NeurobiologieAlfredFessard,FRC2118,LaboratoiredeNeurobiologieetDe ´veloppement,Unite ´PropredeRecherche3294,91198Gif-sur-YvetteCedex,
logical homeostasis, such that calcium-dependent presynaptic and postsynaptic signaling appear functionally normal yet are actually
associated with a shift toward synaptic depression, likely through a reduction in presynaptic vesicle stores and increased postsynaptic
3xTg-AD mice also compensates for an intrinsic predisposition for hippocampal long-term depression (LTD) and reduced long-term
potentiation (LTP). In this study, we detail the impact of disrupted RyR-mediated calcium stores on synaptic transmission properties,
LTD, and calcium-activated membrane channels of hippocampal CA1 pyramidal neurons in presymptomatic 3xTg-AD mice. Using
of vesicle release, and upregulated postsynaptic SK channel activity. Because AD is increasingly recognized as a “synaptic disease,”
tic function by regulating neurotransmission, membrane excit-
ability and synaptic plasticity (Emptage et al., 2001; Bouchard et
al., 2003; Stutzmann et al., 2003; Ross et al., 2005; Raymond and
Redman, 2006; Watanabe et al., 2006). Not surprisingly, ER cal-
erative diseases involving memory loss, including Alzheimer’s
disease (AD) (LaFerla, 2002; Stutzmann, 2007; Bezprozvanny
and Mattson, 2008; Foskett, 2010). For instance, AD-linked pre-
senilin (PS) mutations markedly increase ER calcium release,
resulting in altered presynaptic and postsynaptic synaptic trans-
mission mechanisms (Parent et al., 1999; Chakroborty et al.,
2009; Zhang et al., 2009; Goussakov et al., 2010), intrinsic mem-
al., 2011). Both ryanodine receptor (RyR) and IP3R are involved,
stronger influence within dendrites and presynaptic terminals
(Smith et al., 2005; Cheung et al., 2008, 2010; Rybalchenko et al.,
spine heads, is particularly important for understanding AD pa-
thology, because it is the degree of synaptic dysfunction that best
dependent synaptic plasticity, which serves to encode learning
and memory functions (Bliss and Collingridge, 1993; Martin et
al., 2000; Whitlock et al., 2006).
Although many studies have demonstrated overt impair-
ments in synaptic plasticity coincident with amyloid deposi-
tion (Nalbantoglu et al., 1997; Chapman et al., 1999; Oddo et
al., 2003; Selkoe, 2008), we and others have shown there are
pronounced neuronal signaling deficits that operate “below
the radar” until ER calcium stores are specifically probed
Correspondence should be addressed to Dr. Grace E. Stutzmann, Department of Neuroscience, Rosalind
Franklin University/The Chicago Medical School, 3333 Green Bay Road, North Chicago, IL 60064. E-mail:
TheJournalofNeuroscience,June13,2012 • 32(24):8341–8353 • 8341
(Stutzmann, 2007; Mu ¨ller et al., 2011). For example, basal
synaptic function and plasticity mechanisms appear similar
between young nontransgenic (NonTg) and 3xTg-AD mice,
yet when RyRs are manipulated, striking synaptic transmis-
sion and plasticity aberrations are revealed in the 3xTg-AD
mice, whereas NonTg mice exhibit no observable effects. This
is in large part attributable to an increase in calcium-induced
calcium release (CICR) via RyR (Chakroborty et al., 2009;
Goussakov et al., 2010, 2011). Thus, in 3xTg-AD mouse
brains, compensatory mechanisms mask these early deficits
and maintain a normal physiological phenotype. However,
sustaining synaptic homeostasis and compensating for intra-
cellular calcium dysregulation concurrently can likely com-
promise neuronal function in the long term.
the synaptic pathophysiology evident at later disease stages. This
is manifested as increased postsynaptic small-conductance
calcium-activated potassium SK2 channel function and in-
creased presynaptic spontaneous vesicle release, both of which
likely contribute to synaptic depression and increased long-term
depression (LTD). Overall, we are suggesting that subtle but in-
sidious calcium-mediated pathogenic mechanisms can exist be-
synaptic signaling dysfunction in AD.
Transgenic mice. Six- to 8-week-old triple transgenic (3xTg-AD) mice
and age-matched NonTg controls (males and females) were used in all
experiments. The 3xTg-AD (APPswe, TauP301L, and PS1M146VKI) mice
for several reasons. First, the deficits in PS function and the associated
calcium dysregulations can be applicable to both familial and sporadic
AD (Takami et al., 1997). Second, studies comparing mutant PS, PS1/
APP, APP/Tau, and 3xTg-AD mouse models indicate that the exagger-
ated ER calcium signaling at young ages is associated with mutant PS1,
whereas mutant APP and Tau do not significantly affect ER calcium
signals at these early stages. These studies also show similar synaptic
phenotypes in these AD mouse models at the presymptomatic ages used
here (Oddo et al., 2003; Stutzmann et al., 2006, 2007; Dewachter et al.,
2008; Auffret et al., 2009; Goussakov et al., 2010).
Hippocampal slice preparation. Transverse hippocampal slices were
prepared in accordance with protocols approved by the Institutional
Animal Care and Use Committee at Rosalind Franklin University. In
for extracellular field potential recordings and 300 ?m for patch-clamp
experiments) were cut with a vibrating microtome (Campden Instru-
composition (in mM): 125 NaCl, 2.5 KCl, 1.25 KH2PO4, 1.2 MgSO4, 2
CaCl2, 10 dextrose, and 25 NaHCO3(Chakroborty et al., 2009). Intact
slices were placed in a holding chamber containing aCSF at room tem-
perature (27°C) and oxygenated with 95% O2/5% CO2.
ml/min) at room temperature (27°C), and covered with a continuous
using pClamp 9.2 software with an AxoClamp 2B amplifier and a Digi-
Data 1322A board for digitization (Molecular Devices). Field EPSPs
(fEPSPs) were recorded in the stratum radiatum of the CA1 subfield of
the hippocampus using recording microelectrodes (2–6 M?) filled with
aCSF. Microelectrodes were pulled from glass capillaries (Harvard Ap-
paratus) on a P-2000 pipette puller (Sutter Instruments). Synaptic re-
sponses were evoked by stimulation of the Schaffer collateral/
commissural pathway with a bipolar stimulating electrode. Baseline
line fEPSPs were recorded for 1 h at 0.05 Hz after the induction of LTD.
Input/output (I/O) curves were generated using stimulus intensities
was assessed using interstimulus intervals of 50 ms. Ten successive re-
sponse pairs were recorded at 0.05 Hz intervals. For experiments with
B (XeB) (IP3R antagonist, 5 ?M, extracted and purified from the marine
sponge Xestospongia exigua as described by Jaimovich et al., 2005), slices
were incubated in the drug for 2–3 h before recording. For experiments
with dantrolene (RyR antagonist, 10 ?M), a 20 min baseline at 0.05 Hz
used to record a 20 min baseline in dantrolene, after which LTD was
induced, followed by a 1 h baseline in dantrolene. For experiments with
(RS)-3,5-dihydroxyphenylglycine (DHPG) (mGluR1/5 agonist, 100 ?M;
Tocris Bioscience), a 20 min baseline at 0.05 Hz was recorded in aCSF
containing 2-amino-5-phosphonopentanoic acid (APV) (NMDAR an-
tagonist, 50 ?M, Sigma-Aldrich), after which LTD was induced by wash-
aCSF containing APV.
For analysis, responses were measured offline using pClamp 9.2 soft-
ware and analyzed using Origin Pro8 and Microsoft Excel software. For
LTD recordings, fEPSP slopes recorded 40–60 min after LFS were aver-
aged and expressed as a percentage of the average slope from 20 min
of the second fEPSP over the first fEPSP. For I/O curves, the first 5 ms of
the fEPSP slopes were measured.
and perfused with oxygenated aCSF (2 ml/min) at room temperature.
Data were acquired in current-clamp mode (unless otherwise stated) at
a DigiData 1440 board for digitization (Molecular Devices). Hippocam-
pal CA1 pyramidal neurons were identified visually by infrared-
differential interference contrast optics and electrophysiologically by
their passive membrane properties. Whole-cell patch recordings were
performed from CA1 pyramidal neurons of the hippocampus using re-
mM): 135 K-methylsulfonate, 10 HEPES, 10 Na-phosphocreatine, 2
MgCl2, 4 Na-ATP, and 0.4 Na-GTP, pH adjusted to 7.3–7.4 with KOH.
Microelectrodes were pulled from glass capillaries (Harvard Apparatus)
on a P-87 pipette puller (Sutter Instruments). Synaptic responses were
evoked by stimulation of the Schaffer collateral/commissural pathway
pulses of 500 ms duration, varying in amplitude from ?200 to ?200 pA
1 min. To confirm that the spontaneous postsynaptic miniature poten-
tials we recorded did not reflect action-potential-generated vesicle re-
lease, we also recorded spontaneous events with TTX (1 ?M; Sigma-
potentials with and without TTX and found no significant differences
t(1,3)? 2.09, p ? 0.13; amplitude, t(1,3)? 1.37, p ? 0.26; 3xTg-AD:
4 for both). The lack of a TTX effect likely reflects an absence, or low
aration. For clarity, the data shown include only non-TTX conditions.
PPF was assessed using interstimulus intervals of 25, 50, 100, 200, 500,
and 1000 ms. Five successive responses were recorded for each inter-
ization (mAHP) current was measured in voltage-clamp mode at ?55
8342 • J.Neurosci.,June13,2012 • 32(24):8341–8353Chakrobortyetal.•SynapticDepressioninADMice
pulses at 70 Hz. Five successive response pairs at 0.05 Hz intervals were
recorded for each interstimulus interval. Experiments were performed
with dantrolene (RyR antagonist, 10 ?M) or apamin (SK channel antag-
onist, 1 ?M; Sigma-Aldrich) in the bath solution. Responses were mea-
sured using pClamp 10.2 and further analyzed using Origin Pro8 and
Microsoft Excel. The amplitude of mAHP current was measured 50 ms
50 ms after the offset of the 10th depolarizing pulse for the 10-pulse
protocol. Spontaneous postsynaptic potentials were analyzed offline us-
ing MiniAnalysis software (version 6.0.9; Synaptosoft), and cumulative
probability histograms were generated to compare amplitudes and
Immunoblot analysis. Hippocampal tissue was harvested from 6- to
8-week-old 3xTg-AD and NonTg mice (n ? 6 each). Tissue was ho-
mogenized on ice in Tissue Protein Extraction Reagent (Invitrogen)
containing protease inhibitors (Roche). To-
tal hippocampal protein was quantified and
separated by SDS-PAGE on 3–8% Tris-acetate
NuPAGE gradient gels (Invitrogen). Protein
was transferred onto polyvinylidene difluoride
membranes (Hybond-P; GE Healthcare) at 30
V for 2 h under reducing conditions. Mem-
branes were blocked with 5% nonfat milk in
TBS for 1 h at room temperature. Rabbit anti-
KCNN2 (29 kDa; short form) and rabbit anti-
?-tubulin primary antibodies were diluted
1:1000 in 2.5% nonfat milk and applied to the
respective half of the membranes for 72 h at 4°C
form) was diluted 1:1000 and applied for 48 h
after azide quench. HRP-conjugated goat sec-
ondary antibodies were applied for 1 h at room
temperature. All antibodies were obtained from
Abcam. KCNN2 (short and long forms) density
levels are represented relative to ?-tubulin. Im-
ing system and quantified using NIH ImageJ
Data analysis and statistics.Data are expressed
way or two-way ANOVA with Scheffe ´’s post hoc
analysis, where n denotes the number of slices
examined in extracellular field experiments and
probability histograms for spontaneous poten-
tials, the nonparametric Kolmogorov–Smirnov (K–S) test was performed
The rationale driving this study stems from previous work dem-
onstrating abnormal and potentially disruptive aberrations in
ple, our previous studies in young presymptomatic 3xTg-AD
mice demonstrated a significantly reduced threshold for induc-
ing RyR-mediated CICR during basal synaptic transmission, ex-
spine heads, and enhanced basal neurotransmission and altered
short- and long-term plasticity with RyR block (Chakroborty et
al., 2009; Goussakov et al., 2010, 2011). These data led to ques-
tions regarding possible underlying shifts toward synaptic de-
pression resulting from aberrant RyR-mediated calcium
signaling. The phenomenon of LTD is little studied in AD, and
here we seek to identify alterations in synaptic depression and
investigate contributing factors in an effort to define deficits in
synaptic function in early AD.
Because RyR-mediated CICR is necessary for the induction of
long-term plasticity (Obenaus et al., 1989; Alford et al., 1993;
Harvey and Collingridge, 1993; Reyes and Stanton, 1996), we
further explored the consequences of dysregulated ER calcium
signaling on synaptic physiology in young 3xTg-AD mice by ex-
amining NMDAR-dependent LTD induced by a LFS (see Mate-
rials and Methods). Under control conditions LTD appeared
similar between both 3xTg-AD (n ? 7, 40.4 ? 1.2% decrease)
and NonTg (n ? 5, 40.5 ? 1.1% decrease) mice (p ? 0.05; Fig.
1A), as did basal synaptic transmission as measured with I/O
functions and short-term presynaptic plasticity as measured by
aCSF (1), baseline in dantrolene (2), and after LFS (3) in the NonTg (black) and 3xTg-AD (gray) mice. Right, Bar graph shows
Treatment NonTg 3xTg-AD
Chakrobortyetal.•SynapticDepressioninADMiceJ.Neurosci.,June13,2012 • 32(24):8341–8353 • 8343
PPF ratios (3xTg-AD: n ? 19, 13, respec-
tively; NonTg: n ? 12, 9 respectively; p ?
0.05; data not shown). This I/O and PPF
observations (Chakroborty et al., 2009).
However, when RyRs were blocked
with 10 ?M dantrolene, different signal-
ing patterns emerged, suggesting that
3xTg-AD mice use alternative signaling
mechanisms distinct from RyR pathways.
In NonTg mice, dantrolene had no effect
on I/O or PPF functions (n ? 6, p ? 0.05;
data not shown; Chakroborty et al., 2009)
in 3xTg-AD mice (n ? 5, t(1,4)? ?5.3;
not shown; Chakroborty et al., 2009; Ta-
both a control and a drug treatment base-
line. Here, dantrolene abolished LTD in
NonTg mice (n ? 9, p ? 0.05) but gener-
ated very different responses in 3xTg-AD
mice. Dantrolene increased baseline re-
sponses by 50.8 ? 1.4% (n ? 5, t(1,4)?
to 72.5 ? 1.2% below dantrolene baseline
and 58.5 ? 1.2% below control aCSF
baseline (Fig. 1B).
We next explored calcium-dependent sig-
tor in the 3xTg-AD responses. Calcineurin
is a calcium-dependent protein phos-
phatase that contributes to NMDAR-
dependent LTD by dephosphorylating
AMPA receptor subunits and facilitating
their internalization and removal from the
mice and thereby underlying the divergent
plied the same synaptic stimulus protocols
as described in Figure 1. Blocking calcineu-
rin did not affect I/O functions (p ? 0.05;
Fig. 2A) or PPF (p ? 0.05; Fig. 2D) in either NonTg (n ? 12, 16
respectively) or 3xTg-AD (n ? 11, 14 respectively) mice. FK506
literature (Mulkey and Malenka, 1992; Mulkey et al., 1994), and in
RyR (Brillantes et al., 1994; Xiao et al., 1997; Ozawa, 2008), what
effects it may have on murine neuronal RyR are unclear. In the
present experiments, however, FK506 did not differentially affect
We next examined patterns of synaptic plasticity when both
calcineurin and RyR were blocked. Coapplication of dantrolene
p ? 0.05; Fig. 2B) or PPF (n ? 7; p ? 0.05; Fig. 2E) in NonTg
mice but, consistent with RyR antagonism alone, increased both
PPF (n ? 5, t(1,4)? ?4.1; p ? 0.05; Fig. 2F) in 3xTg-AD mice.
With LTD, blocking both RyR and calcineurin in NonTg mice
0.05). However, in 3xTg-AD mice, blocking both RyR and cal-
cineurin still generated a synaptic phenotype similar to blocking
RyR alone. Baseline responses increased (26.2 ? 0.8%, n ? 7,
0.6% relative to dantrolene/FK506 baseline and 19.2 ? 0.6% rel-
ative to aCSF baseline; Fig. 2H), although the magnitude of LTD
mice with FK506 before and after treatment with 10 ?M dantrolene; and F, from 3xTg-AD mice with FK506 before and after
on right show averaged percentage change in post-LFS baseline relative to pre-LFS baseline from NonTg and 3xTg-AD mice in
trolene (3) from NonTg (black) and 3xTg-AD (gray) mice. Bar graphs on right show averaged percentage change in post-LFS
pretreated for 2 h with drug before recording. Baseline fEPSPs were recorded at 0.05 Hz for 20 min before and 60 min after
8344 • J.Neurosci.,June13,2012 • 32(24):8341–8353Chakrobortyetal.•SynapticDepressioninADMice
was less than during RyR blockade alone
(t(1,11)? ?31.83; p ? 0.05). Thus, it ap-
pears that a signaling pathway for LTD
can be recruited in 3xTg-AD mice that
ity. We therefore examined alternative
signaling cascades, such as the following
mGluR-mediated mechanism for LTD.
Because there is some functional overlap
we examined whether aberrant interac-
tions exist between these ER calcium
with 5 ?M XeB had little effect on I/O or
PPF functions (p ? 0.05; Fig. 3A,D) in
NonTg (n ? 16, 19, respectively) or
3xTg-AD (n ? 13, 17, respectively) mice,
NonTg and 3xTg-AD responses (p ?
0.05; data not shown). Administering the
LFS while blocking IP3R resulted in a po-
larity shift from LTD to long-term poten-
tiation (LTP) in both NonTg (n ? 6,
134.19 ? 2.6% over baseline) and 3xTg-
AD (n ? 9, 100.4 ? 3.4% over baseline)
mice (Fig. 3G), although the degree of
LTP is significantly less in 3xTg-AD mice
(t(1,14)? 7.8; p ? 0.05). This is consistent
with previous observations demonstrat-
ing that IP3R-mediated calcium release
suppresses LTP (Fujii et al., 2000; Taufiq
et al., 2005).
When blocking both IP3R and RyR,
I/O (n ? 10; p ? 0.05; Fig. 3B) and PPF
(n ? 13; p ? 0.05; Fig. 3E) functions were
not altered in NonTg mice. However, in
3xTg-AD mice, I/O (n ? 8, t(1,7)? ?6.0;
from the following: D, NonTg and 3xTg-AD mice with 5 ?M XeB; E, NonTg mice with XeB before and after treatment with
NonTg and 3xTg-AD mice in XeB. H, Left, Graph shows averaged time course of LTD in XeB with dantrolene from NonTg and
in post-LFS baseline relative to pre-LFS baseline from NonTg and 3xTg-AD mice in XeB with dantrolene. I, Left, Graph shows
averaged time course of LTD (chemical induction, mGluR-dependent) in 100 ?M DHPG from NonTg and 3xTg-AD mice; insets
Blunted IP3R-dependent LTD expression in 3xTg-AD mice. A–C, I/O function shows changes in fEPSP slope with
on right show averaged percentage change in post-DHPG
baseline relative to pre-DHPG baseline from NonTg and
3xTg-AD mice. J, Left, Graph shows averaged time course of
Insets (above) show representative fEPSP baseline traces in
percentage change in post-DHPG baseline relative to pre-
The arrow indicates the time of LFS. For DHPG experiments,
of APV to block NMDAR-dependent LTD. *p ? 0.05, signifi-
cantly different from 3xTg-AD before dantrolene treatment;
Chakrobortyetal.•SynapticDepressioninADMice J.Neurosci.,June13,2012 • 32(24):8341–8353 • 8345
to effects observed with RyR block alone.
Blocking RyR and IP3R while administer-
plasticity expression (Fig. 3H). In NonTg
mice, baseline responses were not altered
(n ? 8; p ? 0.05) and a polarity shift to
LTP was still observed (69.7 ? 1.8% over
aCSF baseline and 67.1 ? 1.8% over dan-
trolene/XeB baseline). In contrast, in
3xTg-AD mice, blocking both channels
greatly increased baseline responses
(98.0 ? 2.0%, t(1,8)? ?39.1; p ? 0.05)
and blocked LTP relative to drug baseline
(p ? 0.05). Because evoked baseline re-
sponses are increased in the presence of
dantrolene, likely because of the removal
of SK2 channel activity (Chakroborty et
of the postsynaptic response.
To identify RyR-independent mech-
anisms that could account for the
enhanced LTD observed during RyR
blockade in 3xTg-AD mice, we next ex-
amined mGluR-dependent LTD, which invokes Gqactivation
the mGlu receptors with 100 ?M DHPG while simultaneously
(Palmer et al., 1997; Huber et al., 2001). These conditions gener-
ated a robust mGluR–LTD in both NonTg (n ? 5, 62.6 ? 0.6%
LTD magnitude being modestly but significantly lower in
3xTg-AD mice (t(1,9)? ?20.0; p ? 0.05; Fig. 3I). The possible
role of RyR in mGluR–LTD has not been examined previously,
and it is feasible that the increased CICR in the 3xTg-AD mice
may result in aberrant recruitment of RyR-sensitive calcium
stores in mGluR–LTD. To test this hypothesis, we stimulated the
mGluR while simultaneously inhibiting NMDAR and RyR (with
APV and dantrolene, respectively). Interestingly, this treatment
5, 72.2 ? 2.0% over baseline) and 3xTg-AD (n ? 6, 21.9 ? 0.9%
over dantrolene baseline) mice, although LTP was still relatively
blunted in the 3xTg-AD mice compared with NonTg (t(1,10)?
22.93; p ? 0.05; Fig. 3J). These intriguing findings suggest that
RyR-sensitive calcium stores make an active contribution to
mGluR–LTD and serve to suppress synaptic potentiation. This
observation has not been reported previously and appears anal-
ogous to the phenomenon of IP3R activation suppressing synap-
tic potentiation (Fujii et al., 2000; Taufiq et al., 2005). The
polarity shift to LTP during RyR blockade in mGluR–LTD is
similar in the NonTg and 3xTg-AD mice, albeit the magnitude
continues to be lower in the AD mice, suggesting that separate
signaling pathways are not recruited in the mouse strains. How-
response to the upregulation of RyR function. The blunted LTD
and LTP, induced by DHPG and DHPG ? dantrolene, respec-
This hypothesis is based on studies in 3xTg-AD neurons and
mutant PS-expressing cells demonstrating altered IP3R gating
properties, resulting in increased Poand reduced threshold for
IP3-evoked responses, such that endogenous IP3levels can be
sufficient to trigger calcium release at the single-channel level.
This ER calcium leak constitutively upregulates kinases and sig-
(Cheung et al., 2008, 2010; Mu ¨ller et al., 2011). Because these
kinase activity in their resting state, subsequent IP3-mediated
signaling cascades will not generate the same magnitude of plas-
ticity. Similar plasticity occlusion effects have been observed in
neuronal circuits in which mGluR-mediated signaling cascades
are upregulated (McCutcheon et al., 2011).
from NonTg and 3xTg-AD neurons with 10 ?M dantrolene. *p ? 0.05, significantly different from 3xTg-AD before dantrolene
8346 • J.Neurosci.,June13,2012 • 32(24):8341–8353Chakrobortyetal.•SynapticDepressioninADMice
RyR-mediated CICR can facilitate spontaneous neurotransmit-
ter vesicle release from presynaptic terminals and alter their re-
lease probability and PPF functions (Emptage et al., 1999; Llano
et al., 2000; Bardo et al., 2002). Spontaneous neurotransmission
in the maturation and stabilization of synaptic connections
(McKinney et al., 1999; Verhage et al., 2000), the modulation of
protein synthesis in dendrites (Sutton et al., 2004), and driving
mitter within the synaptic cleft and thereby shape postsynaptic
responses (Wasser and Kavalali, 2009). Here, we examined
whether altered ER calcium release affects spontaneous vesicle
release properties and calcium-dependent short-term plasticity
in CA3 terminals. Using whole-cell patch-clamp recordings, we
control aCSF and dantrolene from NonTg (n ? 13; Fig. 4A) and
3xTg-AD (n ? 12; Fig. 4B) CA1 pyramidal neurons.
In control aCSF, the frequency distribution of spontaneous
EPSPs in 3xTg-AD neurons was significantly different than
NonTg neurons, indicative of a higher release frequency (K–S
test; p ? 0.05 for 3xTg-AD/aCSF vs
NonTg/aCSF; Fig. 4C). RyR block nor-
malized the frequency of spontaneous
events in 3xTg-AD neurons (p ? 0.05 for
3xTg-AD/aCSF vs 3xTg-AD/dantrolene),
with little effect in NonTg neurons (p ?
common feature in 3xTg-AD and other
Salminen et al., 2009; Quiroz-Baez et al.,
2011; Stutzmann and Mattson, 2011), in-
creases the sensitivity of presynaptic cal-
neurotransmitter release (Nosyreva and
Kavalali, 2010). In addition, although
evoked and spontaneous vesicle release
involves calcium sensors, there can be
distinct mechanisms and binding affin-
ities supporting each. For example, the
synaptotagmin-1 calcium-binding do-
release are different from the binding
domains required for evoked release
(Xu et al., 2009), whereas the doc2b and
not recruited for evoked vesicle release
(Maximov et al., 2007; Groffen et al.,
appears to more stringently regulate
evoked release over spontaneous release
in a calcium-dependent manner (Dea ´k et
al., 2006). Any of these mechanisms may
cium sensitivity of spontaneous release
machinery coupled with the aberrant
CICR and ER stress in 3xTg-AD neurons
spontaneous events and render neurons
and Regehr, 2002; Cavelier and Attwell, 2005; Wasser and Kava-
In our studies, PPF measured across a range of interstimulus
intervals (25–1000 ms) suggests there are mechanisms compen-
sating for the increased probability of vesicle release (Fig. 5). In
aCSF, PPF trended lower in 3xTg-AD neurons when compared
with NonTg neurons but does not achieve statistical significance
(p ? 0.05 for all interstimulus intervals; n ? 10 for both). How-
ever, blocking the RyR increases PPF in 3xTg-AD neurons rela-
tive to NonTg, consistent with a reduction in release probability,
particularly at longer interstimulus intervals (p ? 0.05 for 50,
200, and 1000 ms). The amplitude of spontaneous EPSCs was
also significantly increased in 3xTg-AD neurons on RyR block
relative to 3xTg-AD responses in aCSF and NonTg responses in
aCSF and dantrolene (K–S test; p ? 0.05; Fig. 4D). This may
reflect, in part, increased membrane input resistance when RyRs
are blocked in 3xTg-AD neurons (see below), as well as compen-
synaptic vesicles. Because the number of vesicles in the readily
releasable pool is limited, the availability of synaptic vesicles for
exocytosis becomes the limiting step during action potential fir-
SK2-L (right) from the hippocampus of NonTg and 3xTg-AD mice. Density levels are relative to ?-tubulin levels. *p ? 0.05,
Chakrobortyetal.•SynapticDepressioninADMice J.Neurosci.,June13,2012 • 32(24):8341–8353 • 8347
ing, which can then lead to synaptic de-
pression if vesicles are rapidly depleted
(Schneggenburger et al., 2002; Fioravante
and Regehr, 2011). This depletion would
likely be accelerated in the 3xTg-AD neu-
rons because of the increased RyR-
calcium tone and increased spontaneous
The SK channels underlie the mAHP and
modulate neuronal excitability by limit-
al., 1999; Bond et al., 1999; Sah and Da-
vies, 2000). RyR-evoked calcium release
can contribute to the activation of the
mAHP (Sah, 1996; Pineda et al., 1999;
Shah and Haylett, 2000; Stutzmann et al.,
2003; van de Vrede et al., 2007), and pre-
vious studies suggest an enhanced cou-
pling efficiency between the SK channels
and RyR in AD mice (Stutzmann et al.,
2006). Thus, we examined whether dys-
regulated RyR calcium signaling altered
postsynaptic SK channel function in
3xTg-AD CA1 neurons. Under basal con-
ditions, 3xTg-AD neurons (n ? 15) had a
significantly greater mAHP current am-
plitude than NonTg neurons (n ? 12) in
response to a single depolarizing voltage
step (t(1,26)? ?3.5; p ? 0.05; Fig. 6A), as
(t(1,26)? ?2.3; p ? 0.05; Fig. 6B). RyR
block normalized the mAHP amplitude
in 3xTg-AD neurons to NonTg levels
(F(3,26)? 6.6; p ? 0.05, Scheffe ´’s post hoc
analysis, p ? 0.05 for 3xTg-AD/aCSF vs
3xTg-AD/dantrolene, NonTg/aCSF, and
NonTg/dantrolene). Apamin reduced the
mAHP current in both NonTg (t(1,11)? 6.1; p ? 0.05) and
3xTg-AD (t(1,14)? 6.4; p ? 0.05) neurons, confirming involve-
icant differences in hippocampal SK2 channel expression levels,
0.05; Fig. 6D).
To measure the impact of calcium signaling disruptions on
membrane properties, we next measured peak and steady-state
voltage responses to a range of injected current steps. RyR block
0.05; Fig. 7C). This increased voltage response in 3xTg-AD neu-
rons could reflect the increase in membrane resistance during
ences in voltage-gated calcium channel activity have been ob-
served in presymptomatic 3xTg-AD mice in previous studies
(Stutzmann et al., 2004, 2006; Goussakov et al., 2010, 2011).
Steady-state voltage responses were not affected by RyR block in
either NonTg (t(1,14)? 1.4; p ? 0.05) or 3xTg-AD (t(1,13)? 3.2;
p ? 0.05) neurons (Fig. 7D). Measured at resting membrane
potential (?65 mV), membrane input resistance was also signif-
AD is increasingly referred to as a “synaptic disease” because
of the central role synaptic breakdown has in the devastating
cognitive deficits (Selkoe, 2002; Coleman and Yao, 2003;
Chong et al., 2011). Here we propose that aberrant ER calcium
signaling plays an early role in AD synaptic dysfunction, be-
fore the onset of histopathology or cognitive decline. Our pre-
from NonTg and 3xTg-AD CA1 pyramidal neurons. C, Graph showing current–voltage relationship at peak response between
NonTg and 3xTg-AD neurons before and after treatment with 10 ?M dantrolene (Dant). D, Graph showing current–voltage
8348 • J.Neurosci.,June13,2012 • 32(24):8341–8353Chakrobortyetal.•SynapticDepressioninADMice
vious studies in presymptomatic 3xTg-AD mice demonstrate
markedly reduced CICR threshold in synaptic compartments,
evoke greatly elevated ER calcium responses within presynap-
tic and postsynaptic compartments in which ER calcium re-
lease is typically negligible to small (Chakroborty et al., 2009;
Goussakov et al., 2010, 2011). Despite this deviant calcium
release under basal conditions, synaptic transmission appears
normal. In this study, we uncover markedly altered synaptic
blunted or depressed hippocampal network. These early defi-
cits may drive the synaptic breakdown associated with AD-
linked cognitive impairment.
To explore mechanisms accounting for synaptic depression
in 3xTg-AD mice, we examined two prominent LTD signal-
ing pathways: NMDAR–LTD and mGluR–LTD (Table 3).
NMDAR–LTD is generated by LFS of the NMDAR and is me-
diated by calcineurin and RyR-evoked calcium release
(Mulkey et al., 1994; Reyes and Stanton, 1996). mGluR–LTD
involves mGluR1/5 activation, which generates IP3and subse-
quent ER calcium release (Oliet et al., 1996). Although block-
ing calcineurin (also called protein phosphatase 2B) abolished
LTD in both NonTg and 3xTg-AD mice, blocking both cal-
cineurin and RyR revealed a separate LTD induction mecha-
nism in 3xTg-AD mice that is independent of both these
signaling factors. These may reflect a reduction in presynaptic
vesicle stores and increased calcium-activated K?currents,
each of which are facilitated by sensitized CICR in 3xTg-AD
mice. IP3R contributions to NMDAR- dependent LTD were
also compared between NonTg and 3xTg-AD mice. Blocking
IP3R resulted in a shift to LTP in both mouse strains, consis-
tent with previous reports (Fujii et al., 2000; Nishiyama et al.,
2000), yet the magnitude of plasticity was blunted in 3xTg-AD
mice, perhaps as a result of constitutive upregulation of
calcium-regulated kinases (see Results; Mu ¨ller et al., 2011).
Blocking both RyR and IP3R abolished LTP in 3xTg-AD mice
relative to the dantrolene baseline, yet plasticity is maintained
in the control mice. This may reflect an additional “ceiling
in 3xTg-AD mice by suppressing inhibitory SK2 channels
(Chakroborty et al., 2009). These findings demonstrate a reca-
libration toward synaptic depression when RyRs are blocked
in 3xTg-AD mice.
LTD can reflect both presynaptic and postsynaptic changes in
synaptic efficacy, with the former reflecting a reduction in the
probability of calcium-dependent neurotransmitter release
(Stanton et al., 2003; Enoki et al., 2009). Because RyR can
trigger spontaneous vesicle release (Emptage et al., 1999;
Llano et al., 2000; Bardo et al., 2002), the sensitized CICR
responses in 3xTg-AD neurons can diminish vesicle reserves
and impose long-term consequences for synaptic transmis-
sion. Under “normal” conditions, CICR mobilizes vesicles
from the reserve pool to the readily releasable pool, thereby
maintaining a responsive vesicle population primed for rapid
release (Kuromi and Kidokoro, 2002; Zucker and Regehr,
2002); however, upregulated CICR can accelerate vesicle pool
release of neurotransmitter vesicles. During high-frequency activity, CICR can be evoked by voltage-gated calcium influx to increase the residual calcium levels and release probability. In AD,
Chakrobortyetal.•SynapticDepressioninADMiceJ.Neurosci.,June13,2012 • 32(24):8341–8353 • 8349
normalized initially by increasing neurotransmitter synthesis
and vesicle cycling, sustaining these compensatory mecha-
nisms can introduce metabolic stress, promoting synapse loss
(Verkhratsky, 2002; Stutzmann, 2007; Mattson, 2010). Re-
duced vesicle reserves and cycling capacity may also underlie
the homeostatic shift to LTD in 3xTg-AD mice. The effects of
increased RyR calcium on facilitating vesicle release are sus-
tained over longer intervals in 3xTg-AD mice. Residual cal-
cium levels continue to support PPF with interstimulus
intervals up to 1 s, suggesting that calcium release can expend
vesicle pool contents and drive a network into a state of syn-
aptic depression. Conversely, blocking RyR increased PPF in
3xTg-AD mice, consistent with a reduction in presynaptic cal-
cium levels and lowered vesicle release probability.
The calcium-activated SK2 channels underlie the mAHP in
brane excitability, and plasticity (Sah, 1996; Stocker et al.,
1999; Sah and Davies, 2000). Suppressing SK2 channel func-
tion facilitates memory, whereas upregulating SK2 function
impairs LTP and hippocampal-dependent learning and mem-
ory (Fournier et al., 2001; Stackman et al., 2002; Hammond et
al., 2006; Allen et al., 2011). Here we demonstrate that, under
basal conditions, upregulated RyR function underlies in-
creased SK2 currents in 3xTg-AD neurons, which is distinct
from the activity-dependent trigger through L-type voltage-
gated calcium channels. The broader implications of this phe-
nomenon are uncovered when RyR are blocked, resulting in
increased I/O functions. The recruitment of SK2 channels via
RyR-mediated calcium release is further supported by apamin
(an SK2-specific channel blocker), increasing the I/O function
similarly to dantrolene in 3xTg-AD mice but having no effect
in NonTg mice (Chakroborty et al., 2009). Although the aber-
rant RyR calcium release can initially serve to offset postsyn-
aptic excitation resulting from increased neurotransmitter
release, this same phenomenon can shift homeostatic set
points for membrane excitability and spiking thresholds
(Bear, 1995; Sah and Bekkers, 1996; Hirschberg et al., 1999).
The increased SK2 currents are not attributable to increased
SK2 expression; rather, because SK2 channels are expressed in
CA1 dendritic spines (Allen et al., 2011), it is likely that the
deviant RyR-evoked calcium is more tightly coupled to SK2
channels as a result of physical proximity.
An additional process to consider is that of homeostatic
synaptic scaling, which is a calcium-dependent compensatory
form of plasticity that maintains neuronal excitability within
optimal ranges. Neurons adapt to sustained changes in net-
work activity by adjusting their synaptic strengths to stabilize
firing rates and neural networks coordinating complex func-
tions, such as memory encoding. Pratt et al. (2011) uncovered
that mutant PS-expressing neurons had impaired synaptic
scaling responses and concluded that PS1 is required for scal-
ing up excitatory synaptic strengths in response to a suppres-
sion of network activity but is not needed for scaling down in
response to enhanced network activity or setting mEPSC am-
plitude. As this relates to our findings, mutant PS may impair
the ability of the hippocampal network to increase responsiv-
ity and increased RyR function may be an attempt to compen-
sate for this deficit; therefore, when RyRs are blocked, the
network reverts to a depressed synaptic state. Although this
idea will require additional refinement, it is consistent with
our overall hypothesis that deficits in synaptic homeostasis
may contribute to brain dysfunction in AD.
exhibit RyR-mediated calcium deficits early in AD, before
?-amyloid and tau deposition and cognitive impairments. This
alters synaptic transmission properties in a way that is initially
difficult to detect because of the recruitment of signaling mech-
anisms that maintain the appearance of “normalcy” (Fig. 8).
These mechanisms may initially stabilize synaptic activity, but
prolonged metabolic demands and aberrant neuronal activity
may ultimately stress neurotransmission and plasticity func-
tions. For example, mutant PS upregulates RyR expression,
which in turn increases calcium-dependent presynaptic neu-
rotransmitter release as well as postsynaptic SK channel activ-
ity. Alternatively, preexisting synaptic deficits may generate
depressed synaptic networks, and increased RyR expression
serves as the compensatory mechanism to “stabilize” the net-
work. As an example, RyR3 expression is upregulated in late-
stage AD pathology, possibly as a neuroprotective mechanism
against A?1–42deposition (Supnet et al., 2010). Similar alter-
ations in RyR expression are also observed in human brain
studies. Patients with mild cognitive impairment demonstrate
elevated levels of RyR2 linked with cognitive decline and syn-
aptic loss (Kelliher et al., 1999; Bruno et al., 2011), consistent
with early increases in RyR2 expression in several AD mouse
models. However, regardless of fundamental cause, the early
dyshomeostasis in synaptic transmission and plasticity mech-
anisms likely contribute to the cognitive deficits in later AD
stages. For example, disruptions in LTD introduce destabiliz-
ing effects on synaptic networks. This is because LTD opti-
mizes the flexibility and efficiency of synapses by facilitating
the acquisition of new associations while rapidly suppressing
old or irrelevant ones (Dayan and Willshaw, 1991; Kemp and
Manahan-Vaughan, 2004). This has direct implications for
episodic memory, which is greatly affected in AD patients and
results from deficiencies in encoding and storing new infor-
mation. In AD patients, this is manifested as a reduced capac-
ity to learn new material and retain memories of recent events
(Walker et al., 2007), and as discussed previously, this aspect
of late-stage memory loss is highly correlated with earlier syn-
aptic pathology (Selkoe, 2002; Coleman and Yao, 2003; Chong
et al., 2011). Thus, although targeting late-stage AD features is
an attractive goal, the increasing body of evidence demon-
strating the contribution of early pathogenic mechanisms to
late-stage AD symptoms warrants the consideration of new
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