INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

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 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 low-Ca²⁺ 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.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Slice preparation

The experiments were performed as described previously (Schuchmann et al. 1999). Briefly, brain slices (400 µm) 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.

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 µm) 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)
where [TEA⁺]_{submerged/interface} describes the extracellular TEA⁺ concentration at submerged or interface conditions.

Intracellular pH measurements

Intracellular pH (pH_i) 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 µM 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 2 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; 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

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Low-Mg²⁺-induced epileptiform activity under submerged and interface conditions

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 min¹; 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 min¹, P = 0.22). Compared to interface conditions, the duration of the SLEs was significantly shorter (21.2 ± 2.7 s, n = 30; P = 4·10⁴). 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).

View larger version (24K): [in this window] [in a new window]   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 low-Mg2+ 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.

In contrast, at room temperature no epileptiform activity could be induced with low-Mg²⁺ ACSF under submerged or 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 low-Ca²⁺ 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.

View larger version (39K): [in this window] [in a new window]   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.

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 min¹; 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 min¹, 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 = 2·10⁴). [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

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 (pH_o) under submerged and interface conditions. pH-sensitive microelectrodes placed in the entorhinal cortex and area CA1 of untreated slices (recording depth, 100-150 µm; n = 8) showed no significant differences in basal pH_o between submerged and interface conditions (submerged pH_o 7.32 ± 0.02, interface pH_o 7.34 ± 0.02; P = 0.43). The typical slight extracellular acidotic pH_o shift from the slice surface (pH_ACSF 7.40 ± 0.02) to the recording depth of ~100 µm 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 (room temperature) under submerged conditions caused a slight extracellular acidification of 0.08 pH units (35°C: pH_o 7.32 ± 0.02, 22°C: pH_o 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: pH_o 7.34 ± 0.02, 22°C: pH_o 7.30 ± 0.02, n = 6; Fig. 3B). The reduced pH_o sensitivity to temperature under interface conditions compared with submerged conditions underlines the importance of the gas phase in this chamber type.

View larger version (26K): [in this window] [in a new window]   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.

Changes in intracellular pH under submerged conditions

To investigate the effect of temperature, low-Mg²⁺ and low-Ca²⁺ ACSF on intracellular pH (pH_i), 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 pH_i were measured in the entorhinal cortex (n = 3) and in the area CA1 (n = 6) of untreated slices (Fig. 4A). No differences in basal pH_i were found between the two regions (entorhinal cortex: pH_i 7.17 ± 0.1, area CA1: pH_i 7.18 ± 0.12). Temperature reduction from 35 ± 1°C to room temperature (23 ± 1°C) caused a slight, but not significant, decrease in basal pH_i (entorhinal cortex: pH_i 7.15 ± 0.1, P = 0.38; area CA1: pH_i 7.16 ± 0.14, P = 0.4).

View larger version (22K): [in this window] [in a new window]   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).

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 pH_i at 35°C (7.18 ± 0.1, n = 6) nor the pH_i shift following temperature reduction (pH_i 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 pH_i to 7.29 ± 0.09 (n = 6, Fig. 4B). This intracellular alkalization was enlarged to 7.43 ± 0.1 (n = 12, vs. normal ACSF pH_i 7.17 ± 0.11, P = 8.2·10³) by lowering the temperature to room temperature in presence of low-Ca²⁺ ACSF (Fig. 4C).

Changes in extracellular space volume under submerged and interface conditions

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 conditions, 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.

View larger version (22K): [in this window] [in a new window]   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 1 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).

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 = 3.5·10⁴). 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 = 4.3·10⁴). 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

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

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 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, lowering pH_o blocks low-Mg²⁺-induced epileptiform activity, which has been suggested to be due to blockade of NMDA receptor by protons (Traynelis and Cull-Candy 1990; Vilisek et al. 1994). However, a complete blockade of low-Mg²⁺-induced epileptiform activity needs lowering pH_o to 6.2 (Vilisek et al. 1994). Therefore beside the slight extracellular acidification following temperature reduction, further causes are needed to suppress low-Mg²⁺-induced epileptiform activity at room temperature. Osmolality-induced increased extracellular space volume has been reported to reduce epileptiform activity (Baran et al. 1986; Rosen and Andrew 1990). Therefore the reported increased extracellular space volume in submerged slices may has part in reducing or blocking low-Mg²⁺-induced epileptiform discharges, also indicated by the significantly shorter duration of low-Mg²⁺-induced SLE under submerged conditions compared with interface conditions.

The reduction of Ca²⁺ concentration has been known to modulate neuronal activity and induce spontaneous field bursts by several types of nonsynaptic interactions between neurons: electrical coupling by ephaptic transmission between neuronal elements, fluctuations in extracellular ions such as K⁺, and coupling by gap junctions (Jefferys 1995). The present study shows that low-Ca²⁺-induced nonsynaptic epileptiform activity occurs at room temperature under submerged conditions but not under interface conditions. The results indicate altering influences of nonsynaptic communication types for the induction of low-Ca²⁺ epileptiform activity under submerged and interface conditions.

The volume of the extracellular space has been suggested to be different in slices under interface compared with submerged conditions (Croning and Haddad 1998; Haas and Büsselberg 1992). In the present study, using TEA⁺-sensitive microelectrodes, we demonstrated a significant increase in extracellular space volume under submerged conditions compared with interface conditions. This leads to differences in several factors important for nonsynaptic communication, such as ephaptic field effects and fluctuations in extracellular K⁺ concentration. These effects may explain in part the more stable low-Ca²⁺-induced field bursts in interface slices compared with submerged slices in the current study as well as in other studies (Bikson et al. 1999; Ghai et al. 2000; Taylor and Dudek 1982, 1984; Watson and Andrew 1995). Osmolality induced reduction of extracellular space volume has been reported to enhance epileptiform activity independent of chemical synapses in rat hippocampal slices (Dudek et al. 1990). Therefore reduced extracellular space volume and more flattened slices may cause stronger ephaptic field effects. These stronger ephaptic field effects promote synchrony in interface slices indicated by increased field potential amplitude and duration during SLEs as shown in this study.

Equal or increased, due to enhanced epileptiform activity, amounts of released K⁺ should alter peak and kinetic of [K⁺]_e in reduced extracellular space volume during low-Ca²⁺ field bursts. The present study shows in interface slices significant longer low-Ca²⁺ SLEs and prolonged extracellular K⁺ fluctuation, but only a slight not significant increased peak in [K⁺]_e compared with submerged slices. However, low-Ca²⁺ SLEs are measured at 35 ± 1°C under submerged conditions and at room temperature under interface conditions. Therefore comparison of [K⁺]_e peak during low-Ca²⁺ field bursts in interface and submerged slices is complicate to interpret. At similar temperature the [K⁺]_e peak may be greater in interface slices compared with submerged slices.

Temperature is known to affect the activities of mechanisms regulating pH_i (Baxter and Church 1996). The present study shows a significant intracellular alkalization in presence of low-Ca²⁺ ACSF at room temperature under submerged conditions. Intracellular alkalization is known to increase gap junction conductance (Spray et al. 1981). Increased gap junction conductance has been suggested to contribute to the synchronization of neuronal firing during low-Ca²⁺-induced epileptiform activity (Perez-Velazquez et al. 1994) and gap junction blockers has been demonstrated to suppress low-Ca²⁺-induced field bursts (Schweitzer et al. 2000). Furthermore, reduction in temperature has been reported to cause an increased activation of gap junctions (Yuste et al. 1995). Because gap junctions are assumed to play an important role in low-Ca²⁺ discharges, increasing temperature may prevent low-Ca²⁺ discharges by suppressing this nonsynaptic communication.

In conclusion, the present study shows, that synaptic and nonsynaptic ictogenesis occurs at different temperatures under gas-liquid interface and total-liquid submerged conditions. The increased pH_o at 35 ± 1°C compared with room temperature is assumed to contribute the induction of low-Mg²⁺ spontaneous epileptiform activity under submerged and interface conditions. Furthermore, we suggest that, reduced extracellular space volume under interface conditions and increased gap junctional conductance caused by intracellular alkalization under submerged conditions support the induction of low-Ca²⁺ 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.