J Neurophysiol 92: 2714 –2724, 2004;
10.1152/jn.00464.2004.
Developmental Changes in Release Properties of the CA3-CA1 Glutamate
Synapse in Rat Hippocampus
P. Wasling, E. Hanse, and B. Gustafsson
Institute of Physiology and Pharmacology, Department of Physiology, Göteborg University, 405 30 Göteborg, Sweden
Submitted 5 May 2004; accepted in final form 14 June 2004
synapses (Hessler et al. 1993; Huang and Stevens 1997).
During the first postnatal week, however, the synapse population has been described both to have a uniformly high Pr
(Bolshakov and Siegelbaum 1995) and to display, as the
mature population, a considerable Pr heterogeneity (Hanse and
Gustafsson 2001b). Likewise, conflicting data exist regarding a
developmental change in Pr in that both a developmental
decrease in Pr (Bolshakov and Siegelbaum 1995; Muller et al.
1989), and the absence of such a decrease (Hsia et al. 1998),
have been reported. Whether there is a developmental decrease
in Pr among these hippocampal synapses is thus uncertain.
Moreover, if there is one, do the synapses change from a
uniformly high Pr state to a lower one, or is there a more
general shift in Pr within a heterogeneous Pr population?
The aim of this study is to examine to what extent the
CA3-CA1 synapse changes its release properties in the early
postnatal period, and if so, if this change is homogenous or
heterogeneous among the synapses. Moreover, if such a change
in Pr occurs, is it explained by a decrease in the number of
vesicles that are immediately available for release (Dobrunz
and Stevens 1997) and/or by a change in Pves, the probability
of release of the vesicles themselves (Hanse and Gustafsson
2001b). Finally, is such a developmental change in Pr affected
by an altered neural activity during the critical period?
INTRODUCTION
METHODS
Following their formation, synapses undergo various collective changes, commonly referred to as synaptic maturation.
One salient example of such maturation is a developmental
decrease in release probability to a single action potential (Pr).
This decrease in Pr has been documented among glutamate
synapses in several regions, including the developing neocortex (Kumar and Huguenard 2001; Reyes and Sakmann 1999),
calyx of Held (Iwasaki and Takahashi 2001; Taschenberger et
al. 2002), striatum (Choi and Lovinger 1997), and hippocampus (Bolshakov and Siegelbaum 1995; but see Hsia et al. 1998;
Muller et al. 1989), suggesting that it represents an essential
step in the maturation of the glutamate synapse. In the striatum,
patterned neural activity supports the induction of a long-term
depression (LTD) that is expressed as a decrease in Pr (Choi
and Lovinger 1997), suggesting that neural activity may underlie this form of maturation. However, whether inhibition of
neural activity or of LTD during a critical period interferes
with the maturation of Pr has never been directly tested.
Studies of hippocampal CA3-CA1 glutamate synapses have
shown a considerable Pr heterogeneity among the mature
Experiments were performed on hippocampal slices from 3- to
27-day-old Wistar rats (n ⫽ 142). The animals were killed in accordance with the guidelines of the local ethical committee for animal
research. Rats older than 8 days were anesthetized with isoflurane
(Abbott) prior to decapitation. The brain was removed and placed in
an ice-cold solution containing (in mM) 124 NaCl, 3 KCl, 0.5 CaCl2,
6 MgCl2, 26 NaHCO3, 1.25 NaH2PO4, and 10 D-glucose. In the whole
cell experiments, the solution was composed of (in mM) 140 cholineCl, 2.5 KCl, 0.5 CaCl2, 7 MgCl2, 25 NaHCO3, 1.25 NaH2PO4, 1.3
ascorbic acid, and 7 dextrose. Transverse hippocampal slices (300 –
400 ␮m thick) were cut with a vibratome (Campden Instruments; or
Slicer HR 2, Sigmann Elektronik) in the same ice-cold solution, and
they were subsequently stored in artificial cerebrospinal fluid (ACSF)
containing (in mM) 124 NaCl, 3 KCl, 2 CaCl2, 2 MgCl2, 26 NaHCO3,
1.25 NaH2PO4, 0.5 ascorbic acid, 3 myo-inositol, 4 D,L-lactic acid,
and 10 D-glucose at 25°C. After ⱖ30 min of storage, a single slice was
transferred to a recording chamber where it was kept submerged in a
constant flow (⬃2 ml/min) at 30 –32°C. The perfusion ACSF contained (in mM) 124 NaCl, 3 KCl, 4 CaCl2, 4 MgCl2, 26 NaHCO3,
1.25 NaH2PO4, and 10 D-glucose. Bicuculline methiodide (20 ␮M)
Address for reprint requests and other correspondence: P. Wasling, Inst. of
Physiology and Pharmacology, Dept. of Physiology, Göteborg Univ., Box 432,
405 30 Göteborg, Sweden (E-mail: pontus.wasling@physiol.gu.se).
The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
2714
Slice preparation and solutions
0022-3077/04 $5.00 Copyright © 2004 The American Physiological Society
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Wasling, P., E. Hanse, and B. Gustafsson. Developmental changes
in release properties of the CA3-CA1 glutamate synapse in rat
hippocampus. J Neurophysiol 92: 2714 –2724, 2004; 10.1152/jn.00464.
2004. Developmental changes in release probability (Pr) and paired–
pulse plasticity at CA3-CA1 glutamate synapses in hippocampal
slices of neonatal rats were examined using field excitatory postsynaptic potential (EPSP) recordings. Paired-pulse facilitation (PPF) at
these synapses was, on average, absent in the first postnatal week but
emerged and became successively larger during the second postnatal
week. This developmental increase in PPF was associated with a
reduction in Pr, as indicated by the slower progressive block of the
N-methyl-D-aspartate (NMDA) EPSP by the noncompetitive NMDA
receptor antagonist MK-801. This developmental reduction in Pr was
not homogenous among the synapses. As shown by the MK-801
analysis, the Pr heterogeneity observed among adult CA3-CA1 synapses is present already during the first postnatal week, and the
developmental Pr reduction was found to be largely selective for
synapses with higher Pr values, leaving Pr of the vast majority of the
synapses essentially unaffected. A reduction in Pves, the release
probability of the individual vesicle, possibly caused by reduction in
Ca2⫹ influx, seems to explain the reduction in Pr. In vivo injection of
tetanus toxin at the end of the first postnatal week did not prevent the
increase in PPF, indicating that this developmental change in release
is not critically dependent on normal neural activity during the second
postnatal week.
DEVELOPMENTAL CHANGE IN Pr AT THE CA3–CA1 SYNAPSE
was always present in the perfusion ACSF to block GABAA receptor–
mediated activity. All solutions were continuously bubbled with 95%
O2-5% CO2 (pH ⬃ 7.4). A surgical cut between the CA1 and CA3
regions and the higher than normal Ca2⫹ and Mg2⫹ concentrations
were used to block spontaneous network activity.
Recording and analysis
J Neurophysiol • VOL
follow this frequency without failure as judged from the stability of
the amplitude of the fiber volley. Thus the ratio between the 10th and
1st volley in a 50-Hz train was 0.96 ⫾ 0.05 in experiments (n ⫽ 4) in
which the volley was obtained in isolation during blockade of AMPA
receptors (10 ␮M NBQX). To calculate average release probability
based on MK-801– dependent decrease in NMDA field EPSP magnitude, the following equation was used: Pr ⫽ [1 ⫺ exp(⫺1/␶)]/FB,
where FB is the fraction of NMDA receptors blocked by MK-801
after transmitter release at a synapse (Hessler et al. 1993). Data are
expressed as means ⫾ SE. Statistical significance for paired and
independent samples was evaluated using Student’s t-test.
Tetanus toxin experiments
A previous study from our laboratory (Groc et al. 2003) has shown
that injection of tetanus toxin into the hippocampus in vivo (at P1)
leads to an ⬃80% reduction of spontaneous transmitter release for
several days; the reduction still was ⬎50% at P6 –P8. The same
degree of reduction was found in slices taken close to the injection site
as in those taken ⬎600 ␮m away, indicating that the toxin had spread
over a large part of the hippocampus. Nevertheless, in this study, only
the two slices closest (on either side) to the injection site were taken
from each tetanus toxin-injected hippocampus, to ascertain an as
extensive blockade as possible.
To examine the possible effect of neural activity on the developmental change in Pr, tetanus toxin was injected into the hippocampus
of rat pups at P1 or at P6 –P7, by the same person and using the same
procedures as previously (Groc et al. 2003). The rats were anesthetized by inhalation of isoflurane, and their heads were placed in a
surgical mask to maintain the skull stable. A constant flux of a mixture
isoflurane/air was applied inside the surgical mask. A heat sheet was
used to maintain body temperature. A midline incision was made on
the head and a hole was drilled in the skull (OD, 0.4 mm). The
stereotactic coordinates for injection were (P1/P6) as follows: anterioposterior, –1.4/–2.0 mm; mediolateral, ⫹2.0/⫹2.9 mm, coordinates
relative to the bregma; dorsoventral, –2.0/–2.9 mm from the cortical
surface. Tetanus toxin was dissolved in a phosphate-buffered saline
(0.1 M, pH 7.4) to the final concentration of 10 ␮g/ml. Five nanograms of tetanus toxin was injected into the hippocampus at ⬃125
nl/min using a fused silica needle (ID, 75 ␮m; OD, 150 ␮m; Skandinavia Genetec AB). After injection, the needle was left in situ for 5
min to reduce reflux. The incision was chemically sutured (Vet-Seal,
B. Braun Med).
Drugs
Chemicals were from Sigma-Aldrich, except for D-AP5, ␻-conotoxin VIA, MK-801, NBQX, and QX-314 (Tocris Cookson, Bristol,
UK). Cytochrome C (0.2 mg/ml; bovine heart) was included in all
␻-conotoxin GVIA experiments to block nonspecific peptide binding
sites in the perfusion system.
RESULTS
PPF increases during the second postnatal week
PPF (interstimulus interval, 50 ms), i.e., the synaptic response following the second stimulus in a stimulus pair is
larger than that following the first one, is considered a sensitive
measure of the release probability (Pr) of a synapse (Zucker
and Regehr 2002). When examined for field EPSPs in slices
from P3–P6 rats, PPF was, on average, found to be absent (Fig.
1A). PPF then emerges and becomes successively larger during
the second postnatal week, reaching a value of about 1.6 at P15
(Fig. 1A). This result would suggest that the second postnatal
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Electrical stimulation of Schaffer collateral/commissural afferents
and recordings of synaptic responses were carried out in the CA1
hippocampal region. Stimuli consisted of 0.2-ms negative or biphasic
constant current pulses (15– 80 ␮A) delivered through bipolar tungsten wires (resistance ⬃0.1 M⍀, custom-made stimulator) or a glass
pipette (resistance ⬃0.5 M⍀; STG 1002, Multi Channel Systems,
Reutlingen, Germany), in field and whole cell recordings, respectively. Stimulation electrodes were positioned in the stratum radiatum.
Inputs received a test stimulus every 10 (field recordings) or 5 s
(whole cell recordings). The stimulation intensity was set not to evoke
firing in the postsynaptic neurons as evidenced by the absence of a
population spike distorting the field excitatory postsynaptic potential
(EPSP).
Field EPSP recordings were made by means of a glass micropipette
(filled with 1 M NaCl) in s. radiatum. Field EPSPs were amplified
with an Axoclamp-2A (Axon Instruments) and filtered at 3 kHz. Data
were digitized (10-kHz sampling rate) and collected using a PC
computer. Whole cell patch-clamp recordings were performed on
visually identified CA1 pyramidal cells, using infrared-differential
interference contrast videomicroscopy (CV-M50 IR, JAI Corp.)
mounted on a Nikon E600FN microscope (Nikon). The pipette solution contained (in mM) 130 Cs-methanesulfonate, 2 NaCl, 20 HEPES,
0.2 EGTA, 5 QX-314, 4 Mg-ATP, and 0.4 GTP (pH ⬃ 7.2 and
osmolality 290 –300 mOsm). Patch pipette resistances were 3–5 M⍀.
Excitatory postsynaptic currents (EPSCs) were recorded at a sampling
frequency of 10 kHz and filtered at 1 kHz, using an EPC-9 amplifier
(HEKA Elektronik, Lambrecht, Germany). Cells were held in voltageclamp mode at –70 mV for AMPA EPSC recordings. Series resistance
was monitored using a 5-ms 10-mV hyperpolarizing pulse. Series
resistance was not allowed to change more than ⬃10% during an
experiment; otherwise the experiment was discarded. Evoked responses were analyzed off-line using custom-made IGOR Pro (WaveMetrics, Lake Oswego, OR) software. Field EPSP magnitude was
estimated by linear regression over the first 0.8 ms of the initial slope.
Field NMDA receptor–mediated responses were measured by the
initial 40-ms area. The presynaptic volley was measured as the
peak-to-peak amplitude of the initial positive-negative deflection, and
it was not allowed to change by ⬎5% during the experiment. AMPA
EPSCs were measured as the difference between the baseline level
immediately preceding the stimulation artifact and the mean amplitude during a 1-ms time window around the negative peak between 3
and 8 ms after the stimulation artifact. Slope measurements (leastsquare linear regression from 20 to 80% of the rising phase) of EPSCs
confirmed the results obtained with amplitude measurements (data not
shown). In Fig. 3, all experiments were performed in 50 ␮M D-AP5
and 100 ␮M Ni2⫹ to block induction of long-lasting changes of
synaptic strength (Wasling et al. 2002). In experiments in which the
sensitivity of the field EPSP and paired-pulse facilitation (PPF) to
extracellular [Ca2⫹] was examined, the sum of [Ca2⫹] and [Mg2⫹]
was kept at 10 mM to minimize changes in membrane excitability
while altering Ca2⫹ ion concentration. We also did not observe any
changes in the amplitude of the presynaptic volley while changing the
divalent ion concentrations in this manner (data not shown). When
using extracellular [Ca2⫹] above 4 mM, recordings always started at
the higher [Ca2⫹] solution before changing to the lower [Ca2⫹]
solution. When field EPSPs were compared when changing from 4/0
to 4/4 Ca2⫹/Mg2⫹, the change in volley was corrected for. In experiments estimating the releasable pool of vesicles, high-frequency (20
and 50 Hz) stimulation was used (Fig. 6). The axons appeared to
2715
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P. WASLING, E. HANSE, AND B. GUSTAFSSON
week constitutes a period of substantial decrease in Pr among
these synapses.
To examine whether the change in PPF during the second
postnatal week is driven by neural activity, tetanus toxin was
injected in vivo into the hippocampus of P6 –P7 rats (n ⫽ 9; see
METHODS). When examined in slices taken from rats at P12, PPF
averaged 1.48 ⫾ 0.03 (n ⫽ 31) compared with 1.55 ⫾ 0.04
(n ⫽ 25) in slices from noninjected rats (Fig. 1B). It thus
appears that the change in PPF proceeds unabated, despite the
considerable activity reduction caused by such an injection
(Groc et al. 2003). However, the tetanus toxin injection may,
either by a differential action on synapses with different Pr or
by the activity reduction itself, have caused an increase in PPF,
thus masking an effect on the second week developmental
change in PPF. The effect of tetanus toxin was thus examined
prior to the second week when Pr appears to be rather stable
(see also Hanse and Gustafsson 2001b). Injection of tetanus
toxin and examination of PPF at P5–P6, did, however, not
reveal significantly larger PPFs (1.15 ⫾ 0.06, n ⫽ 9) than in
age-matched controls (1.08 ⫾ 0.04, n ⫽ 21; Fig. 1B).
Release probability changes during the second postnatal
week in a nonuniform manner among the synapses
Since Pr is a major determinant of PPF, with a decrease in Pr
leading to an increased PPF, the simplest explanation for the
observed change in PPF is a second week decrease in Pr. The
neonatal synapse was originally reported to have (uniformly) a
very high Pr, approaching 1.0 (Bolshakov and Siegelbaum
1995), and a reduction of this Pr during the second postnatal
week would then seem a likely explanation for the increase in
PPF. However, more recent studies have shown that Pr in the
neonatal synapse, as in the more adult one, is very heterogeneous, with values from close to one to close to zero (Hanse
and Gustafsson 2001b). With such a heterogeneous population,
it is less clear in what manner the synapses may have changed,
uniformly or not, to explain the PPF change. To explore this
matter, we examined the MK-801–induced decay of pharmaJ Neurophysiol • VOL
cologically isolated NMDA receptor–mediated field EPSPs (10
␮M NBQX). Figure 2A shows the average decay curves
obtained from such experiments on P6 (E, n ⫽ 9) and P12 (F,
n ⫽ 9) rats, respectively. In both cases, the NMDA EPSP
decays with a rapid initial phase followed by a slower phase.
The decay time course obtained in MK-801 experiments was
originally fitted by a double exponential indicating two major
categories of CA3-CA1 synapses, a high and a low Pr group
(Hessler et al. 1993; Rosenmund et al. 1993). When examined
for the individual experiments, the present decay curves were
also better fitted by a double than by a single exponential [sum
of squares for the single exponential fits were at P6 7.4 ⫾ 2.2
(n ⫽ 9) and at P12 7.6 ⫾ 2.35 (n ⫽ 9) times larger than for the
double exponential fits]. This result is consistent with a similar
kind of Pr heterogeneity among the synapses during the first as
well as after the second postnatal week (Dobrunz and Stevens
1997; Hanse and Gustafsson 2001b). However, the decay is, on
average, substantially slower for the P12 synapses (Fig. 2A, F)
than for the P6 ones (Fig. 2A, E), suggesting a developmental
decrease in Pr. As judged from the parameters of the double
exponential fits obtained from the individual experiments, the
decrease in Pr was mainly restricted to the high Pr group of
synapses. Thus, whereas the time constant of the fast component changed considerably from 3.7 ⫾ 0.2 (n ⫽ 9) at P6 to
7.8 ⫾ 0.7 (n ⫽ 9) at P12 (P ⬍ 0.001), there was no significant
difference between the time constants of the slow component:
55.2 ⫾ 1.8 (n ⫽ 9, P6) and 63.8 ⫾ 3.6 (n ⫽ 9, P12).
Knowing the fraction of NMDA receptors that are blocked at
each trial (when glutamate is released), the (average) Pr of the
high and low Pr groups of synapses can be calculated (Hessler
et al. 1993). Moreover, the proportion of synapses belonging to
the high and low Pr groups, respectively, can be obtained from
the relative area of the fast and slow exponential components
(exponential amplitude ⫻ time constant) (Rosenmund et al.
1993). Assuming a value of 0.3 for the fraction of NMDA
receptors blocked/trial (Hessler et al. 1993; Huang and Stevens
1997; Rosenmund et al. 1993), the synaptic population at P6
would consist of a high Pr group with an average Pr value of
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FIG. 1. Paired-pulse (PP) ratio increases
in a largely activity-independent manner
during the 2nd postnatal week. A: summary
graph showing PP ratio as a function of
postnatal day (n ⫽ 86). Individual experiments (E) and mean (■) at indicated postnatal day. Insets: example PP field excitatory
postsynaptic potential (EPSP) responses
from P6 (left) and P13 (right) rat. Calibration
bars: 100 ␮V/10 ms. B: average PP ratio in
control and tetanus toxin-injected animals at
P5–P6 (injected at P1) and P12 (injected at
P6 –P7), respectively. *P ⬍ 0.001. Inset:
schematic view of in vivo injection site of
tetanus toxin in the hippocampus. See METHODS for further details.
DEVELOPMENTAL CHANGE IN Pr AT THE CA3–CA1 SYNAPSE
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0.88 ⫾ 0.04 (n ⫽ 9) and of a low Pr group averaging 0.067 ⫾
0.003 (n ⫽ 9) that would constitute 10 and 90% of the
population, respectively. At P12, the corresponding Pr values
would be 0.52 ⫾ 0.02 (n ⫽ 9) and 0.068 ⫾ 0.004 (n ⫽ 9),
respectively, for 13 and 87% of the population. Thus Pr
changes during the second week, but this change is restricted to
the high Pr synapses, constituting a minority of the synapses.
Nonetheless, these synapses will produce more than one-half
of the field EPSP, and the decrease in their Pr value would
result in an overall reduction of Pr (at P12) to 85% of the P6
value (Pr ⫽ weighted average of the average Pr of the high and
of the low Pr group).
The question arises whether such a nonuniform change in Pr
can alter PPF in the manner observed above. The MK-801
experiments were performed in 4/0 extracellular Ca2⫹/Mg2⫹,
implying different release conditions from those in which the
values in Fig. 1A were obtained (4/4 Ca2⫹/Mg2⫹). When PP
ratio for the (non-NMDA) field EPSP was examined in 4/0
Ca2⫹/Mg2⫹, it averaged 0.90 (n ⫽ 12) and 1.25 (n ⫽ 6) for P6
and P12 synapses, respectively. The PP ratio difference to be
explained is thus one from 0.90 to 1.25. Using data from
experiments using minimal stimulation to examine the relation
between PP ratio and Pr for single CA3-CA1 synapses during
the first postnatal week (Hanse and Gustafsson 2001a), the PP
ratios for high Pr (0.88) and low Pr (0.07) synapses at P6 would
be about 0.25 and 2.20, respectively, resulting in an overall
PPF of 1.04. Corresponding values for the P12 synapses would
be PP ratios of about 0.60 and 2.20, respectively, resulting in
an overall PPF of 1.32. The observed change in PPF from P6
to P12 can thus be accounted for by the rather selective
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decrease in Pr of high Pr synapses indicated from the MK-801
experiments.
Possible complicating factors with the MK-801 experiments
The NMDA EPSP observed when stimulation commenced
after a 10-min-long stimulus interruption was substantially
larger than the baseline level present before stimulus interruption (Fig. 2B). The NMDA EPSP decay in MK-801 should then
include a decay of this enhancement, which is unrelated to the
MK-801 block of open channels. However, the decay of this
enhancement is blocked by NMDA receptor antagonists (Niu
et al. 1999), implying that it will only occur following NMDA
receptor activation. Since NMDA receptor activation will lead
to block by MK-801, it seems reasonable to believe that the
decay of this enhancement will not occur in the presence of
MK-801. Another possible complication is that the MK-801–
induced decay is not only a function of the Pr of a synapse, but
also of the open probability of the NMDA receptor channels.
The NMDA EPSPs observed in the P12 synapses were found
to decay faster than those in P6 synapses (P6, ␶ ⫽ 66 ⫾ 5 ms,
n ⫽ 9; P12 ⫽ 42 ⫾ 4 ms, n ⫽ 9; P ⬍ 0.05; Fig. 2C), possibly
reflecting a developmental shift in NMDA receptor subunit
composition to an increasing proportion of NR2A subunits
(Kirson et al. 1999). However, such a shift from NR2B to
NR2A subunits should not be expected to affect the total open
probability of the NMDA receptor channels during an EPSP,
since the reduced duration is compensated for by a higher peak
open channel probability (Chen et al. 1999).
It should also be noted that the good double exponential fit
to the MK-801 data not necessarily implies the existence of
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FIG. 2. Developmental difference in MK-801
induced decrease of N-methyl-D-aspartate
(NMDA) field EPSP magnitude. A: summary
graph of the decrease of NMDA field EPSPs in
the presence of 20 ␮M MK-801. Field EPSP
magnitudes are plotted against stimulus number
and are normalized to the 1st response. The
progressive block at P6 (E, n ⫽ 9 experiments)
and at P12 (F, n ⫽ 9 experiments) was fitted with
a double exponential equation: A1exp(–t/␶1) ⫹
A2exp(–t/␶2). The best fit (at P6; P12) was obtained with A1 ⫽ 62.9; 59.0, ␶1 ⫽ 3.4; 7.2, A2 ⫽
37.1; 41.0, and ␶2 ⫽ 52.5; 59.3. Note that these
values represent the best fit to the average decay
and differ somewhat from the values in the text
that are based on averaging the values obtained
from best fits to the decay from each individual
experiment. B: stimulus interruption (in the absence of MK-801) for 10 min caused an increase
in NMDA field EPSP magnitude that subsequently decayed back to baseline values. Values
for the subsequent decay normalized to the 1st
field EPSP following the resumption of stimulation are shown for P6 (E) and P12 (F) experiments. C: averaged NMDA field EPSPs, taken
from the experiments in A, recorded before the
addition of MK-801 (P6, gray lines; P12, black
lines). NMDA field EPSP from each experiment
represents the average from 15 consecutive responses. Average NMDA field EPSPs from each
experiment were normalized to the same peak
amplitude.
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P. WASLING, E. HANSE, AND B. GUSTAFSSON
two separate homogeneous groups of synapses, but may also
be compatible with more continuous Pr distributions (Huang
and Stevens 1997). The high and low Pr groups of synapses
may thus represent the upper and lower parts of a continuous
distribution rather than two distinct groups.
Does the sensitivity of Pr and PPF to Ca2⫹/Mg2⫹ change
during the second postnatal week?
FIG. 3. Age difference in the sensitivity to changes in
extracellular Ca2⫹ and Mg2⫹ concentrations. A and B:
summary graphs showing changes in field EPSP magnitude and PP ratio as a function of [Ca2⫹]o and [Mg2⫹]o at
P6 (open symbols) and at P12 (closed symbols). C:
changes in field EPSP magnitude plotted against PP ratio.
Arrows on the P6 curve indicate PPF values at P6 (䊐) and
at P12 (■) in 4:4 [Ca2⫹]/[Mg2⫹] solution. In A and C, field
EPSP magnitude is normalized to that obtained at 4:6
[Ca2⫹]/[Mg2⫹]. Note that some error bars are within
symbols. For the various [Ca2⫹]/[Mg2⫹] concentrations
(1/9, 2/8, 3/7, 4/6, 7/3, and 10/0) the n values were 3, 5, 7,
12, 5, and 5 (P6) and 0, 6, 6, 10, 7, and 6 (P12).
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The change in Pr indicated by the MK-801 experiments can
be explained by a change in Pves as well as in the number of
vesicles that are immediately available for release. To explore
whether a Pves change is involved, the effect of changes in the
extracellular Ca2⫹/Mg2⫹ concentration on field EPSPs was
examined at both P6 and P12. To minimize changes in axonal
excitability, divalent ion concentration was kept constant at 10
mM, and Ca2⫹/Mg2⫹ concentrations used were from 1/9 ⱕ
10/0. Field EPSP values obtained at these different concentrations, normalized to the value obtained at 4/6 Ca2⫹/Mg2⫹,
show that P6 and P12 synapses respond differently to these
changes in Ca2⫹ influx (Fig. 3A). Thus, while reductions in
Ca2⫹ influx decrease the EPSPs of P6 and P12 synapses in a
rather similar manner, an increase in Ca2⫹ influx causes a
considerably larger increase of the EPSP at P12 than at P6.
This result suggests that P6 synapses are closer to saturation of
their release machinery to Ca2⫹ ions, indicative of either a
larger influx of Ca2⫹ ions or of a shift in the Ca2⫹ sensitivity
curve. Nonetheless, this result seems compatible with a reduction in Pves as underlying the change in Pr from P6 to P12.
These synapses also responded differently with respect to PPF
to changes in the Ca2⫹/Mg2⫹ relation (Fig. 3B). Thus, while a
larger Ca2⫹ influx resulted in a substantial PPF decrease in P12
synapses, there was little change at P6. On the other hand, a
decrease in Ca2⫹ influx caused a large PPF increase at P6 and
a small one at P12. These results are also compatible with the
release machinery being closer to saturation by Ca2⫹ ions at P6
than at P12.
Can a reduced Ca2⫹ influx quantitatively account for both
the decrease in Pr and increase in PPF? The plot in Fig. 3C
between (relative) field EPSP amplitude and PPF shows that
the field EPSP (at P6) has to decrease to 40% of control for
PPF to increase from its control value (1.11 ⫾ 0.04, n ⫽ 12) to
the value found at P12 (1.55 ⫾ 0.04, n ⫽ 16). As indicated
from the MK-801 experiments, overall Pr at P12 would be 85%
of that at P6. However, these experiments were performed at
4/0 Ca2⫹/Mg2⫹. A change from 4/0 to 4/4 Ca2⫹/Mg2⫹ decreased the field EPSP on average to 67 (n ⫽ 12) and 52% (n ⫽
6) (after volley correction, see METHODS) for P6 and P12
synapses, respectively. This would indicate that, at 4/4 Ca2⫹/
Mg2⫹, Pr at P12 should be 66% [85% ⫻ (52/67)] of that at P6.
This value (66%) differs substantially from the decrease to
40% expected from the data in Fig. 3C. Thus a reduction in
Ca2⫹ influx (by altered Ca2⫹/Mg2⫹ ratio) does not replicate the
developmental change in release properties. A close match
may, however, not be expected since the developmental change
in release properties only affects high Pr synapses. Moreover,
the alteration of the Ca2⫹/Mg2⫹ ratio may have effects on the
DEVELOPMENTAL CHANGE IN Pr AT THE CA3–CA1 SYNAPSE
release machinery unrelated to the change in Ca2⫹ influx per se
(such as changes in resting intracellular Ca2⫹ levels).
Is the expression of presynaptic voltage-gated Ca2⫹
channels altered?
Variation in action potential duration with age
During the second postnatal week, there is a substantial
change in action potential characteristics, at least as observed
from recordings from CA1 pyramidal cell somata and from
Calyx of Held (Spigelman et al. 1992; Taschenberger and von
Gersdorff 2000). The amplitude is increased, and the duration
is substantially reduced, likely as a consequence of changes in
the expression of voltage-gated sodium and potassium channels. If such a change also occurs for the action potential
invading the presynaptic bouton, one may expect a developmental decrease in Ca2⫹ influx associated with the reduced
duration of the spike. As previously described (Berg-Johnsen
and Langmoen 1992; Laerum and Storm 1994), procedures
that alter the action potential duration, such as TEA and
temperature, have significant effects on the fiber volley duration. Following the total blockade of the NMDA receptor–
mediated EPSP by MK-801, the fiber volley could be observed
in isolation and thereby be accurately measured with respect to
its duration. The volley observed at the end of the first postnatal week averaged 1.59 ⫾ 0.03 (n ⫽ 24) ms compared with
1.32 ⫾ 0.02 (n ⫽ 42) ms for that observed 1 wk later, i.e., there
was a substantial shortening (Fig. 5). To examine the activity
dependence of this shortening, the fiber volley was also measured in slices from P12 rats that had been injected with tetanus
toxin at P6. The volley duration in these slices averaged 1.28 ⫾
0.03 (n ⫽ 13), indicating that the action potential shortening
proceeds independent of neural activity.
Does the immediately releasable pool of vesicles change
with age?
Pr is determined not only by Pves but also by the number of
vesicles that are immediately available for release, i.e., the
preprimed pool (Hanse and Gustafsson 2001b). On one hand,
a developmental decrease in pool size could contribute to the
developmental decrease in Pr in high Pr synapses. On the other
hand, a developmental increase in pool size that parallels the
developmental decrease in Pves may explain the nonuniform
developmental change in Pr indicated by the MK-801 data.
This is because an increased pool size will have a larger impact
on the Pr of low than of high Pves synapses (Hanse and
Gustafsson 2001b), and may thus counteract a developmental
decrease in Pr in low but not in high Pr synapses.
An estimate of the preprimed pool can be obtained by
evoking EPSCs by a brief high-frequency train and plotting
the cumulative EPSC amplitude against train length
(Schneggenburger et al. 1999). The rationale is that when
the cumulative amplitude rises in a linear manner, release is
no longer coming from vesicles in the preprimed pool but
only from newly recruited ones (that are assumed to be
recruited/released at the same rate). By assuming that this
recruitment starts at zero time, the intersection of the linear
relation with the y-axis will give an estimate of the preprimed pool. This estimate will not be in absolute terms, but
will be the relation between the preprimed pool and the
2⫹
FIG. 4. Effect of Ca
channel blocker on synaptic transmission is not age dependent. A: example experiment (P6) of
the reduction in field EPSP magnitude following application of
10 ␮M ␻-conotoxin GVIA. Insets: average field EPSPs (n ⫽
10) taken at indicated time-points. Calibration bars: 100 ␮V/10
ms. B: summary graph of the ␻-conotoxin GVIA-sensitive
fraction at P6 and P12.
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In interpreting the above results as indicating a developmental decrease in Pves, possibly related to a decrease in Ca2⫹
influx, the question arises of what may underlie such a change.
One possibility is a developmental change in the type of
voltage-gated Ca2⫹ channels that is expressed in the presynaptic membrane and whose openings underlie the Ca2⫹ influx
leading to release. In fact, at some synapses, including hippocampal glutamate synapses in culture, there is a developmental reduced contribution from N-type voltage-gated channels (see Iwasaki et al. 2000). We thus examined the effect of
␻-conotoxin GVIA, a blocker of these channels, on field
EPSPs from P6 and P12 synapses, respectively. As shown from
one such experiment (P6 rat), this toxin had a substantial effect
on release, reducing the field EPSP to less than one-half its
amplitude (Fig. 4A). On average, the ␻-conotoxin–sensitive
fraction of the EPSP was 55 ⫾ 4% (n ⫽ 4) at P6. This fraction
was not significantly different (51 ⫾ 2%, n ⫽ 6) at P12,
indicating that there is no major shift in N-type channel
expression during this time period (Fig. 4B). This fraction is
also similar to what has been reported in 3- to 4-wk-old
animals (Wheeler et al. 1994). The reduction in the field EPSP
by ␻-conotoxin was associated with an increase in PPF [from
1.15 ⫾ 0.04 to 1.72 ⫾ 0.02 at P6 (n ⫽ 4) and from 1.62 ⫾ 0.08
to 2.12 ⫾ 0.10 at P12 (n ⫽ 6)]. Thus (by linear approximation)
the EPSP, i.e., Pr, at P6 has to be reduced to 60% to produce
a PPF increase corresponding to that at P12. Such a reduction
is in line with that calculated above to occur between P6 and
P12 (66%), suggesting that a reduction in Ca2⫹ influx, by
blockade of N-type channels, can essentially replicate the
developmental change in release properties.
2719
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P. WASLING, E. HANSE, AND B. GUSTAFSSON
FIG. 5. The presynaptic volley duration shortens during development. A: summary graph of volley duration at different
postnatal days and following tetanus toxin injection at P6. *P ⬍
0.001. Inset: example volleys at P6 (thin line) and at P12 (thick
line). The example volley at P6 is scaled to the same peak
amplitude as the P12 volley (dotted line). Calibration: 0.2 mV,
0.5 ms. B: volley duration histogram for P4 –P7 (open bars) and
P10 –P14 (filled bars) with Gaussian fits superimposed (dashed
lines). In these experiments, both AMPA and NMDA receptors
were blocked (10 ␮M NBQX and 20 ␮M MK-801).
were representative of P6 and P12 synapses, respectively.
As calculated above, at 4/4 Ca2⫹/Mg2⫹, Pr at P12 should be
66% of that at P6. However, during the initial stages of the
train activation (5–10 1st of the 30 trains given at 0.2 Hz)
release conditions were altered in that the EPSC amplitude
and/or PPF changed. Thus the pool estimations were performed after a steady state had been reached, during which
the EPSC (following the 1st stimulus of the train) of P6
synapses had decreased (on average) to 0.68 of the initial
value, whereas that of P12 synapses was less altered (0.93).
Thus, under these conditions, Pr of the P12 synapses should
be 90% [66% ⫻ (0.93/0.68)] of that of the P6 ones. The pool
estimates of 1.64 ⫾ 0.11 (n ⫽ 15) and 2.07 ⫾ 0.09 (n ⫽ 13)
given above should be translated into 1.64 ⫾ 0.11 and
1.86 ⫾ 0.08 (90% of 2.07) after correcting for this estimated Pr difference, indicating no significant change in
the overall value of preprimed pool during the second
postnatal week.
DISCUSSION
These results show that the second postnatal week constitutes a period of substantial alterations in the release properties
of CA3-CA1 hippocampal synapses manifested as a large
increase in paired-pulse plasticity. Hence, these results do not
substantiate a previous report that paired-pulse plasticity is
unaltered throughout this time period (Hsia et al. 1998), but
rather those reports indicating such a change (Bolshakov and
Siegelbaum 1995; Muller et al. 1989). However, the results
FIG. 6. Estimation of vesicle pool size. A: example experiment showing cumulative excitatory postsynaptic current
(EPSC) amplitudes (normalized with respect to the amplitude
of the 1st response) obtained by 20- (E) and 50-Hz (■)
stimulation. Dashed lines are extrapolations from linear regression over the last 5 data points. Intersection between
dashed lines and the y-axis represents the estimated pool size
as a function of initial release probability (indicated by
arrow). B: mean relative pool size at P4 –P7, at P12–P15, and
at P12–P15, with mean value corrected for lowered Pr.
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number of vesicles released by the first stimulus in the train,
i.e., it will be a function of Pr. Such estimates of the
preprimed pool at P6 and P12 were obtained by evoking
synaptic activity by 10 impulse trains at 20 and 50 Hz. Since
the estimate should be independent of train frequency, the
use of both frequencies can be seen as an internal control. In
these experiments, synaptic activity was recorded as EPSCs
using whole cell recording (rather than field recording)
primarily to avoid buildup of depolarization (and subsequent spike activation) during the train. An example of such
cumulative EPSC curves, normalized to the peak amplitude
of the first EPSC, given by 20- and 50-Hz trains, respectively, are shown in Fig. 6A. The latter half of both curves
is well described by a linear relation; these linear relations
intersect with the y-axis at the same value, about twice that
of the first EPSC. Averaged data from such experiments
showed similar pool size estimates using 20- and 50-Hz
trains (1.64 and 1.66 at P6; 2.06 and 2.11 at P12), as should
be expected. However, compared for P6 and P12 synapses,
there was a significant age group difference, the values
being 1.64 ⫾ 0.11 (n ⫽ 15) and 2.07 ⫾ 0.09 (n ⫽ 13) for
P6 and P12 synapses, respectively (Fig. 6B). Since these
estimated pool values are a function of Pr, they will,
however, only be useful if one knows the relation between
the Pr values of the P6 and P12 synapses when these pool
estimates were performed. It should first be noted that PPF
of the synapses involved averaged 1.07 (P6) and 1.65 (P12)
(estimated from the 1st of the 30 trains given for each
synapse; see following text), indicating that these synapses
DEVELOPMENTAL CHANGE IN Pr AT THE CA3–CA1 SYNAPSE
J Neurophysiol • VOL
changing the estimated average Pr from 0.88 to 0.52 (blocking
fraction ⫽ 0.3). Calculations using PP ratio–Pr data from
minimal stimulation experiments (Hanse and Gustafsson
2001a) suggested (see RESULTS) that such a selective Pr change
would produce an overall change in PPF consistent with that
observed experimentally. However, whether this developmental Pr change is a distinct shift within a homogenous group of
synapses with high Pr, or a variable shift in Pr among the
synapses with the higher Pr values within a continuous distribution is, as indicated above, uncertain. Nevertheless, the
maturation is not a switch of release properties from one group
to another but an adjustment of the release probability within
one group, or population, of synapses. This second week
maturation thus differs from that observed in cultured hippocampal neurons where a proportion of the high Pr synapses
converts into low Pr ones (Chavis and Westbrook 2001). On
the other hand, it agrees with the developmental change within
the Calyx of Held synapse (Iwasaki and Takahashi 2001). In
this synapse, containing hundreds of release sites, a doubleexponential fit of MK-801 data also suggested a shift within a
high Pr group of release sites from much the same values and
to much the same extent as shown presently for the hippocampal synapses, whereas little change was observed in the low Pr
group. Thus, interestingly, the developmental change observed
among a population of heterogeneous hippocampal synapses,
containing a single release site, is mirrored by that occurring in
a heterogeneous population of release sites within a single
synapse.
What underlies the decrease in Pr in the high Pr group?
Controversy exists to what extent heterogeneity in Pr is
determined solely by a variation in the immediately releasable
vesicle pool (Dobrunz and Stevens 1997), or in addition, by
variation in Pves among the synapses (Hanse and Gustafsson
2001b). According to the first viewpoint, the developmental
change in Pr should be explained by a reduced vesicle pool in
high Pr synapses. If Pr is decided by the pool alone, the high Pr
group would produce the majority of the releasable vesicles,
and a reduction of its pool size would be detectable. However,
this study found no evidence for a developmental reduction in
pool size, estimated using the cumulative EPSC amplitude
arising from train stimulation (Schneggenburger et al. 1999).
On the other hand, high Pr synapses that differ from the low Pr
synapses mainly with respect to Pves will produce only a
minority fraction of the releasable pool, and a reduction in this
fraction may go undetected in our pool estimations. However,
a pool decrease in high Pr synapses would not be expected to
lead to a developmental increase in PPF (Hanse and Gustafsson
2001a).
Evidence for the involvement of a Pves change, rather than a
pool change, in the developmental Pr reduction is the differential effect of changes in Ca2⫹/Mg2⫹ ratio on the EPSPs
evoked from P6 and P12 synapses, respectively. Specifically,
high Ca2⫹/Mg2⫹ ratios increased the EPSP of P6 synapses
considerable less than that of P12 synapses, suggesting that the
P6 synapses are closer to saturation with respect to Ca2⫹, i.e.,
operate higher up on the upper limb of the Pr–Ca2⫹ relation.
This suggestion seems reasonable with respect to the high Pr
group of synapses at P6. However, considering the large
number of low Pr synapses in the P6 population, one may have
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also suggest that the alteration takes place in only a subpopulation of the synapses, those with high Pr values, and leaves
most of the population largely unaffected. This may explain the
failure of Hsia et al. (1998) to observe the developmental
change in paired-pulse plasticity, since fewer synapses will be
sampled in whole cell than in field recordings. The alteration in
paired-pulse plasticity was found to take place despite a profound blockade of synaptic transmission during this time period, by local in vivo tetanus toxin injection, suggesting that
normal neural activity during the second postnatal week is not
a prerequisite for the alteration in paired-pulse plasticity to
occur. Our results suggest that the observed alteration in
release properties is explained by a large reduction in the
release probability of the individual vesicles (Pves) in the
affected synapses, possibly mediated by a reduced Ca2⫹ influx.
Since the observed modification in release largely occurs
within a few days, it is not likely a consequence of synaptogenesis per se, which occurs continuously throughout the first
4 wk (Steward and Falk 1991).
The CA3-CA1 synapses in the first neonatal week have
previously been described as uniformly characterized by a
Pr close to one and by paired-pulse depression (Bolshakov
and Siegelbaum 1995). This behavior was in contrast to that
found in 2- to 3-wk-old animals, where Pr was substantially
lower and there was PPF (Bolshakov and Siegelbaum 1995),
indicating a switch from high to low Pr synapses in the
intervening period. More recently, using minimal stimulation technique, a more heterogeneous first week Pr distribution was described (Hanse and Gustafsson 2001b). The
present results support the existence of such first week
heterogeneity. As in older animals (Hessler et al. 1993), the
NMDA EPSP decayed, following MK-801 application, with
an initial rapid and a later slower exponential phase that
indicates the presence of both high and low Pr synapses.
There is presently no clear understanding of the exact nature
of the Pr distribution in older (⬎2 wk) animals. That is,
MK-801 data taken to indicate the existence of two distinct
groups of synapses (Hessler et al. 1993; Rosenmund et al.
1993) can equally well be accounted for by more continuous
Pr distributions (Huang and Stevens 1997). Nevertheless,
these results suggest that the neonatal population consists of
synapses with higher and lower Pr, the low Pr synapses
constituting the majority of the population. Assuming the
fraction of NMDA receptors channels to be blocked following vesicular release to be 0.3– 0.4 (Hessler et al. 1993;
Huang and Stevens 1997; Rosenmund et al. 1993), the high
Pr group would have an average Pr of 0.88 – 0.66 (at 4/0 mM
Ca2⫹/Mg2⫹) and the low Pr group would average 0.07–
0.05. These deduced Pr values are not incompatible with
data from the minimal stimulation experiments (Hanse and
Gustafsson 2001b). Although these latter data show a continuous distribution of Pr values, they indicate two peaks, at
low (⬍0.1) Pr and high (0.4 – 0.6) Pr that suggests the
presence of two broad categories of synapses. These peaks
may represent the presently deduced high and low Pr
groups, considering the uncertain value of the blocking
fraction, and the fact that the minimal stimulation data were
obtained at 4/4 Ca2⫹/Mg2⫹, and using train stimulation,
both procedures that would decrease Pr.
A main finding in this study was that the second week
synaptic maturation in Pr only affects the high Pr synapses,
2721
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P. WASLING, E. HANSE, AND B. GUSTAFSSON
J Neurophysiol • VOL
buffering capacity of Ca2⫹ ions in the high Pr group (Blatow et
al. 2003; Oleskevich and Walmsley 2002). However, whereas
an increase of slow, as well as fast, buffering would lead to a
decreased Pr, it would also lead to a decreased PPF (Rozov et
al. 2001), and can thus not explain the developmental change
in release. Moreover, a fast buffer, if present, is expected to
lead to an increase in PP ratio when extracellular calcium
levels are increased (Blatow et al. 2003). It was presently noted
(Fig. 3B) that, at the lowest Ca2⫹ levels, P12 synapses did not
increase their PPF when Ca2⫹ was lowered, indicative of a
small concentration of fast buffers in these synapses. However,
since, for P12 synapses, the overall dominating relation between Pr and PP plasticity is an inverse one, fast buffering is
likely to have only a minor effect on Pr and PPF in these
synapses.
An alternative explanation to a reduced amount of Ca2⫹
reaching the Ca2⫹ acceptors is a developmental change of the
distance between Ca2⫹ channels and the Ca2⫹ acceptors or of
the affinity of Ca2⫹ acceptors. Such changes would manifest
themselves in the same manner as a reduced Ca2⫹ influx, and
if occurring selectively in high Pr synapses, may potentially
explain the developmental switch in release properties.
Activity dependence of PPF maturation?
We used in vivo injections of tetanus toxin into the
hippocampal CA1 area to address the question of to what
extent the second postnatal week synaptic maturation depends on neural activity. Such injections have been found to
cause a profound decrease, for several days, of synaptic
release over a large part of the hippocampus (Groc et al.
2003), and when applied at P1, to impair dendritic growth
(Groc et al. 2002). However, tetanus injection at P6 did not
affect the second week increase in PPF, indicating that this
increase does not require the neural activity that normally
exists during the second postnatal week. This result differs
from work on cultured neurons in which NMDA receptor
blockade prevents the developmental decrease in Pr (Chavis
and Westbrook 2001). However, this latter Pr change differs
from the present one, in that synapses changed from high Pr
to low Pr, rather than shifting within the high Pr group.
Moreover, there may be a difference when reducing activity
in different manners (AP5 vs. tetanus toxin). At anteroventral cochlear nucleus (AVCN) glutamate synapses, Pr is, at
P11–P16, lower in control than in deaf mice, possibly
reflecting an activity dependent lowering of Pr (Oleskevich
and Walmsley 2002). However, whether these synapses
actually start out with a high Pr is not known. Moreover, the
synapses differed only with respect to Pr and not with
respect to PPF.
In the striatum, there is a temporal correlation between
the developmental increase in PPF and a decreased susceptibility for the induction of an LTD based on Pr reduction
(Choi and Lovinger 1997). Neural activity leading to induction of LTD may then cause the developmental increase in
PPF. In the hippocampus, LTD may also show age dependence, being more easily induced early on, and be presynaptically expressed (Li et al. 2002; Wasling et al. 2002).
Since these LTDs are evoked by rather weak neural activity,
it cannot be excluded that tetanus toxin blockade does not
interfere with their induction. However, these LTDs do not
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expected greater latitude for increased EPSP amplitude with
increased Ca2⫹/Mg2⫹ ratios. Previous MK-801 experiments
(Hessler et al. 1993) have, however, indicated a smaller effect
of Ca2⫹ influx changes (induced by the addition of Cd2⫹ to the
perfusion solution) on the low than on the high Pr synapses.
The low Pr group (at the high Ca2⫹/Mg2⫹ ratios used) may
also operate on the upper limb of its Pr–Ca2⫹ relation. That is,
the maximal Pr value of these synapses is substantially ⬍1, as
has been observed for cortico-geniculate synapses (Granseth
and Lindstrom 2003, 2004). The effect of changes in Ca2⫹/
Mg2⫹ ratio on PPF was also consistent with P6 synapses
operating higher up on the upper limb of the Pr–Ca2⫹ relation.
These data point toward a second week reduction in Pves that
should be largely restricted to high Pr synapses, possibly
explained by a reduction in the amount of Ca2⫹ reaching the
Ca2⫹ acceptors (triggering release). In support of this explanation, a decrease in Ca2⫹ influx induced by a Ca2⫹ channel
blocker could quantitatively replicate the combined Pr and PPF
shift occurring from P6 to P12.
It should be noted, however, that a restriction of the developmental Pr reduction to the high Pr synapses not necessarily
implies that the Pves reduction is so restricted. Since an increased pool size will have more impact on Pr in low than in
high Pves synapses (Hanse and Gustafsson 2001b), a developmental increase in preprimed pool size for all synapses could
counteract a decrease in Pves in low but not in high Pves
synapses, specifically if, as noted above, Pves in low Pr synapses is less affected by changes in calcium influx than is Pves
in high Pr synapses. However, this scenario is not supported by
our pool estimations, which did not indicate any significant
increase in preprimed pool size.
These results cannot explain why the amount of Ca2⫹
reaching the Ca2⫹ acceptors should decrease with age. A
possible explanation is a developmental decrease in action
potential duration that results in a reduced Ca2⫹ influx. The
action potential in the CA1 neuron somata undergoes a second
week maturation characterized by increased amplitude and
shortened duration (Spigelman et al. 1992) that likely reflects
an increased density of voltage-gated sodium and potassium
channels. We also observed a substantial developmental shortening of the fiber volley duration that possibly reflects a
shortening of the axonal (and bouton) action potential. In
common with PPF, this shortening was not affected by tetanus
toxin-induced activity blockade. However, whether such a
shortening should lead to a decreased Ca2⫹ influx that selectively affects high Pr synapses is unknown. Another possibility
is a developmental shift in the type of voltage-gated Ca2⫹
channel that is involved in the release. N-type voltage-gated
Ca2⫹ channels differ from P/Q and R types in their coupling to
release (Qian and Noebels 2001; Urbano et al. 2003), and
developmental changes in N-type channels have been described for some synapses (Iwasaki et al. 2000). However, we
found no evidence for such a developmental shift in that
␻-conotoxin, a N-type channel blocker, did not differentially
affect EPSPs at P6 and P12. This lack of developmental shift
in N-type channel contribution agrees what has been reported
for another cortical glutamate synapse, a thalamo-cortical one
(Iwasaki et al. 2000).
Alternatively, a reduction in the amount of Ca2⫹ reaching
the Ca2⫹ acceptors may relate to an increase in presynaptic
DEVELOPMENTAL CHANGE IN Pr AT THE CA3–CA1 SYNAPSE
ACKNOWLEDGMENTS
We thank Dr. Laurent Groc for help with the tetanus toxin in vivo injections.
GRANTS
This project was supported by the Swedish Medical Research Council
(Project nos. 05180 and 12600) and Göteborgs Läkaresällskap.
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