Playfair Neuroscience Unit, Toronto Hospital Research Institute, Department of Medicine (Neurology), Bloorview Epilepsy Program, University of Toronto, Toronto, Ontario M5T 2S8, Canada
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ABSTRACT |
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Ouanonou, A.,
Y. Zhang, and
L. Zhang.
Changes in the Calcium Dependence of Glutamate Transmission in
the Hippocampal CA1 Region After Brief Hypoxia-Hypoglycemia.
J. Neurophysiol. 82: 1147-1155, 1999.
Using
the model of hypoxia-hypoglycemia (HH) in rat brain slices, we asked
whether glutamate transmission is altered following a brief HH episode.
The HH challenge was conducted by exposing slices to a glucose-free
medium aerated with 95% N2-5% CO2, for ~4
min, and glutamate transmission in the hippocampal CA1 region was
monitored at different post HH times. In slices examined 8 h post HH,
CA1 synaptic field potentials are comparable in amplitude to controls,
but are less sensitive to experimental manipulations designed to
attenuate intracellular Ca2+ signals, as compared with
controls. Reducing calcium influx, by applying a nonspecific calcium
channel blocker Co2+ or lowering external Ca2+,
attenuated CA1 synaptic potentials much less in challenged slices than
in controls. Buffering intracellular Ca2+ by
bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic
acid-AM (BAPTA-AM) attenuated CA1 synaptic potentials in control but
not in slices post HH. Furthermore, minimally evoked excitatory
postsynaptic currents displayed a lower failure rate in post-hypoxic
CA1 neurons compared with controls. Based on these convergent
observations, we suggest that evoked CA1 glutamate transmission is
altered in the first several hours after brief hypoxia, likely
resulting from alterations in intracellular Ca2+
homeostasis and/or Ca2+-dependent processes governing
transmitter release.
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INTRODUCTION |
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Alteration in glutamate synaptic activity is the
key component of pathophysiology in ischemic brain damage
(Luhmann 1996; Xu and Pulsinelli 1996
).
Brief exposure of brain slices to hypoxia produces dramatic changes in
synaptic responses in the hippocampal CA1 region (Fujiwara et
al. 1987
; Hansen et al. 1982
; Leblond and
Krnjevi
1989
; Lobner and Lipton 1993
).
CA1 synaptic events mediated by ionotropic glutamate receptors are
initially enhanced in ~1 min after starting the hypoxic episode, and
then subsequently suppressed as the hypoxic episode continues. Synaptic
responses recover fully if reoxygenation occurs in ~4 min. The early
hypoxic enhancement likely results from the facilitation of glutamate release, via Ca2+ release from intracellular
stores and subsequent increase in intracellular
Ca2+ (Belousov et al. 1995
;
Katchman and Hershkowitz 1993a
). The later suppression
may be due to a failure in energy supply (Chung et al.
1998
; Kass and Lipton 1989
) and/or
adenosine-dependent inhibition of glutamate release (Gribkoff
and Bauman 1992
; Hershkowitz et al. 1993
;
Katchman and Hershkowitz 1993b
; Martin et al.
1994
; Wu and Saggau 1994
). Interestingly,
synaptic responses mediated by
N-methyl-D-aspartate (NMDA) or
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors
have been shown to be potentiated in slices after brief hypoxia
(Gozlan et al. 1994
; Hammond et al. 1994
; Hsu and Huang 1997
) or after temporary block of
glycolysis (Tekkök and Krnjevi
1995
,
1996
). Because hypoxia or ischemia is known to cause
elevation of intracellular Ca2+ (Choi
1995
; Haddad and Jian 1993
;
Lobner and Lipton 1993
; Meyer 1989
;
Mitani et al. 1994
; Siesjö and Bengtsson
1989
; Silver and Erecinska 1990
), a critical
factor for synaptic plasticity (Bliss and Collingridge
1993
), it is of interest to know whether alterations of
glutamate transmission occur in intermediate times after brief hypoxia.
To test the above possibility, we conducted a brief
hypoxic-hypoglycemic (HH) episode in adult rat brain slices, by
exposing slices to a glucose-free medium aerated with 95%
N2-5% CO2 rather than 95% O2-5%
CO2, for ~4 min. Different from previous studies that
monitor CA1 synaptic responses during and shortly (2 h) after brief
hypoxia, we made recordings in slices at the intermediate times post-HH
(
8 h). To study the Ca2+-dependent glutamate release, CA1
synaptic potentials were evoked during perfusing slices with modified
media that contained either the Ca2+ channel blocker
Co2+, high Mg2+-low Ca2+, or the
membrane permeant calcium chelator
bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid-AM (BAPTA-AM). We show that in slices recorded
8 h
postchallenge, CA1 synaptic responses were comparable in amplitude to
those of controls but reacted differently from controls to the
treatments designed to alter intracellular Ca2+ signals.
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METHODS |
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Hypoxic challenge and electrophysiological recordings in brain
slices have been previously described (Chung et al.
1998; Perez-Velazequez and Zhang 1994
;
Zhang and Krnjevi
1993
). Briefly, male Wistar rats (200-300 g) were deeply anesthetized with halothane and
decapitated. To minimize the consequence of decapitation hypoxia, the
brain was quickly dissected out (within 45 s) and maintained in an
ice-cold, oxygenated artificial cerebrospinal fluid (ACSF) for 3-20
min before slicing. Hemisectioned brain was then mounted on an aluminum block, and cut transversely (400 µm thicknesses) using a vibrotom (series 1000, Tech. Prod. International, St. Louis, MO). The ACSF contained (in mM) 125 NaCl, 2.5 KCl, 1.25 NaH2PO4, 2 CaCl2, 1.3 MgCl2, 26 NaHCO3, and 10 glucose, with pH 7.4 when aerated
with CO2 5%-O2 95% and
300 ± 5 mosM. For conducting a HH challenge, glucose in
the ACSF was replaced equimolarly with sucrose, and 5%
CO2-95% N2 rather than 5%
CO2-95% O2 was used for aeration.
After slicing, brain sections were kept in a glass container filled
with the warm (32-33°C), oxygenated (5% CO2-95%
O2) ACSF for at least 1 h before further
manipulations. After 1 h of stabilization, about half of the
slices were gently transferred to another container filled with the
glucose-free ACSF and aerated with 5% CO2-95% N2 (HH), for a period of 3.5-4 min. After the HH
challenge, slices were returned to the original container and
maintained there together with the control slices. To control effects
due to mechanical disturbance associated with the HH challenge and for
identification, each control slice was placed on a small piece of
filter paper using a fine painting brush while kept in the original
oxygenated container. By holding the filter paper, the slice was then
taken out of the ACSF for a few seconds, and an incision was made
cutting the thalamo-brain stem portion through the filter paper. After cutting, the slices were returned to the original container
immediately, and the attached filter paper was removed. We found no
evident change in CA1 electrophysiological responses recorded from the cut slices as compared with nontreated slices (Ouanonou et al. 1996; Zhang et al. 1994
). Both control and
challenged slices were maintained in the same container, and during the
in vitro maintenance 20-30% of the medium in the container was
replaced with the fresh ACSF every 2 h. The ACSF temperature in
the containers was maintained at 32-33°C using a water bath.
For electrophysiological recordings, slices were transferred to a fully submerged chamber and perfused with oxygenated (5% CO2-95% O2) ACSF continuously. Humidified, warm air of 5% CO2-95% O2 was also applied over the perfusate to increase oxygen tension in the local environment. All recordings were done at the bath temperatures of 32-33°C. Synaptic field potentials were recorded extracellularly using a glass pipette filled with 150 mM NaCl. The recording pipette was placed in the stratum pyramidale (soma) or stratum radiatum (dendrite) of the CA1 region, respectively. The Schaffer collateral pathway was stimulated electrically, by placing a bipolar tungsten electrode in the stratum radiatum at the CA1-CA2 border. Constant current pulses of 0.1 ms duration were generated through a Grass S88 stimulator and delivered through an isolation unit every 15-20 s. To examine paired pulse facilitation (PPF) in dendritic synaptic responses, twin stimuli at 50% of the maximal intensity were delivered at an interpulse interval of 50 ms. The PPF was determined by measuring the percent increase in the peak amplitude of the second response, taking the first response as 100%.
For whole cell recordings, we used a patch pipette solution containing
(in mM)150 potassium methylsulfate, 2 HEPES, 0.5 ATP, and 0.1 EGTA,
with pH 7.25 adjusted with KOH and 280 ± 10 mosM (Zhang et
al. 1994). Filled with this solution, the tip resistance of the
patch pipette was ~4 M
. Electrical signals were recorded using an
Axoclamp 2A amplifier or Axopatch amplifier 200 B (Axon Instruments,
Foster City, CA). The low-pass single-pole filter of the Axoclamp
amplifier was set at 1 kHz, and the Bessel filter of the Axopatch
amplifier (200B) was set at 2 or 5 kHz. After breaking through
membrane, series resistance was usually <15 M
, and resistance
compensation of the patch amplifier was set near 80%. Data were
acquired, stored, and analyzed with Pclamp software (Version 5.5 or
6.3, Axon Instruments), through a 12-bit A/D interface (TL-1 or
Digitata 1200, Axon Instruments).
To elicit minimal synaptic responses, Schaffer collateral afferent
fibers were stimulated by a glass pipette filled with 150 mM NaCl. The
location and intensity of afferent stimulation were adjusted until
all-or-none excitatory postsynaptic currents (EPSCs) were observed.
Evoked minimal EPSCs were sampled in the whole cell voltage-clamp mode
at holding potentials more negative than 70 mV. Minimal EPSCs were
included into data analysis if they fulfilled the following criteria:
1) EPSCs displayed small amplitudes and fast decay;
2) failure rate and amplitude distribution of EPSCs were
independent of stimulation intensity in the range of approximately
fivefold above the threshold stimulus; and 3) EPSCs were
stable for at least 7-8 min when evoked at 0.1 Hz (cf. Allen and Stevens 1994
; Raastad et al. 1992
). Due to
the limitation of the signal/noise ratio, failure of synaptic
transmission was considered if the evoked responses were <5 pA and/or
lack of a fast rising phase. The failure rate of EPSCs (%) for each
individual neuron was calculated from >50 consecutive measurements.
To examine the amplitude distribution of minimal EPSCs, the EPSCs collected from a group of neurons were pooled together (n = 16 or 19 from control or challenged slices) and binned every 5 pA. The mean amplitude of each binned group was plotted versus the number of EPSCs included in the group (Fig. 6). The decay time course of the EPSC was determined by fitting a single exponential function to each individual event, using the Pclamp software (version 6.3, Axon Instruments).
All solutions were made with deionized sterile water (pH 5-6,
resistance 18.2 M/cm) from a Milli-Q UV plus system. Chemicals for
making the patch pipette solutions were purchased from Fluka (New York,
NY), except potassium methylsulfate (ICN, New York). BAPTA-AM was
purchased from Molecular Probes (Eugene, OR). BAPTA-AM was dissolved
initially in DMSO as a stock solution and then appropriately diluted to
the ACSF. The final concentration of DMSO in the ACSF was
0.2%. To
control the effect of DMSO, equal amount of DMSO was included in the
control solution before the application of BAPTA-AM. Other drugs were
purchased from Sigma (St. Louis, MO) or Tocris Cookson (Ballwin, MO).
Means ± SE are given throughout the text.
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RESULTS |
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CA1 synaptic field potentials measured from control or HH challenged slices
In control slices incubated in the ACSF at 32-33°C for 8 h,
stimulation of the Schaffer collateral pathway induced synaptic field
potentials in both somatic and dendritic areas of the CA1 neurons. In
response to afferent stimulation at maximum intensity, the amplitudes
of the somatic population spike and dendritic field excitatory
postsynaptic potential (EPSP) were 4.5 ± 0.5 mV and 1.6 ± 0.5 mV, respectively (Table 1, Fig.
1A). When stimulated with the
PPF paradigm (see METHODS), the dendritic field EPSP following the second stimulus was enhanced by 35.2 ± 3.2%
(n = 7, Table 1, Fig. 1B). Perfusion of
slices with 10 µM CNQX, a potent AMPA/kainate receptor antagonist,
attenuated the dendritic field EPSPs by 83.4 ± 9.2%
(n = 4), suggesting their mediation by non-NMDA
glutamate receptors (Shinno et al. 1997
; Zhang et al. 1997
).
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We then recorded CA1 synaptic field potentials in slices at 6-8 h
post-HH. In response to the maximal afferent stimulation, the
amplitudes of CA1 synaptic responses measured post HH were not
significantly different from those of control slices, but the PPF in
the dendritic field EPSPs was significantly greater (56.8 ± 10.1%, n = 5) than controls (Table 1; Fig. 1,
C and D). The field EPSPs recorded post-HH showed
similar suppression as controls following applications of 10 µM CNQX
(by 89.1 ± 9.3%, n = 4), suggesting that the
pharmacological properties of the CA1 field EPSPs were not
substantially altered in slices post-HH challenge. Because multiple
presynaptic mechanisms are involved in the PPF (Clark et al.
1994; Nathan and Lambert 1991
), including elevated Ca2+ in presynaptic terminals (Wu
and Saggau 1993
), we further examined whether there are changes
in the calcium dependence of CA1 glutamate transmission in slices
recovered for
8 h after the HH challenge.
Relation between CA1 synaptic responses and calcium influx
We first examined the effects of CoCl2, a
nonspecific calcium channel blocker, on CA1 dendritic field EPSPs.
Slices were initially perfused with standard ACSF (zero added
CoCl2), and CA1 field EPSPs evoked by maximal
afferent stimulation were collected as the baseline control. The
dendritic field EPSPs rather than somatic population spikes were
monitored because they are mediated by CNQX-sensitive AMPA/kainate
glutamate receptors and measurements of their amplitudes are less
complicated as compared with synchronized somatic population spikes.
After stable baseline recordings were achieved, slices were perfused
sequentially with the ACSF that contained 0.01, 0.1, 0.3, 0.5, 1, or 2 mM CoCl2. Each concentration of
CoCl2 was applied 5 min until the change in the
field EPSPs was stabilized. In control slices following 3-6 h of in
vitro incubation, CA1 field EPSPs of 1.8 ± 0.1 mV
(n = 8) were observed before
CoCl2 application. Following applications of
CoCl2, these EPSPs showed a
concentration-dependent decrease in amplitude, with a significant
decrease starting from 0.3 mM CoCl2 and a
calculated EC50 of 0.5 mM for external
CoCl2 (Fig. 2,
A1 and A3).
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In slices recorded 3-6 h after the HH challenge
(n = 9), CA1 field EPSPs were comparable with those in
the control group, with a mean amplitude of 1.9 ± 0.2 mV measured
in the standard ACSF. However, significant decrease in the field EPSPs
was not observed until external CoCl2 reached the
concentration of 0.5 mM. The dose-response plot calculated for the
challenged slices showed a rightward shift as compared with the
control, with an EC50 of 1.2 mM for external
CoCl2 (Fig. 2, A2 and A3).
Neither control nor challenged slices showed proportional decreases in presynaptic volleys following applications of
CoCl2 (Fig. 2, A1 and A2),
suggesting that decreases in synaptic potentials by
CoCl2 result largely from reduction in
Ca2+-dependent glutamate release rather than
attenuation of local excitability of afferent axon fibers.
To examine effects of external Ca2+ on the
CA1 synaptic potentials and to control possible nonspecific effects of
CoCl2, we modified the ACSF by keeping external
Mg2+ constant at 4 mM and reducing external
Ca2+ from 2 mM to 1, 0.5, or 0.1 mM,
respectively. The aim of this approach was to reduce transmembrane
Ca2+ gradient and hence the
Ca2+ influx associated with the afferent impulse.
Slices were exposed sequentially to the low-Ca2+
ACSF for 8 min to ensure the steady-state response. In control slices
incubated 2-7 h after dissection, CA1 field EPSPs evoked by the
maximal afferent stimulation had amplitudes of 1.7 ± 0.2 mV in
the presence of 2 mM external Ca2+. Lowering
external Ca2+ from 2 mM to 1, 0.5, or 0.1 mM
decreased CA1 field EPSPs by 21.7 ± 5.0%, 58.5 ± 6.0%, or
90.2 ± 1.8%, respectively (n = 10, Fig. 2,
B1 and B3).
In slices examined 2-6 h post-HH challenge, amplitudes of CA1 field EPSPs were comparable with the controls, with a mean value of 1.5 ± 0.1 mV (n = 12) in the presence of 2 mM external Ca2+. However, lowering external Ca2+ caused smaller changes in the post-HH EPSPs than controls, and the mean decreases were 6.4 ± 5.3%, 33.4 ± 11.2%, or 62.9 ± 6.5% as external Ca2+ was reduced to 1, 0.5, or 0.1 mM, respectively (Fig. 2, B2 and B3). Neither control nor challenged slices showed attenuation in presynaptic volleys when perfused with low-Ca2+ medium (Fig. 2, B1 and B2). These observations, when taken together with the differential effects of CoCl2 mentioned above, suggest an alteration in Ca2+-dependent glutamate transmission in slices following a HH challenge.
Failure of BAPTA-AM to attenuate CA1 synaptic potentials in slices post-HH
We then examined the effects of BAPTA-AM, a
membrane-permeant calcium chelator, on CA1 dendritic field EPSPs, in an
attempt to buffer intracellular Ca2+ associated
with the afferent impulse. To promote intracellular accumulation of
BAPTA, 0.5-1 mM probenecid, an anion transporter inhibitor, was
included in the perfusate before and during the applications of
BAPTA-AM (Ouanonou et al. 1996, 1999
). At
the concentrations we used, probenecid caused no consistent change in
the CA1 field EPSPs, but transient decreases in field EPSPs were
observed in some slices with unknown mechanisms. To control side
effects of this agent, slices were perfused with 0.5-1 mM probenecid
for
15 min until stable field EPSPs were achieved, and the same
concentration of probenecid was present in the BAPTA-containing perfusate throughout the application period.
In control slices incubated 4-8 h in vitro, the mean amplitude of CA1
dendritic field EPSPs was 0.9 ± 0.1 mV (n = 16)
following the afferent stimulation near 50% of the maximum strength.
After applications of BAPTA-AM (10 µM, 20-25 min), these field EPSPs were decreased by 43.6 ± 9.4% (P < 0.001, paired t-test; Fig. 3,
A and C), as measured at the end of BAPTA-AM
application. The decreases in field EPSPs were partially reversible
after washing BAPTA-AM for 30 min, suggesting a suppression of
synaptic potentials by the calcium chelator rather than a
time-dependent deterioration in synaptic responses.
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In slices (n = 11) that had recovered for 4-8 h after the HH challenge, CA1 field EPSPs were comparable in amplitude to controls, with a mean value of 0.8 ± 0.1 mV evoked by the half-maximal stimulation. However, in contrast to the control response, applications of 10 µM BAPTA-AM caused no decrease, but rather lead to a small increase by 16.1 ± 4.7% (P < 0.05, paired t-test) in the field EPSPs (Fig. 3, B and C). These changes induced by BAPTA-AM were significantly different from those in controls (P < 0.01, nonpaired t-test). Small increases in CA1 field EPSPs by BAPTA-AM were also observed at other post-HH times, with a mean change of 19.8 ± 10.1% (n = 7) or 21.3 ± 7.3% (n = 6) in slices recovered 1-3 and 9-12 h after the HH challenge, respectively.
Minimal EPSCs in CA1 neurons post-HH
The above data were obtained by using extracellular
recordings of synaptic field potentials, which represent summed
responses from a population of glutamate synapses. To examine the
post-HH changes in separate glutamate synapses, CA1 pyramidal neurons were recorded in the whole cell voltage-clamp mode, and EPSCs were
evoked using a minimal stimulation paradigm (Allen and Stevens 1994; Raastad et al. 1992
) (see also
METHODS). To block the voltage-dependent and
Mg2+-sensitive synaptic responses mediated by
NMDA receptors, CA1 neurons were held at potentials of
70 to
80 mV.
Bicuculline methiodide (10 µM) was bath applied throughout the
recording period to block synaptic currents mediated by
GABAA receptors. Once the whole cell recordings
were achieved, the stimulation intensity and position were adjusted
until small, all-or-none responses were obtained, such that the
amplitude and failure rate of the evoked EPSCs were independent from
the stimulation intensity in a certain range (see METHODS).
Two identical stimuli separate by 30 ms were delivered every 10 s.
It is expected that the second EPSC would have kinetics similar to that
of the first one if they originate from the same, but limited release
sites. Control neurons (n = 16) were recorded from
slices maintained in vitro for 2-8 h after sectioning, and challenged
neurons (n = 19) were recorded from slices 2-6 h after
the HH challenge.
In control CA1 neurons (n = 16), minimal EPSCs
displayed a large variability in their amplitude, ranging from 5 to 120 pA. To reveal the amplitude distribution of the minimal EPSCs, synaptic currents evoked by the first or second stimulus were pooled together and binned every 5 pA. Then the mean amplitudes were plotted versus the
number of events included in each bin (Fig.
4A). Of the 576 events evoked
after the first stimulus, EPSCs with amplitude of 55 pA accounted for
28.3% of the total events (Fig. 6A). In response to
50
consecutive afferent stimuli in each neuron examined, minimal EPSCs
exhibited mean failure rates of 69.9 ± 2.1% and 48.6 ± 3.3% (Fig. 4, B and C) as evoked by the twin
stimuli. We measured the rise time and the decay time constant of
EPSCs. The latter was determined by computing a single exponential fit
to each individual event. Following the twin minimal stimuli, EPSCs
displayed a mean amplitude of 40.8 ± 0.9 and 53.4 ± 0.9 pA,
rise time of 2.8 ± 0.1 and 3.0 ± 0.1 ms, and decay time
constant of 7.2 ± 0.2 and 8.3 ± 0.4 ms, as measured from
576 and 796 events, respectively. The comparable kinetics between the
twin EPSCs implies that a small number of synapses are activated after
the minimal stimulation. When the amplitudes of EPSCs evoked by the
first stimulus were plotted versus their rise time or decay time
constant, no significant correlation was found between these parameters
(linear correlation coefficient factor
R2 < 0.1). These observations are
consistent with previous studies (Allen and Stevens
1994
; Raastad et al. 1992
), suggesting that the
measurement errors owing to the space-clamp limitation is not the
dominant factor responsible for the variability in the EPSC amplitude
observed.
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Minimal EPSCs were readily evoked from CA1 neurons
(n = 19) in slices post-HH. The amplitude distribution
of these EPSCs was slightly different from that observed in controls,
such that of a total of 416 events evoked by the first stimulus, only
41 EPSCs (10%) had amplitudes of 55 pA (Fig. 4D). The
failure rate of first or second EPSCs was 59.4 ± 1.9% or
37.5 ± 2.6%, which is slightly but significantly lower than that
in controls (69.9 ± 2.1% or 48.6 ± 3.3%,
P < 0.05, Student's t-test). In addition, spontaneous EPSCs or delayed EPSCs following afferent stimulation (Fig.
4E) were often observed in CA1 neurons post-HH challenge. As
found in control neurons, EPSCS evoked by the first and second stimulus
shared similar kinetic parameters, with amplitudes of 29.1 ± 0.9 and 32.1 ± 0.9 pA, rise times of 3.6 ± 0.1 and 3.3 ± 0.1 ms, and decay time constants of 9.8 ± 0.4 and 8.1 ± 0.5 ms (n = 381 and 416), respectively. No significant
correlation was found between the amplitude and rise time or decay time
constant of these EPSCs (linear regression coefficient factor
R2 < 0.1). However, the decay time
constants of first EPSCs were significantly longer in post-HH neurons
than controls (P < 0.05), which may reflect changes in
postsynaptic AMPA receptors as suggested in postischemic neurons
(Gorter et al. 1997
;
Pellegrini-Giampietro et al. 1993
; Rump et al.
1996
; Tsubokawa et al. 1994
; Urban et al. 1989
).
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DISCUSSION |
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In the present experiments, brief HH was made by exposure of adult
rat brain slices to glucose-free ACSF aerated with nitrogen rather than
oxygen for ~4 min. We chose this in vitro HH model because the
challenge shares some common features with ischemic insults in vivo,
particularly an over-stimulation of glutamate NMDA receptors and
disruption of normal Ca2+ homeostasis
(Kass and Lipton 1986; Michaels and Rothman
1990
). In previous studies the similar HH paradigm has been
shown to cause a reversible depression in CA1 synaptic potentials
(Hammond et al. 1994
; PerezVelazquez and Zhang
1994
; Small et al. 1997
), an elevation of
intracellular Ca2+ (Lobner and Lipton
1993
; Mitani et al. 1994
; Silver and
Erecinska 1990
), and alterations in gene expression in
hippocampal slices postchallenge (Charriaut-Marlangue et al.
1992
; PerezVelazquez and Zhang 1994
).
Extracellular recordings of dendritic field potentials were used in
most of the present experiments because these responses represent the
summed activity resulting from a population of glutamate synapses and
they are much more stable as compared with single-cell recordings
during experimental manipulations. We present convergent evidence
suggesting that a brief HH challenge may cause significant alterations
in the Ca2+ dependence of glutamate transmission
in hippocampal CA1 region.
In an attempt to attenuate the Ca2+ influx
associated with the afferent impulse, slices were perfused with a
modified ACSF containing Co2+ or high
Mg2+-low Ca2+. We found
that these two treatments attenuated CA1 synaptic potentials in both
control and post-HH slices, but the extent of attenuation was smaller
in the challenged slices than in controls. The
EC50 for external Co2+ to
attenuate CA1 synaptic potentials was increased from 0.5 mM in control
to 1.2 mM in challenged slices (Fig. 2A). Similarly, lowering external Ca2+ from 2 to 0.1 mM decreased
CA1 synaptic response by 90% in control, but only 60% in slices
postchallenge (Fig. 2B). Neither applying Co2+ nor lowering external
Ca2+ changed the presynaptic volley in both
groups of slices, suggesting that the differences observed between
these two groups are largely due to alterations in the
Ca2+-dependent glutamate release. However,
possible alterations of postsynaptic glutamate receptors, as observed
in postischemic neurons (Gorter et al. 1997;
Pellegrini-Giampietro et al. 1993
; Rump et al.
1996
; Tsubokawa et al. 1994
; Urban et al.
1989
), and their responses to the Co2+ treatment
may also be factors yet to be determined.
BAPTA-AM was used as an alternative to attenuate intracellular calcium
signals without blocking calcium channels or reducing transmembrane
Ca2+ gradient. Previous studies have shown attenuated
synaptic transmission by BAPTA-AM in mammalian CNS neurons and other
neural preparations, largely due to buffering intracellular
Ca2+ responsible for the transmitter release processes
(Adler et al. 1991; Niesen et al. 1991
;
Ouanonou et al. 1996
, 1999
;
Spigelman et al. 1996
; Tymianski et al.
1994
). We show here that external BAPTA-AM is effective in
decreasing the CA1 field EPSPs in control slices, in keeping with
previous studies mentioned above, but the similar BAPTA-AM application
was ineffective to decrease CA1 field EPSPs in slices post-HH. The
later observation is consistent with resistance of the challenged CA1
synaptic potentials to the Co2+ or low Ca2+
treatment. Collectively, these observations suggest altered dynamics between intracellular Ca2+ and release processes in CA1
glutamate synapses post-HH, by which these synapses become insensitive
to a moderate decrease in intracellular Ca2+ at levels
sufficient to attenuate glutamate release in controls. It remains to be
clarified whether BAPTA loading or distribution in challenged
presynaptic terminals is comparable with that in control. A direct
measurement of Ca2+ signals in presynaptic terminals may
provide some clues regarding this issue (Wu and Saggau
1993
, 1994
).
To demonstrate the hypoxic alterations occurring at individual CA1
glutamate synapses, we monitored glutamate EPSCs from singly recorded
CA1 pyramidal neurons using a minimal stimulation paradigm (Allen and Stevens 1994; Raastad et al.
1992
). The minimal EPSCs displayed an all-or-none like
response, presumably reflecting glutamate release following the
impulses generated from a few afferent fibers. If the processes
responsible for the Ca2+-dependent glutamate release are
promoted after the HH challenge such that intracellular
Ca2+ signals are more efficient than normal to induce
functional transmission, one may expect a lower EPSC failure rate in
challenged neurons. In keeping with this view, minimal EPSCs were
readily observed in recordings from post-HH neurons, with the failure
rate slightly but significantly lower than that of controls.
However, the data regarding minimal EPSCs must be interpreted with
caution. First, because of the limitations in the signal/noise ratio
and the effectiveness of the space clamp, particularly for synapses
electrotonically distant from the somatic recording, the amplitude
and/or kinetics of EPSCs may not be accurately measured. EPSCs with
small amplitudes may not be detected under our recording conditions,
therefore causing measurement errors in assessing the failure of
minimal EPSCs. Second, the failure rate of minimal EPSCs represents not
only the probability in transmitter release, but also the functional
state of axon conduction and/or intrinsic axonal excitability
(Allen and Stevens 1994). In extracellular recordings,
CA1 presynaptic volleys with large amplitude seem to be frequently
observed in slices post-HH but with large variability. No intention was
made in the present study to compare the amplitude/waveform of CA1
presynaptic volleys between control and challenged slices, because of
difficulty in controlling the precise transverse slicing plan hence the
preservation of Schaffer collateral afferent fibers in each individual
slice. However, the issue remains as to whether there are HH-induced
changes in the intrinsic excitability of Schaffer collateral fibers.
Third, previous studies have revealed a wide heterogeneity in
morphology and functionality of CA1 glutamate synapses (see review by
Edwards 1995
). For example, at least two classes of
glutamate synapses with a sixfold difference in their release
probability have been noted in the Schaffer collateral-CA1 pathway. The
synapses with lower release probability contribute over half of the
transmission examined in the standard slice recording conditions
(Hessler et al. 1993
). Also, glutamate synapses may undergo activity-dependent changes in release probability (Goda and Steven 1994
) and alterations in postsynaptic AMPA/kainate receptors following the ishemic insult (Benke et al.
1998
; Gorter et al. 1997
; Hu et al.
1998
; Pellegrini-Giampietro et al. 1993
; Rump et al. 1996
; Tsubokawa et al.
1994
; Urban et al. 1989
). Thus it may well be
that synapses with altered release probability and/or postsynaptic
receptors are involved in our samplings in control and/or post-HH
slices. In viewing the above complications, it is tentatively suggested
that there may be an increase in the likelihood of transmitter release
at CA1 glutamate synapses post-HH challenge.
We propose that the brief HH challenge causes alterations in
Ca2+-dependent processes that govern the evoked transmitter
(glutamate) release, such that these processes become more sensitive to
the intracellular Ca2+ signals associated with the afferent
impulse. Thus a moderate reduction of intracellular Ca2+
signals, by buffering intracellular Ca2+, blocking
voltage-gated Ca2+ channels or lowering the transmembrane
Ca2+ gradient, would have weaker influence on glutamate
synaptic potentials in challenged slices than controls. Several factors
may be responsible for the altered synaptic physiology, including
1) elevated intracellular Ca2+ owing to
enhanced entry, retarded buffering, and/or removal, 2)
increased sensitivity of synaptic proteins or release processes to
corresponding intracellular Ca2+ signals, 3)
involvement of more synapses with higher release probability. We have
no direct evidence supporting these possibilities at present.
Considering the dynamic interaction between the intracellular Ca2+ and synaptic proteins (Sheng et al.
1996) and the resulting differential modulations on transmitter
release (Mochida et al. 1996
), it is conceivable that
the multimechanisms may be involved in the post-HH alterations in CA1
glutamate transmission.
Potentiation of NMDA or AMPA receptor-mediated EPSPs has been
found in the CA1 region of slices after brief hypoxia or temporary inhibition of glycolysis with 2-deoxyglucose, termed "anoxic
long-term potentiation" (anoxic LTP) (Hammond et al.
1994; Hsu and Huang 1997
) or "2-DG-LTP"
(Tekkök and Krnjevi
1995
). Complexes of cellular mechanisms are involved in inducing the anoxic LTP or 2-DG-LTP, including activation of NMDA receptors, Ca2+
entry and subsequent increase in intracellular Ca2+, and
nitric oxide production (Crepel and Ben-Ari 1996
;
Huang and Hsu 1997
; Tekkök and
Krnjevi
1996
). Although we have not examined the nature
of NMDA receptor activation and the rise of intracellular
Ca2+ involved in the HH episode we employed, it is likely
that similar signal transduction cascades may also account for the
apparent facilitation of CA1 glutamate transmission post-HH. Our data
support the previous studies mentioned above, further suggesting that the apparent alteration of CA1 glutamate transmission can persist for
several hours after brief HH.
In summary, the present experiments show that brief HH causes a
substantial alteration in CA1 glutamate potentials that last several
hours postchallenge. This is manifested by the findings that challenged
CA1 synaptic potentials are less sensitive to procedures reducing
intracellular Ca2+, and unitary glutamate EPSCs are more
persistent as compared with controls. We propose that the evoked
glutamate transmission is facilitated post-HH, as the result of
alteration of Ca2+-dependent processes that govern the
release processes. It remains to be shown whether similar alterations
take place in CA1 glutamate synapses after transient ischemia in vivo,
and if so, how they are related to the delayed CA1 neuronal
degeneration (Kirino 1982; Pulsinelli et al.
1982
; Shinno et al. 1997
, Zhang et al.
1997
).
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ACKNOWLEDGMENTS |
---|
A. Ouanonou and Y. Zhang made equal contributions to this project. L. Zhang is a research scholar of Canadian Heart and Stroke Foundation. The authors thank Dr. Peter L. Carlen for support and criticism during the course of this work.
This work was supported by the Heart and Stroke Foundation of Canada and Ontario.
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FOOTNOTES |
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Address for reprint requests: L. Zhang, Playfair Neuroscience Unit, Toronto Hospital (Western Division), Room McL. 13-411, 399 Bathurst St., Toronto, Ontario M5T 2S8, Canada.
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.
Received 11 May 1998; accepted in final form 7 April 1999.
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REFERENCES |
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