Free
Meeting Abstracts  |   August 1998
Thiopental Inhibits Increases in [Ca2+]iInduced by Membrane Depolarization, NMDA Receptor Activation, and Ischemia in Rat Hippocampal and Cortical Slices 
Author Notes
  • (Zhan) Visiting Scientist.
  • (Fujiwara, Endoh, Taga) Assistant Professor.
  • (Yamakura) Clinical Lecturer.
  • (Fukuda) Associate Professor.
  • (Shimoji) Professor and Chairman.
Article Information
Meeting Abstracts   |   August 1998
Thiopental Inhibits Increases in [Ca2+]iInduced by Membrane Depolarization, NMDA Receptor Activation, and Ischemia in Rat Hippocampal and Cortical Slices 
Anesthesiology 8 1998, Vol.89, 456-466. doi:
Anesthesiology 8 1998, Vol.89, 456-466. doi:
THE brain is susceptible to anoxia and ischemia, with a regionally differential vulnerability. Substantial evidence exists that ischemic neuronal injury is mediated in part by increases in intracellular calcium ([Ca2+]i). [1,2] Accordingly, the change in [Ca2+]iper se has been considered an indicator of neuronal injury. [3] 
Although barbiturates reduce ischemic neuronal injury, the mechanisms of this reduction remain unclear. [4,5] The protective effect of barbiturates might be involved in stabilizing cellular calcium homeostasis during anoxia and ischemia. Indeed, thiopental reduced anoxia-mediated calcium influx in rat hippocampal slices in vitro. [6] However, because anoxia is a milder insult than ischemia (aglycemic anoxia), the effects of barbiturates such as thiopental on increases in [Ca2+]iduring ischemia should be clarified. Thus the first aim of this study was to determine whether thiopental inhibits increases in [Ca2+]icaused by ischemia in slice preparations, and, if it does, to identify the mechanisms involved. To this end, the effects of thiopental on [Ca2+](i) changes caused by membrane depolarization and N-methyl-D-aspartate (NMDA) receptor activation were examined. Because evidence exists that the protective effect of thiopental against ischemia might be region specific, [7] our second aim was to compare the effects of thiopental on increases of [Ca2+]icaused by membrane depolarization and NMDA receptor activation in the hippocampus and cortex in vitro.
Materials and Methods
This study was approved by the Committee for the Guidelines on Animal Experimentation of Niigata University. Experiments were done on brain slices obtained from male Wistar rats that weighed 100 - 130 g and were 30 - 35 days old. Rats were anesthetized with isoflurane and decapitated, and their brains were quickly removed and placed into ice-cold oxygenated Krebs solution, which contained 117 mM NaCl, 3.6 mM KCl, 1.2 mM NaH2PO4, 2.5 mM CaCl2, 1.2 mM MgCl2, 25 mM NaHCO3, 11.5 mM glucose, and had a pH of 7.35 (at 23 [degree sign]C equilibrated with 95% oxygen and 5% carbon dioxide. Brains were coronally cut into 350 [micro sign]m-thick sections using a vibrating tissue slicer (DTK-1000, Dosaka, Kyoto, Japan). Hippocampi and somatosensory cortices were isolated.
To load fura-2, slices were incubated in oxygenated Krebs solution containing 10 [micro sign]M acetoxymethyl ester of fura-2 (fura-2/AM; Dojindo, Kumamoto, Japan) and 0.1% Pluronic F-127 (Boehringer Diagnostics, La Jolla, CA) for 90 min at room temperature using an interface chamber. These slices were rinsed twice and incubated again in oxygenated Krebs solution for an additional 20 - 30 min to allow hydrolysis of the acetoxymethyl ester.
Fura-2 - loaded slices were transferred individually into a cylindrical chamber glued onto a microscope glass slide that was mounted on the stage of an inverted fluorescence microscope (Diaphot, Nikon, Tokyo, Japan). The chamber had an effective volume of 0.3 ml, and the slices were superfused with Krebs solution, equilibrated with 95% oxygen and 5% carbon dioxide. The flow rate was 5 ml/min, and the temperature was maintained within 36.5 to 37.5 [degree sign]C using a heating bath. The levels of [Ca (2+)]iin the CA1 pyramidal cell layer of hippocampal slices or layers II to III of cortical slices were monitored as the fluorescence of fura-2 using a microspectrofluorimeter (JASCO CAM-500, Japan Spectroscopic Co., Tokyo, Japan), as previously described. [8] Excitation lights were obtained from a xenon lamp (150 W) equipped with a rotating wheel filter and two monochromators. The intensity of fura-2 fluorescence at 510 nm emission was obtained at 340 and 380 nm excitation wavelengths. Changes in the intensities of fluorescence at 340 (I340) and 380 nm (I380) collected from a 100 [micro sign]m x 25 [micro sign]m area of slices with an adjustable diaphragm inserted in the light path were detected using a photo-multiplier at 1 Hz. The I340and I380values and their ratio (I340/I380, referred to as R340/380) were recorded simultaneously using a chart writer. Estimates of [Ca2+]iwere represented by R340/380.
Aglycemic anoxia in slices was made by switching the 95% oxygen and 5% carbon dioxide-gassed Krebs solution to glucose-free Krebs solution (supplemented with equimolar sucrose) that was preequilibrated with 95% nitrogen and 5% carbon dioxide (this decreased the oxygen tension of the perfusate in the recording chamber from 484 +/- 20 mmHg to 30 +/- 5 mmHg). Changes in [Ca2+]icaused by aglycemic anoxia were evaluated based on (1) the latency from the onset of aglycemic anoxia to the appearance of the [Ca2+]iplateau, and (2) the changes in [Ca2+]i8, 10, and 15 min after the onset of aglycemic anoxia, which are expressed as the normalized changes in R340/380 calculated by dividing R340/380 at those points to just before the onset of aglycemic anoxia. The [Ca2+]iplateau indicates the period from the finish of rapid [Ca2+]ielevated to the point at which 15 min of ischemia was stopped.
Membrane depolarization and NMDA receptor activation in slices were produced by exposure to 60 mM K+(60 s) and 100 [micro sign]M NMDA (30 s), respectively. To block membrane depolarization-generated sodium influx, 0.5 [micro sign]M tetrodotoxin was added to the bathing media before, during, and after exposure to high K+or NMDA. Because the [Ca2+](i) response caused by 60 mM K+was completely repeatable in the same slice, the change in [Ca2+]icaused by first exposure to 60 mM K (+) was used as a control in each slice. The high K+-mediatedincreases in [Ca2+]iin the presence of thiopental were normalized to the control levels by the net increase in R340/380 in each slice. The changes in [Ca2+]ievoked by 100 [micro sign]M NMDA were represented by the net increase in R340/380. Interventions were started 5 min before the onset of aglycemic anoxia, 60 mM K+or 100 [micro sign]M NMDA exposure, and were continued at the same concentration during aglycemic anoxia.
The effect of thiopental on the NMDA-mediated change in [Ca2+]iwas also examined in cultured cortical neurons. Cortical neurons were prepared from 18- to 19-day-old embryos of Wistar rats according to the procedure of Mizuno et al. [9] Whole neocortices were mechanically dissociated and plated onto glass coverslips (1 cm in diameter and 0.17 mm thick) that were precoated with poly-L-lysine and laminin at a density of 1 [tilde operator] 2 x 103cells/mm2. Dissociated cells were first grown for 24 h with Dulbecco's modified Eagle's medium containing 2 mM purified glutamine, 2% fetal bovine serum, a nutrient mixture of N2 (100 [micro sign]g/ml transferrin, 5 [micro sign]g/ml bovine insulin, 100 nM putrescine, 20 nM progesterone, and 30 nM sodium selenite), 10 mM HEPES (pH 7.3), and 100 [micro sign]g/100 ml bovine serum albumin (all from Sigma Chemical Co., St. Louis, MO). The next day, the original medium was replaced by the medium lacking serum (serum-free N2 medium), which topically contained 20 ng/ml purified human recombinant neurotrophines (i.e., NGF, BDNF, NT-3, or NT-5). Cultures were maintained at 37 [degree sign]C in humidified 6% carbon dioxide-94% air atmosphere, and the media were replaced every 2 or 3 days. After 7 - 9 days in culture, cells were washed with Krebs solution and then incubated in Krebs solution containing 5 [micro sign]M fura-2-AM and 0.1% Pluronic F-127 for 45 min at 37 [degree sign]C to load fura-2. After the loading period, the cells were incubated in fura-2 - free Krebs solution for an additional 30 min at 21 - 23 [degree sign]C. The coverslips were then attached to a chamber (effective volume, 0.4 ml) and placed on the stage of a Nikon Diaphot inverted microscope equipped with a 100 x oil immersion objective (numeric aperture, 1.3). Cells were superfused continuously with oxygenated Krebs solution at a rate of 4 ml/min, and the temperature was maintained at 37 [degree sign]C. Cells were illuminated alternately with 340 and 380 nm light from a xenon lamp (150 W) and were recorded using an SIT video camera (model C2400–8; Hamamatsu Photonics, Hamamatsu, Japan) controlled by software (ARGUS 200/CA, Hamamatsu Photonics) running on a personal computer (model A2008; Canon Inc., Tokyo, Japan). Hardware gain and black settings were optimized so that the somatic region of the neurons could be seen clearly. Images were sampled and collected at 1-min intervals for 44 min. Three or four neurons were monitored simultaneously in each coverslip. The protocol for eliciting the effect of thiopental on the NMDA-mediated increase in [Ca2+]iwas similar to that in the slice preparation, except the concentration of NMDA was 200 [micro sign]M and the duration of exposure was increased to 2 min.
Drugs were applied by superfusion. DL-2-amino-5-phosphonovaleric acid, NMDA, tetrodotoxin, bicuculline, and [small gamma, Greek]-amino-butyric acid (GABA) were purchased from Sigma Chemical Company. Lanthanum chloride was obtained from WAKO Chemical Industries (Osaka, Japan). Sodium thiopental was a gift of Tanabe Pharmaceutical Co. (Osaka, Japan).
Statistical Analysis
Data are expressed as mean +/- SD and were analyzed, as appropriate, by Student's t test, one- or two-way analysis of variance, with statistical significance determined by Dunnett's post hoc test. A P value <or= to 0.05 was considered significant.
Results
Control Experiments
Control experiments were performed in the fura-2 - unloaded hippocampal slices to determine how autofluorescence would affect the R340/380 after the onset of aglycemic anoxia, or application of 60 mM K+and 100 [micro sign]M NMDA. Figure 1A shows that exposing slices to aglycemic anoxia caused an increase in R340/380 with concomitant increases in both I340and I380. The increases in R340/380 began within 45 s after the onset of aglycemic anoxia and reached a plateau within 1 min. In seven slices examined, the increases in R340/380 caused by aglycemic anoxia are 110 - 117% at 2 min and 115 - 125% at 15 min after the onset of aglycemic anoxia (R340/380 at the two time points compared with those before the onset of aglycemic anoxia). Based on this, it is possible to subtract autofluorescence-related changes in R340/380 in the fura-2 - loaded slices. Exposure of slices to 60 mM K+(n = 5) and 100 [micro sign]M NMDA (n = 5) did not affect R340/380 significantly (Figure 1B and Figure 1C).
Figure 1. Typical traces showing the changes in the intensities of fluorescence at 340 (I340) and 380 nm (I380) and their ratio (R340/380) in the fura-2 - unloaded hippocampal slices responding to aglycemic anoxia (indicated by the bar below the trace), 60 mM K+(60 s) or 100 [micro sign]M N-methyl-D-aspartate (NMDA; 30 s). Closed triangles indicate the onset of 60 mM K+or 100 [micro sign]M NMDA. Experiments were done at 37 [degree sign]C. (A) Aglycemic anoxia simultaneously increased I340, I380, and R340/380. The values of I340and I380(arbitrary units) at several time points are presented above the I340and I380traces. (B) Exposure of a slice to 60 mM K+for 1 min did not significantly affect either I340, I380, or R340/380. (C) Exposure of a slice to NMDA for 30 s slightly decreased both I340and I (380) without changing the R340/380.
Figure 1. Typical traces showing the changes in the intensities of fluorescence at 340 (I340) and 380 nm (I380) and their ratio (R340/380) in the fura-2 - unloaded hippocampal slices responding to aglycemic anoxia (indicated by the bar below the trace), 60 mM K+(60 s) or 100 [micro sign]M N-methyl-D-aspartate (NMDA; 30 s). Closed triangles indicate the onset of 60 mM K+or 100 [micro sign]M NMDA. Experiments were done at 37 [degree sign]C. (A) Aglycemic anoxia simultaneously increased I340, I380, and R340/380. The values of I340and I380(arbitrary units) at several time points are presented above the I340and I380traces. (B) Exposure of a slice to 60 mM K+for 1 min did not significantly affect either I340, I380, or R340/380. (C) Exposure of a slice to NMDA for 30 s slightly decreased both I340and I (380) without changing the R340/380.
Figure 1. Typical traces showing the changes in the intensities of fluorescence at 340 (I340) and 380 nm (I380) and their ratio (R340/380) in the fura-2 - unloaded hippocampal slices responding to aglycemic anoxia (indicated by the bar below the trace), 60 mM K+(60 s) or 100 [micro sign]M N-methyl-D-aspartate (NMDA; 30 s). Closed triangles indicate the onset of 60 mM K+or 100 [micro sign]M NMDA. Experiments were done at 37 [degree sign]C. (A) Aglycemic anoxia simultaneously increased I340, I380, and R340/380. The values of I340and I380(arbitrary units) at several time points are presented above the I340and I380traces. (B) Exposure of a slice to 60 mM K+for 1 min did not significantly affect either I340, I380, or R340/380. (C) Exposure of a slice to NMDA for 30 s slightly decreased both I340and I (380) without changing the R340/380.
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Aglycemic Anoxia Produced a Characteristic Change in [Ca2+]iin Brain Slices
In the fura-2 - loaded slices, the intensities of fluorescence at 340 nm and 380 nm were 8 - 10 times higher than those in the fura-2 - unloaded slices. The use of 0.1% Pluronic F-127 did not harm slices within 6 h, as evaluated by population spikes (data not shown). As shown in Figure 2A, in hippocampal or cortical slices responding to aglycemic anoxia for 15 min, a characteristic increase in R340/380 was observed: an initial slow phase was followed by a rapid elevation and then reached a plateau phase. The initial increases in R340/380 may not reflect changes in [Ca2+]ibecause they were observed with increases in both I340and I380. The rapid increases of [Ca2+]iin the pyramidal cell layer of the CA1 region began 4.5 to 6.5 min after the onset of aglycemic anoxia and reached the plateau phase within 1 min. In layers II to III of cortical slices, latencies to the rapid increase in [Ca2+]iwere shorter than those in hippocampal slices, ranging from 3.5 to 5.5 min. The latencies to the occurrence of the [Ca2+]iplateau after the onset of aglycemic anoxia are 416.0 +/- 23.2 s in hippocampal slices (n = 9) and 348 +/- 60.8 s in cortical slices (n = 6; P < 0.05 by unpaired t test), respectively. Table 1lists the normalized increases in R340/380 at 8 (R (8)), 10 (R10), and 15 min (R15) after the onset of aglycemic anoxia in both hippocampal and cortical slices. The R8, R10, and R (15) values were calculated by dividing R340/380 at 8, 10, and 15 min after the onset of aglycemic anoxia by R340/380 at 2 min after the onset of aglycemic anoxia but not that before the onset of aglycemic anoxia in each slice to offset autofluorescence-related changes in R340/380.
Figure 2. (A) Typical traces show changes in the intensities of fluorescence at 340 (I340) and 380 nm (I380) and their ratio (R340/380) caused by aglycemic anoxia and (B) the inhibitory effects of thiopental on aglycemic anoxia-mediated changes in R340/380 in hippocampal (Hippocampus) and cortical slices (Cortex). The values of I340and I380(arbitrary units) at several time points are given above the I340and I380traces. Aglycemic anoxia is indicated by bars below each trace. The vertical line indicates the normalized increase in R340/380. There are three phases of increases in R340/380 after the onset of 15 min of aglycemic anoxia: a slow increase phase (SP), a rapid increase phase (RP), and a plateau phase (PP). The [Ca2+]iplateau indicates the period between the two vertical dotted lines. Thiopental was present 5 min before and during the period of ischemia, as indicated by the bar above each trace.
Figure 2. (A) Typical traces show changes in the intensities of fluorescence at 340 (I340) and 380 nm (I380) and their ratio (R340/380) caused by aglycemic anoxia and (B) the inhibitory effects of thiopental on aglycemic anoxia-mediated changes in R340/380 in hippocampal (Hippocampus) and cortical slices (Cortex). The values of I340and I380(arbitrary units) at several time points are given above the I340and I380traces. Aglycemic anoxia is indicated by bars below each trace. The vertical line indicates the normalized increase in R340/380. There are three phases of increases in R340/380 after the onset of 15 min of aglycemic anoxia: a slow increase phase (SP), a rapid increase phase (RP), and a plateau phase (PP). The [Ca2+]iplateau indicates the period between the two vertical dotted lines. Thiopental was present 5 min before and during the period of ischemia, as indicated by the bar above each trace.
Figure 2. (A) Typical traces show changes in the intensities of fluorescence at 340 (I340) and 380 nm (I380) and their ratio (R340/380) caused by aglycemic anoxia and (B) the inhibitory effects of thiopental on aglycemic anoxia-mediated changes in R340/380 in hippocampal (Hippocampus) and cortical slices (Cortex). The values of I340and I380(arbitrary units) at several time points are given above the I340and I380traces. Aglycemic anoxia is indicated by bars below each trace. The vertical line indicates the normalized increase in R340/380. There are three phases of increases in R340/380 after the onset of 15 min of aglycemic anoxia: a slow increase phase (SP), a rapid increase phase (RP), and a plateau phase (PP). The [Ca2+]iplateau indicates the period between the two vertical dotted lines. Thiopental was present 5 min before and during the period of ischemia, as indicated by the bar above each trace.
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Table 1. The Latencies to the [Ca2+]iPlateau (Tplateau) and Normalized Increases in R340/380 at 8 (R8), 10 (R10), and 15 min (R (15)) after the Onset of Aglycemic Anoxia in Hippocampal and Cortical Slices in the Absence and Presence of Thiopental 
Image not available
Table 1. The Latencies to the [Ca2+]iPlateau (Tplateau) and Normalized Increases in R340/380 at 8 (R8), 10 (R10), and 15 min (R (15)) after the Onset of Aglycemic Anoxia in Hippocampal and Cortical Slices in the Absence and Presence of Thiopental 
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The Effect of Thiopental on Aglycemic Anoxia-induced Change in [Ca2+]i
Pretreatment with thiopental (50 - 400 [micro sign]M) did not affect basal [Ca2+]iin either hippocampal or cortical slices. The pattern of increase in [Ca2+]icaused by aglycemic anoxia, however, was dose dependently altered by thiopental, which slowed the rapid increase phase, prolonged the [Ca2+]iplateau, and reduced R8, R10, and R15(Figure 2B). Table 1summarizes the [Ca2+]iplateau and R8, R10, and R15in the hippocampal and cortical slices responding to aglycemic anoxia in the presence of thiopental. The calculations of R8, R10, and R15were identical to those in thiopental-untreated slices, as mentioned before.
The Effect of Thiopental on the Membrane Depolarization-evoked Increase in [Ca2+]i
In both hippocampal and cortical slices, the [Ca2+]iresponse caused by membrane depolarization with 60 mM K+(60 s) could be completely repeated in the same slice at least three times, as evaluated by the net increase in R340/380 (Figure 3A and Figure 3A'). Thus changes in [Ca2+]iinduced by 60 mM K+in the presence of thiopental were normalized with respect to the first exposure (referred to as control) by the net change in R340/380 in each slice. Pretreatment with thiopental for 5 min dose dependently reduced the 60 mM K+-mediatedincreases in [Ca2+](i) in either hippocampal or cortical slices (Figure 3B, Figure 3C, Figure 3D and Figure 3B', Figure 3C', Figure 3D'). At the same concentration, the inhibition of thiopental on the 60 mM K+-inducedincrease in [Ca2+]iwas not significantly different for hippocampal and cortical slices (Figure 4). In addition, the depressant effect of thiopental on the 60 mM K+-inducedincrease in [Ca2+]iwas not affected by bicuculline (20 [micro sign]M), a selective GABAAreceptor antagonist (Figure 5). The peak increase in [Ca2+]i(% control) caused by 60 mM K+is 40.5 +/- 8.9% after pretreatment with 400 [micro sign]M thiopental alone and 40.2 +/- 9.1% after pretreatment with a combination of 400 [micro sign]M thiopental and 20 [micro sign]M bicuculline (P > 0.05 by paired t test; n = 4).
Figure 3. Typical traces showing high K+-mediatedincreases in [Ca2+]iin the CA1 pyramidal cell layer of hippocampal slices (left) and layers II to III of cortical slices (right) in the absence and presence of thiopental (Thio). The vertical line indicates the net change in the ratio of fluorescence intensities at 340 nm and 380 nm. Closed triangles indicate the onset of 60 mM K+(60 s). Thiopental was present 5 min before the onset of high K+and was absent during the application of high K+.
Figure 3. Typical traces showing high K+-mediatedincreases in [Ca2+]iin the CA1 pyramidal cell layer of hippocampal slices (left) and layers II to III of cortical slices (right) in the absence and presence of thiopental (Thio). The vertical line indicates the net change in the ratio of fluorescence intensities at 340 nm and 380 nm. Closed triangles indicate the onset of 60 mM K+(60 s). Thiopental was present 5 min before the onset of high K+and was absent during the application of high K+.
Figure 3. Typical traces showing high K+-mediatedincreases in [Ca2+]iin the CA1 pyramidal cell layer of hippocampal slices (left) and layers II to III of cortical slices (right) in the absence and presence of thiopental (Thio). The vertical line indicates the net change in the ratio of fluorescence intensities at 340 nm and 380 nm. Closed triangles indicate the onset of 60 mM K+(60 s). Thiopental was present 5 min before the onset of high K+and was absent during the application of high K+.
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Figure 4. A comparison of changes in [Ca2+]imediated by 60 mM K+ in the presence of thiopental (50, 200, and 400 [micro sign]M) for the pyramidal cell layer of hippocampal slices (Hippocampus) and layers II to III of cortical slices (Cortex). The statistical analysis was done using an unpaired t test (n = number of slices, NS = no significance).
Figure 4. A comparison of changes in [Ca2+]imediated by 60 mM K+ in the presence of thiopental (50, 200, and 400 [micro sign]M) for the pyramidal cell layer of hippocampal slices (Hippocampus) and layers II to III of cortical slices (Cortex). The statistical analysis was done using an unpaired t test (n = number of slices, NS = no significance).
Figure 4. A comparison of changes in [Ca2+]imediated by 60 mM K+ in the presence of thiopental (50, 200, and 400 [micro sign]M) for the pyramidal cell layer of hippocampal slices (Hippocampus) and layers II to III of cortical slices (Cortex). The statistical analysis was done using an unpaired t test (n = number of slices, NS = no significance).
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Figure 5. Bicuculline (Bicu), a [small gamma, Greek]-amino-butyric acid A receptor antagonist, does not affect the inhibition of thiopental (Thio) on the high K+-mediatedincrease in [Ca2+]iin a hippocampal slice. The vertical bar indicates real change in the ratio of fluorescence intensities at 340 nm and 380 nm.
Figure 5. Bicuculline (Bicu), a [small gamma, Greek]-amino-butyric acid A receptor antagonist, does not affect the inhibition of thiopental (Thio) on the high K+-mediatedincrease in [Ca2+]iin a hippocampal slice. The vertical bar indicates real change in the ratio of fluorescence intensities at 340 nm and 380 nm.
Figure 5. Bicuculline (Bicu), a [small gamma, Greek]-amino-butyric acid A receptor antagonist, does not affect the inhibition of thiopental (Thio) on the high K+-mediatedincrease in [Ca2+]iin a hippocampal slice. The vertical bar indicates real change in the ratio of fluorescence intensities at 340 nm and 380 nm.
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The Effect of Thiopental on the N-methyl-D-aspartate - induced Increase in [Ca2+]i
In the presence of magnesium (1.2 mM), exposure to 100 [micro sign]M NMDA (for 30 s) caused a transient increase in [Ca2+]iin both hippocampal and cortical slices, and the responses were not completely repeatable in the same slice, as evaluated by the net change in R340/380 (Figure 6A and Figure 6A'). The ratio of second to first responses is 0.63 +/- 0.09 in hippocampal slices (n = 8) and 0.55 +/- 0.10 in cortical slices (n = 6). The NMDA-mediated increases in [Ca2+]iwere reversibly abolished by DL-2-amino-5-phosphonovaleric acid (50 [micro sign]M), a competitive NMDA receptor antagonist, but rarely were affected by lanthanum chloride (30 [micro sign]M), a nonspecific voltage-gated calcium channel (VGCC) blocker, [10,11] in either hippocampal or cortical slices (Figure 6B and Figure 6C and Figure 6B' and Figure 6C'). Thiopental also dose dependently reduced the NMDA-induced increase in [Ca2+]iin both the pyramidal cell layer of the CA1 region and layers II to III of cortical slices, as shown in Figure 7and Figure 8. The [Ca2+]iresponses caused by a second exposure to NMDA were significantly enhanced by increasing the concentration of thiopental in either hippocampal or cortical slices. The net increases in R340/380 produced by 100 [micro sign]M NMDA are 0.239 +/- 0.028 in hippocampal slices (n = 8) and 0.237 +/- 0.029 in cortical slices (n = 6; P > 0.05 by unpaired t test). The inhibition of thiopental on the NMDA-induced increases in [Ca2+]iwere pronounced in layers II to III of cortical slices (Figure 7and Figure 8). In the presence of 400 [micro sign]M thiopental, the net increase in R340/380 caused by 100 [micro sign]M NMDA was 0.134 +/- 0.011 in the CA1 pyramidal cell layer (n = 6), compared with 0.018 +/- 0.014 in layers II to III of cortical slices (n = 6; P < 0.0001 by unpaired t test).
Figure 6. Typical traces showing the characteristics of N-methyl-D-aspartate (NMDA)-mediated increases in [Ca2+]iin the CA1 pyramidal cell layer of hippocampal slices (left) and layers II to III layer of cortical slices (right). The vertical line indicates the net change in the ratio of fluorescence at 340 nm and 380 nm. Closed triangles indicate the onset of 100 [micro sign]M NMDA (30 s). (A and A'): In both slices, the changes in [Ca2+]icaused by 100 [micro sign]M NMDA for 30 s could not be reproduced. (B and B'): DL-2-amino-5-phosphonovaleric acid reversibly blocked the NMDA-mediated changes in [Ca2+]i. (C and C'): pretreatment with 30 [micro sign]M lanthanum chloride (La+3) did not significantly reduce the increases in [Ca2+]icaused by NMDA. The effect of La3+was examined in 20 mM HEPES buffered solution omitting NaH2PO4and NaHCO3(pH 7.35, adjusted by NaOH), because La (3+) could react with either HCO3-or H2PO4-.
Figure 6. Typical traces showing the characteristics of N-methyl-D-aspartate (NMDA)-mediated increases in [Ca2+]iin the CA1 pyramidal cell layer of hippocampal slices (left) and layers II to III layer of cortical slices (right). The vertical line indicates the net change in the ratio of fluorescence at 340 nm and 380 nm. Closed triangles indicate the onset of 100 [micro sign]M NMDA (30 s). (A and A'): In both slices, the changes in [Ca2+]icaused by 100 [micro sign]M NMDA for 30 s could not be reproduced. (B and B'): DL-2-amino-5-phosphonovaleric acid reversibly blocked the NMDA-mediated changes in [Ca2+]i. (C and C'): pretreatment with 30 [micro sign]M lanthanum chloride (La+3) did not significantly reduce the increases in [Ca2+]icaused by NMDA. The effect of La3+was examined in 20 mM HEPES buffered solution omitting NaH2PO4and NaHCO3(pH 7.35, adjusted by NaOH), because La (3+) could react with either HCO3-or H2PO4-.
Figure 6. Typical traces showing the characteristics of N-methyl-D-aspartate (NMDA)-mediated increases in [Ca2+]iin the CA1 pyramidal cell layer of hippocampal slices (left) and layers II to III layer of cortical slices (right). The vertical line indicates the net change in the ratio of fluorescence at 340 nm and 380 nm. Closed triangles indicate the onset of 100 [micro sign]M NMDA (30 s). (A and A'): In both slices, the changes in [Ca2+]icaused by 100 [micro sign]M NMDA for 30 s could not be reproduced. (B and B'): DL-2-amino-5-phosphonovaleric acid reversibly blocked the NMDA-mediated changes in [Ca2+]i. (C and C'): pretreatment with 30 [micro sign]M lanthanum chloride (La+3) did not significantly reduce the increases in [Ca2+]icaused by NMDA. The effect of La3+was examined in 20 mM HEPES buffered solution omitting NaH2PO4and NaHCO3(pH 7.35, adjusted by NaOH), because La (3+) could react with either HCO3-or H2PO4-.
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Figure 7. Typical traces showing the inhibitory effects of thiopental (Thio) on the N-methyl-D-aspartate (NMDA)-mediated increases in [Ca2+](i) in the CA1 pyramidal cell layer of hippocampal slices (left) and layers II to III of cortical slices (right). The vertical line indicates the net change in the ratio of fluorescence at 340 nm and 380 nm. Closed triangles indicate the onset of 100 [micro sign]M NMDA (30 s). Thiopental was present 5 min before the onset of NMDA and was absent during NMDA application.
Figure 7. Typical traces showing the inhibitory effects of thiopental (Thio) on the N-methyl-D-aspartate (NMDA)-mediated increases in [Ca2+](i) in the CA1 pyramidal cell layer of hippocampal slices (left) and layers II to III of cortical slices (right). The vertical line indicates the net change in the ratio of fluorescence at 340 nm and 380 nm. Closed triangles indicate the onset of 100 [micro sign]M NMDA (30 s). Thiopental was present 5 min before the onset of NMDA and was absent during NMDA application.
Figure 7. Typical traces showing the inhibitory effects of thiopental (Thio) on the N-methyl-D-aspartate (NMDA)-mediated increases in [Ca2+](i) in the CA1 pyramidal cell layer of hippocampal slices (left) and layers II to III of cortical slices (right). The vertical line indicates the net change in the ratio of fluorescence at 340 nm and 380 nm. Closed triangles indicate the onset of 100 [micro sign]M NMDA (30 s). Thiopental was present 5 min before the onset of NMDA and was absent during NMDA application.
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Figure 8. A comparison of the effects of thiopental on the N-methyl-D-aspartate (NMDA)-mediated increases in [Ca2+]iin the pyramidal cell layer of hippocampal slices (Hippocampus) and layers II to III of cortical slices (Cortex). The difference between the hippocampus and cortex was analyzed using unpaired t test (NS = no significance), * P < 0.01 and ** P < 0.001 indicate significant differences from zero dose in hippocampus; and #P = 0.01 and ##P < 0.001 indicate significant differences from zero dose in the cortex, as calculated by analysis of variance followed by multiple comparisons with Dunnett's post hoc test (n = number of slices).
Figure 8. A comparison of the effects of thiopental on the N-methyl-D-aspartate (NMDA)-mediated increases in [Ca2+]iin the pyramidal cell layer of hippocampal slices (Hippocampus) and layers II to III of cortical slices (Cortex). The difference between the hippocampus and cortex was analyzed using unpaired t test (NS = no significance), * P < 0.01 and ** P < 0.001 indicate significant differences from zero dose in hippocampus; and #P = 0.01 and ##P < 0.001 indicate significant differences from zero dose in the cortex, as calculated by analysis of variance followed by multiple comparisons with Dunnett's post hoc test (n = number of slices).
Figure 8. A comparison of the effects of thiopental on the N-methyl-D-aspartate (NMDA)-mediated increases in [Ca2+]iin the pyramidal cell layer of hippocampal slices (Hippocampus) and layers II to III of cortical slices (Cortex). The difference between the hippocampus and cortex was analyzed using unpaired t test (NS = no significance), * P < 0.01 and ** P < 0.001 indicate significant differences from zero dose in hippocampus; and #P = 0.01 and ##P < 0.001 indicate significant differences from zero dose in the cortex, as calculated by analysis of variance followed by multiple comparisons with Dunnett's post hoc test (n = number of slices).
×
The effect of thiopental on the NMDA-mediated [Ca2+]iresponse was also examined in 15 cultured cortical neurons of four sister coverslips. In the presence of 400 [micro sign]M thiopental, 7 of 15 neurons did not respond to NMDA (200 [micro sign]M), whereas 14 of 15 neurons responded to NMDA after thiopental was washed out. Figure 9shows the effect of thiopental on the NMDA-mediated increases in [Ca2+]iin the somatic region of four neurons.
Figure 9. Traces show the average changes in the ratio of fluorescence at 340 nm and 380 nm in the somatic region of individual cortical neurons induced by 200 [micro sign]M N-methyl-D-aspartate (NMDA; indicated by horizontal bars below traces) in the presence of 400 [micro sign]M (indicated by a bar above the traces) and after a wash of thiopental.
Figure 9. Traces show the average changes in the ratio of fluorescence at 340 nm and 380 nm in the somatic region of individual cortical neurons induced by 200 [micro sign]M N-methyl-D-aspartate (NMDA; indicated by horizontal bars below traces) in the presence of 400 [micro sign]M (indicated by a bar above the traces) and after a wash of thiopental.
Figure 9. Traces show the average changes in the ratio of fluorescence at 340 nm and 380 nm in the somatic region of individual cortical neurons induced by 200 [micro sign]M N-methyl-D-aspartate (NMDA; indicated by horizontal bars below traces) in the presence of 400 [micro sign]M (indicated by a bar above the traces) and after a wash of thiopental.
×
Discussion
We found that thiopental could attenuate increases in [Ca2+](i) induced by membrane depolarization, NMDA receptor activation, and ischemia in both the hippocampus and cortex in vitro, and its attenuation of the NMDA-mediated increases in [Ca2+]iwas more profound in layers II to III of the cortex.
The excessive increases in [Ca2+]ihave been hypothesized to be responsible for neuronal injury under excitotoxic, anoxic, and ischemic insults. [1,2] Several studies [12,13] have revealed that net intracellular Ca2+accumulation measured by the radioactive45Ca2+isotope correlates with neuronal injury, whereas most attempts have failed to show a correlation between neuronal injury and the levels of [Ca (2+)]i, measured using Ca2+-sensitivedyes. [14,15] Given that a large amount of Ca2+can be sequestrated by internal organelles (such as mitochondria) and the peak levels of [Ca2+]icould not be estimated precisely using some Ca2+-sensitivedyes, [16] the results obtained by the radioactive45Ca2+isotope and by Ca2+-sensitivedyes actually may not be in conflict. On the other hand, intramitochondrial Ca2+accumulation would cause the permeability transition pore to open, [17,18] further leading to mitochondrial dysfunction, which may be a key step preceding apoptotic cell death. [19] 
Using the fura-2 fluorescent technique for brain slice preparations enabled us to determine an average change in [Ca2+]iof a heterogeneous population, including neuronal somata, dendrites, axons, and glial cells. A true elevation of [Ca2+]ican be identified as an increase in R340/380, with mirror changes of I340and I380. In both hippocampal and cortical slices, ischemia caused a typical increase in [Ca2+]i. The ischemia-induced [Ca2+]ioverload was shown to result from several routes, including the opening of both voltage-gated and NMDA-gated calcium channels, reverse of Na+-Ca2+exchange, [20] release of Ca2+from internal stores, and activation of nonselective ion conductance. [1,21] Thiopental delayed the occurrence of the [Ca2+]iplateau and reduced the magnitude of increase in [Ca (2+)]iduring ischemia. This result is consistent with the report that thiopental was effective in reducing anoxia-caused calcium influx in the CA1 region of hippocampal slices. [6] Because the occurrence of the [Ca2+]iplateau was associated with the irreversible loss of synaptic activity, [22] attenuation of ischemia-induced increases in [Ca2+](i) may be an important mechanism for thiopental to protect neurons against ischemia.
It is conceivable that attenuation of ischemia-induced increases in [Ca2+]iby thiopental may be related to its ability to reduce the cellular metabolic rate. Nevertheless, studies in vitro [6] and in vivo [23] have not shown that thiopental decreases energy consumption during anoxia or ischemia. Even in a slice model, thiopental accelerated the decrease in adenosine triphosphate level during anoxia. [6] Thus mechanisms other than decreases in energy consumption appear to be involved in its attenuation of ischemia-induced increases in [Ca2+]i. In the current study, we have provided evidence that attenuation of ischemia-induced increases in [Ca2+]iby thiopental is parallel to its inhibitory effects of both VGCCs and NMDA receptors.
A potential explanation for the inhibition of thiopental on high K (+-evoked) increases in [Ca2+]imay be its potentiation of GABA (A) receptor activity. However, direct exposure of GABA (100 [micro sign]M) had no effect on the high K+-evokedincrease in [Ca2+]i(data not shown), and bicuculline, a GABAAreceptor antagonist, did not reduce thiopental-mediated inhibition, suggesting that activation of GABAAreceptors is not the cause. It is well known that increases in [Ca2+](i) induced by high K+are the result of membrane depolarization, which opens VGCCs. In non-neuronal cell lines, thiopental has been known as a VGCC blocker. [24,25] In rat striatal synaptosomes, thiopental was found to reduce high K+-inducedGABA release. [26] When these observations are combined with our findings, we would expect that thiopental also works as a VGCC blocker to neurons, and its inhibition of VGCCs should contribute in part to its attenuation of ischemia-induced increases in [Ca2+]i.
Barbiturates at higher concentrations can block the NMDA receptor. [27] Because NMDA receptor channels are highly permeable to Ca2+, recording NMDA-mediated [Ca2+]iresponses can be used as an indicator to assess the activity of NMDA receptors. In the presence of 1.2 mM Mg2+, NMDA caused a transient increase in [Ca2+]iin either pyramidal cell layer of the CA1 region or layers II to III of cortical slices, and the response could be abolished by DL-2-amino-5-phosphonovaleric acid, indicating that the [Ca2+]iresponses caused by NMDA initially resulted from NMDA receptor activation. Given that glial cells lack NMDA receptors [28] and their [Ca2+]ilevels are insensitive to NMDA, [29] the NMDA-induced increases in [Ca2+]iin both hippocampal and cortical slices appear to occur in neurons but not in glial cells. Indeed, the NMDA-mediated increase in [Ca2+]iin a pattern that is comparable to the NMDA-induced inward current recorded from neostriatal neurons. [30] Although it is possible that the increases in [Ca2+]iafter NMDA receptor activation may result in part from the opening of VGCCs secondary to membrane depolarization, we found that lanthanum chloride, a nonspecific VGCC blocker, [10,11] did not reduce the NMDA-mediated increase in [Ca2+]i.
In both hippocampal and cortical slices, the [Ca2+]iresponse caused by a second exposure to NMDA was smaller than that by the first exposure in each slice, which was reversed by thiopental at higher concentrations. Two explanations are possible: First, thiopental might actually protect slices from NMDA-induced damage [31]; and second, a larger increase in [Ca2+]icaused by NMDA receptor activation may lead to long-term inactivation of NMDA receptors. [32,33] Interestingly, the effect of thiopental on the NMDA-induced increases in [Ca2+]ivaries regionally; in the presence of 200 or 400 [micro sign]M thiopental, the NMDA-induced increases in [Ca2+]iwere reduced to a greater extent in layers II to III of cortical slices than were those in the pyramidal cell layer of the CA1 region. The potent inhibition of thiopental on the NMDA-induced increases in [Ca2+]iwas confirmed in cultured cortical neurons. In addition, recording the NMDA-induced currents in CA1 pyramidal neurons by blind whole-cell patch revealed that the inhibitory effect of thiopental (400 [micro sign]M) is only partial (data not shown), according to its effect on the NMDA-mediated increases in [Ca2+]i.
It is now clear that multiple NMDA receptor subtypes exist in the brain and differ in their anatomic distribution, pharmacologic profiles, and regulatory and physiologic properties. [34,35] Barbiturates differentially affecting NMDA receptors in the cortex and hippocampus have been suggested indirectly by two investigations, [36,37] in which pentobarbital and barbital increased [(3) H] dizocilpine binding sites in the cortex but not in the hippocampus. Because overstimulation of NMDA receptors is considered an important factor in causing ischemic neuronal injury, [1,2] it is reasonable to speculate that thiopental protecting neurons against ischemia would be region specific. Indeed, Sano et al. [7] showed that thiopental reduces ischemic neuronal injury in the cortex but not in the hippocampus.
The significance of our results might be as follows. First, a combined inhibition of both VGCCs and NMDA receptors may be one of the mechanisms by which thiopental protects neurons against ischemia. In an earlier study, Raley-Susman and Lipton [38] found that neither Ca2+removal nor ketamine alone, but combining Ca2+removal with ketamine, could prevent the inhibition of protein synthesis and the morphological changes caused by ischemia in the CA1 pyramidal neurons in vitro. Similarly, an in vivo investigation [39] also found that neither VGCC blocker nor NMDA receptor antagonist, but rather a combination of VGCC blocker and NMDA receptor antagonist could reduce ischemic neuronal injury. Furthermore, the protective effect of sigma ligands such as dextromethorphan in neurons against ischemia in vivo [40,41] also has been related to the inhibition of both VGCCs and NMDA receptors. [41] Second, although thiopental attenuated the increases in [Ca2+]icaused by membrane depolarization, NMDA receptor activation, and ischemia dose dependently, its inhibitions at lower concentrations were not significant, indicating that high doses of thiopental would be needed to achieve appreciable protection. Third, because calcium influx through NMDA receptor channels is especially neurotoxic, [42] a regionally differential inhibition of NMDA receptors by thiopental may result in a region-specific action against ischemia.
In conclusion, thiopental attenuates increases in [Ca2+]icaused by ischemia in both CA1 pyramidal cell layer of the hippocampus and layers II and III of the cortex in vitro, probably by its inhibition of VGCCs and NMDA receptors. The inhibition of thiopental on NMDA receptors is more profound in the cortex than in the hippocampus. These results indicate that stabilizing Ca2+homeostasis by inhibiting VGCCs and NMDA receptors may be one of the mechanisms through which thiopental protects neurons against ischemia, and its protection would be region specific.
The authors thank Professor Hiroyuki Nawa and Dr. Huabao Xiong, Department of Molecular Neurobiology, Brain Institute, Niigata University, for technical assistance in preparing the cultured cortical neurons; and Dr. Manabu Okamoto, Department of Anesthesiology, for performing the blind whole-cell patch in hippocampal slices.
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Figure 1. Typical traces showing the changes in the intensities of fluorescence at 340 (I340) and 380 nm (I380) and their ratio (R340/380) in the fura-2 - unloaded hippocampal slices responding to aglycemic anoxia (indicated by the bar below the trace), 60 mM K+(60 s) or 100 [micro sign]M N-methyl-D-aspartate (NMDA; 30 s). Closed triangles indicate the onset of 60 mM K+or 100 [micro sign]M NMDA. Experiments were done at 37 [degree sign]C. (A) Aglycemic anoxia simultaneously increased I340, I380, and R340/380. The values of I340and I380(arbitrary units) at several time points are presented above the I340and I380traces. (B) Exposure of a slice to 60 mM K+for 1 min did not significantly affect either I340, I380, or R340/380. (C) Exposure of a slice to NMDA for 30 s slightly decreased both I340and I (380) without changing the R340/380.
Figure 1. Typical traces showing the changes in the intensities of fluorescence at 340 (I340) and 380 nm (I380) and their ratio (R340/380) in the fura-2 - unloaded hippocampal slices responding to aglycemic anoxia (indicated by the bar below the trace), 60 mM K+(60 s) or 100 [micro sign]M N-methyl-D-aspartate (NMDA; 30 s). Closed triangles indicate the onset of 60 mM K+or 100 [micro sign]M NMDA. Experiments were done at 37 [degree sign]C. (A) Aglycemic anoxia simultaneously increased I340, I380, and R340/380. The values of I340and I380(arbitrary units) at several time points are presented above the I340and I380traces. (B) Exposure of a slice to 60 mM K+for 1 min did not significantly affect either I340, I380, or R340/380. (C) Exposure of a slice to NMDA for 30 s slightly decreased both I340and I (380) without changing the R340/380.
Figure 1. Typical traces showing the changes in the intensities of fluorescence at 340 (I340) and 380 nm (I380) and their ratio (R340/380) in the fura-2 - unloaded hippocampal slices responding to aglycemic anoxia (indicated by the bar below the trace), 60 mM K+(60 s) or 100 [micro sign]M N-methyl-D-aspartate (NMDA; 30 s). Closed triangles indicate the onset of 60 mM K+or 100 [micro sign]M NMDA. Experiments were done at 37 [degree sign]C. (A) Aglycemic anoxia simultaneously increased I340, I380, and R340/380. The values of I340and I380(arbitrary units) at several time points are presented above the I340and I380traces. (B) Exposure of a slice to 60 mM K+for 1 min did not significantly affect either I340, I380, or R340/380. (C) Exposure of a slice to NMDA for 30 s slightly decreased both I340and I (380) without changing the R340/380.
×
Figure 2. (A) Typical traces show changes in the intensities of fluorescence at 340 (I340) and 380 nm (I380) and their ratio (R340/380) caused by aglycemic anoxia and (B) the inhibitory effects of thiopental on aglycemic anoxia-mediated changes in R340/380 in hippocampal (Hippocampus) and cortical slices (Cortex). The values of I340and I380(arbitrary units) at several time points are given above the I340and I380traces. Aglycemic anoxia is indicated by bars below each trace. The vertical line indicates the normalized increase in R340/380. There are three phases of increases in R340/380 after the onset of 15 min of aglycemic anoxia: a slow increase phase (SP), a rapid increase phase (RP), and a plateau phase (PP). The [Ca2+]iplateau indicates the period between the two vertical dotted lines. Thiopental was present 5 min before and during the period of ischemia, as indicated by the bar above each trace.
Figure 2. (A) Typical traces show changes in the intensities of fluorescence at 340 (I340) and 380 nm (I380) and their ratio (R340/380) caused by aglycemic anoxia and (B) the inhibitory effects of thiopental on aglycemic anoxia-mediated changes in R340/380 in hippocampal (Hippocampus) and cortical slices (Cortex). The values of I340and I380(arbitrary units) at several time points are given above the I340and I380traces. Aglycemic anoxia is indicated by bars below each trace. The vertical line indicates the normalized increase in R340/380. There are three phases of increases in R340/380 after the onset of 15 min of aglycemic anoxia: a slow increase phase (SP), a rapid increase phase (RP), and a plateau phase (PP). The [Ca2+]iplateau indicates the period between the two vertical dotted lines. Thiopental was present 5 min before and during the period of ischemia, as indicated by the bar above each trace.
Figure 2. (A) Typical traces show changes in the intensities of fluorescence at 340 (I340) and 380 nm (I380) and their ratio (R340/380) caused by aglycemic anoxia and (B) the inhibitory effects of thiopental on aglycemic anoxia-mediated changes in R340/380 in hippocampal (Hippocampus) and cortical slices (Cortex). The values of I340and I380(arbitrary units) at several time points are given above the I340and I380traces. Aglycemic anoxia is indicated by bars below each trace. The vertical line indicates the normalized increase in R340/380. There are three phases of increases in R340/380 after the onset of 15 min of aglycemic anoxia: a slow increase phase (SP), a rapid increase phase (RP), and a plateau phase (PP). The [Ca2+]iplateau indicates the period between the two vertical dotted lines. Thiopental was present 5 min before and during the period of ischemia, as indicated by the bar above each trace.
×
Figure 3. Typical traces showing high K+-mediatedincreases in [Ca2+]iin the CA1 pyramidal cell layer of hippocampal slices (left) and layers II to III of cortical slices (right) in the absence and presence of thiopental (Thio). The vertical line indicates the net change in the ratio of fluorescence intensities at 340 nm and 380 nm. Closed triangles indicate the onset of 60 mM K+(60 s). Thiopental was present 5 min before the onset of high K+and was absent during the application of high K+.
Figure 3. Typical traces showing high K+-mediatedincreases in [Ca2+]iin the CA1 pyramidal cell layer of hippocampal slices (left) and layers II to III of cortical slices (right) in the absence and presence of thiopental (Thio). The vertical line indicates the net change in the ratio of fluorescence intensities at 340 nm and 380 nm. Closed triangles indicate the onset of 60 mM K+(60 s). Thiopental was present 5 min before the onset of high K+and was absent during the application of high K+.
Figure 3. Typical traces showing high K+-mediatedincreases in [Ca2+]iin the CA1 pyramidal cell layer of hippocampal slices (left) and layers II to III of cortical slices (right) in the absence and presence of thiopental (Thio). The vertical line indicates the net change in the ratio of fluorescence intensities at 340 nm and 380 nm. Closed triangles indicate the onset of 60 mM K+(60 s). Thiopental was present 5 min before the onset of high K+and was absent during the application of high K+.
×
Figure 4. A comparison of changes in [Ca2+]imediated by 60 mM K+ in the presence of thiopental (50, 200, and 400 [micro sign]M) for the pyramidal cell layer of hippocampal slices (Hippocampus) and layers II to III of cortical slices (Cortex). The statistical analysis was done using an unpaired t test (n = number of slices, NS = no significance).
Figure 4. A comparison of changes in [Ca2+]imediated by 60 mM K+ in the presence of thiopental (50, 200, and 400 [micro sign]M) for the pyramidal cell layer of hippocampal slices (Hippocampus) and layers II to III of cortical slices (Cortex). The statistical analysis was done using an unpaired t test (n = number of slices, NS = no significance).
Figure 4. A comparison of changes in [Ca2+]imediated by 60 mM K+ in the presence of thiopental (50, 200, and 400 [micro sign]M) for the pyramidal cell layer of hippocampal slices (Hippocampus) and layers II to III of cortical slices (Cortex). The statistical analysis was done using an unpaired t test (n = number of slices, NS = no significance).
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Figure 5. Bicuculline (Bicu), a [small gamma, Greek]-amino-butyric acid A receptor antagonist, does not affect the inhibition of thiopental (Thio) on the high K+-mediatedincrease in [Ca2+]iin a hippocampal slice. The vertical bar indicates real change in the ratio of fluorescence intensities at 340 nm and 380 nm.
Figure 5. Bicuculline (Bicu), a [small gamma, Greek]-amino-butyric acid A receptor antagonist, does not affect the inhibition of thiopental (Thio) on the high K+-mediatedincrease in [Ca2+]iin a hippocampal slice. The vertical bar indicates real change in the ratio of fluorescence intensities at 340 nm and 380 nm.
Figure 5. Bicuculline (Bicu), a [small gamma, Greek]-amino-butyric acid A receptor antagonist, does not affect the inhibition of thiopental (Thio) on the high K+-mediatedincrease in [Ca2+]iin a hippocampal slice. The vertical bar indicates real change in the ratio of fluorescence intensities at 340 nm and 380 nm.
×
Figure 6. Typical traces showing the characteristics of N-methyl-D-aspartate (NMDA)-mediated increases in [Ca2+]iin the CA1 pyramidal cell layer of hippocampal slices (left) and layers II to III layer of cortical slices (right). The vertical line indicates the net change in the ratio of fluorescence at 340 nm and 380 nm. Closed triangles indicate the onset of 100 [micro sign]M NMDA (30 s). (A and A'): In both slices, the changes in [Ca2+]icaused by 100 [micro sign]M NMDA for 30 s could not be reproduced. (B and B'): DL-2-amino-5-phosphonovaleric acid reversibly blocked the NMDA-mediated changes in [Ca2+]i. (C and C'): pretreatment with 30 [micro sign]M lanthanum chloride (La+3) did not significantly reduce the increases in [Ca2+]icaused by NMDA. The effect of La3+was examined in 20 mM HEPES buffered solution omitting NaH2PO4and NaHCO3(pH 7.35, adjusted by NaOH), because La (3+) could react with either HCO3-or H2PO4-.
Figure 6. Typical traces showing the characteristics of N-methyl-D-aspartate (NMDA)-mediated increases in [Ca2+]iin the CA1 pyramidal cell layer of hippocampal slices (left) and layers II to III layer of cortical slices (right). The vertical line indicates the net change in the ratio of fluorescence at 340 nm and 380 nm. Closed triangles indicate the onset of 100 [micro sign]M NMDA (30 s). (A and A'): In both slices, the changes in [Ca2+]icaused by 100 [micro sign]M NMDA for 30 s could not be reproduced. (B and B'): DL-2-amino-5-phosphonovaleric acid reversibly blocked the NMDA-mediated changes in [Ca2+]i. (C and C'): pretreatment with 30 [micro sign]M lanthanum chloride (La+3) did not significantly reduce the increases in [Ca2+]icaused by NMDA. The effect of La3+was examined in 20 mM HEPES buffered solution omitting NaH2PO4and NaHCO3(pH 7.35, adjusted by NaOH), because La (3+) could react with either HCO3-or H2PO4-.
Figure 6. Typical traces showing the characteristics of N-methyl-D-aspartate (NMDA)-mediated increases in [Ca2+]iin the CA1 pyramidal cell layer of hippocampal slices (left) and layers II to III layer of cortical slices (right). The vertical line indicates the net change in the ratio of fluorescence at 340 nm and 380 nm. Closed triangles indicate the onset of 100 [micro sign]M NMDA (30 s). (A and A'): In both slices, the changes in [Ca2+]icaused by 100 [micro sign]M NMDA for 30 s could not be reproduced. (B and B'): DL-2-amino-5-phosphonovaleric acid reversibly blocked the NMDA-mediated changes in [Ca2+]i. (C and C'): pretreatment with 30 [micro sign]M lanthanum chloride (La+3) did not significantly reduce the increases in [Ca2+]icaused by NMDA. The effect of La3+was examined in 20 mM HEPES buffered solution omitting NaH2PO4and NaHCO3(pH 7.35, adjusted by NaOH), because La (3+) could react with either HCO3-or H2PO4-.
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Figure 7. Typical traces showing the inhibitory effects of thiopental (Thio) on the N-methyl-D-aspartate (NMDA)-mediated increases in [Ca2+](i) in the CA1 pyramidal cell layer of hippocampal slices (left) and layers II to III of cortical slices (right). The vertical line indicates the net change in the ratio of fluorescence at 340 nm and 380 nm. Closed triangles indicate the onset of 100 [micro sign]M NMDA (30 s). Thiopental was present 5 min before the onset of NMDA and was absent during NMDA application.
Figure 7. Typical traces showing the inhibitory effects of thiopental (Thio) on the N-methyl-D-aspartate (NMDA)-mediated increases in [Ca2+](i) in the CA1 pyramidal cell layer of hippocampal slices (left) and layers II to III of cortical slices (right). The vertical line indicates the net change in the ratio of fluorescence at 340 nm and 380 nm. Closed triangles indicate the onset of 100 [micro sign]M NMDA (30 s). Thiopental was present 5 min before the onset of NMDA and was absent during NMDA application.
Figure 7. Typical traces showing the inhibitory effects of thiopental (Thio) on the N-methyl-D-aspartate (NMDA)-mediated increases in [Ca2+](i) in the CA1 pyramidal cell layer of hippocampal slices (left) and layers II to III of cortical slices (right). The vertical line indicates the net change in the ratio of fluorescence at 340 nm and 380 nm. Closed triangles indicate the onset of 100 [micro sign]M NMDA (30 s). Thiopental was present 5 min before the onset of NMDA and was absent during NMDA application.
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Figure 8. A comparison of the effects of thiopental on the N-methyl-D-aspartate (NMDA)-mediated increases in [Ca2+]iin the pyramidal cell layer of hippocampal slices (Hippocampus) and layers II to III of cortical slices (Cortex). The difference between the hippocampus and cortex was analyzed using unpaired t test (NS = no significance), * P < 0.01 and ** P < 0.001 indicate significant differences from zero dose in hippocampus; and #P = 0.01 and ##P < 0.001 indicate significant differences from zero dose in the cortex, as calculated by analysis of variance followed by multiple comparisons with Dunnett's post hoc test (n = number of slices).
Figure 8. A comparison of the effects of thiopental on the N-methyl-D-aspartate (NMDA)-mediated increases in [Ca2+]iin the pyramidal cell layer of hippocampal slices (Hippocampus) and layers II to III of cortical slices (Cortex). The difference between the hippocampus and cortex was analyzed using unpaired t test (NS = no significance), * P < 0.01 and ** P < 0.001 indicate significant differences from zero dose in hippocampus; and #P = 0.01 and ##P < 0.001 indicate significant differences from zero dose in the cortex, as calculated by analysis of variance followed by multiple comparisons with Dunnett's post hoc test (n = number of slices).
Figure 8. A comparison of the effects of thiopental on the N-methyl-D-aspartate (NMDA)-mediated increases in [Ca2+]iin the pyramidal cell layer of hippocampal slices (Hippocampus) and layers II to III of cortical slices (Cortex). The difference between the hippocampus and cortex was analyzed using unpaired t test (NS = no significance), * P < 0.01 and ** P < 0.001 indicate significant differences from zero dose in hippocampus; and #P = 0.01 and ##P < 0.001 indicate significant differences from zero dose in the cortex, as calculated by analysis of variance followed by multiple comparisons with Dunnett's post hoc test (n = number of slices).
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Figure 9. Traces show the average changes in the ratio of fluorescence at 340 nm and 380 nm in the somatic region of individual cortical neurons induced by 200 [micro sign]M N-methyl-D-aspartate (NMDA; indicated by horizontal bars below traces) in the presence of 400 [micro sign]M (indicated by a bar above the traces) and after a wash of thiopental.
Figure 9. Traces show the average changes in the ratio of fluorescence at 340 nm and 380 nm in the somatic region of individual cortical neurons induced by 200 [micro sign]M N-methyl-D-aspartate (NMDA; indicated by horizontal bars below traces) in the presence of 400 [micro sign]M (indicated by a bar above the traces) and after a wash of thiopental.
Figure 9. Traces show the average changes in the ratio of fluorescence at 340 nm and 380 nm in the somatic region of individual cortical neurons induced by 200 [micro sign]M N-methyl-D-aspartate (NMDA; indicated by horizontal bars below traces) in the presence of 400 [micro sign]M (indicated by a bar above the traces) and after a wash of thiopental.
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Table 1. The Latencies to the [Ca2+]iPlateau (Tplateau) and Normalized Increases in R340/380 at 8 (R8), 10 (R10), and 15 min (R (15)) after the Onset of Aglycemic Anoxia in Hippocampal and Cortical Slices in the Absence and Presence of Thiopental 
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Table 1. The Latencies to the [Ca2+]iPlateau (Tplateau) and Normalized Increases in R340/380 at 8 (R8), 10 (R10), and 15 min (R (15)) after the Onset of Aglycemic Anoxia in Hippocampal and Cortical Slices in the Absence and Presence of Thiopental 
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