80 Z
[8]. This was possible since cultures from post-natal day session, the cultures were placed in slice culture media
10 rats resist the hypoxia that occurs without perfusion containing the antibiotic streptomycin and returned to a
P.J.B., unpublished data. We believe that perfusing the 328C incubator. CA1 neuronal loss, a delayed response to
cultures resulted in better retention of the dye. The acidosis in slice cultures, was analyzed 1 and 2 days after
continuous perfusion of the cultures in this study rapidly the recording session Fig. 1C. Propidium iodide epi-
removes dye that leaked to the extracellular space. For this fluorescence in the cultures following low bicarbonate
reason, the extracellular contamination of pH values is acidosis treatment did not differ significantly from controls
likely minimal. suggesting that the 30-min acidosis treatment did not
induce neuronal loss. These results are consistent with the 2.6. Statistics
earlier findings of Ding et al. [6], that demonstrated that low bicarbonate acidosis was nontoxic when performed at
Statistical comparisons were performed using one-way 328C.
ANOVA or Student’s t-test using a significant level of Acidosis-induced neuronal loss was suppressed at 328C
0.05. All values are presented as mean6S.E.M. Fig. 1C; [8]. Intracellular pH, synaptic depression and
neuronal loss were examined at 37 or 328C. A 20-min low bicarbonate acidosis at 378C induced acidification of
3. Results intracellular pH 7.2660.24; n54 that did not differ
significantly from acidosis at 328C Fig. 1A and D. The 3.1. Synaptic depression and neuronal death following
amount of depression of synaptic response during low low bicarbonate acidosis
bicarbonate acidosis at 378C was similar to depression at 328C Fig. 1B and E. Synaptic depression, however,
Hippocampal slice cultures were prepared from 20 to 30 recovered more rapidly at 37 than 328C. The cultures were
day old rats and maintained in vitro for 2 weeks. These returned to the incubator and neuronal loss assayed 1 and 2
cultures were subjected to low bicarbonate acidosis. Using days later Fig. 4F. Neuronal loss was not observed
the pH sensitive dye BCECF, intracellular pH was mea- following the 328C low bicarbonate acidosis treatment. In
sured before, during, and after exposure to low bicarbonate contrast, neuronal loss was induced 1 and 2 days following
aCSF Fig. 1A. In normal aCSF, pH in the CA1
the 378C acidosis treatment. While temperature had mini-
i
pyramidal layer at 328C 7.3160.12; n57 was compar- mal effects on acidosis-induced synaptic depression, neu-
able to other reports Table 1, [4,16,30]. Average pH did ronal loss was observed at 378C but was absent at 328C.
i
not change in control cultures during optical recording. The differences in the effect of temperature on delayed
Upon exposure to low bicarbonate aCSF pH 6.70, pH in neuronal loss as compared to synaptic depression suggest
i
the pyramidal cell layer cells acidified rapidly. After a 20 that they are independent phenomena. Synaptic depression
min exposure to low bicarbonate aCSF, pH stabilized at and neuronal loss were also examined in slice cultures
i
6.5360.08, a value slightly more acidic than the pH 6.70 receiving a 60-min low bicarbonate acidosis treatment
of the perfusing solution. Upon reperfusion with normal Fig. 2. Synaptic depression was induced during the low
aCSF, pH quickly returned the level seen before induction bicarbonate aCSF perfusion that did not recover following
i
of acidosis. These data suggest that low bicarbonate aCSF perfusion with normal aCSF Fig. 2A. Immediately
induced intracellular acidosis that rapidly recovered at the following the recording session, cultures were assayed
end of low bicarbonate aCSF perfusion Fig. 1A. with propidium iodide to determine if the irreversible
Synaptic depression, a well-established rapid response to synaptic depression was associated with neuronal loss. No
acidosis, was also analyzed. Schaffer collateral evoked significant difference between cultures receiving low bicar-
responses were measured extracellularly in the CA1 bonate acidosis or mock acidosis was observed suggesting
pyramidal layer before, during and after perfusion with low the absence of neuronal loss immediately after the record-
bicarbonate aCSF. Field EPSP fEPSP rapidly depressed ing session Fig. 2B. Cultures were returned to the
during the 30-min perfusion of low bicarbonate aCSF Fig. incubator and neuronal death assayed 1 and 2 days
1B. Within 10 min of exposure to low bicarbonate aCSF, following the acidosis treatment. A significant increase in
fEPSP decreased to 50 of baseline level and remained neuronal loss was observed in low bicarbonate group 1 and
depressed for the duration of the exposure to low bicarbon- 2 days after the acidosis treatment Fig. 2B. These data
ate aCSF. Upon reperfusion with normal aCSF, fEPSP suggest that the 60-min acidosis treatment at 328C leads to
recovered from 60 to 100 of fEPSP recorded before low neuronal loss. These data agree with the previous study of
bicarbonate aCSF perfusion. These data suggest that Ding et al. [8] showing that prolonging the acidosis
acidosis induced by low bicarbonate aCSF is accompanied treatment increases acidosis toxicity.
by transient synaptic depression. Using this experimental paradigm, both extracellular and intracellular pH became
3.2. Synaptic depression and neuronal loss following acidic, so the relative contributions of each to synaptic
lactic acid acidosis depression could not be determined.
After completion of the electrophysiological recording The neurotoxicity of low bicarbonate acidosis suggests
Z .-M. Xiang, P.J. Bergold Brain Research 881 2000 77 –87
81
Fig. 1. Analysis of 30 min of low bicarbonate acidosis at 32 and 378C. Panel A: intracellular acidification during low bicarbonate acidosis at 328C. Slice cultures n57 were loaded with the pH sensitive dye BCECF. The cultures were perfused for 30 min with normal aCSF pH 7.35. At time zero, cultures
were perfused for 30 min with low bicarbonate aCSF pH 6.70 bar, followed by 30 min of perfusion with normal aCSF. Intracellular pH mean6S.E.M. was continuously measured. Panel B: synaptic depression during low bicarbonate acidosis at 328C. Mock acidosis n56 cultures were perfused with aCSF
for 90 min. Low bicarbonate acidosis cultures n54 were perfused in a similar manner as panel A with continuous recording of Schaeffer collateral evoked fEPSPs. A bar indicates the time of perfusion with low bicarbonate aCSF pH 6.70. Values are presented as the percent change from
baseline6S.E.M. The two curves are significantly different ANOVA, P,0.001. Panel C: neuronal loss does not follow low bicarbonate acidosis at 328C. Slices were perfused as described in panel B with normal aCSF n53 or low bicarbonate aCSF n54. After the recording session, neuronal loss was
assayed 1 h 0 days, 1 day and 2 days later. Neuronal loss in the CA1 pyramidal cell layer is presented as arbitrary propidium iodide fluorescence units PI index, mean6S.E.M.. Panel D: intracellular acidification during a 20-min low bicarbonate acidosis treatment at 378C. Slice cultures n54 were loaded
with BCECF. The cultures were perfused for 30 min with normal aCSF pH 7.35. At time zero, cultures were perfused for 30 min with low bicarbonate aCSF pH 6.70 bar, followed by 30 min of perfusion with normal aCSF. Intracellular pH mean6S.E.M. was continuously measured. The change of pH
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induced by low bicarbonate acidosis did not significantly differ at 32 and 378C. Panel E: synaptic depression following a 20-min acidosis treatment at 32 and 378C. Values are percent difference6S.E.M. from the average field potential amplitude recorded for 10 min prior to acidosis treatment. The two curves
were not significantly different. Panel F: summary of neuronal loss at 32 and 378C. An asterisk indicates a significant increase of propidium iodide epifluorescence as compared to day zero ANOVA, P,0.001, Student–Neuman–Keuls, P,0.001.
that neuronal loss should be induced by other methods to When the perfusion was switched to lactate aCSF pH 6.70,
induce intracellular acidosis. To test this prediction, slice the intracellular pH rapidly acidified, reaching 6.4360.07,
cultures were treated with lactate containing aCSF to test a value more acidic than the pH of the lactate aCSF pH
the potential neurotoxicity of lactate acidosis. Intracellular 6.70 perfusion Fig. 3A. Upon reperfusion with lactate
pH, synaptic depression and neuronal loss were examined. aCSF pH 7.35, the intracellular pH rapidly returned basal
BCECF was used to monitor intracellular pH. Average pH levels. The rates of acidification and recovery of intracellu-
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was pH 7.1960.13 n56 for normal lactate aCSF at 328C. lar pH by lactate acidosis were similar.
82 Z
Fig. 1. continued
Synaptic depression was also examined during lactic exclude a possibility that the test stimulation contributed to
acidosis Fig. 3B. fEPSP rapidly decreased following the synaptic depression, test stimulation was stopped
treatment with lactate aCSF pH 6.70. Upon reperfusion during recovery period in some experiments, yet no
with lactate aCSF pH 7.35, fEPSP remained depressed. To significant improvement was observed data not shown.
Table 1
a
Summary of the results of this study Acidosis
Time Temperature
pH pH
Synaptic Neuronal
o i
treatment min
8C depression
loss? Low bicarbonate
30 32
6.70 6.5360.08
Reversible No
Low bicarbonate 30
37 6.70
6.4660.08 Reversible
Yes Low bicarbonate
60 32
6.70 NP
Irreversible Yes
Lactic acid 30
32 6.70
6.4360.07 Irreversible
Yes
a
Acidosis was induced by two different means — low bicarbonate and lactic acid and at two different temperatures, 32 and 378C. Each of these treatments resulted in the statistically similar extracellular pH pH and intracellular pH pH . Evoked responses were depressed during the acidosis treatment that
o i
recovered reversible or remained depressed irreversible. The cultures were returned to the incubation and assayed for delayed neuronal loss 1 or 2 days following the acidosis treatment. NP, not performed.
Z .-M. Xiang, P.J. Bergold Brain Research 881 2000 77 –87
83
Fig. 2. Analysis of 60 min of low bicarbonate acidosis. Panel A: synaptic responses following 60 min of low bicarbonate acidosis. Slice cultures n56 were perfused at 328C with low bicarbonate aCSF for 30 min. At time zero, the cultures were perfused with low bicarbonate aCSF for 60 min followed by
normal aCSF for 30 min. Mock acidosis cultures n55 were perfused for 2 h with normal aCSF. Schaeffer collateral evoked responses were recorded continuously through the perfusion. Shown is the average of the last 5 min of each episode. An asterisk indicates that the fEPSP in the acidosis cultures
was significantly different than in mock acidosis cultures during and after the acidosis treatment Student’s t-test, P,0.05. Panel B: Neuronal loss following prolonged low bicarbonate acidosis. Neuronal loss was measured by quantitative propidium iodide staining 1 h day 0, 1 and 2 days after the
acidosis treatment. Propidium iodide fluorescence was measured in arbitrary units PI index, mean6S.E.M.. An asterisk indicates significant neuronal loss in day 1 and day 2 in acidosis group compared to day 0 as well as corresponding time points in control group ANOVA, P,0.01; Student–Neuman–Keuls
post test, P,0.05.
Recovery of synaptic responses was not observed even stable synaptic transmission when lactate replaces glucose
when the reperfusion was prolonged to 90 min. In cultures [27,35]. To address this issue, synaptic responses were
receiving mock lactate acidosis, fEPSP remained stable for recorded in slice cultures that were perfused with aCSF
60 min during perfusion with lactate aCSF pH 7.35. containing 10 mM lactate. Synaptic depression was com-
Between 60 and 80 min, fEPSP reduced to 80 of baseline plete after 1 h n53.
Fig. 3B. This value remained significantly higher than No recovery of synaptic responses was observed follow-
cultures receiving lactic acidosis. At 100 min, fEPSP in ing treatment with lactate aCSF pH 6.70. This may be due
control slices reduced to 50 of baseline and no longer to rapid neuronal loss. To test this, neuronal loss was
significantly differed from fEPSP in cultures receiving assayed immediately after the recording session Fig. 3C.
lactic acidosis. fEPSP was undetectable after 2 h of Neuronal loss was not observed in the pH 7.35 or the pH
perfusion with lactate aCSF, pH 7.35. These data suggest 6.7 groups. Cultures were returned to the incubator and
that lactate acidosis induces irreversible synaptic depres- neuronal loss assayed 1 day later. Propidium iodide
sion. These data also suggest that synaptic responses of epifluorescence was significantly increased in the lactate
slice culture in lactate aCSF, pH 7.35 slowly depress as acidosis cultures when compared to control cultures sug-
well. These data suggest a depressing effect of lactate gesting that lactate acidosis induces neuronal loss. In
anion on synaptic transmission [44]. Other reports suggest lactate acidosis, synaptic depression that did not reverse
84 Z
Fig. 3. Analysis of lactic acidosis. Panel A: intracellular acidification during lactate acidosis. Slice cultures n56 were loaded with BCECF. The cultures were perfused for 30 min with HEPES aCSF containing 20 mM lactate pH 7.35. At time zero, cultures were perfused for 30 min with HEPES aCSF
containing 20 mM lactate pH 6.70 bar. The recovery period was 30 min of perfusion with HEPES aCSF containing 20 mM lactate pH 7.35. Intracellular pH mean6S.E.M. was continuously measured. Panel B: synaptic depression following lactate acidosis. Lactate acidosis slices n54 were
perfused with lactate aCSF pH 6.70. Slices receiving mock lactate acidosis n53 were perfused with aCSF pH 7.30. Shown is the average of the last 5 min of each episode. The acidosis and mock acidosis curves are significantly different ANOVA, P,0.001. Panel C, neuronal loss following lactate
acidosis. Neuronal loss was measured by quantitative propidium iodide staining 1 h day 0, and 1 day after the acidosis treatment. Propidium iodide fluorescence was measured in arbitrary units PI index, mean6S.E.M.. An asterisk indicates significant difference from the neuronal loss on day zero
ANOVA, P,0.05; Student–Neuman–Keuls post test, P,0.05.
during the recording session was associated with delayed The results of these studies are summarized in Table 1.
neuronal loss in the pH 6.7 group. In contrast irreversible Both low bicarbonate and lactic acid resulted in intracellu-
synaptic depression was not associated with neuronal loss lar acidosis of similar extent and duration Figs. 1 and 3.
in the pH 7.35 group. Both rapidly induced intracellular acidosis that recovered
to baseline pH within minutes after the completion of the acidosis treatment Figs. 1 and 3. Synaptic responses were
4. Discussion also depressed during the time of intracellular acidosis.
