- Email: [email protected]
Aging-related increase in hippocampal calcium channels
Life Sciences, Vd. 59, No.s S/6, pp. 399404,1996 Coppight o 19% F!lscvicr Science Inc. Printed in the USA. All rights reserved w24-32051% $15.co t .oo PI1 SOOM-3205(96)00318-9
ELSEVIER
AGING-RELATED
INCREASE
IN HIPPOCAMPAL
CALCIUM
CHANNELS
Philip W. Landfield Department
of Pharmacology,
University
of Kentucky, Lexington,
KY 40536-0084
This paper briefly reviews more than 10 years of our studies on brain aging and voltage-activated calcium (Ca) currents in rat hippocampal CA1 neurons. Initial studies in the hippocampal slice preparations found that synaptic plasticity was impaired with aging, apparently due to excess Ca influx. In subsequent analyses it was found that the Cadcpcndcnt afierhyperpolarization, the Ca action potential and voltage-activated Ca currents were all increased in aged CA 1 neurons. This was not due to impaired inactivation processes. Multiple types of Ca channels appear to be affected by aging. A long Ca tail current was also found in these studies, which seems to represent an unrecognized and significant Ca entry pathway at resting potential. In primary cell cultures, Ca currents and single Ca channels increase steadily over the life cycle of the cultured neurons and are correlated with cell death. Single L-type Ca channels were also studied in brain neurons of an aged mammal (rat), using the partially dissociated (“zipper”) hippocampal slice preparation. A substantial increase in the density of functionally available Ca channels was present in CA1 neurons of aged rats, similar to the increase seen in cultured neurons. Thus, a gradual increase in the density of Ca channels appears to be a consistent property of hippocampal neuronal aging and might well be a factor in the vulnerability of aged neurons to Alzheimer’s disease and other neurodegenerativeltraumatic conditions. Key Word: aging, hippocampus, calcium channels, calcium currents, Alzheimer ’ s disease
Brain aging is intimately involved with the development of Alzheimer’s disease (AD) and other forms of dementia. A number of epidemiologic studies have shown that there is an exponential increase in the incidence of Alzheimer’s dementia after age 65, with some studies finding an incidence of AD of nearly 50% at age 85 and above. Thus, although normal brain aging is probably not simply an early and nonvirulent form of AD, it clearly is among the primary risk factors for AD and therefore appears to induce processes that predispose neurons to neurodegencration. Our research interests have focused on these subtle early processes of brain aging, and the mechanisms through which they gradually enhance neuronal susceptibility. In our electrophysiological studies of aging we have primarily utilized the rat hippocampal slice preparation, which retains its synaptic organization and normal physiological responses. The slice can be used to perform extracellular, intracellular and voltage clamp recording. In addition, the human hippocampal CA1 field is among the brain regions most affected by aging and AD, and the rodent CA1 field is also affected by aging. Consequently, the hippocampal slice appears to be
400
Calcium Channels
inAging
Vol. 59,No.s 516,1996
a good animal model system in which to study brain aging processes and the factors which contribute to neuronal susceptibility. More than 10 years ago, we began to find initial evidence in the slice that voltage-activated calcium (Ca) influx was altered in hippocampal neurons of aged rats. Over this period a number of other laboratories also have found evidence of altered Ca homeostasis in neuronal aging (see reviews in this volume). However, using electrophysiological methods, our laboratory has focused specifically upon the role of voltage-activated Ca influx in the disruption of Ca homeostasis. This chapter briefly reviews the development of our studies on voltage-gated Ca influx, from current clamp analyses up to our most recent findings at the single channel level.
The slow afierhyperpolarization (AHP), which follows a burst of sodium (Na) action potentials, is mediated by Ca influx and activation of a Ca-dependent potassium (K) current. In several studies, we have found the AHP to be increased substantially in CA1 neurons with aging (1, 2). In addition, modestly elevated magnesium (Mg)-to-calcium ratios in the extracellular medium significantly counteracted aging-dependent impairments in frequency facilitation, a pronounced form of hippocampal synaptic plasticity (3). As Mg blocks Ca influx through several pathways, and the AHP is directly dependent upon the submembrane concentration of Ca, these findings were consistent with other evidence of altered neuronal Ca homeostasis that was being uncovered at about the same time (4,5). These and subsequent results from a number of laboratories, have contributed to the formal development of the Ca hypothesis of brain aging and dementia (6,7) . However, for conclusive studies, more direct indicants of voltage-gated Ca influx than the AHP were needed. One means of directly measuring voltage-activated Ca influx is to assess the Ca action potential. Blocking Na action potentials with tetrodotoxin and the slow AHP with internal caesium (Cs) allows large calcium action potentials to emerge. The Ca action potential lasts several hundred milliseconds, compared with one or two milliseconds for a Na action potential, and closely reflects Ca influx. As with the Cadependent AI-II’,Ca action potentials also were longer and larger in the aged neurons (8), providing strong evidence that the aging change in the AHP was due to changes in the properties of Ca channels rather than in the K channels that mediate the Ca-activated AHP. Highly analogous results have been found by another major laboratory working with rabbit hippocampal slices (9). A still more direct method of investigating voltage-gated Ca currents is to use voltage clamp techniques. The voltage clamp approach allows a more careful analysis of the voltage sensitivity and other properties of Ca currents affected by aging. However, voltage clamping CNS neurons in the slice presents a number of major technical hurdles. One approach that allows voltage clamp procedures to be applied with minimal disruption of the neurons, however, is the single electrode, discontinuous voltage clamp technique. This method utilizes an intracellular sharp, high resistance electrode with discontinuous sampling of voltage. In this procedure tetraethylanmronium is also added to block additional K ChaMels that are not fully blocked by Cs. Under voltage clamp conditions, Ca currents were again found to be larger in CA 1 neurons from aged than from young rats. Nimodipine reduced Ca current by approximately one-third in young and aged neurons, indicating that L-type Ca currents are increased with aging, but that other high threshold (N,P) Ca currents also may be increased. One other important observation in these studies, moreover, was that the larger currents with aging were not due to impaired inactivation because the rate and degree of inactivation were at least as great in aged neurons (10, and in preparation).
Vol. 59, No.s S/6, 1%
Calcium Channels in Aging
401
Thus, multiple approaches to recording Ca influx, from physiological intracellular potential recordings (AHP, synaptic potentials) and measures of Ca action potentials, to whole cell voltage clamp analyses, have indicated that rodent CA1 neurons, which are among the most vulnerable to aging, exhibit increased voltage-gated Ca influx with aging. However, not all investigators have observed similar results (see Lamour, Verkhratsky, this volume). With one exception (Lamour, this volume), studies finding no increase in Ca currents have used cell types other than hippocampal pyramidal neurons (peripheral, cerebellar, dentate gyms), from regions for which there is less evidence of major decline with AD or aging. Some preparations have also been very different from In addition, these are extremely difficult preparations to study ours (dissociated cells). quantitatively and extreme efforts must be made to control variability. Strict criteria for healthy neurons and comparable quality of recordings are necessary. Although we cannot account for all discrepancies in the literature, it should be emphasized that four separate studies performed by different investigators in our laboratory (1,2,8,10) have found consistent evidence of enhanced AHP, Ca action potentials or Ca currents in CA1 neurons. New studies are also consistent with these results (see below). Moreover, independent studies by Disterhoft and colleagues (cf. this volume), have found similar results in CA1 cells of another mammalian species. Thus, the evidence of aging-dependent increases in Ca influx appears to be strong and consistent, at Last for the CA1 neurons that are among the most susceptible to aging/AD.
Single Ca channel approaches The advent of the patch clamp technique in the early 1980s in which a pipette tip is placed onto a small patch (1-3 um2) of cell membrane and suction is used to form a high resistance seal has allowed investigators to record the activity of a single ion channel, and thereby to investigate changes in ionic currents at a more basic, mechanistic level. The “whole cell” patch clamp method breaks through the membrane patch to gain access to the cell’s interior and record the activity of the whole cell. This yields currents that are generally analogous to those obtained with the intracellular sharp electrode methods described above. In addition, however, patch clamp methods from an intact membrane patch allow single channels to be recorded from the small patch, either in the cellattached mode or in the cell-free mode (patch pulled away from cell while still on the pipette). These relatively new single channel methods provide an important opportunity to analyze aging changes at a more molecular level than has previously been feasible as described below.
Long Ca tail currents in hippocampal neurons In addition to the basic results on aging differences, the whole-cell voltage clamp studies in hippocampal slices revealed a very long inward tail current (hundreds of milliseconds) that followed the depolarization pulse. Although many electrophysiologists view such long tails as space clamp artifacts, several aspects of the long tail current did not seem consistent with the artifact interpretation. We have therefore spent quite a few years attempting to determine whether or not this was a true Ca current, and have recently obtained evidence at the single channel (11) and whole cell (in preparation) levels indicating that the long tail current arises from an unusual mode of Ca channel activity that permits channel opening after rcpolarization. This evidence therefore suggests that the long tail current reflects a substantial unrecognized pathway of Ca entry and may be a site of aging-related change.
Devel~t
.
of Calcuun Cu rrents in Cuiturg
Calcium channel currents also change with the age of the cells in culture On day 2, after plating, hippocampal neurons manifest a very small voltage-activated Ca current and do not have a long tail current, Over the life cycle of the neurons in culture (~30 days) the voltage-activated Ca current increases substantially. Moreover, on day 3 the tail current first appears, and also increases
402
Calcium Channels
inAging
Vol. 59,No.s 516,1996
over the life cycle of a cell. Single channel studies show a similar pat&m. Single channel (L-type) activity increases steadily over the life of the neurons. Few repolarization openings (ROs) are observed up to day 2; they first appear on day 3, and then become increasingly more prevalent as the neurons mature. Although calcium channel current increases with age in culture, this is not simply a reflection of the fact that the cells grow in size, because even with corrections for cell capacitance (a direct indicant of cell size), Ca current density increases rapidly in the first week and then continues to rise more slowly. Correlations with neuron survival Calcium current density also correlates with hippocampal neuron survival in culture. Quantitative counts of neurons over time show that periods of cell death correlate with a higher rate of increase of Ca current density. Moreover, with nimodipine chronically present in the culture bath, almost twice as many cells survive by day 15 as survive under control conditions (12). Our working hypothesis is that the increase in Ca current density seems to be correlated with and may be causally related to cell death. . [email protected] Cmual
Neuron6
As intriguing as the cell culture model is for studying the relationship between gradual increases in Ca channel activity and cell survival and/or function, aging of embryonic cells in culture clearly cannot be assumed to be the same as aging of neurons in vivo. Thus, functional studies at the level of a single molecule (channel), specifically in aging mammalian brain cells, are necessary to dissect out the mechanisms underlying the increase in macroscopic Ca currents. Whole cell studies alone do not clarify whether macroscopic currents increase because each channel exhibits larger conductance, or a greater open probability, or whether there are more functionally available channels. However, in order to determine the single channel basis for the aging changes in whole cell Ca currents and potentials, we needed a preparation in which we could apply single channel patch clamp methods to brain neurons of an aging mammal. The standard adult neuron preparation for patch clamp studies at the single channel level is acutely dissociated cells, but this approach appears too traumatic for studies of aged brain cells. In our hands, the yield of healthy neurons from aged animals, using the dissociation technique, was very low, raising the possibility of sampling bias ( 13). Moreover, while non-dissociated slices presently are being used widely for whole cell patch clamp studies, they are difficult to use on a large scale for single channel patch clamp analyses. For these reasons, we modified a technique of partial dissociation of the hippocampal slice, originally developed by Gray, Johnston and colleagues (14). This method is referred to as the “zipper slice” because of the tendency for the hippocampal slice to “unzip” along the CA1 cell layer, thereby exposing the neuronal somata for formation of a high resistance seal with a pipette. This preparation provides the same yield of good recordings from young and aged animals, in cells that retain their dendrites and appear normal in shape and size (13). We used this method to conduct the first large scale quantitative analyses of single channel activity in brain neurons of aged mammals (over 75 total neurons from young, mid-aged and aged rats) (Thibault and Landfield, in preparation). Multiple depolarizations were used to construct an average ensemble current for each patch in young, mid-aged and aged rat neurons, from which average current in each patch was calculated. In multichannel patches on neurons from aged rats, we found a large increase in the pseudomacroscopic (averaged) current during depolarization as well as an increase in repolarization openings relative to young or mid-aged rat neurons. Currentvoltage curves showed that this increase was not due to a shift in voltage dependence. These studies focused on L-type Ca channels, and the L-type agonist, Bay K 8644, was added to the pipette to
Vol. 59, No.s 5/6, 19%
Calcium Channelsin Aging
403
enhance L-type activity in the recordings. However, measures of mean open time and voltage dependence indicated no age differences in the sensitivity of L-type channels to the agonist. An increase in average patch current with aging could be due to more functionally available channels, to an increase in the single channel conductance, or to an increased open probability. To separate these possibilities, we measured the amplitude of current in single L-type Ca channels at multiple voltages, and calculated slope conductance. Single calcium channel conductance did not change with aging. The amplitude of repolarization openings also did not change with age. Estimates of the number of available channels in each patch, obtained by the method of maximal simultaneous openings also were made for each patch. These measures showed that the number of Ca channels was substantially increased in aged neurons. Estimates of patch size, based on patch pipette resistance showed no age differences, indicating that the increase in channel numbers reflected an increase in the density of Ca channels. The increase in density of L-type Ca channels was roughly proportional to the increase in average patch current, and was generally similar to the changes in the AHP, the calcium spike and macroscopic currents that we have seen in earlier studies. Consequently it seems reasonable to conclude that most of the aging changes in Ca currents observed previously are due to an aging-related increase in the density of Ca channels in the membranes of CA1 hippocampal neurons. Moreover, several weeks prior to the electrophysiological studies, we ran eight of the aged animals in a water maze (a spatial learning task dependent on hippocampal function and affected by aging). Channel density in hippocampal neurons was found to be inversely correlated with maze performance in the aged animals. Young and mid-aged animals perform uniformly well in this task. These results raise the possibility that the reported positive effects of the L-type channel blocker, nimodipine, on learning tasks in aged animals (15 17) may be in part related to the increase in the density of L-type channels. . Conclus~~ After many years of study of voltage-activated Ca potentials and currents in hippocampal neurons of aging animals, it appears that we are now beginning to focus in on a quintessential change at the molecular level, namely an increase in the density of Ca channels per unit membrane. Very possibly, this change accounts for most of the whole cell alterations we have observed previously. As described by other participants here, many changes occur in Ca regulation with aging, including impairments of Ca buffering and extrusion processes. A chronic elevation of Ca influx through increased numbers of Ca channels, as described above, would further pressure regulatory mechanisms, perhaps resulting in an accelerating interactive disruption of Ca regulation. The concentration of Ca in a neuron is regulated extraordinarily tightly, indicating that such regulation is highly important to the cell’s well-being. In turn, this suggests that even a modest increase in Ca intlux, if persistently maintained over long periods, would continually challenge and activate multiple regulatory systems and might well exert a wide variety of disruptive and deleterious actions. Although neuronal aging in culture is unlikely to directly mimic neuronal aging in vivo, it nevertheless seems intriguing that Ca channel density increases in both culture and in vivo aging systems over time. These observations raise the possibility that a gradual increase in Ca channel density may be a consistent time-dependent correlate of brain cell aging and/or maturation under very different conditions. Additional studies will be needed to determine the molecular biological basis of the increase in available Ca channels in either aging or long-term cultured neurons. Several possibilities exist, including the synthesis of new channels or the recruitment of previously silent channels through altered phosphorylation of channel subunits or changes in membrane structure.
404
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.
Calcium Channels
inAging
Vol. 59, Nos S/6,1996
P.W. LANDFIELD and T.A. PITLER, Science 226 1089-1092 (1984). D.S. KERR, L.W. CAMPBELL, S-Y HA0 and P.W. LANDFIELD, Science 2411505-1509 (1989). P.W. LANDFIELD, T.A. PITLER and M.D APPLEGATE .I. Neurophysiol x 797-811 (1986). G.E. GIBSON and C. PETERSON, Neurobiol. Aging, 8 329-344 (1987). M.L. MICHAELIS, K. JOHE and T.E. KITOS Mech. Aging Dev. 2 215-225 (1984). Z.S. KHACHATURIAN, Handbook of Studies on P&,&&y and Old plge, D.S. Kay and G.W. Burrow (eds), 7-30 (1984). Z.S KHACHATURIAN, Aging 1 17-34 (1989). T.A. PITLER and P.W. LANDFIELD, Brain Res. m 1-6 (1990). J.F. DISTERHOFT, J.R. MOYER, Jr., L.T. THOMPSON and M. KOWALSKA, Clin. Neuropharmacol., 16 S12-S24 (1993). L.W. CAMPBELL, S-Y HA0 and P.W. LANDFIELD, Sot. Neurosci. Abstr., 1. 260 (1989). 0. THIBAULT, N.M. PORTER and P.W. LANDFIELD, Proc. Natl. Acad. Sci. USA, %I 11792-l 1796 (1993). N.M. PORTER, 0. THIBAULT, V. THIBAULT, J.L. GEDDES and P.W. LANDFIELD, Sot. Neurosci. Abstr., .2Q900 (1995). O.THIBAULT, M.L. MAZZANTI, E.M. BLALOCK, N.M. PORTER, AND P.W. LANDFIELD: J. Neurosci. Meth., 5p 77-83 (1995). R. GRAY, R. FISHER, N. SPRUSTON and D. JOHNSTON, &pa&ions of Vertabra& Central Nervous System In V’tr~ H. Jahnsen (ed), 3-24 (1990). R.A. DEYO, K.T. STRAUBE and J.F. DISTERHOFT, Science 241809-811 (1989). A. SCRIABINE, T. SCHUURMAN and J. TRABER, FASEB J., 1 1799-1806 (1989). K. MCMONAGLE-STRUCK0 and R.J. FANELLI, Pharmacol. Biochem. Behav., 44 827835 (1993).
ELSEVIER
AGING-RELATED
INCREASE
IN HIPPOCAMPAL
CALCIUM
CHANNELS
Philip W. Landfield Department
of Pharmacology,
University
of Kentucky, Lexington,
KY 40536-0084
This paper briefly reviews more than 10 years of our studies on brain aging and voltage-activated calcium (Ca) currents in rat hippocampal CA1 neurons. Initial studies in the hippocampal slice preparations found that synaptic plasticity was impaired with aging, apparently due to excess Ca influx. In subsequent analyses it was found that the Cadcpcndcnt afierhyperpolarization, the Ca action potential and voltage-activated Ca currents were all increased in aged CA 1 neurons. This was not due to impaired inactivation processes. Multiple types of Ca channels appear to be affected by aging. A long Ca tail current was also found in these studies, which seems to represent an unrecognized and significant Ca entry pathway at resting potential. In primary cell cultures, Ca currents and single Ca channels increase steadily over the life cycle of the cultured neurons and are correlated with cell death. Single L-type Ca channels were also studied in brain neurons of an aged mammal (rat), using the partially dissociated (“zipper”) hippocampal slice preparation. A substantial increase in the density of functionally available Ca channels was present in CA1 neurons of aged rats, similar to the increase seen in cultured neurons. Thus, a gradual increase in the density of Ca channels appears to be a consistent property of hippocampal neuronal aging and might well be a factor in the vulnerability of aged neurons to Alzheimer’s disease and other neurodegenerativeltraumatic conditions. Key Word: aging, hippocampus, calcium channels, calcium currents, Alzheimer ’ s disease
Brain aging is intimately involved with the development of Alzheimer’s disease (AD) and other forms of dementia. A number of epidemiologic studies have shown that there is an exponential increase in the incidence of Alzheimer’s dementia after age 65, with some studies finding an incidence of AD of nearly 50% at age 85 and above. Thus, although normal brain aging is probably not simply an early and nonvirulent form of AD, it clearly is among the primary risk factors for AD and therefore appears to induce processes that predispose neurons to neurodegencration. Our research interests have focused on these subtle early processes of brain aging, and the mechanisms through which they gradually enhance neuronal susceptibility. In our electrophysiological studies of aging we have primarily utilized the rat hippocampal slice preparation, which retains its synaptic organization and normal physiological responses. The slice can be used to perform extracellular, intracellular and voltage clamp recording. In addition, the human hippocampal CA1 field is among the brain regions most affected by aging and AD, and the rodent CA1 field is also affected by aging. Consequently, the hippocampal slice appears to be
400
Calcium Channels
inAging
Vol. 59,No.s 516,1996
a good animal model system in which to study brain aging processes and the factors which contribute to neuronal susceptibility. More than 10 years ago, we began to find initial evidence in the slice that voltage-activated calcium (Ca) influx was altered in hippocampal neurons of aged rats. Over this period a number of other laboratories also have found evidence of altered Ca homeostasis in neuronal aging (see reviews in this volume). However, using electrophysiological methods, our laboratory has focused specifically upon the role of voltage-activated Ca influx in the disruption of Ca homeostasis. This chapter briefly reviews the development of our studies on voltage-gated Ca influx, from current clamp analyses up to our most recent findings at the single channel level.
The slow afierhyperpolarization (AHP), which follows a burst of sodium (Na) action potentials, is mediated by Ca influx and activation of a Ca-dependent potassium (K) current. In several studies, we have found the AHP to be increased substantially in CA1 neurons with aging (1, 2). In addition, modestly elevated magnesium (Mg)-to-calcium ratios in the extracellular medium significantly counteracted aging-dependent impairments in frequency facilitation, a pronounced form of hippocampal synaptic plasticity (3). As Mg blocks Ca influx through several pathways, and the AHP is directly dependent upon the submembrane concentration of Ca, these findings were consistent with other evidence of altered neuronal Ca homeostasis that was being uncovered at about the same time (4,5). These and subsequent results from a number of laboratories, have contributed to the formal development of the Ca hypothesis of brain aging and dementia (6,7) . However, for conclusive studies, more direct indicants of voltage-gated Ca influx than the AHP were needed. One means of directly measuring voltage-activated Ca influx is to assess the Ca action potential. Blocking Na action potentials with tetrodotoxin and the slow AHP with internal caesium (Cs) allows large calcium action potentials to emerge. The Ca action potential lasts several hundred milliseconds, compared with one or two milliseconds for a Na action potential, and closely reflects Ca influx. As with the Cadependent AI-II’,Ca action potentials also were longer and larger in the aged neurons (8), providing strong evidence that the aging change in the AHP was due to changes in the properties of Ca channels rather than in the K channels that mediate the Ca-activated AHP. Highly analogous results have been found by another major laboratory working with rabbit hippocampal slices (9). A still more direct method of investigating voltage-gated Ca currents is to use voltage clamp techniques. The voltage clamp approach allows a more careful analysis of the voltage sensitivity and other properties of Ca currents affected by aging. However, voltage clamping CNS neurons in the slice presents a number of major technical hurdles. One approach that allows voltage clamp procedures to be applied with minimal disruption of the neurons, however, is the single electrode, discontinuous voltage clamp technique. This method utilizes an intracellular sharp, high resistance electrode with discontinuous sampling of voltage. In this procedure tetraethylanmronium is also added to block additional K ChaMels that are not fully blocked by Cs. Under voltage clamp conditions, Ca currents were again found to be larger in CA 1 neurons from aged than from young rats. Nimodipine reduced Ca current by approximately one-third in young and aged neurons, indicating that L-type Ca currents are increased with aging, but that other high threshold (N,P) Ca currents also may be increased. One other important observation in these studies, moreover, was that the larger currents with aging were not due to impaired inactivation because the rate and degree of inactivation were at least as great in aged neurons (10, and in preparation).
Vol. 59, No.s S/6, 1%
Calcium Channels in Aging
401
Thus, multiple approaches to recording Ca influx, from physiological intracellular potential recordings (AHP, synaptic potentials) and measures of Ca action potentials, to whole cell voltage clamp analyses, have indicated that rodent CA1 neurons, which are among the most vulnerable to aging, exhibit increased voltage-gated Ca influx with aging. However, not all investigators have observed similar results (see Lamour, Verkhratsky, this volume). With one exception (Lamour, this volume), studies finding no increase in Ca currents have used cell types other than hippocampal pyramidal neurons (peripheral, cerebellar, dentate gyms), from regions for which there is less evidence of major decline with AD or aging. Some preparations have also been very different from In addition, these are extremely difficult preparations to study ours (dissociated cells). quantitatively and extreme efforts must be made to control variability. Strict criteria for healthy neurons and comparable quality of recordings are necessary. Although we cannot account for all discrepancies in the literature, it should be emphasized that four separate studies performed by different investigators in our laboratory (1,2,8,10) have found consistent evidence of enhanced AHP, Ca action potentials or Ca currents in CA1 neurons. New studies are also consistent with these results (see below). Moreover, independent studies by Disterhoft and colleagues (cf. this volume), have found similar results in CA1 cells of another mammalian species. Thus, the evidence of aging-dependent increases in Ca influx appears to be strong and consistent, at Last for the CA1 neurons that are among the most susceptible to aging/AD.
Single Ca channel approaches The advent of the patch clamp technique in the early 1980s in which a pipette tip is placed onto a small patch (1-3 um2) of cell membrane and suction is used to form a high resistance seal has allowed investigators to record the activity of a single ion channel, and thereby to investigate changes in ionic currents at a more basic, mechanistic level. The “whole cell” patch clamp method breaks through the membrane patch to gain access to the cell’s interior and record the activity of the whole cell. This yields currents that are generally analogous to those obtained with the intracellular sharp electrode methods described above. In addition, however, patch clamp methods from an intact membrane patch allow single channels to be recorded from the small patch, either in the cellattached mode or in the cell-free mode (patch pulled away from cell while still on the pipette). These relatively new single channel methods provide an important opportunity to analyze aging changes at a more molecular level than has previously been feasible as described below.
Long Ca tail currents in hippocampal neurons In addition to the basic results on aging differences, the whole-cell voltage clamp studies in hippocampal slices revealed a very long inward tail current (hundreds of milliseconds) that followed the depolarization pulse. Although many electrophysiologists view such long tails as space clamp artifacts, several aspects of the long tail current did not seem consistent with the artifact interpretation. We have therefore spent quite a few years attempting to determine whether or not this was a true Ca current, and have recently obtained evidence at the single channel (11) and whole cell (in preparation) levels indicating that the long tail current arises from an unusual mode of Ca channel activity that permits channel opening after rcpolarization. This evidence therefore suggests that the long tail current reflects a substantial unrecognized pathway of Ca entry and may be a site of aging-related change.
Devel~t
.
of Calcuun Cu rrents in Cuiturg
Calcium channel currents also change with the age of the cells in culture On day 2, after plating, hippocampal neurons manifest a very small voltage-activated Ca current and do not have a long tail current, Over the life cycle of the neurons in culture (~30 days) the voltage-activated Ca current increases substantially. Moreover, on day 3 the tail current first appears, and also increases
402
Calcium Channels
inAging
Vol. 59,No.s 516,1996
over the life cycle of a cell. Single channel studies show a similar pat&m. Single channel (L-type) activity increases steadily over the life of the neurons. Few repolarization openings (ROs) are observed up to day 2; they first appear on day 3, and then become increasingly more prevalent as the neurons mature. Although calcium channel current increases with age in culture, this is not simply a reflection of the fact that the cells grow in size, because even with corrections for cell capacitance (a direct indicant of cell size), Ca current density increases rapidly in the first week and then continues to rise more slowly. Correlations with neuron survival Calcium current density also correlates with hippocampal neuron survival in culture. Quantitative counts of neurons over time show that periods of cell death correlate with a higher rate of increase of Ca current density. Moreover, with nimodipine chronically present in the culture bath, almost twice as many cells survive by day 15 as survive under control conditions (12). Our working hypothesis is that the increase in Ca current density seems to be correlated with and may be causally related to cell death. . [email protected] Cmual
Neuron6
As intriguing as the cell culture model is for studying the relationship between gradual increases in Ca channel activity and cell survival and/or function, aging of embryonic cells in culture clearly cannot be assumed to be the same as aging of neurons in vivo. Thus, functional studies at the level of a single molecule (channel), specifically in aging mammalian brain cells, are necessary to dissect out the mechanisms underlying the increase in macroscopic Ca currents. Whole cell studies alone do not clarify whether macroscopic currents increase because each channel exhibits larger conductance, or a greater open probability, or whether there are more functionally available channels. However, in order to determine the single channel basis for the aging changes in whole cell Ca currents and potentials, we needed a preparation in which we could apply single channel patch clamp methods to brain neurons of an aging mammal. The standard adult neuron preparation for patch clamp studies at the single channel level is acutely dissociated cells, but this approach appears too traumatic for studies of aged brain cells. In our hands, the yield of healthy neurons from aged animals, using the dissociation technique, was very low, raising the possibility of sampling bias ( 13). Moreover, while non-dissociated slices presently are being used widely for whole cell patch clamp studies, they are difficult to use on a large scale for single channel patch clamp analyses. For these reasons, we modified a technique of partial dissociation of the hippocampal slice, originally developed by Gray, Johnston and colleagues (14). This method is referred to as the “zipper slice” because of the tendency for the hippocampal slice to “unzip” along the CA1 cell layer, thereby exposing the neuronal somata for formation of a high resistance seal with a pipette. This preparation provides the same yield of good recordings from young and aged animals, in cells that retain their dendrites and appear normal in shape and size (13). We used this method to conduct the first large scale quantitative analyses of single channel activity in brain neurons of aged mammals (over 75 total neurons from young, mid-aged and aged rats) (Thibault and Landfield, in preparation). Multiple depolarizations were used to construct an average ensemble current for each patch in young, mid-aged and aged rat neurons, from which average current in each patch was calculated. In multichannel patches on neurons from aged rats, we found a large increase in the pseudomacroscopic (averaged) current during depolarization as well as an increase in repolarization openings relative to young or mid-aged rat neurons. Currentvoltage curves showed that this increase was not due to a shift in voltage dependence. These studies focused on L-type Ca channels, and the L-type agonist, Bay K 8644, was added to the pipette to
Vol. 59, No.s 5/6, 19%
Calcium Channelsin Aging
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enhance L-type activity in the recordings. However, measures of mean open time and voltage dependence indicated no age differences in the sensitivity of L-type channels to the agonist. An increase in average patch current with aging could be due to more functionally available channels, to an increase in the single channel conductance, or to an increased open probability. To separate these possibilities, we measured the amplitude of current in single L-type Ca channels at multiple voltages, and calculated slope conductance. Single calcium channel conductance did not change with aging. The amplitude of repolarization openings also did not change with age. Estimates of the number of available channels in each patch, obtained by the method of maximal simultaneous openings also were made for each patch. These measures showed that the number of Ca channels was substantially increased in aged neurons. Estimates of patch size, based on patch pipette resistance showed no age differences, indicating that the increase in channel numbers reflected an increase in the density of Ca channels. The increase in density of L-type Ca channels was roughly proportional to the increase in average patch current, and was generally similar to the changes in the AHP, the calcium spike and macroscopic currents that we have seen in earlier studies. Consequently it seems reasonable to conclude that most of the aging changes in Ca currents observed previously are due to an aging-related increase in the density of Ca channels in the membranes of CA1 hippocampal neurons. Moreover, several weeks prior to the electrophysiological studies, we ran eight of the aged animals in a water maze (a spatial learning task dependent on hippocampal function and affected by aging). Channel density in hippocampal neurons was found to be inversely correlated with maze performance in the aged animals. Young and mid-aged animals perform uniformly well in this task. These results raise the possibility that the reported positive effects of the L-type channel blocker, nimodipine, on learning tasks in aged animals (15 17) may be in part related to the increase in the density of L-type channels. . Conclus~~ After many years of study of voltage-activated Ca potentials and currents in hippocampal neurons of aging animals, it appears that we are now beginning to focus in on a quintessential change at the molecular level, namely an increase in the density of Ca channels per unit membrane. Very possibly, this change accounts for most of the whole cell alterations we have observed previously. As described by other participants here, many changes occur in Ca regulation with aging, including impairments of Ca buffering and extrusion processes. A chronic elevation of Ca influx through increased numbers of Ca channels, as described above, would further pressure regulatory mechanisms, perhaps resulting in an accelerating interactive disruption of Ca regulation. The concentration of Ca in a neuron is regulated extraordinarily tightly, indicating that such regulation is highly important to the cell’s well-being. In turn, this suggests that even a modest increase in Ca intlux, if persistently maintained over long periods, would continually challenge and activate multiple regulatory systems and might well exert a wide variety of disruptive and deleterious actions. Although neuronal aging in culture is unlikely to directly mimic neuronal aging in vivo, it nevertheless seems intriguing that Ca channel density increases in both culture and in vivo aging systems over time. These observations raise the possibility that a gradual increase in Ca channel density may be a consistent time-dependent correlate of brain cell aging and/or maturation under very different conditions. Additional studies will be needed to determine the molecular biological basis of the increase in available Ca channels in either aging or long-term cultured neurons. Several possibilities exist, including the synthesis of new channels or the recruitment of previously silent channels through altered phosphorylation of channel subunits or changes in membrane structure.
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P.W. LANDFIELD and T.A. PITLER, Science 226 1089-1092 (1984). D.S. KERR, L.W. CAMPBELL, S-Y HA0 and P.W. LANDFIELD, Science 2411505-1509 (1989). P.W. LANDFIELD, T.A. PITLER and M.D APPLEGATE .I. Neurophysiol x 797-811 (1986). G.E. GIBSON and C. PETERSON, Neurobiol. Aging, 8 329-344 (1987). M.L. MICHAELIS, K. JOHE and T.E. KITOS Mech. Aging Dev. 2 215-225 (1984). Z.S. KHACHATURIAN, Handbook of Studies on P&,&&y and Old plge, D.S. Kay and G.W. Burrow (eds), 7-30 (1984). Z.S KHACHATURIAN, Aging 1 17-34 (1989). T.A. PITLER and P.W. LANDFIELD, Brain Res. m 1-6 (1990). J.F. DISTERHOFT, J.R. MOYER, Jr., L.T. THOMPSON and M. KOWALSKA, Clin. Neuropharmacol., 16 S12-S24 (1993). L.W. CAMPBELL, S-Y HA0 and P.W. LANDFIELD, Sot. Neurosci. Abstr., 1. 260 (1989). 0. THIBAULT, N.M. PORTER and P.W. LANDFIELD, Proc. Natl. Acad. Sci. USA, %I 11792-l 1796 (1993). N.M. PORTER, 0. THIBAULT, V. THIBAULT, J.L. GEDDES and P.W. LANDFIELD, Sot. Neurosci. Abstr., .2Q900 (1995). O.THIBAULT, M.L. MAZZANTI, E.M. BLALOCK, N.M. PORTER, AND P.W. LANDFIELD: J. Neurosci. Meth., 5p 77-83 (1995). R. GRAY, R. FISHER, N. SPRUSTON and D. JOHNSTON, &pa&ions of Vertabra& Central Nervous System In V’tr~ H. Jahnsen (ed), 3-24 (1990). R.A. DEYO, K.T. STRAUBE and J.F. DISTERHOFT, Science 241809-811 (1989). A. SCRIABINE, T. SCHUURMAN and J. TRABER, FASEB J., 1 1799-1806 (1989). K. MCMONAGLE-STRUCK0 and R.J. FANELLI, Pharmacol. Biochem. Behav., 44 827835 (1993).