dyshomeostasis underlies aspects of aging-dependent brain impairment. However, some key predictions of this view remain untested,
with initial signs of cognitive decline. Moreover, blocking a putative common source of dysregulated Ca2?should eliminate aging
differences. Here, we tested these predictions using combined electrophysiological, imaging, and pharmacological approaches in CA1
the increases in the slow afterhyperpolarization, spike accommodation, and [Ca2?]irise during repetitive synaptic stimulation. In
L-type Ca2?channels, provides a common source of dysregulated Ca2?in aging. Results showed that multiple aging biomarkers were
It has long been recognized that aging-dependent changes occur
transmission and plasticity have been found in several brain re-
ber of Ca2?-dependent/mediated processes have been found to
be consistent biomarkers of aging in hippocampal neurons, in-
cluding those in the Ca2?-dependent slow afterhyperpolariza-
tion (sAHP), spike accommodation, the Ca2?action potential,
hoft et al., 1996; Norris et al., 1998; Thibault et al., 1998; Dister-
hoft et al., 2004; Tombaugh et al., 2005). The activity of L-type
the rise of [Ca2?]iduring postsynaptic action potential genera-
tion (Thibault et al., 2001; Hemond and Jaffe, 2005) are also
increased in hippocampal neurons from aging animals.
These and other findings, from different neuronal cell types
and technical approaches, have given rise to several versions of
the general hypothesis that a common mechanism of Ca2?dys-
regulation underlies many aspects of functional aging and, po-
tentially, Alzheimer’s disease (AD) (Landfield and Pitler, 1984;
Verkhratsky and Toescu, 1998; Murchison et al., 2004; Toescu et
al., 2004). However, several critical questions and predictions of
the onset of Ca2?dysregulation should precede or coincide with
cognitive deficits begin to appear in mammals as early in adult-
hood as midlife (Aitken and Meaney, 1989; Fischer et al., 1992;
1999; Knuttinen et al., 2001). Second, multiple Ca2?-related
biomarkers of hippocampal aging should emerge approximately
simultaneously, rather than asynchronously. Third, blocking a
common source of dysregulated Ca2?should eliminate aging
differences in multiple Ca2?-mediated biomarkers.
An increase in Ca2?influx through L-type voltage-gated
Ca2?channels (L-VGCCs) has been implicated previously as a
possible Ca2?source for several hippocampal electrophysiolog-
cology, MS320, University of Kentucky Medical Center, 800 Rose Street, Lexington, KY 40536-0298. E-mail:
3482 • TheJournalofNeuroscience,March29,2006 • 26(13):3482–3490
et al., 2004). However, there are other candidate sources of dys-
regulated Ca2?. In particular, Ca2?-induced Ca2?release
(CICR) from ryanodine receptors (RyRs) on the endoplasmic
reticulum (ER), which can be triggered by Ca2?influx via
L-VGCCs (Chavis et al., 1996; Empson and Galione, 1997; Fagni
et al., 2000; Sukhareva et al., 2002; Verkhratsky, 2005), may be
altered in some models of aging or AD (Gibson et al., 1996;
al., 2002; Kumar and Foster, 2004). Therefore, it seems possible
that amplified CICR also plays a role in generating Ca2?-related
markers of aging. If so, high-dose ryanodine, which selectively
locks RyR channels into a low-conductance state (Bezprozvanny
et al., 1991; Coronado et al., 1994; Humerickhouse et al., 1994),
counteracting the larger CICR components in older age groups.
Here, we tested key questions and predictions of this hypoth-
the age of onset and of the ryanodine sensitivity of multiple
biomarkers of hippocampal aging. The results lend strong new
support to a unified Ca2?dyshomeostasis hypothesis.
Slice preparations. All experiments were conducted in compliance with
Institute on Aging aged rat colony in subsets, each containing rats for all
age points. The subsets were staggered such that, when studied, animals
in the different age groups averaged 4, 10, 12, 14, and 23 months of age.
Animals were anesthetized in a CO2chamber before rapid decapitation.
Brains were rapidly removed and transverse hippocampal slices (350
?m) were cut with a vibratome (TPI, St. Louis, MO) into cold oxygen-
ated artificial CSF (ACF) of the following composition (in mM): 128
NaCl, 1.25 KH2PO4, 10 glucose, 26 NaHCO3, 3 KCl, 0.1 CaCl2, and 2
MgCl2(Thibault et al., 2001). Intact slices were placed in an interface-
type chamber containing ACF with 2 mM CaCl2(Ca-ACF) at 32°C and
recovery, individual slices were then transferred for recording to a per-
fusion chamber (Warner Instruments, Hamden, CT) equipped with a
bottom net for Ca-ACF perfusion beneath the slice. The oxygenated
inline heater (TC2Bip; Cell Micro Controls, Norfolk, VA) positioned 1
cm before the chamber inlet.
Electrophysiology. Recording data were acquired and analyzed using
pCLAMP 8, a sharp-electrode amplifier (Axoclamp 2A), and a DigiData
1320 board for digitization (Molecular Devices, Union City, CA). Sharp
ies (Fisher Scientific, Pittsburgh, PA) on a Sutter Instruments (Novato,
CA) pipette puller, and had tip resistances of 80–120 M? when filled
with 2 M KmeSO4, 10 mM HEPES, and 10 mM bis-Fura-2, pH 7.4. After
impalement and stabilization, neurons were held at ?70 mV in current
clamp with minimal injected current for ?5 min to obtain input resis-
tance measures and to allow indicator filling. All experiments were con-
ducted in current-clamp mode with bridge balance compensation and
capacitance neutralization. Voltage records were digitized at 2–20 kHz
and low-pass filtered at 1 kHz.
The afterhyperpolarization (AHP) was triggered with the membrane
held at ?60 mV using a 100 ms current depolarization pulse delivered
potentials. AHP amplitudes were measured at the negative voltage peak
immediately after the depolarization pulse, reflecting the medium AHP
(mAHP), which typically lasts several hundred milliseconds in hip-
pocampal pyramidal neurons, and at 1 s after the end of the step, during
the sAHP, which has durations in the 1–3 s range (Lancaster and Nicoll,
1987; Storm, 1990; Williamson and Alger, 1990; Sah and Faber, 2002;
Stocker, 2004). Area and duration of the AHP were measured from the
end of the depolarization step until return of the membrane voltage to
baseline (?60 mV). AHPs were elicited every 30 s, and measures were
averaged from 10–15 consecutive AHPs recorded in each cell.
Spike-frequency accommodation was also determined from ?60 mV
using current intensity just sufficient to generate three Na?action po-
modation was measured as the number of action potentials during the
entire current step.
Repetitive synaptic stimulation (RSS) for ratiometric Ca2?imaging
coated stainless steel; A-M Systems, Everett, WA) positioned in the
Schaffer-collaterals/commissural fibers of stratum radiatum ?500 ?m
from the recorded neuron. For all synaptic stimulation, pulse duration
was 100 ?s and was delivered by a SD9K stimulator (Astro Med, Grass
Instruments, Warwick, RI). Input–output (I/O) relationships were de-
termined in every cell during baseline periods (0.2 Hz) before RSS. The
RSS train was delivered at 7 Hz for 20 s. Stimulus intensity during RSS
ensure action potential generation at 7 Hz throughout the 20 s train.
Double spikes to a single pulse were rare and counted as single spikes.
with resting [Ca2?]i?200 nM were excluded from analysis. For record-
ing analyses, neurons with input resistance ?35 M?, action potential
recorded neurons that met all criteria (including those not imaged be-
cause of depth) was approximately two cells per daily animal prepara-
tion. The yield of neurons both imaged and recorded per animal was
somewhat less (?1.5), and neither yield differed across aging. As previ-
ously reported (Thibault et al., 2001), gray values taken at rest for either
with age, nor did cell depth measures (Table 2), indicating that age dif-
ferences in Ca2?indicator loading or tissue opacity did not contribute
significantly to the quantitative measures of [Ca2?]ireported here.
Fluorometric [Ca2?]imeasurements. Individual neurons loaded with
the ratiometric Ca2?indicator bis-Fura-2 (10 mM) were imaged on the
stage of a Nikon (Tokyo, Japan) E600 microscope equipped with a 40?
water immersion objective and a CCD camera (Roper Scientific, Prince-
ton Instruments, Trenton, NJ). The fluorophore was excited using a
wavelength switcher (Sutter Lambda DG-4) and software control (Axon
Imaging Workbench, version 184.108.40.206; Molecular Devices). The 510 nm
wavelength was monitored during both 357 and 380 nm wavelength
speed of image acquisition during concomitant electrophysiology (Jaffe
Gantetal.•Ca2?DysregulationOnsetandAlteredCICRinAging J.Neurosci.,March29,2006 • 26(13):3482–3490 • 3483
naling in the soma of hippocampal neurons by Ca2?release from intra-
cellular stores. J Neurosci 17:4129–4135.
Jaffe DB, Brown TH (1994) Confocal imaging of dendritic Ca2?transients
in hippocampal brain slices during simultaneous current- and voltage-
clamp recording. Microsc Res Tech 29:279–289.
Jaffe DB, Johnston D, Lasser-Ross N, Lisman JE, Miyakawa H, Ross WN
(1992) The spread of Na?spikes determines the pattern of dendritic
Ca2?entry into hippocampal neurons. Nature 357:244–246.
Johnston D, Christie BR, Frick A, Gray R, Hoffman DA, Schexnayder LK,
Watanabe S, Yuan LL (2003) Active dendrites, potassium channels and
synaptic plasticity. Philos Trans R Soc Lond B Biol Sci 358:667–674.
Kadar T, Arbel I, Silbermann M, Levy A (1994) Morphological hippocam-
pal changes during normal aging and their relation to cognitive deterio-
ration. J Neural Transm Suppl 44:133–143.
Khachaturian ZS (1989) Calcium, membranes, aging, and Alzheimer’s dis-
ease. Introduction and overview. Ann NY Acad Sci 568:1–4.
Knuttinen MG, Gamelli AE, Weiss C, Power JM, Disterhoft JF (2001) Age-
related effects on eyeblink conditioning in the F344 ? BN F1hybrid rat.
Neurobiol Aging 22:1–8.
Kovalchuk Y, Eilers J, Lisman J, Konnerth A (2000) NMDA receptor-
mediated subthreshold Ca2?signals in spines of hippocampal neurons.
J Neurosci 20:1791–1799.
KumarA,FosterTC (2004) Enhancedlong-termpotentiationduringaging
is masked by processes involving intracellular calcium stores. J Neuro-
LancasterB,NicollRA (1987) Propertiesoftwocalcium-activatedhyperpo-
larizations in rat hippocampal neurones. J Physiol (Lond) 389:187–203.
Lancaster B, Hu H, Ramakers GM, Storm JF (2001) Interaction between
synaptic excitation and slow afterhyperpolarization current in rat hip-
pocampal pyramidal cells. J Physiol (Lond) 536:809–823.
Landfield PW (1987) “Increased calcium-current” hypothesis of brain ag-
ing. Neurobiol Aging 8:346–347.
Landfield PW (1988) Hippocampal neurobiological mechanisms of age-
related memory dysfunction. Neurobiol Aging 9:571–579.
Landfield PW, Pitler TA (1984) Prolonged Ca2?-dependent afterhyperpo-
larizations in hippocampal neurons of aged rats. Science 226:1089–1092.
Leissring MA, Akbari Y, Fanger CM, Cahalan MD, Mattson MP, LaFerla FM
(2000) Capacitative calcium entry deficits and elevated luminal calcium
content in mutant presenilin-1 knockin mice. J Cell Biol 149:793–798.
Magee JC, Johnston D (1997) A synaptically controlled, associative signal
for Hebbian plasticity in hippocampal neurons. Science 275:209–213.
Magnusson KR (1998) The aging of the NMDA receptor complex. Front
MarkowskaAL (1999) Sexdimorphismsintherateofage-relateddeclinein
spatial memory: relevance to alterations in the estrous cycle. J Neurosci
Michaelis ML, Bigelow DJ, Schoneich C, Williams TD, Ramonda L, Yin D,
Huhmer AF, Yao Y, Gao J, Squier TC (1996) Decreased plasma mem-
brane calcium transport activity in aging brain. Life Sci 59:405–412.
Miller RA, Nadon NL (2000) Principles of animal use for gerontological
research. J Gerontol A Biol Sci Med Sci 55:B117–B123.
Moyer Jr JR, Thompson LT, Black JP, Disterhoft JF (1992) Nimodipine in-
creases excitability of rabbit CA1 pyramidal neurons in an age- and
concentration-dependent manner. J Neurophysiol 68:2100–2109.
MoyerJrJR,ThompsonLT,DisterhoftJF (1996) Traceeyeblinkcondition-
J Neurosci 16:5536–5546.
Murchison D, Zawieja DC, Griffith WH (2004) Reduced mitochondrial
buffering of voltage-gated calcium influx in aged rat basal forebrain neu-
rons. Cell Calcium 36:61–75.
Norris CM, Halpain S, Foster TC (1998) Reversal of age-related alterations
in synaptic plasticity by blockade of L-type Ca2?channels. J Neurosci
Paschen W, Frandsen A (2001) Endoplasmic reticulum dysfunction—a
the brain? J Neurochem 79:719–725.
Potier B, Poindessous-Jazat F, Dutar P, Billard JM (2000) NMDA receptor
activation in the aged rat hippocampus. Exp Gerontol 35:1185–1199.
PowerJM,WuWW,SametskyE,OhMM,DisterhoftJF (2002) Age-related
hippocampal CA1 pyramidal neurons in vitro. J Neurosci 22:7234–7243.
Regehr WG, Tank DW (1992) Calcium concentration dynamics produced
by synaptic activation of CA1 hippocampal pyramidal cells. J Neurosci
Rosenzweig ES, Barnes CA (2003) Impact of aging on hippocampal func-
tion: plasticity, network dynamics, and cognition. Prog Neurobiol
Sabatini BL, Oertner TG, Svoboda K (2002) The life cycle of Ca2?ions in
dendritic spines. Neuron 33:439–452.
Sah P, Faber ES (2002) Channels underlying neuronal calcium-activated
potassium currents. Prog Neurobiol 66:345–353.
Stocker M (2004) Ca2?-activated K?channels: molecular determinants
and function of the SK family. Nat Rev Neurosci 5:758–770.
Storm JF (1990) Potassium currents in hippocampal pyramidal cells. Prog
Brain Res 83:161–187.
SukharevaM,SmithSV,MaricD,BarkerJL (2002) Functionalpropertiesof
ryanodine receptors in hippocampal neurons change during early differ-
entiation in culture. J Neurophysiol 88:1077–1087.
ThibaultO,LandfieldPW (1996) IncreaseinsingleL-typecalciumchannels
in hippocampal neurons during aging. Science 272:1017–1020.
Brewer LD, Landfield PW (1998) Calcium dysregulation in neuronal
aging and Alzheimer’s disease: history and new directions. Cell Calcium
Thibault O, Hadley R, Landfield PW (2001) Elevated postsynaptic [Ca2?]i
and L-type calcium channel activity in aged hippocampal neurons: rela-
tionship to impaired synaptic plasticity. J Neurosci 21:9744–9756.
Thompson LT, Moyer Jr JR, Disterhoft JF (1996) Transient changes in ex-
memory consolidation. J Neurophysiol 76:1836–1849.
Toescu EC, Verkhratsky A (2004) Ca2?and mitochondria as substrates for
deficits in synaptic plasticity in normal brain ageing. J Cell Mol Med
Toescu EC, Verkhratsky A, Landfield PW (2004) Ca2?regulation and gene
expression in normal brain aging. Trends Neurosci 27:614–620.
Tombaugh GC, Rowe WB, Rose GM (2005) The slow afterhyperpolariza-
aged Fisher 344 rats. J Neurosci 25:2609–2616.
Veng LM, Mesches MH, Browning MD (2003) Age-related working mem-
protein alpha1D (Cav1.3) in area CA1 of the hippocampus and both are
ameliorated by chronic nimodipine treatment. Brain Res Mol Brain Res
Verkhratsky A (2005) Physiology and pathophysiology of the calcium store
in the endoplasmic reticulum of neurons. Physiol Rev 85:201–279.
Verkhratsky A, Toescu EC (1998) Calcium and neuronal ageing. Trends
Williamson A, Alger BE (1990) Characterization of an early afterhyperpo-
larization after a brief train of action potentials in rat hippocampal neu-
rons in vitro. J Neurophysiol 63:72–81.
Wu WW, Chan CS, Disterhoft JF (2004) Slow afterhyperpolarization gov-
erns the development of NMDA receptor-dependent afterdepolarization
in CA1 pyramidal neurons during synaptic stimulation. J Neurophysiol
XiongJ,VerkhratskyA,ToescuEC (2002) Changesinmitochondrialstatus
associated with altered Ca2?homeostasis in aged cerebellar granule neu-
rons in brain slices. J Neurosci 22:10761–10771.
ZandiPP,BreitnerJC,AnthonyJC (2002) Ispharmacologicalpreventionof
Alzheimer’s a realistic goal? Expert Opin Pharmacother 3:365–380.
3490 • J.Neurosci.,March29,2006 • 26(13):3482–3490Gantetal.•Ca2?DysregulationOnsetandAlteredCICRinAging