J Neurophysiol
87: 2929 –2935, 2002; 10.1152/jn.00504.2001.
Synaptic and Nonsynaptic Ictogenesis Occurs at Different
Temperatures in Submerged and Interface Rat Brain Slices
S. SCHUCHMANN,1 H. MEIERKORD,2 K. STENKAMP,1 J. BREUSTEDT,1 O. WINDMÜLLER,2
U. HEINEMANN,1 AND K. BUCHHEIM2
1
Institut für Physiologie and 2Neurologische Klinik und Poliklinik, Universitätsklinikum Charité,
Humboldt-Universität Berlin, D-10117 Berlin, Germany
Received 18 June 2001; accepted in final form 16 January 2002
Address for reprint requests: S. Schuchmann, Institut für Physiologie, Universitätsklinikum Charité, Humboldt-Universität Berlin, Tucholskystr. 2,
D-10117 Berlin, Germany (E-mail: [email protected]).
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.
INTRODUCTION
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Epileptiform activity can be induced in the presence and in
the absence of synaptic transmission (Anderson et al. 1986;
Jefferys and Haas 1982; Taylor and Dudek 1982). Epileptiform
activity induced by low-Mg2⫹ artificial cerebrospinal fluid
(ACSF) results from increased synaptic excitation due to facilitated activation of N-methyl-D-aspartate (NMDA) receptors
(Mody et al. 1987). Epileptiform activity induced by low-Ca2⫹
ACSF depends on nonsynaptic mechanisms in regions of
densely packed neurons, including increases in excitability by
field effects, electrical coupling by gap junctions, and fluctuations in extracellular K⫹ concentration (Jefferys 1995; Konnerth et al. 1984; Taylor and Dudek 1984).
Due to its physiological basis, the induction and maintenance of low-Mg2⫹- and low-Ca2⫹-induced epileptiform activity is influenced by temperature, pH, and extracellular volume. Changes in temperature have been shown to modulate
extracellular ionic environment and therefore synaptic transmission in hippocampal slices (Igelmund and Heinemann
1995). Furthermore, extracellular pH has been demonstrated to
modulate low-Mg2⫹-induced epileptiform discharges as well
as nonsynaptic field bursts induced by low-Ca2⫹ (Schweitzer et
al. 2000; Velisek et al. 1994). Finally, changes in extracellular
volume have been shown to alter spontaneous epileptiform
activity in presence of synaptic transmission but also independent of chemical synaptic transmission (Baran et al. 1986;
Dudek et al. 1990; Rosen and Andrew 1990).
Brain slice experiments are differently influenced by
changes in temperature, pH, and extracellular volume depending on the experimental condition, i.e., interface or submerged
condition. Studies using hippocampal brain slices were mainly
carried out by placing the slice on a liquid-gas interface (for
overview, see Aitken et al. 1995; Lipton et al. 1995). However,
a number of questions involving investigations on the slice
metabolism, rapid application of drugs, and microfluorometric
measurements using water-immersion objectives require submerged conditions with a fluid level of 2–3 mm above the brain
slice (Meierkord et al. 1997; Schuchmann et al. 1999). The
major difference of the chamber types is the maintenance of the
slices, i.e., gas-liquid interface compared with total-liquid submerged conditions. Therefore particularly temperature-sensitive O2 and CO2 supply and therefore pH stability and extracellular space volume may differ between interface and
submerged conditions.
The saturation of ACSF with O2 and CO2 is determined by
the pressure of the O2-CO2 gas mixture used to bubble the
ACSF and the solubility coefficient of O2 and CO2 in ACSF,
which depends on the ACSF temperature. Using a gas mixture
Schuchmann, S., H. Meierkord, K. Stenkamp, J. Breustedt, O.
Windmüller, U. Heinemann, and K. Buchheim. Synaptic and nonsynaptic ictogenesis occurs at different temperatures in submerged
and interface rat brain slices. J Neurophysiol 87: 2929 –2935, 2002;
10.1152/jn.00504.2001. To investigate the temperature sensitivity of
low-Ca2⫹-induced nonsynaptic and low-Mg2⫹-induced synaptic ictogenesis under submerged and interface conditions, we compared
changes of extracellular field potential and extracellular potassium
concentration at room temperature (23 ⫾ 1°C; mean ⫾ SD) and at
35 ⫾ 1°C in hippocampal-entorhinal cortex slices. The induction of
spontaneous epileptiform activity under interface conditions occurred
at 35 ⫾ 1°C in both models. In contrast, under submerged conditions,
spontaneous epileptiform activity in low-Mg2⫹ artificial cerebrospinal
fluid (ACSF) was observed at 35 ⫾ 1°C, whereas epileptiform discharges induced by low-Ca2⫹ ACSF occurred only at room temperature. To investigate the different temperature effects under submerged and interface conditions, measurements of extra- and
intracellular pH and extracellular space volume were performed.
Lowering the temperature from 35 ⫾ 1°C to room temperature effected a reduction in extracellular pH under submerged and interface
conditions. Under submerged conditions, temperature changes had no
significant influence on the intracellular pH in presence of either
normal or low-Mg2⫹ ACSF. In contrast, application of low-Ca2⫹
ACSF effected a significant increase in intracellular pH at room
temperature but not at 35 ⫾ 1°C under submerged conditions. Therefore increasing intracellular pH by lowering the temperature in lowCa2⫹ ACSF may push slices to spontaneous epileptiform activity by
opening gap junctions. Finally, extracellular space volume significantly decreased by switching from submerged to interface conditions. The reduced extracellular space volume under interface conditions may lead to an enlarged ephaptic transmission and therefore
promotes low-Mg2⫹- and low-Ca2⫹-induced spontaneous epileptiform activity. The results of the study indicate that gas-liquid interface
and total-liquid submerged slice states impart distinct physiological
parameters on brain tissue.
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METHODS
Slice preparation
The experiments were performed as described previously (Schuchmann et al. 1999). Briefly, brain slices (400 ␮m) containing the
temporal cortex area 3, the perirhinal cortex, the entorhinal cortex, the
subiculum, the dentate gyrus, and the ventral hippocampus were
prepared in a nearly horizontal plane from Wistar rats (150 –200 g)
after decapitation under deep ether anesthesia. The slices were stored
in an interface holding chamber at room temperature in oxygenated
(95% O2-5% CO2) ACSF, which contained (in mM) 124 NaCl, 3 KCl,
1.8 MgSO2, 1.6 CaCl2, 1.25 NaH2PO4, 26 NaHCO3, and 10 glucose
(pH 7.4). For the experiments, slices were individually transferred to
an interface-type recording chamber or a submersion-type recording
chamber (for review, see Haas and Büsselberg 1992). Under interface
conditions, slices were placed on lens paper (Eastman Kodak, Rochester, NY) and continuously perfused (1.5–2 ml/min) with oxygenated
(95% O2-5% CO2) ACSF. Additionally, humidified carbogen gas
mixture (95% O2-5% CO2) was directed over the surface of the slices.
Under submerged conditions, slices were placed on lens paper and
fixed with a thin thread grid. The slices were continuously superfused
(4 –5 ml/min) by flooding with 2–3 mm oxygenated (95% O2-5%
CO2) ACSF. Experiments were performed at room temperature (23 ⫾
1°C) and at 35 ⫾ 1°C.
J Neurophysiol • VOL
Ion-sensitive microelectrodes
Ion-selective microelectrodes were prepared using the method described by Heinemann and Arens (1992). Double-barreled glass was
filled on one side with 154 mM NaCl operating as reference. The
silanized ion-sensitive barrel (5% trimethyl-1-chlorosilane in 95%
dichloromethane) was front-filled by dipping the microelectrode into
potassium-potassium ionophore I cocktail A 60031 Fluka (K⫹-sensitive microelectrode), pH-hydrogen ionophore I cocktail A 95291
Fluka (pH-sensitive microelectrode), or tetraethylammonium (TEA⫹)potassium ion exchanger cocktail 477317 Corning (TEA⫹-sensitive
microelectrode). The method produces ion-sensitive microelectrodes
with short columns (⬍100 ␮m) of the respective ion sensor. Recordings of these short-column electrodes have been shown to possess
good stability against changes in temperature and fluctuation in the
level of the bath solution (Vaughan-Jones and Kaila 1986). The
electrodes used in the present study showed a temperature sensitivity
of 0.08 ⫾ 0.04 mV/°C in the interval from 22 to 37°C. A modified
Nernst equation was employed to obtain extracellular K⫹, TEA⫹
concentrations or pH values from recorded slices.
Changes in extracellular space volume were measured by adding 2
mM tetraethylammonium (TEA⫹, Sigma) to normal ACSF (Dietzel et
al. 1980). At this concentration, the Corning ion-exchanger containing
microelectrode is no longer sensitive to changes in extracellular K⫹
concentration (Huang and Karwoski 1992). Changes in extracellular
TEA⫹ concentration reflect inverse proportional alterations in extracellular space volume, and therefore relative changes in extracellular
space volume were obtained using the equation (Dietzel et al. 1980)
Change in extracellular space volume 共%兲
⫽ 共关TEA ⫹ 兴 submerged /关TEA ⫹ 兴 interface ⫺ 1兲 ⫻ 100
where [TEA⫹]submerged/interface describes the extracellular TEA⫹ concentration at submerged or interface conditions.
Intracellular pH measurements
Intracellular pH (pHi) was measured using the dual-wavelength
fluormetric hydrogen ion-sensitive dye 2⬘,7⬘-bis-(2-carboxyethyl)-5(and-6)-carboxyfluorescein (BCECF, Molecular Probes Europe, Leiden, Netherlands). Slices were loaded with dye by incubation in
normal, gassed ACSF containing 10 ␮M of the acetoxymethyl ester
form of BCECF for 20 –30 min at room temperature. After washing
the slices for 15 min using fresh ACSF, the dye was retained for 2–3
h. Fluorescence measurements were carried out under submerged
conditions using an imaging system based on a Zeiss Axioskop
microscope with ⫻10 and ⫻40 water-immersion objectives (numerical aperture 0.3 and 0.75, respectively; Zeiss, Jena, Germany), a
xenon light source with a combination of two monochromators (Photon Technology Instruments, Wedel, Germany) and a photomultiplier
(Seefelder Messtechnik, Seefeld, Germany). Ratio measurements
were calculated from emitted BCECF fluorescence (520 –550 nm,
dichroic mirror ,505 nm; longpass filter, 515) for excitation at 490 and
440 nm. Calibration was performed from separated slices incubated in
nigericin containing solutions of pH 6.0, 6.5, 7.0, 7.5, and 8.0 (Duchen
1992; Thomas et al. 1979).
Induction of epileptiform activity and data analysis
The viability of the slices was tested and monitored by observing
the extracellular field potential in the medial entorhinal cortex layer
III/IV following stimulation of the lateral entorhinal cortex. Slices
were accepted for study if extracellular field potentials were ⱖ2 mV
in amplitude. Epileptiform activity was induced by omitting Mg2⫹
(which contained, in mM: 124 NaCl, 3 KCl, 1.6 CaCl2, 1.25
NaH2PO4, 26 NaHCO3, and 10 glucose; pH 7.4) or Ca2⫹ (in mM: 124
NaCl, 5 KCl, 1.8 MgSO2,1.25 NaH2PO4, 26 NaHCO3 and 10 glucose;
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containing 95% O2-5% CO2 under normal air pressure (pO2 ⬃
96 kPa, pCO2 ⬃ 5 kPa), a reduction of the ACSF temperature
from 37 to 20°C causes an increase in the solubility coefficient
of ⬃25% for O2 and 35% for CO2 (Schmidt and Thews 1993).
These changes increase the concentration of O2 from ⬃1.0 to
⬃1.3 mM/l and the concentration of CO2 from ⬃1.3 to ⬃0.9
mM/l. Using the Henderson-Hasselbalch equation (bicarbonate
concentration, 26 mM; pK ⫽ 6.1), the change in CO2 concentration results a pH shift from 7.40 at 37°C to 7.22 at 20°C.
Under submerged conditions, pCO2 is determined by the
ACSF that is saturated with CO2, whereas in interface chambers the gas phase dominates the setting of pCO2 within the
slice (Voipio 1998). Therefore one would expect a reduced
temperature sensitivity of pH under interface conditions compared with submerged conditions.
Brain slices tend to loss thickness when placed on the
interface between gas and liquid due to the surface tension
(Haas and Büsselberg 1992). A compacting effect of brain
slices may in part effect a decrease in extracellular space
volume and therefore influence ephaptic transmission (Croning
and Haddad 1998).
To further clarify the role of temperature on spontaneous
synaptic and nonsynaptic epileptiform activity under interface
and submerged conditions, we have studied the effect of 35 ⫾
1°C and room temperature in presence of low-Mg2⫹ and lowCa2⫹ ACSF in hippocampal slices. In the present study, we
report that low-Mg2⫹-induced spontaneous epileptiform activity occurs at 35 ⫾ 1°C under interface and submerged conditions, whereas low-Ca2⫹-induced spontaneous epileptiform activity occurs only at room temperature under submerged
conditions and at 35 ⫾ 1°C under interface conditions. Measurements of extra- and intracellular pH and extracellular space
volume discovered differences under submerged and interface
conditions in presence of low-Mg2⫹ and low-Ca2⫹ ACSF,
which may modulate synaptic and nonsynaptic transmission
and therefore be in part responsible for the distinct conditions
to induce spontaneous epileptiform activity.
ICTOGENESIS AT DIFFERENT TEMPERATURES
pH 7.4) from the ACSF. In the low-Ca2⫹ ACSF, the induction of
spontaneous discharges required an elevation of K⫹ concentration to
5 mM (Watson and Andrew 1995). Low-Mg2⫹ discharges were
recorded in the medial entorhinal cortex layer III/IV, which has been
reported to represent a “focus” for generation of low-Mg2⫹-induced
seizure like events (Dreier and Heinemann 1991). Epileptiform activity induced by low-Ca2⫹ ACSF depends on nonsynaptic mechanisms
in regions of densely packed neurons (Jefferys 1995), and therefore
low-Ca2⫹ discharges were recorded in the somatic layer of the CA1
region. Data were stored on chart recorder or computer hard disk and
subsequently analyzed off-line. All values are given as means ⫾ SE.
Statistical differences were assessed by ANOVA and Bonferroni/
Dunn contrast.
RESULTS
Low-Mg2⫹-induced epileptiform activity under submerged
and interface conditions
interface conditions (Fig. 1, A and C). When the temperature
was lowered to room temperature, the discharges disappeared
under interface conditions within 134 ⫾ 28 s (n ⫽ 7) and under
submerged conditions within 72 ⫾ 22 s (n ⫽ 15; P ⫽ 0.041
interface vs. submerged conditions).
Low-Ca2⫹-induced epileptiform activity under submerged
and interface conditions
Nonsynaptic ictogenesis induced by omitting extracellular
Ca2⫹ occurred at room temperature under submerged conditions and at 35 ⫾ 1°C under interface conditions. No spontaneous epileptiform activity could be induced using low-Ca2⫹
ACSF at room temperature under interface conditions (Fig.
2D) and at 35 ⫾ 1 °C under submerged conditions (Fig. 2B).
Lowering the temperature from 35 ⫾ 1 °C to room temperature
during spontaneous low-Ca2⫹-induced epileptiform activity
under interface conditions caused the disappearance of the
discharges within 97 ⫾ 31 s (n ⫽ 7). Elevating the temperature
from room temperature to 35 ⫾ 1°C during spontaneous lowCa2⫹ induced epileptiform activity under submerged conditions resulted in the disappearance of discharges within 21 ⫾
7 s (n ⫽ 12). Figure 2C demonstrates the reversibility of
temperature depending loss and onset of low-Ca2⫹ induced
activity.
Under interface conditions in nine of nine slices, spontaneous series of SLEs induced by lowering extracellular Ca2⫹
concentration at 35 ⫾ 1°C could be recorded in the pyramidal
cell layer of area CA1 (Fig. 2E). The frequency of low-Ca2⫹induced SLEs was 2.03 ⫾ 0.18 min⫺1; their duration was
16.2 ⫾ 1.1 s (n ⫽ 30). During SLEs, extracellular potassium
concentration [K⫹]e increased to 8.0 ⫾ 0.3 mM (n ⫽ 30).
Under submerged conditions at room temperature (12 from 12
slices; Fig. 2A), the frequency of low-Ca2⫹-induced SLEs was
significantly higher (3.68 ⫾ 0.26 min⫺1, P ⫽ 0.032). When
compared with interface conditions at 35 ⫾ 1°C, the duration
of low-Ca2⫹-induced SLEs was significantly shorter under
submerged conditions at room temperature (8.4 ⫾ 2.2 s, n ⫽
30; P ⫽ 2䡠10⫺4). [K⫹]e increased to 7.7 ⫾ 0.8 mM (n ⫽ 30)
during SLEs under submerged conditions at room temperature
and showed no significant difference when compared with
interface conditions at 35 ⫾ 1°C (P ⫽ 0.53).
Changes in extracellular pH under submerged and interface
conditions
FIG. 1. Synaptic ictogenesis under submerged and interface conditions. A
and B: time course of changes in field potentials (f.p., top trace) and extracellular K⫹ concentration ([K⫹]e, bottom trace) during application of low-Mg2⫹
ACSF at room temperature (RT) and at 35°C under submerged conditions. C
and D: time course of changes in field potentials (f.p., top trace) and extracellular K⫹ concentration ([K⫹]e, bottom trace) during application of lowMg2⫹ ACSF at room temperature (RT) and at 35°C under interface conditions.
Note that low-Mg2⫹-induced epileptiform activity was only observed at 35°C
under both submerged and interface conditions.
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To investigate possible reasons for the induction of synaptic
and nonsynaptic epileptiform activity at room temperature and
35 ⫾ 1°C, we studied changes in extracellular pH (pHo) under
submerged and interface conditions. pH-sensitive microelectrodes placed in the entorhinal cortex and area CA1 of untreated slices (recording depth, 100 –150 ␮m; n ⫽ 8) showed
no significant differences in basal pHo between submerged and
interface conditions (submerged pHo 7.32 ⫾ 0.02, interface
pHo 7.34 ⫾ 0.02; P ⫽ 0.43). The typical slight extracellular
acidotic pHo shift from the slice surface (pHACSF 7.40 ⫾ 0.02)
to the recording depth of ⬃100 ␮m reflects the “respiratory
acidosis” of hippocampal brain slices (Voipio and Kaila 1993).
Figure 3 summarizes temperature dependent changes in extracellular pH under submerged and interface conditions. Temperature reduction of the perifused ACSF from 35 to 22°C
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Synaptic ictogenesis induced by omitting extracellular
Mg2⫹ occurred at a temperature of 35 ⫾ 1°C under both
submerged and interface conditions. Under interface conditions, in nine of nine slices, spontaneous series of seizure-like
events (SLEs) could be recorded in the medial entorhinal
cortex (Fig. 1D). The frequency of low-Mg2⫹-induced SLEs
was 0.43 ⫾ 0.14 min⫺1; their duration was 48.1 ⫾ 7.5 s (n ⫽
30). During SLEs extracellular potassium concentration [K⫹]e
increased to 7.6 ⫾ 0.4 mM (n ⫽ 30). Under submerged
conditions (15 from 15 slices; Fig. 1B), the frequency of
low-Mg2⫹-induced SLEs was slightly, but not significantly
higher (0.52 ⫾ 0.21 min⫺1, P ⫽ 0.22). Compared to interface
conditions, the duration of the SLEs was significantly shorter
(21.2 ⫾ 2.7 s, n ⫽ 30; P ⫽ 4䡠10⫺4). The [K⫹]e increases under
submerged conditions during SLEs showed no significant difference compared with interface conditions (6.8 ⫾ 0.3 mM,
n ⫽ 30; P ⫽ 0.46).
In contrast, at room temperature no epileptiform activity
could be induced with low-Mg2⫹ ACSF under submerged or
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temperature (23 ⫾ 1°C) caused a slight, but not significant,
decrease in basal pHi (entorhinal cortex: pHi 7.15 ⫾ 0.1, P ⫽
0.38; area CA1: pHi 7.16 ⫾ 0.14, P ⫽ 0.4).
In a further step, intracellular pH was investigated following
the application of low-Mg2⫹ ACSF (entorhinal cortex) and
low-Ca2⫹ ACSF (area CA1). In the presence of low-Mg2⫹
ACSF, neither the basal pHi at 35°C (7.18 ⫾ 0.1, n ⫽ 6) nor
the pHi shift following temperature reduction (pHi 7.16 ⫾ 0.19,
n ⫽ 6) showed differences compared with normal ACSF. A
different situation was observed in the presence of low-Ca2⫹
ACSF. Switching to low-Ca2⫹ ACSF at 35°C caused a slight,
but not significant increase of pHi to 7.29 ⫾ 0.09 (n ⫽ 6, Fig.
4B). This intracellular alkalization was enlarged to 7.43 ⫾ 0.1
(n ⫽ 12, vs. normal ACSF pHi 7.17 ⫾ 0.11, P ⫽ 8.2䡠10⫺3) by
lowering the temperature to room temperature in presence of
low-Ca2⫹ ACSF (Fig. 4C).
FIG. 2. Nonsynaptic ictogenesis under submerged and interface conditions.
A and B: time course of changes in field potentials (f.p., top trace) and
extracellular K⫹ concentration ([K⫹]e, bottom trace) during application of
low-Ca2⫹ ACSF at room temperature (RT) and at 35°C under submerged
conditions. Low-Ca2⫹ induced spontaneous epileptiform activity was observed
under submerged conditions only at room temperature (23 ⫾ 1°C). C: changes
in field potentials (f.p., top trace) and extracellular K⫹ concentration ([K⫹]e,
bottom trace) during low-Ca2⫹-induced activity during changes in temperature
under submerged conditions. Note the reversible loss of the activity following
a rise in temperature. D and E: time course of changes in field potentials (f.p.,
top trace) and extracellular K⫹ concentration ([K⫹]e, bottom trace) during
application of low-Ca2⫹-free ACSF at room temperature (RT) and at 35°C
under interface conditions. Low-Ca2⫹ induced spontaneous epileptiform activity was observed under interface conditions only at 35°C.
Extracellular space volume plays an important role in synaptic and nonsynaptic ictogenesis. To investigate differences in
extracellular space volume between submerged and interface
conditions, the extracellular space marker TEA⫹ was used.
Experiments were performed in the submerged recording
chamber (Fig. 5). To obtain interface conditions, the ACSF
level was lowered to the slice surface. Under submerged con-
(room temperature) under submerged conditions caused a
slight extracellular acidification of 0.08 pH units (35°C: pHo
7.32 ⫾ 0.02, 22°C: pHo 7.24 ⫾ 0.03, n ⫽ 6; Fig. 3A). A similar
temperature reduction only induced an extracellular decrease
of 0.04 pH units under interface conditions (35°C: pHo 7.34 ⫾
0.02, 22°C: pHo 7.30 ⫾ 0.02, n ⫽ 6; Fig. 3B). The reduced pHo
sensitivity to temperature under interface conditions compared
with submerged conditions underlines the importance of the
gas phase in this chamber type.
Changes in intracellular pH under submerged conditions
To investigate the effect of temperature, low-Mg2⫹ and
low-Ca2⫹ ACSF on intracellular pH (pHi), microfluorometric
measurements were performed using the fluorescence dye
BCECF. Due to the conditions required for microfluorometric
techniques, the measurements were only made under submerged conditions. Using normal ACSF, temperature-dependent changes in pHi were measured in the entorhinal cortex
(n ⫽ 3) and in the area CA1 (n ⫽ 6) of untreated slices (Fig.
4A). No differences in basal pHi were found between the two
regions (entorhinal cortex: pHi 7.17 ⫾ 0.1, area CA1: pHi
7.18 ⫾ 0.12). Temperature reduction from 35 ⫾ 1°C to room
J Neurophysiol • VOL
FIG. 3. Temperature-dependent changes in pHo and pHi in presence of
normal ACSF. A: measurement of pHo using pH-sensitive microelectrodes
during stepwise reduction of the temperature of the superfusate from 35 to
22°C under submerged conditions. B: measurement of pHo using pH-sensitive
microelectrodes during continuously reduction of the temperature of the perifusate from 35 to 22°C under interface conditions. C: changes in pHi following
temperature reduction under submerged conditions. The left trace demonstrates a typical measurement in area CA1. The right diagram summarizes the
changes in pHi from 6 slices. Lowering the temperature from 35°C to room
temperature effected a slight but not significant decrease in intracellular pH.
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Changes in extracellular space volume under submerged and
interface conditions
ICTOGENESIS AT DIFFERENT TEMPERATURES
ditions, TEA⫹-sensitive microelectrodes were placed in the
somatic layer of the CA1 region and normal ACSF plus 2 mM
TEA⫹ was applied until a stable [TEA⫹]e baseline was
reached. Changes in extracellular space volume can be detected as a transient signal in [TEA⫹]e with a relaxation time
constant that reflects the rate of net diffusion between the
extracellular volume and the perifusate. In the used submerged
recording chamber, the halftime of the relaxation time constant
was 6 ⫾ 2 min. To avoid artificial influences by TEA⫹ diffusion between the extracellular volume and the perifusate, the
switch from submerged to interface and back to submerged
conditions was implemented within 1 min.
Following the change from submerged to interface conditions [TEA⫹]e increased significantly from 2.0 ⫾ 0.02 to
2.16 ⫾ 0.03 mM (n ⫽ 8, submerged vs. interface condition
P ⫽ 3.5䡠10⫺4). This increase in [TEA⫹]e indicates a reduction
in extracellular space volume by 7.3 ⫾ 0.6% under interface
conditions compared with submerged conditions. The return
from interface to submerged conditions effected a significant
decrease in [TEA⫹]e to 2.02 ⫾ 0.02 mM (n ⫽ 8, interface
vs. submerged condition P ⫽ 4.3䡠10⫺4). This reduction in
[TEA⫹]e indicates an increase in extracellular space volume by
6.9 ⫾ 0.7% under submerged conditions compared with interface conditions.
DISCUSSION
The present study demonstrates the following findings: 1)
under interface conditions, both low-Mg2⫹ and low-Ca2⫹ induced ictogenesis occurred at 35 ⫾ 1°C. 2) Under submerged
conditions, low-Mg2⫹-induced ictogenesis occurred at 35 ⫾
1°C, whereas discharges induced by low-Ca2⫹ ACSF occurred
at room temperature. 3) Ongoing spontaneous epileptiform
activity vanishes reversibly by changing the corresponding
temperature. 4) Using either normal or low-Mg2⫹ ACSF, the
reduction of temperature causes a slight extracellular acidosis
J Neurophysiol • VOL
but has no significant effect on the intracellular pH. 5) Application of low-Ca2⫹ ACSF causes a significant intracellular
alkalosis only at room temperature under submerged conditions. And 6) extracellular space volume is significantly reduced under interface conditions compared with submerged
conditions.
The reduction of extracellular Mg2⫹ concentration has been
known to enhance neuronal excitability and induce spontaneous epileptiform activity by decreasing membrane surface
charge screening and the removal of the voltage-dependent
Mg2⫹ block from the NMDA receptor (Mody et al. 1987). The
present study shows that reducing temperature to room temperature suppresses low-Mg2⫹-induced synaptic epileptiform
activity under interface and submerged conditions. Lowering
temperature causes considerable changes in ionic conductances, such as the activation of Na⫹, K⫹, and Ca2⫹ channels
(McAllister-Williams and Kelly 1995) and ionic transporters,
such as the Na2⫹/K⫹-ATPase and the Na⫹/Ca2⫹ exchanger
(Fraser and MacVicar 1996; Hansford 1980). Furthermore,
reducing temperature increases pCO2 and decreases extracellular pH (see table). Extracellular acidification has been shown
to depress synaptic transmission and neuronal excitability in
vivo and in vitro (Balestrino and Somjen 1988). Indeed, low-
FIG. 5. Change in extracellular space volume comparing submerged and
interface conditions. TEA-sensitive microelectrodes were used to demonstrate
differences in extracellular space volume in the somatic layer of the CA1
region between submerged and interface conditions. TEA⫹ was applied in
concentration of 2 mM, a concentration at which the microelectrode is no
longer sensitive to changes in extracellular potassium. After establishing the
steady state, transient changes in [TEA⫹]e reflect alterations in extracellular
space volume. To avoid artificial influences by TEA⫹ diffusion between
extracellular volume and perifusate, the switch between submerged and interface conditions was implemented within ⱕ1 min. A: the trace showes the
characteristic change in [TEA⫹]e following the switch from submerged to
interface and back to submerged conditions. Following the change to interface
conditions, the [TEA⫹]e signal increased and after return to submerged conditions, the [TEA⫹]e signal decreased. B: the graph summarizes [TEA⫹]e
signals from 8 slices following changes from submerged to interface and back
to submerged conditions. The significant [TEA⫹]e increase following the
change to interface conditions indicates a reduction in extracellular space
volume. The corresponding significant [TEA⫹]e reduction after return to
submerged conditions indicates the re-increase of extracellular space volume
(***P ⬍ 0.01).
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FIG. 4. Temperature-dependent changes in intracellular pH in presence of
low-Ca2⫹ ACSF under submerged conditions. A: changes in pHi following
application of low-Ca2⫹ ACSF at 35°C. Left: a typical measurement in area
CA1. Right: the changes in pHi from 6 slices. At 35°C, low-Ca2⫹ ACSF
effected a slight, but not significant increase in intracellular pH. B: changes in
pHi following application of low-Ca2⫹ ACSF at room temperature (RT). Left:
a typical intracellular alkalization in area CA1. Right: the changes in pHi from
12 slices. At room temperature, low-Ca2⫹ ACSF effected a significant increase
in intracellular pH (**P ⬍ 0.01).
2933
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TABLE
SCHUCHMANN ET AL.
1. Summary of extracellular pH data
36 ⫾ 1°C
Room temperature (23 ⫾ 1°C)
Submerged
Conditions
Interface
Conditions
Physiological
Conditions
7.32 ⫾ 0.02
7.24 ⫾ 0.03
7.34 ⫾ 0.02
7.30 ⫾ 0.02
7.15–7.35
—
Table summarizes extracellular pH data under submerged and interface
conditions at temperatures and under physiological conditions. Bulk solution
pH under submerged and interface conditions was adjusted to 7.4. The data
under physiological conditions were quoted from Balestrion and Somjen
(1988) and correspond to the interstitial pH measured using ion-sensitive
microelectrodes in the hippocampus formation of anaesthetized rats. Therefore, extracellular pH under experimental conditions reported in the present
study corresponds well with the broad range of extracellular pH under physiological conditions.
J Neurophysiol • VOL
We thank K. Kaila and J. Voipio for constructive comments on the manuscript and K. Schulze, H. Lindner, H. Siegmund, and H. J. Gabriel for technical
support. We also thank M. Wagner for special help with the study.
This study was supported by the Gotthard-Schettler-Stipendium (Foundation
for Behavior and Environment, VerUm) to S. Schuchmann and Deutsche
Forschungsgemeinschaft Grant Bu1331-1 to K. Buchheim and H. Meierkord.
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space volume under interface conditions and increased gap
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epileptiform activity. The shown differences between gas-liquid interface and total-liquid submerged condition may enable
further investigations of low-Mg2⫹- and low-Ca2⫹-induced
epileptiform activity.
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