.
tions in neurons of aged animals. One possible mechanism might be the calcium-dependent reversible Cachannel inactivation [19]. A further addition of K + (20 mmol/1) to cells which had been depolarized with K + (20 mmol/1) for 60 min revealed that even after this long K + preincubation potassium-induced Ca 2+ influx was still possible and again was to a similar extent higher in neurons of young (105 + 36 nmol/1) than of aged animals (44 + 17 nmol/1, P < 0.01). This suggests that in both preparations prolonged depolarization with K + is not leading to a complete inactivation or down-regulation of voltage-dependent Ca-channels. If one assumes that Ca2÷-dependent mechanisms which terminate Ca2+-influx become more sensitive in aged neurons as possible adaptive response to the lower [Ca2+]i, one is tempted to speculate that other Ca2+-dependent intracellular processes also become more sensitive to calcium or need lower [Ca2÷]i to be activated. To confirm this speculation, we have additionally investigated [Ca2+]~-stimulated phosphoinositide hydrolysis [2]. In agreement with our previous findings (Hartmann and Mfiller, submitted), IP response after depolarization with increasing K + concentration was significantly enhanced in aged animals (Fig. 3), although the same condition (Fig. 1) increases [Ca2+]i significantly less in aged than in young animals. Accordingly, [Ca2+]i-dependent IP hydrolysis is not only enhanced in aging (Fig. 3), which could indicate an increase in the Vmax of the phospholipase C (PLC), but also occurs at considerable smaller rises of [Ca2+]i (Fig. 3, inset). The latter observation is consis-
tent with the assumption of an enhanced Ca 2+ sensitivity of the phospholipase C (PLC) system. Our findings of a reduced [Ca2+]i availability but an enhanced intracellular Ca 2÷ sensitivity can speculatively integrate a large variety of divergent findings obtained for different brain preparations, i.e. initially enhanced C a 2+ inward currents [14] (as a consequence of the elevated gradient) but decreased Ca 2+ influx (see also this study) or decreased 45Ca uptake [16], altered properties of L channels [7, 13] (as possible indicators of an enhanced down-regulation), and enhanced sensitivity of various intracellular [CaZ+]i-dependent mechanisms like inactivation of Ca 2+ inward currents [14] or stimulation of PLC (this paper), impairment of frequency potentiation [12] and prolonged Ca2+-dependent afterhyperpolarization [15]. An explanation for the reduced [Ca2+]iof aged neurons cannot yet be given especially since the etltux is rather decreased than increased [18]. It is however remarkable that synaptosomes show rather elevated than reduced [Ca2+]i levels with aging [17]. Besides differences in metabolic activity in both preparations (see above), Ca 2+ influx in synaptosomes is mainly mediated by N channels, that of neurons by L channels [10]. Furthermore, N channels do not seem to be involved in depolarizationinduced PI hydrolysis [6]. Accordingly, the already mentioned age-related change of L channel properties might not only be a response to the reduced Ca 2+ level but might be alternatively one of its possible causes. Decreased [Ca2+]i and enhanced intracellular Ca 2÷ sensitivity of the aged neuron might compensate each other under normal conditions. However, it seems quite convincible that the ability of the Ca 2+ signal-transduction pathway to adopt to periods of over- and/or understimulation (e.g. stress, hypoxia) might be close to its limits in the aged brain. An enhanced bidirectional vulnerability of the calcium signal-transduction pathway of the aged neuron forms an attractive hypothesis to integrate the large variety of divergent findings linking alterations of Ca 2+ homeostasis with many pathological mechanisms of brain aging. This study was supported by a grant of the Deutsche Forschungsgemeinschaft (SFB 258).
1 Anderson, D.J. and McKinney, M., Muscarinic M 2 receptor-mediated cyclic AMP reduction in mechanically dissociated rat cortex, Brain Res., 475 (1988) 28 34. 2 Eberhardt, D.A. and Holz, R.W., Intracellular Ca z+ activates phospholipase C, Trends Neurosci., 11 (1988) 517-520. 3 E1-Fakahany, E.E. and Lee, J.H., Agonist induced muscarinic acetylcholine receptor down-regulation in intact rat brain cells, Eur. J. Pharmacol., 132 (1986) 21-30.
184 4 Gibson, G.E. and Peterson, C., Calcium and the aging nervous system, Neurobiol. Aging, 8 (1987) 329 344. 5 Giovanelli, L. and Pepeu, G., Effect of age on K+-induced cytosolic changes in rat cortical synaptosomes, J. Neurochem., 53 (1989) 392 398. 6 Gonzales, R.A. and Minor, L.D., Calcium channel involvement in potassium depolarization-induced phosphoinositide hydrolysis in rat brain, Neurochem. Res., 14 (1989) 1067 1074. 7 Govoni, S., Rius, R.A.. Battaini, F., Bianchi, A. and Trabucchi, M., Age-related reduced affinity in [~H]-nitrendipine labeling of brain voltage dependent calcium channels, Brain Res., 333 (1985) 374 377. 8 Grynkiewiecz G., Poenie, M. and Tsien, R.Y., A new generation of CaZ+indicators with greatly improved fluorescence properties, J. Biol. Chem.. 260 (1985) 3440 3450. 9 Hartmann, H., Cohen, S.A. and M/tiler, W.E., Efl'ect of subchronic pyritinol administration on receptor deficits and phosphatidylinositol metabolism in the aged mouse brain, Neuropharmacology, 32 (19931 119 125. 10 Hirning, L.D., Fox, A.D., McCleskey. E.W., Olivera. B.M., Thayer, S.A.. Miller, R.J. and Tsien. R.W., Dominant role of Ntype Ca > chamlels in evoked release of norepinephrine from sympathetic neurons, Science, 239 (1988) 57 61. I 1 ttonegger, P. and Richelson, E., Biochemical differentiation of mechanically dissociated mammalian brain in aggregating cell culture, Brain Res., 1(19(1976) 335 354. 12 Landlleld, RW., Hippocampal neurobiological mechanisms of agerelated memory dysfunction, Neurobiol. Aging, 9 (I 987) 571 579. 13 Landtield, RW., Nimodipine modulation of aging-related increases m hippocampal calcium currents. In J. Traber and WH. Gispen (Eds.i, Nimodipine and Central Nervous System Function: New Vistas, Schattauer, Stuttgart, 1989. pp. 227 238, 14 Landfield. P.W. and Pitier, T.A., Calcium spike duration: prolongation on hippocampal neuron of aged rats, Soc. Neurosci. Abstr.. 13 11987) 718. 15 Landtield, P.W. and Pitler, T.A., Prolonged Ca:~-dependent alL terhyperpolarization in hippocampal neurons of aged rats, Science, 226 11984) 1086 1091.
16 Leslie, S.W., Chandler, J., Barr, E.M. and Farrar, R.R, Reduced calcium uptake by rat brain mitochondria and synaptosomes in response to aging, Brain Res.. 329 (1985) 177 183. 17 Martinez, A., Vitorica, J. and Satrustegui, J., Cytosolic free calcium levels increase with age in rat brain synaptosomes. Neurosci. l,ett., 88 (1988) 336 342. 18 Michaelis, M.L, Johe, K. and Kitos, T.E., Age-dependent alterations in synaptic membrane systems for Ca > regulations, Mech. Ageing Dev., 25 (1984) 215 225. 19 Pitier, T.A. and Landfield, P.W., Probable Ca2+-mediated inactivation of Ca~--currents in mammalian brain neurons, Brain Res., 410 (1987) 147 153. 20 Roth, G.S., Mechanisms of altered hormone-neurotransmitter action during aging: from receptor to calcium mobilization. Annu. Re~. Geront, Geriatr., 10 (1990) 132 146. 21 Schubert, J-.. Stoll, L. and Mfiller, W.E., Therapeutic concentrations of lithium and carbamazepine inhibit c-GMP accumulation in human lymphocytes, Psychopharmacology, 104 (l 991 ) 45 50, 22 Sloll, L. and M61ler, W.E., In vitro down-regulation predicts agonist efficacy at cenlral muscarinic cholinergic receptors, Biochem Pharmacol.,41 (1991) 1963 1966. 23 Stoll, L.. Schubert, T. and Miiller, W.E., Age-related deficits of central muscarinic cholmergic receptor function in the mouse: partial restoration by chronic piracetam treatment, Neurobiol. Aging, 13 (1991) 39 44. 24 Surichamorn, W., Abdallah. ILA. and EI-Fakahany, [i.E., Aging does not alter brain muscarinic receptor-mediated phosphoinositide hydrolysis and its inhibition by phorbol esters, tctrodotoxin and receptor desensitization, J. Pharmacol. Exp. Ther., 251 (1989) 543 549. 25 Villalba, M., Martinez-Serrano, A.. Cristina, B., Blanco, P. and Satrustegui, J., NMDA-induced increase in [Ca>]~ and 4SCa>-up take in acutely dissociated brain cells derived from adult rats, Brain Res., 570 (1992) 347 353.
181
© 1993 ElsevierScientificPublishers Ireland Ltd. All rights reserved0304-3940193/$06.00 NSL 09411
Aging enhances the calcium sensitivity of central neurons of the mouse as an adaptive response to reduced free intracellular calcium H e n r i k e H a r t m a n n , A n n e E c k e r t a n d W a l t e r E. Miiller Department of Psychopharmacology. Central Institute of Mental Health, Mannheim ( FRG)
(Received 30 October 1992; Revised version received 16 December 1992; Accepted 11 January 1993) Key words." Dissociatedneuron; Brain aging; Depolarization;Intracellular calcium; Inositolphosphate
Age-relatedchanges in CaZ+-homeostasishave been investigatedin mechanicallydissociated neurons from young and aged mice. In aged animals, basal intracellular calcium ([Ca2+]i) was significantlyreduced and depolarization (KC1)-inducedrise in [CaZ*]~was lower, probably as a result of increased activationof Ca2+-dependentmechanisms terminating Ca 2+ influx.Additionally,depolarization-inducedinositolphosphate(IP) accumulation in aged animals was found to be significantlyincreased. Both findingssuggest that Ca2+-dependentintracellularprocessesbecomemore sensitive to Ca> in aged animals due to decreasedCa> availability.
Intracellular free calcium ([Ca2+]i) represents the most important intracellular messenger for many signaltransduction pathways of central neurons. Changes of Ca2+-homeostasis have been suggested to play a crucial role for brain aging in general and for age-related deficits of learning, memory and other cognitive functions particularly [4, 20]. Although this hypothesis is quite attractive in many ways, its general acceptance is hampered by conflicting observations which do not allow to draw a final conclusion if [Ca>]j availability in central neurons is decreased or increased in aging [14, 20]. This controversy might originate from the fact that in different studies various tissue preparations have been used and investigations have concentrated on only one of many cellular processes which actually regulate [Ca2+]~. Accordingly, reduced Ca 2÷ influx into synaptosomes [4], but enhanced inward Ca 2+ currents [14] and enhanced calcium-dependent afterhyperpolarization in hippocampal slices [15] are examples for these divergent findings. In an attempt to find a comprehensive answer on age-related changes in neuronal Ca2+-homeostasis, we compared baseline [Ca2+]i and depolarization-induced rise of [Ca2+]i in mechanically dissociated neurons from young and aged mice. Dissociated mouse brain cells were prepared from young (3 months) and aged (22 months) femal N M R I mice (Interfauna, Tuttlingen, F R G ) essentially following Correspondence.. H. Hartmann, Department of Psychopharmacology, Central Institute of Mental Health, 6800 Mannheim, FRG.
the method of Stoll et al. [23]. The protein content of the preparation of young and aged animals was not different and averaged between 60-100/.tg/ml. For measurements of [Ca2+]~, the cell preparation was resuspended in 2 ml Hank's balanced salt solution (HBSS) containing 1 mmol/1 CaCI2 and 1 mmol/1 MgSO4, and incubated with fura-2 AM (Molecular Probes, USA) (10/1moll1) for 45 min in a shaking water bath (37°C). After dye-loading, cells were washed twice and resuspended in 14 ml HBSS (see above). Aliquots of 2 ml were taken and kept at 37°C. For studies on time dependence, depolarization was started by adding 50/11 KC1 (20 mmol/1) and cells kept at 37°C for the according time period. At the end of the appropriate incubation time (15, 30, 45, 60 min), each sample was washed immediate before measurement. Fluorescence was measured with a SLM Aminco 4800 spectrofluorometer, where samples were kept at 37°C under magnetic stirring. After equilibration to get the basal [Ca2+]i KC1 (50 pl) was added. [Ca2+]i was calculated according to Grynkiewiecz et al. [8] (Rma x 0.2% S D S , Rmin EGTA 6 mmol/1, TRIS 30 mmol/1). Inositolphosphate accumulation was determined as described previously by Stoll et al. [23] with slight modifications [9] using a stimulation period of 60 min. Statistical comparison was performed by ANOVA (SAS package) and by t-test as post hoc analysis. The preparation of mechanically dissociated neurons [11] has been increasingly used in the last years to study a variety of functions of central neurons including receptor regulation [3, 22], phosphoinositide hydrolysis [23,
182
1200
O
Et -
900
.
~- =°:
!~"
1
~ Young Aged, T,
:v- i
lC=~]., [Ca=*I., lmmoi 'I
3.1mmol/l
600
! I
O 300
Basal
20
60
80
K*(mmol/I)
Fig. 1. Basal and potassium-induced [Ca:~]~in mechanically dissociated neurons from young (3 months, open bars) and aged (23 months, hatched bars) female NMRI mice. Values represent the maximal initial response (see Fig. 2) and are means + S.D. In = 6 7), each representing an individual animal (ANOVA P<0.001, young vs. aged). Inset: [Ca2+]i([Ca2+]iminus basal [Ca2+]i)in mechanically dissociated neurons from young and aged mice (see above) alter depolarization with K +(20 mmol/l). Extracellular calcium concentration was 1 mmol/I or 0.1 mmoV1, respectively. Values represent means _+S.D. (n = 6 7), each representing an individual animal, ("P < 0.01. *"P < 0.001, t-test).
24] and accumulation o f c A M P and c G M P [1,21]. Moreover, a very recent report has clearly demonstrated the suitability o f this preparation to study [Ca2+]~ [25]. The ATP content o f this preparation has been shown to be lower than in intact brain, but substantially higher than ATP levels f o u n d in synaptosomes. Furthermore, the respiration rate o f dissociated brain cells was found to be in the range o f that f o u n d in cultured neurons, indicating a g o o d preservation o f metabolic activity. In agreement with findings using the same tissue preparation but rat brain [25], [Ca2+]~ o f adult mice averages at about 350+41 nmol/1 (n = 13). Very interestingly, baseline [Ca2+]i was significantly (P < 0.001 ) lower in dissociated neurons o f aged animals (265 + 4 4 nmol/1) (n = 13). The significant difference in baseline [Ca2+]~ was not altered by incubating cells for up to 60 min, since there was only a small increase (about 10%) in baseline [Ca2+]~ during that time in y o u n g and aged neurons. Depolarization o f cells with KCI elevated [Ca2+]~ significantly more in neurons f r o m y o u n g than from aged animals. The increase in [Ca2+]i was dependent on the potassium concentration (Fig. 1). Furthermore, KC1 (20 mmol/1)-induced rise in [Ca2+]i was dependent on the extracellular calcium concentration for neurons of y o u n g and aged animals in a similar fashion (Fig. 1, inset), indicating that different mobilization o f Ca 2+ f r o m intracel-
lular pools might not be the explanation for the a g e - d i f ference given in Fig. 1. Fig. 2 shows the time course o f KC1 (20 mmol/l)-induced rise in [Ca2+]i. The KCl-induced increase in [Ca2+]1 was already seen after a few seconds and remained oil el high level over 60 min. Reduced [Ca2~]i levels in aged animals were seen t h r o u g h o u t the same time period (Fig. 2). However, 15 min after the initial maximal response [Ca2+]~ was significantly reduced in the y o u n g g r o u p (from 613 +_ 65 nmol/1 to 511 _+ 34 nmol/l, P < 0.01), but not in the aged g r o u p (from 385 + 44 nmol/1 to 357 _+ 30 nmol/1, P > 0.05) (Fig. 2). A possible explanation is olfered by the findings o f Michaelis et al. [18] indicating that the affinity o f the Na+/Ca 2+ exchanger is rather decreased than increased in the aging brain. The sustained high [Ca2+]i levels in our preparation even 60 rain after depolarization contrast to observations in rat brain synaptosomes where the potassium-induced rise of [Ca2'], was rapidly reversed to baseline levels, even when K ' was still present [5]. In our experiments, K+-induced elevations of [Ca2+]i remained constant t"oi at least 60 min, indicating the formation o f a new stable equilibrium between increased Ca TM influx, increased inactivation o f calcium channels and stimulation of Ca 2+ extrusion mechanisms. A specific impairment o f the energy metabolism in the aged neurons would presumedly result in a reduced gradient between [Ca2+]~x and [Ca2+]i. This has not been found in the present study. Therefore, the ability of the aged neuron to keep the [Ca2~}~ response lower when c o m p a r e d with neurons of y o u n g animals rather suggests that Ca2~-dependent mechanisms terminating Ca > influx might be activated by lower Ca 2~ concentra-
800
600 O
Et-~-
400
o 200
0
I
L
I
I
I
0
15
30
45
60
time
(rnin}
Fig. 2. Time course of[Ca2+],upon depolarization with KC1 (20 mmol/I) in dissociated neurons from young (open circles) and aged (closed circles) mice. Values represent means -+ S.D. (n = 7), each representing an individual animal (ANOVA P < 0.001, young vs. aged).
183
0.20
0.15
~ ~
0.t0
~0.,0
0.10
~: 0.05
0
10
20
60
80
K*(mmol/I) Fig. 3. Potassium-induced [3H]InsP~ accumulation in dissociated neurons from young (open bars) and aged (hatched bars) mice. Values represent means + S.E.M. (n = 8), each representing an individual animal (ANOVA P < 0.05, young vs. aged). Inset: relationship between KCl-induced IP response (Fig. 3) and increase in [Ca2+], (Fig. 1) in dissociated neurons from young (open circles) and aged (closed circles) female NMRI mice.
tions in neurons of aged animals. One possible mechanism might be the calcium-dependent reversible Cachannel inactivation [19]. A further addition of K + (20 mmol/1) to cells which had been depolarized with K + (20 mmol/1) for 60 min revealed that even after this long K + preincubation potassium-induced Ca 2+ influx was still possible and again was to a similar extent higher in neurons of young (105 + 36 nmol/1) than of aged animals (44 + 17 nmol/1, P < 0.01). This suggests that in both preparations prolonged depolarization with K + is not leading to a complete inactivation or down-regulation of voltage-dependent Ca-channels. If one assumes that Ca2÷-dependent mechanisms which terminate Ca2+-influx become more sensitive in aged neurons as possible adaptive response to the lower [Ca2+]i, one is tempted to speculate that other Ca2+-dependent intracellular processes also become more sensitive to calcium or need lower [Ca2÷]i to be activated. To confirm this speculation, we have additionally investigated [Ca2+]~-stimulated phosphoinositide hydrolysis [2]. In agreement with our previous findings (Hartmann and Mfiller, submitted), IP response after depolarization with increasing K + concentration was significantly enhanced in aged animals (Fig. 3), although the same condition (Fig. 1) increases [Ca2+]i significantly less in aged than in young animals. Accordingly, [Ca2+]i-dependent IP hydrolysis is not only enhanced in aging (Fig. 3), which could indicate an increase in the Vmax of the phospholipase C (PLC), but also occurs at considerable smaller rises of [Ca2+]i (Fig. 3, inset). The latter observation is consis-
tent with the assumption of an enhanced Ca 2+ sensitivity of the phospholipase C (PLC) system. Our findings of a reduced [Ca2+]i availability but an enhanced intracellular Ca 2÷ sensitivity can speculatively integrate a large variety of divergent findings obtained for different brain preparations, i.e. initially enhanced C a 2+ inward currents [14] (as a consequence of the elevated gradient) but decreased Ca 2+ influx (see also this study) or decreased 45Ca uptake [16], altered properties of L channels [7, 13] (as possible indicators of an enhanced down-regulation), and enhanced sensitivity of various intracellular [CaZ+]i-dependent mechanisms like inactivation of Ca 2+ inward currents [14] or stimulation of PLC (this paper), impairment of frequency potentiation [12] and prolonged Ca2+-dependent afterhyperpolarization [15]. An explanation for the reduced [Ca2+]iof aged neurons cannot yet be given especially since the etltux is rather decreased than increased [18]. It is however remarkable that synaptosomes show rather elevated than reduced [Ca2+]i levels with aging [17]. Besides differences in metabolic activity in both preparations (see above), Ca 2+ influx in synaptosomes is mainly mediated by N channels, that of neurons by L channels [10]. Furthermore, N channels do not seem to be involved in depolarizationinduced PI hydrolysis [6]. Accordingly, the already mentioned age-related change of L channel properties might not only be a response to the reduced Ca 2+ level but might be alternatively one of its possible causes. Decreased [Ca2+]i and enhanced intracellular Ca 2÷ sensitivity of the aged neuron might compensate each other under normal conditions. However, it seems quite convincible that the ability of the Ca 2+ signal-transduction pathway to adopt to periods of over- and/or understimulation (e.g. stress, hypoxia) might be close to its limits in the aged brain. An enhanced bidirectional vulnerability of the calcium signal-transduction pathway of the aged neuron forms an attractive hypothesis to integrate the large variety of divergent findings linking alterations of Ca 2+ homeostasis with many pathological mechanisms of brain aging. This study was supported by a grant of the Deutsche Forschungsgemeinschaft (SFB 258).
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