Brain Research 881 (2000) 77–87 www.elsevier.com / locate / bres
Interactive report
Synaptic depression and neuronal loss in transiently acidic hippocampal slice cultures 1 2 Zhong-Min Xiang , Peter J. Bergold*
Department of Physiology and Pharmacology, State University of New York-Downstate Medical Center, 450 Clarkson Avenue, Brooklyn, NY 11203, 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 Intracellular and extracellular pH are highly regulated in the brain. Intracellular neuronal pH ranges from 6.9 to 7.2 and intracellular glial pH ranges between 6.8 and 7.6 [4,7,23]. Larger deviations of intracellular pH occur only during pathological conditions such as cerebral ischemia or trauma [32]. During ischemia, intracellular pH of neurons and glia typically acidify to 6.5 [32,20,13,2]. During
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Published on the World Wide Web on 1 September 2000. *Corresponding author. Tel.: 11-718-270-3927; fax: 11-718-2702241. E-mail address:
[email protected] (P.J. Bergold). 2 Present address: Department of Psychiatry, Mount Sinai School of Medicine, One Gustave Levy Place, New York, NY 10029, USA.
trauma, brain pH acidifies to 6.2 to 6.8 [32]. Even more severe brain acidification occurs if hyperglycemia precedes ischemia or if the ischemia is incomplete so that glucose continues to be delivered to hypoxic cells [32,18]. In these conditions, pH can acidify to as low as 6.0 [20,13,2]. Acidosis of the brain is typically transient. In both ischemia and trauma, pH recovers to physiological levels in minutes to hours. 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 resting membrane potential and excitability of neurons [5]. Synaptic transmission is strongly depressed [1,19,37,17]. The site of synaptic depression is thought to be postsynaptic, since presynaptic fiber volley is more resistant to acidosis than excitatory post-synaptic potential (EPSP)
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[44]. Post-synaptic effects of acidosis include suppression of ionotropic glutamate receptor function and enhanced function of GABA-A receptors [36,39,11]. A potential delayed effect of acidosis is neurotoxicity. Hyperglycemia or hypercapnea preceding ischemia results in more severe acidosis and neuronal loss than ischemia alone [13,18,14]. These data suggest that acidosis is toxic. In contrast, hypercapnea preceding ischemia has been reported to reduce brain injury [33]. Direct application of lactic acid in vivo suggested that acidosis may not be neurotoxic since very acidic lactic acid (pH o 5.3) was needed to induce neuronal damage [15]. Such pH extremes do not occur in vivo. Intracellular pH was not measured in this study so the extent and duration of the acidosis is not known. In vitro studies of acidosis toxicity have not been conclusive. Depending upon the culture system, acidosis has been shown to be either neuroprotective or neurotoxic. The evidence that acidosis is neuroprotective largely comes from embryonic primary neuronal cultures. Inhibition of NMDA receptors at acidic pH suggests a mechanism how acidosis may be neuroprotective. The neurotoxicity of hypoxia or excitotoxicity toward primary embryonic neurons was strongly attenuated by acidosis [37,38,12]. These cultures are highly resistant to acidosis [34,38,10,21]. The resistance of young neurons to acidosis toxicity may underestimate acidosis toxicity factor toward older neurons. Direct evidence for acidosis toxicity comes from studies using hippocampal slice cultures. Moderate and transient acidosis of pH 6.6 induced widespread, delayed neuronal necrosis and apoptosis. Increasing the duration of acidosis increased the amount of neuronal loss [8]. These data support a neurotoxic role of acidosis [8,31]. In these studies, intracellular acidosis was induced by perfusing slice cultures with reduced bicarbonate. To further study the potential neurotoxicity of acidosis, intracellular acidosis was induced by either low bicarbonate or lactic acid perfusion. The mechanism of induction of intracellular acidosis differs using low bicarbonate or lactic acid. Perfusion with low bicarbonate induces acidosis by diminishing the strength of intracellular buffering by HCO 2 3 / CO 2 . Perfusion with acidic solutions of lactic acid acidifies intracellular pH by the free diffusion of lactic acid and active transport of lactate across the cell membrane in addition to diminishing the strength of intracellular buffering by HCO 2 3 / CO 2 [26,43]. In this study, two parameters were measured during the acidosis treatment, acidification of intracellular pH and depression of synaptic responses. Assay of these immediate effects ensure that slice cultures display well-known immediate effects of extracellular acidosis. Following the end of the acidosis treatment cultures were returned to the incubator and delayed neuronal loss assayed. These studies provide additional evidence that moderate acidosis seen in in vivo ischemic conditions is toxic enough to kill neurons.
2. Materials and methods
2.1. Hippocampal slice cultures Hippocampal slice cultures were prepared from Sprague–Dawley rats aged 20–30 days [45]. Rats were treated with the anesthetics ketamine and halothane to ensure that any pain or discomfort were minimized during brain removal. One to three slices were plated on a Millipore-CM filter (Millipore, Woburn, MA). The filters were placed above 1 ml of slice culture media (SCM, 50% Basal Medium Eagle’s, 25% Earles Balanced Salt solution, 25% horse serum, 25 mM N-(2-Hydroxyethyl) piperazineN9-2-ethanesulfonic acid (HEPES), 1 mM glutamine, 28 mM glucose, pH57.2) and incubated at 328C in a 5% CO 2 atmosphere. After 1 week, slices were cultured in slice culture medium containing 5% horse serum. Cultures were fed weekly if the insert contained one slice, every 3 days for two slices and every 2 days for three slices. Cultures were maintained for 2 weeks before use.
2.2. Electrophysiological recording Slice cultures attached to the Millicell-CM filter were transferred to an air-interface slice recording chamber that had been modified to fit the filter insert (Fine Science Tools, Foster City, CA). Cultures were perfused at 1 ml / min with aCSF that was aerated with 95% O 2 and 5% CO 2 , and maintained at 328C or 378C (aCSF: (in mM) NaCl, 124; KCl, 3; MgCl 2 , 1.6; CaCl 2 , 1.7; NaH 2 PO 4 , 1.2; NaHCO 3, 25; glucose, 11; pH57.35). Schaffer collaterals of slice cultures were stimulated every 30 s with bipolar tungsten electrodes. Glass recording electrodes were filled with aCSF (4–8 MV) and placed in the CA1 pyramidal cell layer. Amplified signals were digitized with a McAdios digitizer (GW Instruments, Somerville, MA) and processed using Superscope 2.0 software. Field excitatory post-synaptic potentials (fEPSPs) were recorded in CA1 pyramidal cell layer by maximally stimulating Schaffer collaterals. In all experiments input–output data from stimulation intensities of 50 to 200 mA, fEPSP amplitude was roughly proportional. The maximum stimulation was near 300 mA in all groups. At each stimulation intensity, there were no statistical differences among groups (ANOVA, P.0.05, data not shown). Similar results were obtained if half-maximal stimulation was used (data not shown). Evoked synaptic responses were also tested at 32, 34 and 378C. Increased temperature resulted in larger fEPSP regardless of the stimulation intensity. Prior to sterile recordings, the recording chamber and electrodes were washed with 70% ethanol and perfusion solutions were filtered using 0.2 mm filter (Corning, Bedford, MA). After recording, the slices were cultured in SCM containing streptomycin (50 unit / ml, Sigma). Streptomycin is a cell impermeable antibiotic that did not effect the results of this study.
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2.3. Assay of cell death Cell death was measured using the cell impermeable fluorescent dye propidium iodide (PI). When the cell membrane is damaged, PI enters the cell, binds DNA, and emits fluorescence when excited. Fluorescence intensity is proportional to the amount of DNA binding as well as cell death [8,45,22]. Unlike earlier studies using slice cultures from rats younger than 10 days, this study uses slice cultures from 20-day-old rats. These cultures remain substantially thicker than cultures from 10-day-old rats [45]. Cell counting is difficult since the cell density in the pyramidal cell layer is high and PI-positive cells are in multiple focal planes. Studies from the authors [8] and from other investigators [22,40] have demonstrated that propidium iodide labeling quantitatively measures cell loss. All images were normalized using an image of a calibration slide containing InSpeck red 100% fluorescent beads (Molecular Probes, Eugene, OR). The use of the InSpeck fluorescent beads ensures that the excitation intensity and camera sensitivity are uniform and all readings are in the linear range of the camera. Slice cultures were transferred to serum-free SCM with 4 mg / ml propidium iodide (Sigma, St. Louis, MO), incubated in 5% CO 2 incubator at 328C for 30 min. Fluorescence was observed using a Zeiss Axiovert 100 microscope with a rhodamine filter set. After each image session, slices were rinsed with serum-free SCM, transferred to SCM containing 5% horse serum and returned to the incubator. The fluorescent images were taken using a PTI intensified CCD camera and analyzed using NIH Image v.1.59. Mean pixel values were assayed the CA1 pyramidal layer. The CA1 pyramidal cell layer in organotypic hippocampal slice cultures is known to contain predominantly CA1 pyramidal cells [9]. Interneurons and glia are also present in the CA1 pyramidal cell layer [9]. When PI florescence was observed in the CA1 pyramidal cell layer, PI florescence was minimal in the adjacent regions of stratum radiatum and stratum oriens (data not shown). These regions contain predominantly neuropil, glia, and interneurons [9]. The presence of PI staining in CA1 stratum pyramidale and the absence of staining in adjacent regions suggests that the staining in the CA1 pyramidal cell layer is predominantly neuronal. In all experiments, propidium iodide assay at day zero of staining corresponds to 1 h following the acidosis or mock acidosis treatment. The manipulations of the experiment, including temperature changes, changes in buffers, are thought to induce propidium iodide staining at this time.
2.4. Acidosis treatments In low bicarbonate acidosis, the 25 mM bicarbonate in aCSF was reduced to 5 mM, and NaCl was correspondingly increased by 20 mM. When gassed with 95% O 2 and 5% CO 2 , solution pH was 6.70. Before acidosis induction, the
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slice cultures were perfused for 30 min with aCSF containing 25 mM bicarbonate. The solution pH was 7.35 when gassed with 95% O 2 and 5% CO 2 . Acidosis was induced for 30 or 60 min by perfusing the slice cultures with low bicarbonate aCSF, pH 6.70. A 30-min recovery period of perfusion with aCSF followed the low bicarbonate acidosis treatment. In some experiments, cultures were perfused with lactate aCSF. Lactate aCSF was prepared by replacing 20 mM NaCl with 20 mM lactate in aCSF buffered with 20 mM HEPES instead of 25 mM bicarbonate. The pH of lactate aCSF was adjusted to pH 7.35 or pH 6.70 using HCl. Acidosis was induced using a 30-min perfusion of normal lactate aCSF (pH 7.35), followed by 30 min of acidic lactate aCSF (pH 6.70), followed by a recovery period of 30 min of normal lactate aCSF (pH 7.35).
2.5. Measurement of intracellular pH Slice cultures were loaded for 60 min at 358C in 2 ml of modified Gey’s Balanced Salt solution (in mM, NaCl, 120; KCl, 5; KH 2 PO 4 , 0.2; Na 2 HPO 4 , 0.8; NaHCO 3, 27; MgSO 4 , 0.3; MgCl 2 , 10; CaCl 2 , 1.5; glucose, 5.6; pH5 7.2) containing 10 mM BCECF-AM and 0.001% (v / v) pluronic acid (Molecular Probes, Eugene, OR). Cultures were excised from the filter insert, and transferred to a perfusion chamber assembled on the stage of a Nikon Diaphot inverted fluorescence microscope. The slice cultures were slightly submerged and perfused (1 ml / min) with aCSF or modified aCSF aerated with 95%O 2 , 5%CO 2 . Depending upon the experiment, chamber temperature was maintained either at 32 or 378C. Slice cultures were illuminated with alternating excitation at 440 and 490 nm and emissions at 510 nm were recorded (Photon Technology International, Princeton, NJ). Ratio images of 490 nm / 440 nm were obtained. Calibration curve were generated by exposing cultures to the solutions with 6 pH values ranging from 5.8 to 7.5: (in mM) KCl 70, HEPES 20, sucrose 90, Na 2 HPO 4 2.5, glucose 10, MgSO 4 1, CaCl 2 1, nigericin 10 mM. The acquired equation was used to convert the ratio 490 nm / 440 nm to pHi values: pH i 5 4.0810.93*Ratio, r 2 50.89. (n59). The high K / nigericin method used to calibrate BCECF fluorescence potentially induces a pH-dependent error [3]. To address this issue, a second calibration curve was generated using a buffer that lacked nigericin (in mM; NaCl, 10; KCl, 70; MgCl 2 , 1; HEPES, 20, plus BCECF, 2 mM) at solution pH of pH 5.0, 5.5, 6.0, 6.5, 7.0, 7.5 [46]. A comparison of the pH values generated from these two calibration curves yielded similar pH values except for a higher intracellular pH value (7.4660.15) using aCSF, solution pH 7.35. The conclusions of this study are equivalent using either calibration curve. In this study, slice cultures were continuously perfused during the pH measurements. In a previous paper, slice cultures were imaged under a coverslip without perfusion
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[8]. This was possible since cultures from post-natal day 10 rats resist the hypoxia that occurs without perfusion (P.J.B., unpublished data). We believe that perfusing the cultures resulted in better retention of the dye. The continuous perfusion of the cultures in this study rapidly removes dye that leaked to the extracellular space. For this reason, the extracellular contamination of pH values is likely minimal.
2.6. Statistics Statistical comparisons were performed using one-way ANOVA or Student’s t-test using a significant level of 0.05. All values are presented as mean6S.E.M.
3. Results
3.1. Synaptic depression and neuronal death following low bicarbonate acidosis Hippocampal slice cultures were prepared from 20 to 30 day old rats and maintained in vitro for 2 weeks. These cultures were subjected to low bicarbonate acidosis. Using the pH sensitive dye BCECF, intracellular pH was measured before, during, and after exposure to low bicarbonate aCSF (Fig. 1A). In normal aCSF, pH i in the CA1 pyramidal layer at 328C (7.3160.12; n57) was comparable to other reports (Table 1, [4,16,30]). Average pH i did not change in control cultures during optical recording. Upon exposure to low bicarbonate aCSF (pH 6.70), pH i in the pyramidal cell layer cells acidified rapidly. After a 20 min exposure to low bicarbonate aCSF, pH i stabilized at 6.5360.08, a value slightly more acidic than the pH (6.70) of the perfusing solution. Upon reperfusion with normal aCSF, pH i quickly returned the level seen before induction of acidosis. These data suggest that low bicarbonate aCSF induced intracellular acidosis that rapidly recovered at the end of low bicarbonate aCSF perfusion (Fig. 1A). Synaptic depression, a well-established rapid response to acidosis, was also analyzed. Schaffer collateral evoked responses were measured extracellularly in the CA1 pyramidal layer before, during and after perfusion with low bicarbonate aCSF. Field EPSP (fEPSP) rapidly depressed during the 30-min perfusion of low bicarbonate aCSF (Fig. 1B). Within 10 min of exposure to low bicarbonate aCSF, fEPSP decreased to 50% of baseline level and remained depressed for the duration of the exposure to low bicarbonate aCSF. Upon reperfusion with normal aCSF, fEPSP recovered from 60 to 100% of fEPSP recorded before low bicarbonate aCSF perfusion. These data suggest that acidosis induced by low bicarbonate aCSF is accompanied by transient synaptic depression. Using this experimental paradigm, both extracellular and intracellular pH became acidic, so the relative contributions of each to synaptic depression could not be determined. After completion of the electrophysiological recording
session, the cultures were placed in slice culture media containing the antibiotic streptomycin and returned to a 328C incubator. CA1 neuronal loss, a delayed response to acidosis in slice cultures, was analyzed 1 and 2 days after the recording session (Fig. 1C). Propidium iodide epifluorescence in the cultures following low bicarbonate acidosis treatment did not differ significantly from controls suggesting that the 30-min acidosis treatment did not induce neuronal loss. These results are consistent with the earlier findings of Ding et al. [6], that demonstrated that low bicarbonate acidosis was nontoxic when performed at 328C. Acidosis-induced neuronal loss was suppressed at 328C (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 intracellular pH (7.2660.24; n54) that did not differ significantly from acidosis at 328C (Fig. 1A and D). The amount of depression of synaptic response during low bicarbonate acidosis at 378C was similar to depression at 328C (Fig. 1B and E). Synaptic depression, however, recovered more rapidly at 37 than 328C. The cultures were returned to the incubator and neuronal loss assayed 1 and 2 days later (Fig. 4F). Neuronal loss was not observed following the 328C low bicarbonate acidosis treatment. In contrast, neuronal loss was induced 1 and 2 days following the 378C acidosis treatment. While temperature had minimal effects on acidosis-induced synaptic depression, neuronal loss was observed at 378C but was absent at 328C. The differences in the effect of temperature on delayed neuronal loss as compared to synaptic depression suggest that they are independent phenomena. Synaptic depression and neuronal loss were also examined in slice cultures receiving a 60-min low bicarbonate acidosis treatment (Fig. 2). Synaptic depression was induced during the low bicarbonate aCSF perfusion that did not recover following perfusion with normal aCSF (Fig. 2A). Immediately following the recording session, cultures were assayed with propidium iodide to determine if the irreversible synaptic depression was associated with neuronal loss. No significant difference between cultures receiving low bicarbonate acidosis or mock acidosis was observed suggesting the absence of neuronal loss immediately after the recording session (Fig. 2B). Cultures were returned to the incubator and neuronal death assayed 1 and 2 days following the acidosis treatment. A significant increase in neuronal loss was observed in low bicarbonate group 1 and 2 days after the acidosis treatment (Fig. 2B). These data suggest that the 60-min acidosis treatment at 328C leads to neuronal loss. These data agree with the previous study of Ding et al. [8] showing that prolonging the acidosis treatment increases acidosis toxicity.
3.2. Synaptic depression and neuronal loss following lactic acid acidosis The neurotoxicity of low bicarbonate acidosis suggests
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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 i 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 induce intracellular acidosis. To test this prediction, slice cultures were treated with lactate containing aCSF to test the potential neurotoxicity of lactate acidosis. Intracellular pH, synaptic depression and neuronal loss were examined. BCECF was used to monitor intracellular pH. Average pH i was pH 7.1960.13 (n56) for normal lactate aCSF at 328C.
When the perfusion was switched to lactate aCSF pH 6.70, the intracellular pH rapidly acidified, reaching 6.4360.07, a value more acidic than the pH of the lactate aCSF pH 6.70 perfusion (Fig. 3A). Upon reperfusion with lactate aCSF pH 7.35, the intracellular pH rapidly returned basal levels. The rates of acidification and recovery of intracellular pH by lactate acidosis were similar.
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Fig. 1. (continued)
Synaptic depression was also examined during lactic acidosis (Fig. 3B). fEPSP rapidly decreased following treatment with lactate aCSF pH 6.70. Upon reperfusion with lactate aCSF pH 7.35, fEPSP remained depressed. To
exclude a possibility that the test stimulation contributed to the synaptic depression, test stimulation was stopped during recovery period in some experiments, yet no significant improvement was observed (data not shown).
Table 1 Summary of the results of this study a Acidosis treatment
Time (min)
Temperature (8C)
pH o
pH i
Synaptic depression
Neuronal loss?
Low bicarbonate Low bicarbonate Low bicarbonate Lactic acid
30 30 60 30
32 37 32 32
6.70 6.70 6.70 6.70
6.5360.08 6.4660.08 NP 6.4360.07
Reversible Reversible Irreversible Irreversible
No Yes Yes 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 o ) and intracellular pH (pH i ). Evoked responses were depressed during the acidosis treatment that 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.
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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 when the reperfusion was prolonged to 90 min. In cultures receiving mock lactate acidosis, fEPSP remained stable for 60 min during perfusion with lactate aCSF pH 7.35. Between 60 and 80 min, fEPSP reduced to 80% of baseline (Fig. 3B). This value remained significantly higher than cultures receiving lactic acidosis. At 100 min, fEPSP in control slices reduced to 50% of baseline and no longer significantly differed from fEPSP in cultures receiving lactic acidosis. fEPSP was undetectable after 2 h of perfusion with lactate aCSF, pH 7.35. These data suggest that lactate acidosis induces irreversible synaptic depression. These data also suggest that synaptic responses of slice culture in lactate aCSF, pH 7.35 slowly depress as well. These data suggest a depressing effect of lactate anion on synaptic transmission [44]. Other reports suggest
stable synaptic transmission when lactate replaces glucose [27,35]. To address this issue, synaptic responses were recorded in slice cultures that were perfused with aCSF containing 10 mM lactate. Synaptic depression was complete after 1 h (n53). No recovery of synaptic responses was observed following treatment with lactate aCSF pH 6.70. This may be due to rapid neuronal loss. To test this, neuronal loss was assayed immediately after the recording session (Fig. 3C). Neuronal loss was not observed in the pH 7.35 or the pH 6.7 groups. Cultures were returned to the incubator and neuronal loss assayed 1 day later. Propidium iodide epifluorescence was significantly increased in the lactate acidosis cultures when compared to control cultures suggesting that lactate acidosis induces neuronal loss. In lactate acidosis, synaptic depression that did not reverse
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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 neuronal loss in the pH 6.7 group. In contrast irreversible synaptic depression was not associated with neuronal loss in the pH 7.35 group.
4. Discussion In this study, two methods, low bicarbonate or lactate, were used to induce intracellular and extracellular acidosis.
The results of these studies are summarized in Table 1. Both low bicarbonate and lactic acid resulted in intracellular acidosis of similar extent and duration (Figs. 1 and 3). 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 also depressed during the time of intracellular acidosis. Synaptic depression is a well-known immediate response of hippocampal neurons to acidosis [1,19,37,17]. These data suggest that the immediate response of hippocampal
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slice cultures to acidosis is similar to the response in acute hippocampal slices. Synaptic depression, however, was either reversible or irreversible depending upon the method used and the duration of the acidosis treatment. With low bicarbonate acidosis, synaptic depression was reversible when the acidosis treatment was 30 min long (Fig. 1C). Increasing the acidosis treatment to 60 min resulted in irreversible synaptic depression (Fig. 2A). Thirty minutes of lactic acidosis, in contrast, induced irreversible synaptic depression. (Fig. 3B) In contrast to earlier studies of acidosis, cultured hippocampal slices permit analysis of synaptic depression followed by delayed neuronal loss. Acidification of intracellular pH may or may not result in neuronal loss. Few hippocampal neurons are lost following cerebral acidosis induced by hypercapnea [6]. Acidosis-induced neuronal loss is influenced by three parameters: the method to induce acidosis, the duration of the acidosis, and the temperature of the acidosis treatment. The duration and temperature of the acidosis treatment are important since neuronal loss was induced by increasing low bicarbonate acidosis to 60 min or raising the temperature from 32 to 378C (Figs. 1 and 2). At 328C, 30 min of lactate acidosis was neurotoxic while 30 min of low bicarbonate acidosis was not. These data suggest that acidosis by lactic acid is more neurotoxic to slice cultures than low bicarbonate. Lactate has been proposed to protect against hypoxic injury in acute hippocampal slices [27,29,28]. Due to the limited in vitro life span of the acute slice preparation, these studies are limited to examining a protective effect within hours of the insult. In contrast, when similarly prepared slices that were placed into long-term culture, lactate acidosis induced delayed neurotoxicity (Fig. 3). In vivo, increased lactate acidosis is associated with increased ischemic injury [32,20,13,2,18]. Taken together, these
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observations suggest that the short-term effect of lactate is protective while its long-term effect is neurotoxic. The enhanced neurotoxicity of lactate as compared to low bicarbonate may result from their different effects on intracellular buffering and ion transport. The differing rates of acidification of intracellular pH at the onset of the lactate and low bicarbonate acidosis treatments suggest different mechanisms of acidification. Intracellular pH acidified more rapidly by lactate than low bicarbonate (Fig. 4). In contrast, there was no significant difference in the rate of recovery (Fig. 4). Lactic acid may acidify intracellular pH by freely diffusing across the plasma membrane and by ionizing to lactate and H 1 [26]. Lactate can also enter the cell via the H 1 -lactate cotransporter [43]. Omitting bicarbonate in the extracellular solutions may reduce intracellular buffering power by exiting of intracellular 2 bicarbonate anions through HCO 2 exchanger, and 3 / Cl facilitate intracellular acidification. Unexpectedly, synapses were observed to be depressed in control slice cultures treated with lactate aCSF, pH 7.35 (Fig. 3B). Synaptic depression was slow as fEPSP began to decrease after 1 h into the recording session. This contrasts with the stability of slice culture synaptic responses in normal aCSF. Intracellular pH did not significantly differ between normal lactate aCSF, (pH 7.35) and aCSF, (pH 7.35) suggesting that this depression of synaptic responses is unrelated to acidosis. The possibility exists that lactate has a slowly depressing effect on synaptic responses in slice cultures. In studies using acute hippocampal slices, the issue of lactate supporting synaptic transmission remains unresolved; synaptic responses have been reported to be stable [27,35] or depressed by lactate [44,41,42]. The question if lactate can support synaptic transmission in slice cultures requires additional study. A critical question remains: why is acidosis neurotoxic in slice cultures when similar acidosis has no effect to
Fig. 4. Comparison of the change of pH i during low bicarbonate and lactic acidosis Induction (0–12 min) and recovery (30–42 min) of intracellular pH are compared between low bicarbonate (Fig. 2) or lactic acidosis (Fig. 4) at 328C. The rates of acidification of intracellular pH (low bicarbonate, 20.072 pH units / min; lactate, 20.12 pH units / min) are significantly different (ANOVA, P,0.05). In contrast, the rates of recovery of intracellular pH (low bicarbonate, 0.128 pH units / min; lactate 0.124 pH units / min) do not significantly differ.
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dissociated neuronal cultures? Embryonic and perinatal neurons may have reduced susceptibility to acidosis injury as compared to slice cultures. A critical factor may be the age at which slice culture are isolated and placed into culture. Age effects are a common theme in brain injury following ischemia and trauma. Interestingly, pH regulation becomes more impaired in the aged hippocampus [25,24]. An alternative explanation is that the higher neuronal density or the maintenance of neuronal glial interactions [45] may be critical. The demonstration that acidosis by different means results in neuronal loss provides further evidence for a role of acidosis in brain injury.
Acknowledgements The authors would like to thank Dr. Angel Cinelli for providing assistance for pH imaging experiments. We would also thank Dr. Ilham Muslimov for help with the photography and Mr. Subha Basu for reading the manuscript. This work was supported by an American Heart Association Grant-in-aid to P.J.B.
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