Brain Research, 508 (1990) 1-6
1
Elsevier BRES 15146
Research Reports
Aging-related prolongation of calcium spike duration in rat hippocampal slice neurons Thomas A. Pitier* and Philip W. Landfield Department of Physiology and Pharmacology, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, NC 27103 (U.S.A.)
(Accepted 27 June 1989) Key words: Calcium spike; Aging; l-tippocampus; Calcium inactivation
Calcium (Ca) spike potentials were investigated in cesium-loaded, tetrodotoxin (TTX)-treated CA1 pyramidal cells in hippocampal slices from young-mature and aged rats. The duration of single Ca spike potentials was prolonged in cells from aged rats, indicating that previously observed age-related changes in Ca-dependent mechanisms (e.g. in the K-mediated afterhyperpolarization and in frequency potentiation) may result from an age-related increase of voltage-dependent Ca conductance. Since we recently found that Ca spike duration in hippocampus can be modulated strongly by a form of Ca-dependent inactivation of Ca current, spike inactivation paradigms also were examined. However, following 5- or 10-s-long depolarizing pulses, or during a 2-Hz train of elicited Ca spikes, there were no age differences in percent inactivation. These results do not support (but do not fully rule out) the possibility that impaired Ca-dependent inactivation underlies the increase in the Ca spike with aging. Conceivably, this prolongation of voltage-dependent Ca influx could have implications for our understanding of normal and abnormal brain aging. INTRODUCTION There is increasing evidence that alterations in calcium (Ca) homeostasis may play a significant role in brain aging and/or Alzheimer's disease. However, neither the specific nature nor even the direction of these alterations is well understood (cf. reviews in refs. 8,11), perhaps because there are multiple classes of Ca channels. These several kinds of Ca channels exhibit different degrees of activation and inactivation under various experimental conditions 2°'22'24'2s, and may be affected differentially by brain aging processes t2. In a prior study using intracellular recordings from hippocampal slice neurons, it was found that the Cadependent, K-mediated, slow afterhyperpolarization ( A H P ) was longer in cells from aged than from young rats, following a defined degree of depolarization (e.g. either 2 or 3 Na spikes elicited by current injection) ~6. Moreover, it was found that the aging-related impairment 15 of a robust form of hippocampal synaptic plasticity (frequency potentiation) appears to result from an age-related increase in the amount of Ca influx during repetitive activation is. These findings have led to the hypothesis that brain
aging is associated with an alteration in the properties of voltage-dependent Ca channels and that this alteration might be a key factor in age-related disturbances of Ca homeostasis 12"13. However, a number of factors other than Ca influx per se could influence Ca-dependent processes, including age-related alterations in internal Ca buffering, sequestration, or extrusion s'~1"21'3° or changes in Ca-dependent K channels. Therefore, a more specific test of the hypothesis of age-related alterations in Ca conductance would be to measure Ca potentials directly, following the blockade of confounding Na and K currents. In addition, direct measures of Ca potentials or currents should allow greater resolution in defining the activation/inactivation states of particular classes of channels, which, in turn, might help to clarify the effects of aging on Ca flux in neurons, The hippocampus is characterized by particularly large Ca spike potentials (seen following tetrodotoxin application) and prominent voltage-dependent Ca currents ~' 10,27.33. Further, we found recently that some Ca currents in hippocampal neurons are subject to a strong and rapid form of negative feedback Ca-dependent inactivation 2'25, analogous to the Ca-dependent inactivation of Ca currents present in invertebrate neurons 5. That is, Ca
*Present address: Department of Physiology, University of Maryland School of Medicine, Baltimore, MD 21201, U.S.A. Correspondence: P.W. Landfield, Department of Physiology and Pharmacology, Bowman Gray School of Medicine, Wake Forest University, 300 S. Hawthorne Road, Winston-Salem, NC 27103, U.S.A. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
c o n d u c t a n c e is r e d u c e d as i n t r a c e l l u l a r C a c o n c e n t r a t i o n i n c r e a s e s . A l t e r a t i o n s in t h e s e C a - d e p e n d e n t i n a c t i v a t i o n p r o c e s s e s c l e a r l y s e e m to b e a c a n d i d a t e m e c h a n i s m f o r t h e b a s i s o f a g i n g - r e l a t e d c h a n g e s in v o l t a g e - d e p e n d e n t C a influx t2'13. In t h e p r e s e n t s t u d y , t h e r e f o r e , we m e a s u r e d d u r a t i o n , amplitude
and
inactivation of isolated
Ca
spikes,
in
h i p p o c a m p a l slices o f y o u n g a n d a g e d rats. M e a s u r e s o f C a s p i k e s in t h e slice p r e p a r a t i o n p r o v i d e i n f o r m a t i o n specifically on Ca currents which exhibit both voltagedependent
activation and Ca-dependent
i n a c t i v a t i o n 25,
and which exhibit relatively brief durations under resting c o n d i t i o n s (e.g. g e n e r a l l y less t h a n 1 s). T h e r e s u l t s s h o w t h a t t h e i s o l a t e d C a s p i k e , like t h e A H P , is p r o l o n g e d in a g e d r a t n e u r o n s , a l t h o u g h n o d i f f e r e n c e s w e r e f o u n d in percentage
of Ca-dependent
inactivation.
These
data
p r o v i d e e v i d e n c e c o n s i s t e n t w i t h t h e s u g g e s t i o n of a n a g e - r e l a t e d i n c r e a s e in t h e activity o f at l e a s t o n e class of voltage-dependent
Ca
channels
in t h e
hippocampus.
S o m e o f t h e s e r e s u l t s h a v e b e e n d e s c r i b e d in a b s t r a c t f o r m 17.
MATERIALS AND METHODS Subjects were healthy male Fischer 344 rats of either 4-7 months of age or 26-29 months of age, obtained from the National Institute on Aging's specific pathogen free colony. The young animals were fully mature adults, as the age of sexual maturity in these animals is approximately 2 months of age, and the median lifespan is approximately 27 months of age. While in our facilities, the animals were maintained behind an air barrier isolation system. Data are reported from a total of 27 neurons that met all criteria for health (see below) from 15 young and 7 aged rats. Hippocampal pyramidal neurons in the CA1 field of 450-~um-thick transverse slices were impaled with micropipettes (60-85 MI2) containing 2.0 M CsCI. Cesium has been found to block the slow Ca-dependent K conductance in vertebrate neurons t°, and the effectiveness of Cs loading was assessed by reduction of the AHP. For neurons in which the AHP was blocked substantially while being held by current injection at approximately -60 mV, and which were stable for at least 20 min, tetrodotoxin ('ITX) was administered onto the slice by pressure ejection from another pipette (3-5/A of 10-4 M TTX in bathing medium). Effectiveness of the TI'X was confirmed in each case by complete blockade of synaptic potentials and of intracellularly induced Na spikes. In Cs-loaded, TFX-treated neurons, a prominent Ca spike could be elicited readily by intracellular depolarizing current injection. TI'X-resistant spikes in central neurons are blocked by Ca antagonists and appear to be relatively pure Ca spikes 19"27'33. In TI'X-treated hippocampal neurons, impaled with CsCI pipettes, the spikes are characterized by an initial fast spike of 20-35 ms duration, followed by a slow plateau phase of reduced amplitude and longer duration (e.g. 100-300 ms) (ref. 25 and Fig. 1). In wave form and kinetics, these two phases correspond approximately to Ca spike patterns described for inferior olivary neurons 2°. Only cells which met a series of criteria for health and Cs-loading were used in these analyses. These criteria included: Na spikes of at least 75 mV, input resistance of at least 30 MI2, near-complete blockade of the AHP, and development of a full Ca spike potential after TTX (initial fast spike followed by a longer plateau phase of reduced amplitude). In this study, as in previous studies 16'18, the yield of healthy cells and slices appeared to be highly comparable
in slices from young and aged animals (e.g+ t neurons from 15 young rats and l0 neurons from 7 aged rats) Neurons meeting these criteria were then run through a series ot Ca spike activation and inactivation paradigms, as described below. Neuronal membrane potential was held at --60 mV by stead}', low-level current injection through the pipette (0.1-0.3 nA), and Ca spikes were elicited by depolarizing constant current pulses of 70 ms duration. Threshold for the Ca spike was determined for each cell, and the depolarizing constant current pulse was set at 150% of threshold for the remainder of the experiment. Input resistance and time constant were assessed with a 0.2 nA hyperpolarizing current pulse, since at this current intensity the membrane voltage response is largely linear '+. Duration of the full Ca potential was measured from the peak of the fast spike (and also from the onset of the current pulse) to the return to baseline of the slow plateau phase. Spike and plateau peak amplitudes were measured from holding potential, and latency to peak was measured from the onset of the depolarizing pulse to the peak of the fast spike. Fast spike width was measured from the peak to the widest point above the plateau phase. (cf. ref. 25 for additional technical details). The inactivation paradigms consisted of a baseline resting measure of Ca spike duration ('PRE') followed by a 5-s depolarizing current pulse at 150% of spike threshold, followed by measures of the recovery of Ca spike duration at 1 s and at 4 s after the end of the 5-s depolarizing pulse. After approximately a 5-rain interval, the same procedure was repeated using a 10-s depolarizing current pulse. During the 5- and 10-s pulses used in the above paradigms, Ca spikes fired repetitively. However, the frequency and number of total spikes during each long pulse varied to some degree from cell to cell. Therefore, in order to study inactivation during a controlled number of spikes, inactivation was also studied during a 2-Hz train of 5 constant depolarizing pulses, in a sub-set of 9 young cells and 6 aged cells, from separate animals, that exhibited somewhat less of an age difference in baseline values than the full samples. In this paradigm, hyperpolarizing pulses were administered just before the first depolarizing pulse and 200 ms following the last depolarizing pulse of the 2-Hz train, in order to ensure that apparent inactivation was not due to activation of large outward (e.g. K) currents (cf. Fig. 2 and discussion in ref. 25). As a control for age differences in resting spike duration, inactivation data were analyzed both as percent of baseline and as raw values. The inactivation studies focused on measurement of full spike duration since previous studies found that duration provides the most sensitive measure of inactivation (e.g. during inactivation paradigms, spike amplitude does not change substantially, and slow plateau amplitude varies
a
2
l
Fig. 1. Ca spikes recorded from cesium-loaded, TTX-treated cells in normal medium: 1, Young rat cell; 2, aged rat cell; 3, current monitor trace showing depolarizing current pulse used to trigger the Ca spike. Calibration: 25 mV, 50 ms.
TABLE I
Resting characteristics of CA1 cells from young-mature and aged animals Means ± S.E.M. of passive membrane properties (in response to hyperpolarizing current pulses) and Ca spike potential characteristics (in response to single depolarizing current pulses at 150% of Ca spike threshold), during resting baseline conditions prior to the onset of inactivating stimuli. Only the full spike duration values exhibited a significant difference between young and aged rat neurons (see text for procedures).
Young-mature (n = 17) Full Ca spike duration (ms) Fast spike width (ms) Fast spike amplitude (mV) Plateau amplitude (mV) Input resistance (MD) Time constant (ms) Latency to fast spike peak (ms)
216.4±10.3 25.4±1.9 65.9±1.1 21.9±1.7 47.9±4.2 14.5±0.7 37.7±1.4
(e.g. 5-s, 10-s and, if available, 2-Hz). This provided an assessment of resting Ca spike duration that was more representative of duration over the full course of the experiments. Data were analyzed by two-way analyses of variance, using a split-plot design for repeated measures, in studies with multiple measures (e.g. inactivation paradigms). Individual group contrasts were assessed by ad hoc Bonferroni tests in the ANOVA, or by two-tailed t-tests in the case of single measure comparisons.
RESULTS
Aged (n = 10)
T h e a v e r a g e d r e s t i n g full s p i k e d u r a t i o n v a l u e s for
275.7±16.7"* 28.7±3.1 65.0±1.9 19.9±2.2 52.6±6.7 15.9±1.0 39.0±0.9
** Significantly different from young-mature values (P < 0.01).
e a c h cell e x h i b i t e d a h i g h l y s i g n i f i c a n t e f f e c t o f age ( T a b l e I, also see Fig. 1). T h i s e f f e c t was s i g n i f i c a n t w h e t h e r cells were treated
as i n d e p e n d e n t
samples
(P
1
Elsevier BRES 15146
Research Reports
Aging-related prolongation of calcium spike duration in rat hippocampal slice neurons Thomas A. Pitier* and Philip W. Landfield Department of Physiology and Pharmacology, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, NC 27103 (U.S.A.)
(Accepted 27 June 1989) Key words: Calcium spike; Aging; l-tippocampus; Calcium inactivation
Calcium (Ca) spike potentials were investigated in cesium-loaded, tetrodotoxin (TTX)-treated CA1 pyramidal cells in hippocampal slices from young-mature and aged rats. The duration of single Ca spike potentials was prolonged in cells from aged rats, indicating that previously observed age-related changes in Ca-dependent mechanisms (e.g. in the K-mediated afterhyperpolarization and in frequency potentiation) may result from an age-related increase of voltage-dependent Ca conductance. Since we recently found that Ca spike duration in hippocampus can be modulated strongly by a form of Ca-dependent inactivation of Ca current, spike inactivation paradigms also were examined. However, following 5- or 10-s-long depolarizing pulses, or during a 2-Hz train of elicited Ca spikes, there were no age differences in percent inactivation. These results do not support (but do not fully rule out) the possibility that impaired Ca-dependent inactivation underlies the increase in the Ca spike with aging. Conceivably, this prolongation of voltage-dependent Ca influx could have implications for our understanding of normal and abnormal brain aging. INTRODUCTION There is increasing evidence that alterations in calcium (Ca) homeostasis may play a significant role in brain aging and/or Alzheimer's disease. However, neither the specific nature nor even the direction of these alterations is well understood (cf. reviews in refs. 8,11), perhaps because there are multiple classes of Ca channels. These several kinds of Ca channels exhibit different degrees of activation and inactivation under various experimental conditions 2°'22'24'2s, and may be affected differentially by brain aging processes t2. In a prior study using intracellular recordings from hippocampal slice neurons, it was found that the Cadependent, K-mediated, slow afterhyperpolarization ( A H P ) was longer in cells from aged than from young rats, following a defined degree of depolarization (e.g. either 2 or 3 Na spikes elicited by current injection) ~6. Moreover, it was found that the aging-related impairment 15 of a robust form of hippocampal synaptic plasticity (frequency potentiation) appears to result from an age-related increase in the amount of Ca influx during repetitive activation is. These findings have led to the hypothesis that brain
aging is associated with an alteration in the properties of voltage-dependent Ca channels and that this alteration might be a key factor in age-related disturbances of Ca homeostasis 12"13. However, a number of factors other than Ca influx per se could influence Ca-dependent processes, including age-related alterations in internal Ca buffering, sequestration, or extrusion s'~1"21'3° or changes in Ca-dependent K channels. Therefore, a more specific test of the hypothesis of age-related alterations in Ca conductance would be to measure Ca potentials directly, following the blockade of confounding Na and K currents. In addition, direct measures of Ca potentials or currents should allow greater resolution in defining the activation/inactivation states of particular classes of channels, which, in turn, might help to clarify the effects of aging on Ca flux in neurons, The hippocampus is characterized by particularly large Ca spike potentials (seen following tetrodotoxin application) and prominent voltage-dependent Ca currents ~' 10,27.33. Further, we found recently that some Ca currents in hippocampal neurons are subject to a strong and rapid form of negative feedback Ca-dependent inactivation 2'25, analogous to the Ca-dependent inactivation of Ca currents present in invertebrate neurons 5. That is, Ca
*Present address: Department of Physiology, University of Maryland School of Medicine, Baltimore, MD 21201, U.S.A. Correspondence: P.W. Landfield, Department of Physiology and Pharmacology, Bowman Gray School of Medicine, Wake Forest University, 300 S. Hawthorne Road, Winston-Salem, NC 27103, U.S.A. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
c o n d u c t a n c e is r e d u c e d as i n t r a c e l l u l a r C a c o n c e n t r a t i o n i n c r e a s e s . A l t e r a t i o n s in t h e s e C a - d e p e n d e n t i n a c t i v a t i o n p r o c e s s e s c l e a r l y s e e m to b e a c a n d i d a t e m e c h a n i s m f o r t h e b a s i s o f a g i n g - r e l a t e d c h a n g e s in v o l t a g e - d e p e n d e n t C a influx t2'13. In t h e p r e s e n t s t u d y , t h e r e f o r e , we m e a s u r e d d u r a t i o n , amplitude
and
inactivation of isolated
Ca
spikes,
in
h i p p o c a m p a l slices o f y o u n g a n d a g e d rats. M e a s u r e s o f C a s p i k e s in t h e slice p r e p a r a t i o n p r o v i d e i n f o r m a t i o n specifically on Ca currents which exhibit both voltagedependent
activation and Ca-dependent
i n a c t i v a t i o n 25,
and which exhibit relatively brief durations under resting c o n d i t i o n s (e.g. g e n e r a l l y less t h a n 1 s). T h e r e s u l t s s h o w t h a t t h e i s o l a t e d C a s p i k e , like t h e A H P , is p r o l o n g e d in a g e d r a t n e u r o n s , a l t h o u g h n o d i f f e r e n c e s w e r e f o u n d in percentage
of Ca-dependent
inactivation.
These
data
p r o v i d e e v i d e n c e c o n s i s t e n t w i t h t h e s u g g e s t i o n of a n a g e - r e l a t e d i n c r e a s e in t h e activity o f at l e a s t o n e class of voltage-dependent
Ca
channels
in t h e
hippocampus.
S o m e o f t h e s e r e s u l t s h a v e b e e n d e s c r i b e d in a b s t r a c t f o r m 17.
MATERIALS AND METHODS Subjects were healthy male Fischer 344 rats of either 4-7 months of age or 26-29 months of age, obtained from the National Institute on Aging's specific pathogen free colony. The young animals were fully mature adults, as the age of sexual maturity in these animals is approximately 2 months of age, and the median lifespan is approximately 27 months of age. While in our facilities, the animals were maintained behind an air barrier isolation system. Data are reported from a total of 27 neurons that met all criteria for health (see below) from 15 young and 7 aged rats. Hippocampal pyramidal neurons in the CA1 field of 450-~um-thick transverse slices were impaled with micropipettes (60-85 MI2) containing 2.0 M CsCI. Cesium has been found to block the slow Ca-dependent K conductance in vertebrate neurons t°, and the effectiveness of Cs loading was assessed by reduction of the AHP. For neurons in which the AHP was blocked substantially while being held by current injection at approximately -60 mV, and which were stable for at least 20 min, tetrodotoxin ('ITX) was administered onto the slice by pressure ejection from another pipette (3-5/A of 10-4 M TTX in bathing medium). Effectiveness of the TI'X was confirmed in each case by complete blockade of synaptic potentials and of intracellularly induced Na spikes. In Cs-loaded, TFX-treated neurons, a prominent Ca spike could be elicited readily by intracellular depolarizing current injection. TI'X-resistant spikes in central neurons are blocked by Ca antagonists and appear to be relatively pure Ca spikes 19"27'33. In TI'X-treated hippocampal neurons, impaled with CsCI pipettes, the spikes are characterized by an initial fast spike of 20-35 ms duration, followed by a slow plateau phase of reduced amplitude and longer duration (e.g. 100-300 ms) (ref. 25 and Fig. 1). In wave form and kinetics, these two phases correspond approximately to Ca spike patterns described for inferior olivary neurons 2°. Only cells which met a series of criteria for health and Cs-loading were used in these analyses. These criteria included: Na spikes of at least 75 mV, input resistance of at least 30 MI2, near-complete blockade of the AHP, and development of a full Ca spike potential after TTX (initial fast spike followed by a longer plateau phase of reduced amplitude). In this study, as in previous studies 16'18, the yield of healthy cells and slices appeared to be highly comparable
in slices from young and aged animals (e.g+ t neurons from 15 young rats and l0 neurons from 7 aged rats) Neurons meeting these criteria were then run through a series ot Ca spike activation and inactivation paradigms, as described below. Neuronal membrane potential was held at --60 mV by stead}', low-level current injection through the pipette (0.1-0.3 nA), and Ca spikes were elicited by depolarizing constant current pulses of 70 ms duration. Threshold for the Ca spike was determined for each cell, and the depolarizing constant current pulse was set at 150% of threshold for the remainder of the experiment. Input resistance and time constant were assessed with a 0.2 nA hyperpolarizing current pulse, since at this current intensity the membrane voltage response is largely linear '+. Duration of the full Ca potential was measured from the peak of the fast spike (and also from the onset of the current pulse) to the return to baseline of the slow plateau phase. Spike and plateau peak amplitudes were measured from holding potential, and latency to peak was measured from the onset of the depolarizing pulse to the peak of the fast spike. Fast spike width was measured from the peak to the widest point above the plateau phase. (cf. ref. 25 for additional technical details). The inactivation paradigms consisted of a baseline resting measure of Ca spike duration ('PRE') followed by a 5-s depolarizing current pulse at 150% of spike threshold, followed by measures of the recovery of Ca spike duration at 1 s and at 4 s after the end of the 5-s depolarizing pulse. After approximately a 5-rain interval, the same procedure was repeated using a 10-s depolarizing current pulse. During the 5- and 10-s pulses used in the above paradigms, Ca spikes fired repetitively. However, the frequency and number of total spikes during each long pulse varied to some degree from cell to cell. Therefore, in order to study inactivation during a controlled number of spikes, inactivation was also studied during a 2-Hz train of 5 constant depolarizing pulses, in a sub-set of 9 young cells and 6 aged cells, from separate animals, that exhibited somewhat less of an age difference in baseline values than the full samples. In this paradigm, hyperpolarizing pulses were administered just before the first depolarizing pulse and 200 ms following the last depolarizing pulse of the 2-Hz train, in order to ensure that apparent inactivation was not due to activation of large outward (e.g. K) currents (cf. Fig. 2 and discussion in ref. 25). As a control for age differences in resting spike duration, inactivation data were analyzed both as percent of baseline and as raw values. The inactivation studies focused on measurement of full spike duration since previous studies found that duration provides the most sensitive measure of inactivation (e.g. during inactivation paradigms, spike amplitude does not change substantially, and slow plateau amplitude varies
a
2
l
Fig. 1. Ca spikes recorded from cesium-loaded, TTX-treated cells in normal medium: 1, Young rat cell; 2, aged rat cell; 3, current monitor trace showing depolarizing current pulse used to trigger the Ca spike. Calibration: 25 mV, 50 ms.
TABLE I
Resting characteristics of CA1 cells from young-mature and aged animals Means ± S.E.M. of passive membrane properties (in response to hyperpolarizing current pulses) and Ca spike potential characteristics (in response to single depolarizing current pulses at 150% of Ca spike threshold), during resting baseline conditions prior to the onset of inactivating stimuli. Only the full spike duration values exhibited a significant difference between young and aged rat neurons (see text for procedures).
Young-mature (n = 17) Full Ca spike duration (ms) Fast spike width (ms) Fast spike amplitude (mV) Plateau amplitude (mV) Input resistance (MD) Time constant (ms) Latency to fast spike peak (ms)
216.4±10.3 25.4±1.9 65.9±1.1 21.9±1.7 47.9±4.2 14.5±0.7 37.7±1.4
(e.g. 5-s, 10-s and, if available, 2-Hz). This provided an assessment of resting Ca spike duration that was more representative of duration over the full course of the experiments. Data were analyzed by two-way analyses of variance, using a split-plot design for repeated measures, in studies with multiple measures (e.g. inactivation paradigms). Individual group contrasts were assessed by ad hoc Bonferroni tests in the ANOVA, or by two-tailed t-tests in the case of single measure comparisons.
RESULTS
Aged (n = 10)
T h e a v e r a g e d r e s t i n g full s p i k e d u r a t i o n v a l u e s for
275.7±16.7"* 28.7±3.1 65.0±1.9 19.9±2.2 52.6±6.7 15.9±1.0 39.0±0.9
** Significantly different from young-mature values (P < 0.01).
e a c h cell e x h i b i t e d a h i g h l y s i g n i f i c a n t e f f e c t o f age ( T a b l e I, also see Fig. 1). T h i s e f f e c t was s i g n i f i c a n t w h e t h e r cells were treated
as i n d e p e n d e n t
samples
(P
