Presynaptic Ca2?Entry Is Unchanged during Hippocampal Mossy
Fiber Long-Term Potentiation
Haruyuki Kamiya,1,2Kazumasa Umeda,1,3Seiji Ozawa,2,4and Toshiya Manabe1,5
1Division of Cell Biology and Neurophysiology, Department of Neuroscience, Faculty of Medicine, Kobe University, Kobe,
Hyogo 650-0017, Japan,2Department of Physiology, Gunma University School of Medicine, Maebashi, Gunma
371-8511, Japan,3Department of Applied Biology, Kyoto Institute of Technology, Kyoto 606-8585, Japan,4Core
Research for Evolutional Science and Technology, Japan Science and Technology Corporation, Kawaguchi, Saitama
332-0012, Japan, and5Division of Neuronal Network, Department of Basic Medical Sciences, Institute of Medical
Science, University of Tokyo, Tokyo 108-8639, Japan
The hippocampal mossy fiber (MF)–CA3 synapse exhibits
NMDA receptor-independent long-term potentiation (LTP),
which is expressed by presynaptic mechanisms leading to
persistent enhancement of transmitter release. Recent studies
have identified several molecules that may play an important
role in MF-LTP. These include Rab3A, RIM1?, kainate autore-
ceptor, and hyperpolarization-activated cation channel (Ih).
However, the precise cellular expression mechanism remains to
be determined because some studies noticed essential roles of
release machinery molecules, whereas others suggested mod-
ulation of the ionotropic processes affecting Ca2?entry into the
presynaptic terminals. Using fluorescence recordings of pre-
synaptic Ca2?in hippocampal slices, here we demonstrated
that MF-LTP is not accompanied by an increase in presynaptic
Ca2?influx during an action potential. Whole-cell recordings
from CA3 neurons revealed long-lasting increases in mean
frequency, but not mean amplitude, of miniature EPSCs after
the high-frequency stimulation of MFs. These data indicate that
the presynaptic expression mechanisms responsible for en-
hanced transmitter release during MF-LTP involve persistent
modification of presynaptic molecular targets residing down-
stream of Ca2?entry.
Key words: cAMP; hippocampus; long-term potentiation;
mossy fiber; presynaptic Ca2?influx; transmitter release
The hippocampal mossy fiber (MF) synapse provides major ex-
citatory input onto CA3 pyramidal neurons and exhibits robust
short- and long-term presynaptic plasticity (Zalutsky and Nicoll,
1990; Weisskopf and Nicoll, 1995; Kobayashi et al., 1996; Henze
et al., 2000) independent of activation of NMDA receptors
(Nicoll and Malenka, 1995). Because long-term potentiation
(LTP) at this synapse can be induced without postsynaptic acti-
vation (Castillo et al., 1994; Mellor and Nicoll, 2001) (but see
Yeckel et al., 1999) and is blocked by the inhibitors of protein
kinase A (PKA), it has been proposed that a rise in cAMP
concentration within the presynaptic terminals and subsequent
activation of PKA are essential for induction of MF-LTP (Weiss-
kopf and Nicoll, 1994). Recently, several studies have revealed the
molecular targets of the cAMP signaling pathway. The studies of
mice lacking Rab3A (Castillo et al., 1997) and RIM1? (Castillo
et al., 2002) suggested essential roles of these two proteins in
MF-LTP. Because RIM1? is an active zone protein that interacts
with the synaptic vesicle protein Rab3A, changes in vesicular
mobilization rather than modification of ion entry processes
would be expected. However, this notion was challenged by the
recent report by Mellor et al. (2002) showing that blockers of the
hyperpolarization-activated cation channel (Ih), whose activity is
modulated directly by cAMP, reversed already established MF-
LTP. The authors proposed a hypothesis that cAMP-dependent
modification of Ihresults in long-lasting depolarization of MF
terminals. Depolarization of the terminals would enhance trans-
mitter release by either increasing Ca2?entry via broadening of
action potentials (Geiger and Jonas, 2000) or activating Ca2?
channels to elevate intraterminal basal Ca2?levels (Turecek and
Trussell, 2001). It must be noted that Chevaleyre and Castillo
(2002) put through the new paper reporting strong evidence
against involvement of Ihin the expression of MF-LTP (see
Discussion). Other lines of evidence suggesting involvement of
presynaptic kainate receptors in MF-LTP (Contractor et al.,
2001; Lauri et al., 2001) (but see Nicoll et al., 2000) also imply
modulation of the presynaptic Ca2?dynamics, because kainate
autoreceptors at this particular synapse was demonstrated to
operate by an ionotropic mechanism (Kamiya and Ozawa, 2000;
Schmitz et al., 2001).
Two possibilities suggested by these studies (i.e., modification
of downstream steps involving vesicular mobilization as suggested
by the studies of Rab3A and RIM1? knockout mice, or modula-
tion of presynaptic Ca2?dynamics as suggested by involvement
of Ihor kainate autoreceptors) are apparently contradictory, and
it has been difficult to propose the unifying model at this time. To
directly determine whether Ca2?entry is modified, we adopted
fluorescence measurement of presynaptic Ca2?at MF terminals
(Kamiya and Ozawa, 1999) and found that action potential-
driven presynaptic Ca2?influx is unchanged during the expres-
Received Aug. 16, 2002; revised Sept. 17, 2002; accepted Oct. 7, 2002.
This work was supported by Grants-in-Aid for Science Research (H.K., S.O., and
T.M.), by Special Coordination Funds for Promoting Science and Technology
(T.M.) from the Ministry of Education, Science, Sports, Culture and Technology of
Japan, and by grants from the Ichiro Kanehara Foundation and the Novartis
Foundation (Japan) for the Promotion of Science (T.M.). We thank Prof. Atsu Aiba
for reading this manuscript.
Correspondence should be addressed to Haruyuki Kamiya, Division of Cell
Biology and Neurophysiology, Department of Neuroscience, Faculty of Medicine,
Kobe University, Kobe, Hyogo 650-0017, Japan. E-mail: firstname.lastname@example.org.
Copyright © 2002 Society for Neuroscience 0270-6474/02/2210524-05$15.00/0
The Journal of Neuroscience, December 15, 2002, 22(24):10524–10528
sion of MF-LTP. Our results strongly support the hypothesis that
the presynaptic expression mechanism responsible for MF-LTP is
persistent modification of the release machinery downstream of
MATERIALS AND METHODS
Simultaneous recordings of field EPSPs and presynaptic Ca2?. Transverse
hippocampal slices (?400 ?m thick) were prepared from BALB/c mice
(14–20 d of age). All experiments were performed according to the
guidelines established by the Animal Care and Experimentation Com-
mittees of Gunma University and Kobe University. Slices were contin-
uously superfused with a solution composed of the following (in mM):
127 NaCl, 1.5 KCl, 1.2 KH2PO4, 2.4 CaCl2, 1.3 MgSO4, 26 NaHCO3, and
10 glucose. The solution was equilibrated with 95% O2and 5% CO2.
Electrical stimuli (100 ?sec duration, ?500 ?A intensity) were delivered
through a tungsten concentric bipolar electrode inserted into the stratum
granulosum of the dentate gyrus, and the resultant field EPSPs were
recorded from the stratum lucidum in the CA3 region with glass micro-
electrodes of ?10 ?m tip diameter filled with the standard extracellular
solution (Kamiya et al., 1996). All recordings were made at room tem-
Fluorescence recordings of presynaptic Ca2?within the mossy fiber
terminals were made as described previously (Kamiya and Ozawa, 1999).
Briefly, rhod-2 AM (Dojindo Laboratory, Kumamoto, Japan), a
membrane-permeable Ca2?indicator, was loaded into the MF terminals
without severing the axons. The dye was injected locally into the stratum
lucidum, resulting in selective labeling of the mossy fibers. The fluores-
cence (excitation at 510–560 nm and monitoring above 580 nm) from the
area (?100 ?m diameter) containing the labeled terminals was measured
with a single photodiode (S2281–01; Hamamatsu Photonics, Hamamatsu,
Japan), while the field EPSPs were monitored simultaneously from the
area. The ?F/F value evoked by a single electrical stimulus was used as
a measure of [Ca2?]iincrease during an action potential. Subtraction of
the background fluorescence was not performed, because the fluores-
cence of unlabeled region of the slices at this wavelength was almost
negligible. No attempt was made to relate the ?F/F value to peak [Ca2?]i,
because our methods detect the fluorescence signals from the terminals
as well as the axons, and we could not estimate the relative contribution
to the total signals. The output of the photodiode was I–V converted,
amplified, and filtered at 500 Hz with an eight-pole Bessel filter (FLA-1;
Cygnus Technology, Delaware Water Gap, PA). The signal was then
digitized with a 12 bit analog-to-digital converter (Digidata 1200A; Axon
instruments, Foster City, CA) and acquired at 10 kHz using pClamp8
software (Axon Instruments). Data in the text and figures are expressed
as mean ? SEM (the number of experiments). Statistical analysis was
performed using the paired t test unless otherwise noted, and p ? 0.05
was accepted for statistical significance.
Measurement of fiber volley. The presynaptic fiber volley (FV) was
recorded in the presence of 10 ?M CNQX to avoid contamination of the
field EPSPs. The amplitude of FV was measured as a difference between
the initial positive and the following negative peaks. Field potential was
filtered at 2 kHz and digitized at 20–40 kHz for FV measurement. To
confirm that the responses surely reflect FV, 1 ?M TTX was applied at
the end of all experiments (see Figs. 1C, 2C).
Measurement of miniature EPSCs. Whole-cell recordings were made
from CA3 pyramidal neurons, and miniature EPSCs (mEPSCs) were
recorded at ?70 mV in the presence of 0.5 ?M tetrodotoxin and 100 ?M
picrotoxin (Kamiya and Ozawa, 1999). Patch pipettes were filled with an
internal solution (pH 7.2) containing the following (in mM): 150 Cs
gluconate, 0.2 EGTA, 8 NaCl, 10 HEPES, 2 Mg2?ATP, and 5 QX-314
(lidocaine N-ethyl bromide quaternary salt). The membrane currents
were filtered at 1 kHz and collected for 60 sec in each data point. The
mEPSCs (6 pA amplitude threshold) was analyzed off-line using Mini
Analysis Program 5.1 (Synaptosoft, Decatur, GA). No attempt was made
to group the events by the rise time. The Kolmogorov–Smirnov test was
used to assess the effects on amplitude and interevent interval.
Presynaptic Ca2?entry is unchanged by
First, we examined whether stimulus-evoked presynaptic Ca2?
influx is modified during expression of MF-LTP. High-frequency
stimulation of MF (100 Hz for 1 sec) elicited sustained potenti-
ation of field EPSP amplitudes (195 ? 14% of control at 20 min
after tetanus; n ? 8) (Fig. 1A). In contrast, fluorescent signals
(FCa) recorded simultaneously did not change significantly (94 ?
2% of control) (Fig. 1B). Background fluorescence (F), reflecting
resting Ca2?level, was also not affected (97 ? 3% of control).
The lack of effect on FCamight not be attributable to saturation
of indicator, because raising external Ca2?to 3 mM (125% of 2.4
mM standard solution) enhanced FCasubstantially (119 ? 2% of
control; n ? 5). In separate experiments, we monitored presyn-
aptic FV in the presence of the AMPA receptor antagonist
CNQX (10 ?M), because a previous study demonstrated that
tetanic stimulation produces long-lasting change in presynaptic
excitability by assessing latency of FV (Mellor et al., 2002).
Changes in the FV amplitude are rather small (105 ? 1% of
control) at 20 min after tetanus (n ? 6) (Fig. 1C). These results
suggest that MF-LTP is not accompanied by a change in presyn-
aptic Ca2?dynamics within each terminal but is instead attrib-
utable to enhanced efficacy of exocytotic machinery downstream
from Ca2?influx. Then, we examined the mechanism responsible
for forskolin (FSK)-induced potentiation at this synapse, which
has been proposed to share common expression mechanisms with
tetanus-induced LTP (Weisskopf et al., 1994). Application of
FSK (50 ?M for 20 min) resulted in a gradual increase in field
EPSP amplitudes (480 ? 42% of the control value; n ? 6) (Fig.
2A). Simultaneously recorded FCawas increased by FSK (121 ?
in presynaptic Ca2?entry. A, Amplitudes of field EPSP ( fEPSP) were
plotted against time. Tetanic stimulation (Tet; 100 Hz, 1 sec) was applied
at the time shown by the arrow. Representative traces are those recorded
before (control, thin trace) and 20 min after (LTP, thick trace) tetanic
stimulation. B, Presynaptic Ca2?transients recorded simultaneously
(FCa) were unchanged, whereas clear LTP was observed as in A. Appli-
cation of CNQX (10 ?M) did not decrease FCa, confirming that fluores-
cence signals were originated exclusively from presynaptic structures. C,
Time course of the presynaptic FV amplitude recorded in the presence of
10 ?M CNQX. The FV amplitude was decreased soon after the tetanus,
although it recovered afterward.
LTP at the MF–CA3 synapse is not accompanied by a change
Kamiya et al. • Ca2?-Independent Presynaptic Expression of MF-LTPJ. Neurosci., December 15, 2002, 22(24):10524–10528 10525
4% of control) (Fig. 2B), whereas F value was not affected
significantly (96 ? 2% of control). FV recorded in the presence of
CNQX increased in amplitude by almost the same degree as FCa
(115 ? 3% of control; n ? 7; p ? 0.24) (Fig. 2C). Application of
50 ?M 1,9-dideoxyforskolin, an inactive analog of FSK, affected
neither field EPSP amplitude (103 ? 7% of control) nor FCa
(94 ? 5% of control; n ? 3). These results suggest that the
FSK-induced potentiation is accompanied by the increase in the
number of firing axons that explains the increase in FCaduring
application of FSK but is unlikely attributable to an increase in
action potential-driven Ca2?influx into the individual presynap-
Enhancement of frequency, but not amplitude, of
miniature EPSCs during MF-LTP
To investigate changes in transmitter releasing machinery more
directly, we also examined the effect of tetanic stimulation of MF
on mEPSCs recorded from CA3 neurons (Jonas et al., 1993;
Kamiya and Ozawa, 1999). Some events displayed relatively slow
decay time course (Fig. 3A1, A2), possibly reflecting that kainate
as well as AMPA receptors partly contribute to mEPSCs in CA3
neurons (Cossart et al., 2002). After recording control data in the
presence of TTX, the TTX was removed from the perfusing
solution for 10 min, and then a high-frequency stimulation (100
Hz for 1 sec) was given (Fig. 3B). Substantial evoked MF re-
sponses were replenished at this time (data not shown). Soon
after the tetanus, TTX was added again, and 1 min records were
taken every 5 min thereafter. The increase in mEPSC frequency
was noted even 30 min after the tetanic stimulation (Fig. 3A1)
without significant changes in the distribution of the amplitudes
(Fig. 3A3). The cumulative amplitude histogram was not signifi-
cantly affected, whereas the cumulative plot of interevent intervals
showed a significant difference ( p ? 0.05; Kolmogorov–Smirnov
test) (Fig. 3A4). On average, mean frequency of mEPSCs increased
to 212 ? 50% of the control value, whereas mean amplitude was
little affected (104 ? 9% of control; n ? 14) (Fig. 3B).
The frequency of mEPSCs was also increased during the ap-
plication of 50 ?M FSK (Fig. 3C1) without significant changes in
the amplitude distribution (Fig. 3C2). The cumulative plot of
interevent intervals, but not of amplitude, was affected by FSK
( p ? 0.05; Kolmogorov–Smirnov test) (Fig. 3C3). On average,
mean frequency of mEPSCs increased to 211 ? 25% of the
control value, whereas mean amplitude was little affected (97 ?
5% of control; n ? 11) (Fig. 3D). Application of 50 ?M 1,9-
dideoxyforskolin affected neither mean frequency (104 ? 9% of
control) nor mean amplitude (101 ? 7% of control; n ? 6) of
mEPSCs. Because sustained elevation of basal Ca2?level within
the MF terminals was not accompanied by LTP expression (Re-
gehr and Tank, 1991) and we also did not observe significant
change in F value in this study, the enhancement of frequency of
mEPSCs strongly supports the notion that LTP and FSK-induced
potentiation was accompanied by enhancement of the release
machinery downstream from Ca2?entry. Taking all results to-
gether, we conclude that the expression mechanism of MF-LTP
does not involve an increase in stimulation-dependent Ca2?
influx into the terminals but is instead attributable to an increase
in the efficacy of downstream exocytotic processes.
Using fluorescence measurement of presynaptic Ca2?in mouse
hippocampal slices, we demonstrated here that MF-LTP is not
accompanied by a change in the presynaptic Ca2?transients, as
shown for LTP at the CA1 synapses (Wu and Saggau, 1994).
Instead, sustained increase in the efficacy of release machinery
was suggested by the findings that mean frequency, but not mean
amplitude, of mEPSCs was increased during MF-LTP.
Our results ruled out the possibility that activity-dependent
broadening of the presynaptic action potential and the subsequent
increase in presynaptic Ca2?influx (Geiger and Jonas, 2000) may
underlie the expression of MF-LTP. A previous study revealed
that sustained elevation of the basal Ca2?level within the termi-
nals does not occur during LTP at this synapse (Regehr and
Tank, 1991). Thus, MF-LTP does not involve any change in
presynaptic Ca2?dynamics. Rather, exocytotic machinery down-
stream from Ca2?influx is selectively upregulated, as suggested
by the mEPSC experiments in cultured hippocampal granule cells
(Tong et al., 1996) and by the neurochemical study using MF
synaptosomes (Lonart et al., 1998). Ca2?-independent expres-
sion mechanism of MF-LTP, as demonstrated in this study, is
rather unexpected in light of the recent findings showing that
presynaptic Ih(Mellor et al., 2002) or kainate autoreceptor (Con-
tractor et al., 2001; Lauri et al., 2001) (but see Nicoll et al., 2000)
is essential for MF-LTP, because activation of these channels
would depolarize MF terminals and affect action potential-driven
Ca2?entry processes by modulating voltage-dependent K?chan-
nels and/or Ca2?channels.
It should be mentioned that the very recent paper by Cheva-
leyre and Castillo (2002) reported strong evidence against the
hypothetical roles of Ihin MF-LTP. They found that organic Ih
blockers (ZD7288 and DK-AH269), which had been supposed to
in Ca2?dynamics within MF terminals. A, Enhancement of fEPSPs
during application of 50 ?M FSK. B, Time course of FCain the same
experiments as in A. Representative traces are those recorded before
(control, thin trace) and 20 min after (FSK, thick trace) FSK application. C,
Time course of FV amplitudes. FV was increased in size by almost the
same degree as that of FCa, suggesting that FSK enhanced presynaptic
excitability but is not accompanied by an increase in Ca2?influx into the
FSK-induced synaptic enhancement is independent of changes
10526 J. Neurosci., December 15, 2002, 22(24):10524–10528Kamiya et al. • Ca2?-Independent Presynaptic Expression of MF-LTP
be selective for Ihchannels, exert a nonspecific action to suppress
MF synaptic transmission. They also performed “two-pathway”
experiments to get around this masking effect of the blockers and
clearly demonstrated that these blockers do not affect MF-LTP.
From these results, the authors also put forward their hypothesis
that MF-LTP results from a direct modification of the release
machinery (Castillo et al., 1997, 2002).
Because of the negative nature of the results in this study, one
may argue that saturation of the Ca2?indicator would mask the
changes in the Ca2?transients. However, we tried to minimize
this possible artifact by using the relatively low-affinity Ca2?
indicator rhod-2 (Minta et al., 1989) instead of the higher-affinity
dye fura-2. In fact, the signals increased substantially by several
conditions, e.g., application of phorbol ester (Honda et al., 2000),
paired stimuli at short intervals (Kamiya et al., 2002), or elevated
external Ca2?concentration (this study). We also paid attention
to load the dye at room temperature to reduce the compartmen-
talization into the mitochondria. As a result, the signal displays
monotonic decay after the peak (Kamiya and Ozawa, 1999; Ka-
miya et al., 2002), suggesting minimal contribution of the signal
originated from mitochondria or other organelles.
To examine the changes in the efficacy of release machinery, we
examined the mEPSCs recorded from CA3 neurons. Because we
cannot distinguish the origin of the observed minis (MF termi-
nals or the other presynaptic terminals making contact on CA3
neurons), contamination of those originated from non-MF inputs
might distort the present results in an unevaluated way. There-
fore, it must be emphasized that the effects of tetanic stimulation
or FSK might be somewhat underestimated, because these ma-
nipulations are expected to selectively affect minis originated
from MF terminals (Weisskopf et al., 1994). More importantly,
however, it certainly supports the notion that MF-LTP and FSK
potentiation are accompanied by enhanced efficacy of release
machinery at the MF terminals.
FV amplitude was increased by application of FSK but not by
LTP-inducing tetanic stimulation. These findings might be inter-
preted as follows. The excitability of MF axons is enhanced by
both manipulations via cAMP elevation in the MF terminals
(Mellor et al., 2002). Bath application of FSK would raise cAMP
levels in the whole population of MF terminals and thus increased
fiber volley amplitude by recruitment of surrounding subthreshold
fibers. In contrast, tetanic stimulation might elevate cAMP con-
centration only within the stimulated MF terminals and therefore
does not lead to increase in the number of stimulated axons.
Differential effects of tetanic stimulation and FSK on FV ampli-
tude would be important for answering the question of why FSK
caused robust enhancement of MF responses in knock-out mice
of Rab3A (Castillo et al., 1997), RIM1? (Castillo et al., 2002),
RI? and C?1isoforms of protein kinase A (Huang et al., 1995),
and type 1 adenylyl cyclase (Villacres et al., 1998) in which
tetanus-induced MF-LTP is absent. Our results highlight differ-
ential mechanisms responsible for FSK-induced enhancement
and tetanus LTP, and this difference may explain, at least in part,
why FSK potentiated MF synaptic transmission in those mutant
mice. It should be noted that FSK enhanced both FV and pre-
synaptic Ca2?transient to the same degree in the similar multi-
fiber recordings at the parallel fiber synapses in the cerebellum
frequency of mEPSCs during MF-LTP
(A, B) and FSK-induced enhancement (C,
D). A1, Representative traces recorded
before (control) and 30 min after tetanic
(top) and averaged (bottom) traces of 10
consecutive mEPSCs. A3, Amplitude his-
tograms of miniature EPSCs recorded un-
der control conditions ( filled bars) and 30
min after tetanus (open bars). A4, Cumu-
lative probability plots of amplitudes (left)
and interevent intervals (right) of minia-
ture EPSCs for control (continuous line)
and LTP (dotted line) data. B, Averaged
time course of the mean frequency ( filled
circles) or amplitude (open circles) of
mEPSCs. Tetanic stimulation (Tet; 100
Hz, 1 sec) was applied at the time shown
by the arrow. TTX (0.5 ?M) was perfused
during the periods as indicated. C1, Rep-
resentative traces recorded before (con-
trol) and 20 min after FSK application
(FSK). C2, Amplitude histograms of min-
iature EPSCs in the absence ( filled bars)
and presence (open bars) of 50 ?M FSK.
C3, Cumulative probability plots of ampli-
tudes (left) and interevent intervals (right)
of miniature EPSCs for control (continu-
ous line) and FSK (dotted line) data. D,
Time course of the FSK effect on the
mean frequency ( filled circles) or ampli-
tude (open circles).
Long-lasting enhancement of
Kamiya et al. • Ca2?-Independent Presynaptic Expression of MF-LTPJ. Neurosci., December 15, 2002, 22(24):10524–10528 10527
(Chen and Regehr, 1997), which also display cAMP-dependent
presynaptic LTP (Salin et al., 1996).
In summary, our data clearly demonstrate Ca2?-independent
expression mechanisms for MF-LTP. Our results, together with
the recent evidence reported by Chevaleyre and Castillo (2002),
strongly support the hypothesis that the presynaptic expression
mechanism responsible for MF-LTP is persistent modification of
cellular steps involving the release machinery of synaptic vesicles
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