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Interactive report
Synaptic depression and neuronal loss in transiently acidic
1hippocampal slice cultures
2
*
Zhong-Min Xiang , Peter J. Bergold
Department of Physiology and Pharmacology, State University of New York-Downstate Medical Center, 450 Clarkson Avenue, Brooklyn, NY11203, USA
Accepted 11 August 2000
Abstract
Acidosis is a rapid and inevitable event accompanying cerebral ischemia or trauma. We used hippocampal slice cultures to examine an immediate effect of acidosis, synaptic depression; and a delayed effect, neuronal loss. Exposure to low bicarbonate artificial cerebral spinal fluid (aCSF), pH 6.70 for 30 min at 328C, acidified intracellular pH from 7.3160.12 to 6.5360.08. Accompanying intracellular acidosis was a depression of synaptic responses. Both effects rapidly reversed after treatment with normal aCSF pH 7.35. Death analysis after acidosis treatment revealed no delayed neuronal loss. Increasing the duration of the acidosis to 60 min, however, induced irreversible synaptic depression and delayed neuronal loss. Increasing acidosis temperature to 378C acidified intracellular pH and depressed synaptic responses. Delayed neuronal loss was also observed. Acidosis using lactate aCSF, pH 6.70 for 30 min at 328C acidified intracellular pH from 7.1960.13 to 6.4360.07 and depressed synaptic responses. After reperfusion with lactate containing aCSF pH 7.35, intracellular pH recovered yet synaptic responses remained depressed and delayed neuronal loss was observed. This suggested that, for a 30-min treatment at 328C, lactate acidosis was neurotoxic while low bicarbonate acidosis was not. Increasing the duration or temperature of low bicarbonate acidosis induced neuronal loss. These data provide additional evidence that acidosis contributes to the neurotoxicity during stroke and trauma. 2000 Elsevier Science B.V. All rights reserved.
Theme: Disorders of the nervous system
Topic: Ischemia
Keywords: Intracellular pH; Lactate; Bicarbonate; Hypothermia; Slice culture
1. Introduction trauma, brain pH acidifies to 6.2 to 6.8 [32]. Even more severe brain acidification occurs if hyperglycemia precedes Intracellular and extracellular pH are highly regulated in ischemia or if the ischemia is incomplete so that glucose the brain. Intracellular neuronal pH ranges from 6.9 to 7.2 continues to be delivered to hypoxic cells [32,18]. In these and intracellular glial pH ranges between 6.8 and 7.6 conditions, pH can acidify to as low as 6.0 [20,13,2]. [4,7,23]. Larger deviations of intracellular pH occur only Acidosis of the brain is typically transient. In both during pathological conditions such as cerebral ischemia or ischemia and trauma, pH recovers to physiological levels trauma [32]. During ischemia, intracellular pH of neurons in minutes to hours.
and glia typically acidify to 6.5 [32,20,13,2]. During Both early and delayed effects of acidosis have been described. Early effects of acidosis include depression of neuronal activity [1,44], cell swelling [34] and enhanced production of free radicals [32]. Unless acidosis is severe, these effects are reversible. Acidosis has minor effect on
1
Published on the World Wide Web on 1 September 2000. resting membrane potential and excitability of neurons [5]. *Corresponding author. Tel.: 11-718-270-3927; fax: 11-718-270- Synaptic transmission is strongly depressed [1,19,37,17]. 2241.
The site of synaptic depression is thought to be post-E-mail address: [email protected] (P.J. Bergold).
2 synaptic, since presynaptic fiber volley is more resistant to
Present address: Department of Psychiatry, Mount Sinai School of
Medicine, One Gustave Levy Place, New York, NY 10029, USA. acidosis than excitatory post-synaptic potential (EPSP)
[44]. Post-synaptic effects of acidosis include suppression 2. Materials and methods
of ionotropic glutamate receptor function and enhanced
function of GABA-A receptors [36,39,11]. 2.1. Hippocampal slice cultures A potential delayed effect of acidosis is neurotoxicity.
Hyperglycemia or hypercapnea preceding ischemia results Hippocampal slice cultures were prepared from in more severe acidosis and neuronal loss than ischemia Sprague–Dawley rats aged 20–30 days [45]. Rats were alone [13,18,14]. These data suggest that acidosis is toxic. treated with the anesthetics ketamine and halothane to In contrast, hypercapnea preceding ischemia has been ensure that any pain or discomfort were minimized during reported to reduce brain injury [33]. Direct application of brain removal. One to three slices were plated on a lactic acid in vivo suggested that acidosis may not be Millipore-CM filter (Millipore, Woburn, MA). The filters neurotoxic since very acidic lactic acid (pHo 5.3) was were placed above 1 ml of slice culture media (SCM, 50% needed to induce neuronal damage [15]. Such pH extremes Basal Medium Eagle’s, 25% Earles Balanced Salt solution, do not occur in vivo. Intracellular pH was not measured in 25% horse serum, 25 mM N-(2-Hydroxyethyl) piperazine-this study so the extent and duration of the acidosis is not N9-2-ethanesulfonic acid (HEPES), 1 mM glutamine, 28
known. mM glucose, pH57.2) and incubated at 328C in a 5% CO2
In vitro studies of acidosis toxicity have not been atmosphere. After 1 week, slices were cultured in slice conclusive. Depending upon the culture system, acidosis culture medium containing 5% horse serum. Cultures were has been shown to be either neuroprotective or neurotoxic. fed weekly if the insert contained one slice, every 3 days The evidence that acidosis is neuroprotective largely for two slices and every 2 days for three slices. Cultures comes from embryonic primary neuronal cultures. Inhibi- were maintained for 2 weeks before use.
tion of NMDA receptors at acidic pH suggests a
mecha-nism how acidosis may be neuroprotective. The neuro- 2.2. Electrophysiological recording toxicity of hypoxia or excitotoxicity toward primary
embryonic neurons was strongly attenuated by acidosis Slice cultures attached to the Millicell-CM filter were [37,38,12]. These cultures are highly resistant to acidosis transferred to an air-interface slice recording chamber that [34,38,10,21]. The resistance of young neurons to acidosis had been modified to fit the filter insert (Fine Science toxicity may underestimate acidosis toxicity factor toward Tools, Foster City, CA). Cultures were perfused at 1
older neurons. ml / min with aCSF that was aerated with 95% O and 5%2
Direct evidence for acidosis toxicity comes from studies CO , and maintained at 322 8C or 378C (aCSF: (in mM) using hippocampal slice cultures. Moderate and transient NaCl, 124; KCl, 3; MgCl , 1.6; CaCl , 1.7; NaH PO , 1.2;2 2 2 4
acidosis of pH 6.6 induced widespread, delayed neuronal NaHCO3, 25; glucose, 11; pH57.35). Schaffer collaterals necrosis and apoptosis. Increasing the duration of acidosis of slice cultures were stimulated every 30 s with bipolar increased the amount of neuronal loss [8]. These data tungsten electrodes. Glass recording electrodes were filled support a neurotoxic role of acidosis [8,31]. In these with aCSF (4–8 MV) and placed in the CA1 pyramidal studies, intracellular acidosis was induced by perfusing cell layer. Amplified signals were digitized with a slice cultures with reduced bicarbonate. To further study McAdios digitizer (GW Instruments, Somerville, MA) and the potential neurotoxicity of acidosis, intracellular processed using Superscope 2.0 software. Field excitatory acidosis was induced by either low bicarbonate or lactic post-synaptic potentials (fEPSPs) were recorded in CA1 acid perfusion. The mechanism of induction of intracellu- pyramidal cell layer by maximally stimulating Schaffer lar acidosis differs using low bicarbonate or lactic acid. collaterals. In all experiments input–output data from Perfusion with low bicarbonate induces acidosis by dimin- stimulation intensities of 50 to 200mA, fEPSP amplitude
2
ishing the strength of intracellular buffering by HCO /3 was roughly proportional. The maximum stimulation was CO . Perfusion with acidic solutions of lactic acid acidifies2 near 300 mA in all groups. At each stimulation intensity, intracellular pH by the free diffusion of lactic acid and there were no statistical differences among groups active transport of lactate across the cell membrane in (ANOVA, P.0.05, data not shown). Similar results were addition to diminishing the strength of intracellular buffer- obtained if half-maximal stimulation was used (data not
2
2.3. Assay of cell death slice cultures were perfused for 30 min with aCSF containing 25 mM bicarbonate. The solution pH was 7.35 Cell death was measured using the cell impermeable when gassed with 95% O2 and 5% CO . Acidosis was2
fluorescent dye propidium iodide (PI). When the cell induced for 30 or 60 min by perfusing the slice cultures membrane is damaged, PI enters the cell, binds DNA, and with low bicarbonate aCSF, pH 6.70. A 30-min recovery emits fluorescence when excited. Fluorescence intensity is period of perfusion with aCSF followed the low bicarbon-proportional to the amount of DNA binding as well as cell ate acidosis treatment. In some experiments, cultures were death [8,45,22]. Unlike earlier studies using slice cultures perfused with lactate aCSF. Lactate aCSF was prepared by from rats younger than 10 days, this study uses slice replacing 20 mM NaCl with 20 mM lactate in aCSF cultures from 20-day-old rats. These cultures remain buffered with 20 mM HEPES instead of 25 mM bicarbon-substantially thicker than cultures from 10-day-old rats ate. The pH of lactate aCSF was adjusted to pH 7.35 or pH [45]. Cell counting is difficult since the cell density in the 6.70 using HCl. Acidosis was induced using a 30-min pyramidal cell layer is high and PI-positive cells are in perfusion of normal lactate aCSF (pH 7.35), followed by multiple focal planes. Studies from the authors [8] and 30 min of acidic lactate aCSF (pH 6.70), followed by a from other investigators [22,40] have demonstrated that recovery period of 30 min of normal lactate aCSF (pH propidium iodide labeling quantitatively measures cell 7.35).
loss. All images were normalized using an image of a
calibration slide containing InSpeck red 100% fluorescent 2.5. Measurement of intracellular pH beads (Molecular Probes, Eugene, OR). The use of the
InSpeck fluorescent beads ensures that the excitation Slice cultures were loaded for 60 min at 358C in 2 ml of intensity and camera sensitivity are uniform and all modified Gey’s Balanced Salt solution (in mM, NaCl, 120; readings are in the linear range of the camera. Slice KCl, 5; KH PO , 0.2; Na HPO , 0.8; NaHCO2 4 2 4 3, 27; cultures were transferred to serum-free SCM with 4mg / ml MgSO , 0.3; MgCl , 10; CaCl , 1.5; glucose, 5.6; pH4 2 2 5 propidium iodide (Sigma, St. Louis, MO), incubated in 5% 7.2) containing 10 mM BCECF-AM and 0.001% (v / v) CO2 incubator at 328C for 30 min. Fluorescence was pluronic acid (Molecular Probes, Eugene, OR). Cultures observed using a Zeiss Axiovert 100 microscope with a were excised from the filter insert, and transferred to a rhodamine filter set. After each image session, slices were perfusion chamber assembled on the stage of a Nikon rinsed with serum-free SCM, transferred to SCM con- Diaphot inverted fluorescence microscope. The slice cul-taining 5% horse serum and returned to the incubator. The tures were slightly submerged and perfused (1 ml / min) fluorescent images were taken using a PTI intensified CCD with aCSF or modified aCSF aerated with 95%O ,2
camera and analyzed using NIH Image v.1.59. Mean pixel 5%CO . Depending upon the experiment, chamber tem-2
values were assayed the CA1 pyramidal layer. The CA1 perature was maintained either at 32 or 378C. Slice cultures pyramidal cell layer in organotypic hippocampal slice were illuminated with alternating excitation at 440 and 490 cultures is known to contain predominantly CA1 pyramidal nm and emissions at 510 nm were recorded (Photon cells [9]. Interneurons and glia are also present in the CA1 Technology International, Princeton, NJ). Ratio images of pyramidal cell layer [9]. When PI florescence was observed 490 nm / 440 nm were obtained. Calibration curve were in the CA1 pyramidal cell layer, PI florescence was generated by exposing cultures to the solutions with 6 pH minimal in the adjacent regions of stratum radiatum and values ranging from 5.8 to 7.5: (in mM) KCl 70, HEPES stratum oriens (data not shown). These regions contain 20, sucrose 90, Na HPO2 4 2.5, glucose 10, MgSO4 1, predominantly neuropil, glia, and interneurons [9]. The CaCl 1, nigericin 102 mM. The acquired equation was used presence of PI staining in CA1 stratum pyramidale and the to convert the ratio 490 nm / 440 nm to pHi values: pHi5
2
absence of staining in adjacent regions suggests that the 4.0810.93*Ratio, r 50.89. (n59). The high K / nigericin staining in the CA1 pyramidal cell layer is predominantly method used to calibrate BCECF fluorescence potentially neuronal. In all experiments, propidium iodide assay at day induces a pH-dependent error [3]. To address this issue, a zero of staining corresponds to 1 h following the acidosis second calibration curve was generated using a buffer that or mock acidosis treatment. The manipulations of the lacked nigericin (in mM; NaCl, 10; KCl, 70; MgCl , 1;2
experiment, including temperature changes, changes in HEPES, 20, plus BCECF, 2mM) at solution pH of pH 5.0, buffers, are thought to induce propidium iodide staining at 5.5, 6.0, 6.5, 7.0, 7.5 [46]. A comparison of the pH values
this time. generated from these two calibration curves yielded similar
pH values except for a higher intracellular pH value 2.4. Acidosis treatments (7.4660.15) using aCSF, solution pH 7.35. The conclu-sions of this study are equivalent using either calibration In low bicarbonate acidosis, the 25 mM bicarbonate in curve.
[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, pHi in the CA1 the 378C acidosis treatment. While temperature had mini-pyramidal layer at 328C (7.3160.12; n57) was compar- mal effects on acidosis-induced synaptic depression, neu-able to other reports (Tneu-able 1, [4,16,30]). Average pH didi ronal loss was observed at 378C but was absent at 328C. 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 ini neuronal loss as compared to synaptic depression suggest 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 ati and neuronal loss were also examined in slice cultures 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 inductioni bicarbonate aCSF perfusion that did not recover following 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.
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 pHi
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).
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 pHo pHi Synaptic Neuronal
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 thato i
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. 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
slice cultures to acidosis is similar to the response in acute observations suggest that the short-term effect of lactate is hippocampal slices. protective while its long-term effect is neurotoxic.
Synaptic depression, however, was either reversible or The enhanced neurotoxicity of lactate as compared to irreversible depending upon the method used and the low bicarbonate may result from their different effects on duration of the acidosis treatment. With low bicarbonate intracellular buffering and ion transport. The differing rates acidosis, synaptic depression was reversible when the of acidification of intracellular pH at the onset of the acidosis treatment was 30 min long (Fig. 1C). Increasing lactate and low bicarbonate acidosis treatments suggest the acidosis treatment to 60 min resulted in irreversible different mechanisms of acidification. Intracellular pH synaptic depression (Fig. 2A). Thirty minutes of lactic acidified more rapidly by lactate than low bicarbonate (Fig. acidosis, in contrast, induced irreversible synaptic depres- 4). In contrast, there was no significant difference in the sion. (Fig. 3B) rate of recovery (Fig. 4). Lactic acid may acidify intracel-In contrast to earlier studies of acidosis, cultured hip- lular pH by freely diffusing across the plasma membrane
1
pocampal slices permit analysis of synaptic depression and by ionizing to lactate and H [26]. Lactate can also
1
followed by delayed neuronal loss. Acidification of in- enter the cell via the H -lactate cotransporter [43]. Omit-tracellular pH may or may not result in neuronal loss. Few ting bicarbonate in the extracellular solutions may reduce hippocampal neurons are lost following cerebral acidosis intracellular buffering power by exiting of intracellular
2 2
induced by hypercapnea [6]. Acidosis-induced neuronal bicarbonate anions through HCO / Cl3 exchanger, and loss is influenced by three parameters: the method to facilitate intracellular acidification.
induce acidosis, the duration of the acidosis, and the Unexpectedly, synapses were observed to be depressed temperature of the acidosis treatment. The duration and in control slice cultures treated with lactate aCSF, pH 7.35 temperature of the acidosis treatment are important since (Fig. 3B). Synaptic depression was slow as fEPSP began neuronal loss was induced by increasing low bicarbonate to decrease after 1 h into the recording session. This acidosis to 60 min or raising the temperature from 32 to contrasts with the stability of slice culture synaptic re-378C (Figs. 1 and 2). At 328C, 30 min of lactate acidosis sponses in normal aCSF. Intracellular pH did not sig-was neurotoxic while 30 min of low bicarbonate acidosis nificantly differ between normal lactate aCSF, (pH 7.35) was not. These data suggest that acidosis by lactic acid is and aCSF, (pH 7.35) suggesting that this depression of more neurotoxic to slice cultures than low bicarbonate. synaptic responses is unrelated to acidosis. The possibility Lactate has been proposed to protect against hypoxic exists that lactate has a slowly depressing effect on injury in acute hippocampal slices [27,29,28]. Due to the synaptic responses in slice cultures. In studies using acute limited in vitro life span of the acute slice preparation, hippocampal slices, the issue of lactate supporting synaptic these studies are limited to examining a protective effect transmission remains unresolved; synaptic responses have within hours of the insult. In contrast, when similarly been reported to be stable [27,35] or depressed by lactate prepared slices that were placed into long-term culture, [44,41,42]. The question if lactate can support synaptic lactate acidosis induced delayed neurotoxicity (Fig. 3). In transmission in slice cultures requires additional study. vivo, increased lactate acidosis is associated with increased A critical question remains: why is acidosis neurotoxic ischemic injury [32,20,13,2,18]. Taken together, these in slice cultures when similar acidosis has no effect to
Fig. 4. Comparison of the change of pH during low bicarbonate and lactic acidosis Induction (0–12 min) and recovery (30–42 min) of intracellular pH arei
glutamate antagonists and extracellular acidity, Science 260 (1993) dissociated neuronal cultures? Embryonic and perinatal
1516–1518. neurons may have reduced susceptibility to acidosis injury
[13] K. Katsura, A. Ekholm, B. Asplund, B.K. Siesjo, Extracellular pH in as compared to slice cultures. A critical factor may be the the brain during ischemia: relationship to the severity of lactic age at which slice culture are isolated and placed into acidosis, J. Cereb. Blood Flow Metab. 11 (1991) 597–599. culture. Age effects are a common theme in brain injury [14] K. Katsura, T. Kristian, M.L. Smith, B.K. Siesjo, Acidosis induced
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[15] R.P. Kraig, C.K. Petito, F. Plum, W.A. Pulsinelli, Hydrogen ions kill [25,24]. An alternative explanation is that the higher brain at concentrations reached in ischemia, J. Cereb. Blood Flow neuronal density or the maintenance of neuronal glial Metab. 7 (1987) 379–386.
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[17] J. Lee, T. Taira, P. Pihlaja, B.R. Ransom, K. Kaila, Effects of CO2
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Acknowledgements [18] P.A. Li, B.K. Siesjo, Role of hyperglycaemia-related acidosis in
ischaemic brain damage, Acta Physiol. Scand. 161 (1997) 567–580. [19] Y. Morimoto, O. Kemmotsu, Effect of lactic and CO acidosis on
The authors would like to thank Dr. Angel Cinelli for 2
neuronal function following glucose-oxygen deprivation in rat providing assistance for pH imaging experiments. We
hippocampal slices, Brain Res. 654 (1994) 273–278. would also thank Dr. Ilham Muslimov for help with the
[20] K. Munekata, K.A. Hossmann, Effect of 5-min ischemia on regional photography and Mr. Subha Basu for reading the manu- pH and energy state of the gerbil brain: relation to selective script. This work was supported by an American Heart vulnerability of the hippocampus, Stroke 18 (1987) 412–417.
[21] M. Nedergaard, S.A. Goldman, S. Desai, W.A. Pulsinelli, Acid-Association Grant-in-aid to P.J.B.
induced death in neurons and glia, J. Neurosci. 11 (1991) 2489– 2497.
[22] J. Noraberg, B.W. Kristensen, J. Zimmer, Markers for neuronal degeneration in organotypic slice cultures, Brain Res. Brain Res.
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