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Adenosine antagonists delay hypoxia-induced depression of neuronal activity in hippocampal brain slice
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Brain Research, 490 (1989) 378 384
Elsevicr BRE 23578
Adenosine antagonists delay hypoxia-induced depression of neuronal activity in hippocampal brain slice John C. Fowler Physiology Group, LS-1, Life Sciences Division, Los Alarnos National Laboratory, Los Alamos, NM 87545 (U.S.A.)
(Accepted 13 March 1989) Key words: Adenosine; Hippocampus; Hypoxia; Theophylline; 8-Phenyltheophylline
Submerged rat hippocampal slices were exposed to hypoxic medium prepared with 95% N2/5% CO 2. The population spikes recorded from CA1 cell layer were completely blocked within a range of 5-10 min. The adenosine antagonist theophylline (100/~M) delayed and partially prevented the hypoxia-induced depression. Increasing concentrations of the more potent adenosine antagonist 8-phenyltheophylline (8-PT; 0.1, 1, 10/~M) resulted in progressively less hypoxia-induced depression. The antidromically elicited afterpotentials recorded in the absence of synaptic transmission in low calcium, high magnesium medium were blocked within 8 rain of hypoxia. Theophylline (100/~M) and 8-PT (10/~M) delayed to a similar extent the hypoxia-induced depression of the first afterpotential but did not prevent its complete depression.
Adenosine is a potent neuroactive substance present in the mammalian central nervous system in sufficient concentrations to generate a tonic inhibitory tone 46. This purine and its analogs inhibit both spontaneous and evoked neuronal firing 3°'31. There also exists an inhibitory adenosinergic tone in the isolated hippocampal slice preparation. Thus, competitive adenosine antagonists, such as theophylline and 8-phenyltheophylline (8-PT), increase pyramidal cell excitability and the amplitude of synaptic responses 4-6. Adenosine's inhibitory actions at the cellular level, most completely demonstrated in the hippocampal slice, may be explained by a variety of actions including a decrease in calcium influx (although see refs. 17, 27), a membrane hyperpolarization and a decrease in membrane resistance attributed to an increase in potassium conductance 14" 15,33,41. Adenosine's depressant action in the hippocampus appears to be due to activation of A 1 subtype receptors 34. Adenosine is considered a neuromodulator because its regulation is not directly contingent on calcium-dependent, stimulus-secretion coupling. Thus, unlike the more classical neurotransmitters,
the bulk of purine release in brain slices occurs after the termination of electrical or potassium stimulation 19'21. Even when synaptic transmission is blocked there is sufficient adenosine present in the hippocampal slice to depress neuronal excitability 9' 16 Adenosine levels in the brain are thought to be regulated by the balance between energy supply and demand of the tissue (for review, see ref. 7). For example, hypoxia results in a rapid increase in adenosine levels 32'46. Hypoxia induces a depression of neuronal activity 13'25'28'36'37'4°'42'44. In a manner reminiscent of the actions of adenosine, hypoxiainduced depression of electrical activity is associated with an initial hyperpolarization and a decreased membrane resistance due to an increase in potassium
conductancel3,18,28. The orthodromically evoked population spike recorded from the CA1 stratum pyramidale region of the submerged hippocampal slice is reversibly depressed within minutes of exposure to physiological medium equilibrated with 95% N2/5% CO236'37. Experiments were designed to determine, in the rat hippocampal slice, the contribution of endogenous
Correspondence: J.C. Fowler, Physiology Group, LS-1, MS M-882, Life Sciences Division, Los Alamos National Laboratory, Los Alamos, NM 87545, U.S.A.
0006-8993/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
379 adenosine to this hypoxia-induced depression of neuronal activity. In addition, experiments were performed to determine if, in the absence of synaptic transmission, hypoxia could alter adenosine levels sufficiently to depress neuronal excitability. Transverse hippocampal slices (400 /~m) were prepared from Sprague-Dawley rats (35-45 days old) in a manner similar to that previously described 9'm. Slices were incubated in a static bath containing physiological medium (in mM): NaC1 124, KC! 5.9, NaH2PO 4 1.2, MgSO 4 1.3, CaCI 2 3.1, N a H C O 3 25.6, glucose 10. Slices used to examine the effect of hypoxia in the absence of synaptic transmission were incubated for at least two hours in a modified physiological medium substituted with (in mM): MgSO 4 4.0, CaCI 2 0.24. This medium has a relatively low calcium, high magnesium concentration that blocks synaptic transmission and prevents spontaneous burst discharges in the hippocampal slice 20.3s.43.
Before recording, slices were transferred to a recording chamber where they were submerged and superfused at 2 ml/min with the appropriate medium at a temperature of 33-34 °C. For normoxic conditions the medium was equilibrated with a 95% 02/5% CO 2 gas mixture. Hypoxia was induced by switching to a medium equilibrated with 95% N2/5% C O 2.
Bath pO 2 was measured, using a blood-gas machine, in samples of medium withdrawn from the recording chamber while perfusing with normoxic medium and after 10 min of perfusion with the nominally anoxic medium equilibrated with 95% N2/5% CO 2. Normoxic p O 2 values ranged from 400 to 500 mm Hg. Five measurements of bath p O 2 during hypoxic conditions resulted in a m e a n p O 2 of 155 __+ 17 mm Hg (mean + S.E.M.). This elevated pO 2 value is ascribed to the gain of oxygen from atmosphere through plastic tubing 36"37. Because of the steep diffusion gradient, this bath pO 2 is expected to produce an estimated pO 2 near the center of the 400-/~m slice of 0-20 mm Hg 12. pH in both normoxic and hypoxic medium was 7.3-7.4. In normal medium the monosynaptic population spike recorded in the pyramidal cell layer of CA1 was elicited by stimulating the stratum radiatum. In modified medium stimulation was in the alveus containing the axonal projections from the pyrami-
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Fig. 1. Response of population spikes to hypoxia (open squares), to hypoxia + theophylline (100/aM) (filled circles), and after wash-out of theophylline (filled squares). Amplitude of population spike is expressed as percent of control spike measured at time = 0 min. Error bars are mean + S.E.M. Means are of 7 slices exposed to all 3 conditions.
dal cells. In both cases stimulation was suprathreshold at a frequency of 1 every 30 s. The amplitude of the population spike was measured as described before 1°. In modified medium a stimulation pulse applied to the alvear fibers produced an extracellularly recorded antidromic action potential followed by 2-4 afterpotentials recorded from the CA1 pyramidal cell layer. The amplitudes of the action potential or first spike and the first afterpotential were measured as described previously 9. Theophylline (Sigma, St. Louis, MO) was made up in distilled water at 500 times the desired final concentration and added to the perfusion reservoir to a final concentration of 100/tM. A stock solution of 8-phenyltheophylline (Research Biochemicals Inc., Natick, MA) was prepared in dimethyl sulfoxide and added to the reservoir at dilutions of 10 -4 for 0.1 ~tM and 10 -3 for final concentrations of 1 and 10 ktM. Dimethyl sulfoxide at 10- 3 alone did not affect the time course of the hypoxia-induced depression. Exposing the slice to hypoxic medium completely blocked the population spikes over a range of 5-10 min (Fig. 1). This depression was reversible, with an average maximum population spike amplitude of 5.29 ___ 0.44 mV (mean + S.E.M.; n = 7) before hypoxia and 5.24 + 0.51 mV following reoxygenation. To determine the possible role of endogenous
380 adenosine in hypoxia-induced depression, slices were exposed to the competitive adenosine antagonist, theophylline (100/~M), for at least two min before and then continuously throughout the subsequent hypoxic period. In the presence of this antagonist, the population spike amplitude declined at a slower rate and persisted even after 12 min of exposure to hypoxia as illustrated in Fig. 1. Following 12 min of exposure to hypoxia the mean population spike amplitude measured in 7 slices was 33 _+ 14% of the prehypoxic value. Variance between slices was high: the population spikes in 3 out of 7 of these slices in the presence of theophylline were completely depressed within 8-10 min of hypoxia. A paired comparison between slices exposed to hypoxia in the absence and presence of theophyUine revealed that in each case the time to complete block of the population spike was extended in the presence of the antagonist. After wash-out of theophylline in normoxic medium the same slices were re-exposed to hypoxic medium. As in the predrug hypoxic period, the population spike was completely blocked in all slices within 5-10 min (Fig. 1). The partial protection against hypoxia-induced depression offered by 100/~M theophylline may be due to increasing adenosine levels overcoming theophylline's antagonism. Theophylline above 200 /~M begins to exert significant effects on phosphodiesterase activity and on intracellularly bound calcium 2'22. For this reason the effect of the more selective adenosine antagonist 8-PT6 on hypoxiainduced depression was investigated• Slices were either used once only at a single concentration of the drug in the presence of hypoxia or were serially exposed to hypoxic conditions, first in the absence of drug and then in successively higher concentrations of 0.1, 1, and 10/~M 8-PT. Following each hypoxic period, population spikes recovered under normoxic conditions. Cumulative dose-response exposures were performed as the 8-PT effect persisted after wash-out periods as long as 50 min. The response to 8-PT in an individual slice is illustrated in Fig. 2. Hypoxia completely blocked both the population spike and the synaptic component of the evoked response within 7 min (Fig. 2A, bottom). The slice had been prepared so as to preserve the presynaptic fiber potential (at arrow).
This field potential was unaffected by hypoxia (Fig. 2A, bottom). During reoxygenation the population spike returned to 77% of prehypoxic amplitude (Fig. 2B, top). In the presence of 10 /~M 8-PT, the amplitude of the evoked response was completely preserved even after 15 min of re-exposure to hypoxia (Fig. 2B, bottom). Increasing concentrations of 8-PT resulted in progressively less of an hypoxia-induced depression of the population spike as seen in Fig. 2C. Thus, at the end of 15 min of hypoxia, population spike amplitude was completely blocked in the absence of drug. Spike amplitude was 19 _ 13% (n = 4) of prehypoxic amplitude in the presence of 0.1 ktM
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Time (rain) Fig. 2. Response of population spike to hypoxia and to hypoxia + 8-phenyltheophylline (8-PT). A: population spike recorded under normoxic conditions (top) and 7 min after exposure to hypoxia (bottom). The population spike was completely blocked within 7 min. Note fiber potential is still present. B: population spike recorded from the same slice after return to normoxic conditions (top) and after 15 min in hypoxia + 8-PT (10/~M) (bottom). C: response of population spike to hypoxia alone (open squares, n = 7), to hypoxia + 0.1 /~M 8-PT (filled squares, n = 4), to hypoxia + 1 /~M 8-PT (Open circles, n = 6), and to hypoxia + 10 # M 8-PT (filled circles, n = 8). Population spike amplitudes expressed as percent of control spike at time = 0 min. Scale: 5 mV, 2 ms. Some error bars are removed for clarity.
381 8-PT, 60 + 14% (n = 6) in 1 BM 8-PT, and 85 + 9% (n = 8) in the presence of 10/zM 8-PT. Two aspects of this experimental design need to be addressed. First, it is well known that adenosine antagonists often increase the amplitudes of the synaptic responses 4'5'16. Thus, a slowing of the time course of the hypoxia-induced depression of the population spike could be due to an essentially independent coincident decrease in inhibitory adenosinergic tone. To investigate this possibility the effect of 10/tM 8-PT added to normoxic medium on the population spike evoked with suprathreshold stimulation was determined. Four slices were exposed to 8-PT for 15 min and the population spike was measured at 5, 10, and 15 min. Spike amplitude was 102 + 5%, 102 + 8%, and 92 + 3% of predrug amplitude at 5, 10, and 15 min, respectively. These values are not significantly different from the predrug values. The conclusion is that 8-PT does not affect the amplitude of the suprathreshold-evoked population spike during the 15-min hypoxic periods used to collect the data for Fig. 2. A second concern in the interpretation of these results involves the observation that a brief exposure of hippocampal slices in an interface chamber to anoxia significantly attenuates the depression of the population spike during a subsequent hypoxic exposure 4°. Thus, in the present experiments, the protection provided by adenosine antagonists in submerged slices previously exposed to a hypoxic period could in fact be due to this anoxic adaptation effect. This possibility was investigated in a number of ways. First, the time course of hypoxia-induced depression was found to essentially overlap in two slices exposed to repeated hypoxic periods interrupted by periods of recovery in normoxic medium. Second, 3 slices were exposed to their first hypoxic episode in the presence of 10/~M 8-PT. After 15 min of hypoxia, the population spike amplitudes recorded in these slices was 99 _+ 15% of the prehypoxic amplitude. This value is not significantly different from that observed in slices exposed to hypoxia and 10/xM 8-PT after preceding hypoxic exposures. Thus, the adenosine antagonist prevents hypoxia-induced depression of the population spike during the first exposure to hypoxia. Finally, supportive evidence against an adaptive effect of repeated hypoxic episodes is provided by the data
presented in Fig. 1. These data indicate that after the rinse-out of theophylline, the time course of hypoxia-induced depression during a third exposure to hypoxia does not significantly differ from the first response to hypoxia observed in the absence of the drug. Sufficient endogenous adenosine exists in the absence of synaptic transmission to depress neuronal excitability9. However, it is not clear whether hypoxia can elevate adenosine levels when synaptic transmission is blocked. In modified (low calcium, high magnesium) medium, that blocks synaptic transmission, the antidromically elicited response consists of an extracellularly recorded action potential or first spike followed by a series of afterpotentials recorded in the CA1 pyramidal cell layer9'23. Exogenous adenosine has been shown to block the afterpotentials with little effect on the first spike 23. Hypoxia alone completely blocked the amplitude of the first afterpotential after approximately 8 min (Fig. 3A). The first spike was not consistently affected at this time by the exposure to hypoxia (Fig. 3B). As in the case of the orthodromically evoked population spike, the hypoxic-induced depression was reversible and repeatable. Theophylline (100 /~M) delayed the depression of the first afterpotential but did not prevent its complete block (Fig. 3A). The amplitudes of the first afterpotential were significantly greater in the presence of theophylline 4 min after the start of hypoxia (P < 0.005, unpaired Student's t-test). The more potent antagonist, 8-PT (10/xM), essentially mimicked the effect of theophylline in delaying the loss of the afterpotential during hypoxia (Fig. 3A). The main finding of this study is that adenosine antagonists greatly delay the development of hypoxia-induced depression of neuronal activity in the brain slice. These results are consistent with the interpretation that adenosine plays a central role in the depression of neuronal activity during hypoxia. At a concentration of 10/~M, the adenosine antagonist 8-PT prevents the depression of the population spike observed during early exposure to hypoxia. An estimate of 50/~M for the concentration of adenosine associated with hypoxia is consistent with a potency series done in the hippocampal slice indicating that 5/~M 8-PT completely suppresses the electrophysiological effect of 50 /xM adenosine 6. The greater
382 potency of 8-PT when compared to that of theophylline is apparent from the present study. 10 ~M 8-PT preserved the population spike to a greater extent than did 100 #M theophylline. Previous reports investigating the role of adenosine in hypoxia-induced depression in the hippocampal slice have suggested at least a partial contribution. The adenosine antagonist, 3-isobutyl methylxanthine, was found to slightly delay the loss of the population spike recorded from guinea pig dentate granule cell layer 24. 8-PT (10-20 ~tM) somewhat delayed the fall of the evoked response measured in rat CA1 region during hypoxia ~. This occurred despite the observation that adenosine levels were not appreciably increased until after 14 min of hypoxia. The present results closely resemble those ob-
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Time (min) Fig. 3. Response of first afterpotential and first spike to hypoxia alone and in the presence of two adenosine antagonists. A: first afterpotential expressed as percent of control
afterpotential at time = 0 min was depressed in hypoxia alone (open squares, n = 8). The rate of decline during hypoxia was slowed by the addition of either theophylline (100/~M; closed circles, n = 6) or 8-PT (10 #M; open circles, n = 10). B: the amplitude of the first spike is expressed as percent of control at time = 0 min. The first spike was not consistently affected during hypoxia alone (open squares), or in the presence of either theophylline (100 #M, closed circles) or 8-PT (10 #M, open circles).
served in the isolated, superfused spinal cord. The adenosine antagonist, 8-cyclopentyltheophylline, for the most part prevented the hypoxia-induced depression of the monosynaptic reflex during early hypoxia 2~. The present results also indicate that adenosine levels may be altered by hypoxia in the absence of synaptic transmission. Both 8-PT and theophyUine slow the development of hypoxia-induced depression. However, it is not clear why no potency difference is observed between these two antagonists as both theophylline (100 #M) and 8-PT (10 #M) prolonged the time course of depression to a similar degree. One possible explanation is that somewhat less adenosine is released in the modified medium containing low calcium, high magnesium. Thus, 100 uM theophylline is sufficient to completely block the response to this increase in adenosine concentration. Other as yet unidentified processes must underlie the remaining depression that occurs fairly early during hypoxia. The present results suggest that adenosine may play a protective role during periods of metabolic stress by inhibiting neuronal activity and thus conserving cerebral energy supplies. The stable adenosine analogue, 2-chloroadenosine, protects against ischemic hippocampal CA1 cell loss s while theophylline exacerbates CAI damage 35. Adenosine influences cerebral energy consumption. In mice administration of the adenosine antagonist, aminophyiline, is associated with an increase in cerebral metabolic rate and a decrease in anoxic survival in vivo 45. The protective action of adenosine extends to its ability to depress the release of excitatory neurotransmitters such as glutamate 3. During conditions of metabolic stress glutamate exerts a neurotoxic effect and the neurotoxicity of excitatory transmitters is particularly evident when intracellular energy levels are reduced 29. The role of adenosine is of particular interest because, at least under the present conditions, adenosine inhibits neuronal function during the metabolic stress of hypoxia. The inhibition of central nervous function occurs before such activity is limited by the more severe pathological consequences of cerebral energy failure. It is worth noting that, in the present experiments, the population spike recorded in the submerged hippocampal slice did not experience an increased
383 It has been suggested that anoxic adaptation may be due to elevated glycolysis providing the necessary energy during the subsequent hypoxic period 4°. In the present case, it is certainly of interest to determine the source of energy consumed during the hypoxic period when adenosine antagonists are blocking the hypoxia-induced depression of the population spike. The initial metabolic state of the submerged slice and the slice with a surface directly exposed to oxygenated gas do differ. This difference is evident from the observation that the exposed slice undergoes a decline in energy charge when submerged ~. It is probable that the differences exhibited by submerged slices and by slices in an interface chamber exposed to hypoxia/anoxia represent a physiological response continuum to this form of metabolic stress. An understanding of these differences and of the role of possible endogenous modulators in these responses will further our understanding of the neuronal response to hypoxia and ischemia.
tolerance to hypoxia as a result of repeated exposure to hypoxia. This observation contrasts with that made in slices in an interface chamber. These slices exhibited an anoxic adaptation expressed as an increased resistance to hypoxia-induced depression of the population spike following a brief conditioning exposure to anoxia 4°. The reason for this difference is not clear but may involve the experimental dissimilarities between the submerged and the interface conditions. Slices in an interface chamber, where the humidified gas is passed directly over the exposed surface of the slice, are exposed to very nearly true anoxic conditions (defined as the complete lack of oxygen) in the presence of 95% N J 5 % C O 2, whereas the submerged slice exposed to nominally anoxic medium equilibrated with 95% N2/5% CO2 is actually exposed to hypoxic conditions with bath pO 2 near that of atmospheric pO237. Perhaps reflective of this difference in severity of hypoxic/anoxic conditions is the observation that electrical .activity recorded in slices in an interface chamber is irreversibly abolished after roughly 10 min of exposure to 95% N2/5% CO21'39"44, while submerged slices exhibit recovery after much longer periods of hypoxia 37. Submerged slices recover electrical activity after hypoxic periods as long as 30-45 min 36. The increased sensitivity of slices in an interface chamber may be due to the development of spreading depression which is not seen in submerged slices I .
The review of an earlier manuscript by Drs. John E McEiroy and Donal G. Sinex is appreciated. This work was supported by institutionally supported research funds of the Los Alamos National Laboratory under the auspices of the Department of Energy.
1 Balestrino, M. and Somjen, G.G., Chlorpromazine protects brain tissue in hypoxia by delaying spreading depression-mediated calcium influx, Brain Research, 385 (1986) 219-226. 2 Butcher, R.W. and Sutherland, E.W., Adenosine 3", 5"-phosphate in biological materials. I. Purification and properties of cyclic 3",5"-nucleotide phosphodiesterase and use of this enzyme to characterize adenosine 3",5"-phosphate in human urine, J. Biol. Chem., 237 (1962) 12441250. 3 Dolphin, A.C. and Archer, E.R., An adenosine agonist inhibits and a cyclic AMP analogue enhances the release of glutamate but not GABA from slices of rat dentate gyrus, Neurosci. Len., 43 (1983) 49-54. 4 Dunwiddie, T.V., Endogenously released adenosine regulates excitability in the in vitro hippocampus, Epilepsia, 21 (1980) 541-548. 5 Dunwiddie, T.V. and Hoffer, B.J., Adenine nucleotides and synaptic transmission in the in vitro rat hippocampus, Br. J. Pharmacol., 69 (1980) 59-68. 6 Dunwiddie, T.V., Hoffer, B.J. and Fredholm, B.B., Alkylxanthines elevate hippocampal excitability. Evidence
for a role of endogenous adenosine, Naunyn-Schmiedeberg's Arch. Pharmacol., 316 (1981) 326-330. 7 Dunwiddie, T.V., The physiological role of adenosine in the central nervous system, Int. Rev. NeurobioL, 27 (1985) 63-139. 8 Evans, M.C., Swan, J.H. and Meldrum, B.B., An adenosine analogue, 2-chloroadenosine, protects against longterm development of ischaemic cell loss in rat hippocampus, Neurosci. Lea., 83 (1987) 287-292. 9 Fowler, J.C., Modulation of neuronal excitability by endogenous adenosine in the absence of synaptic transmission, Brain Research, 463 (1988) 368-373. 10 Fowler, J.C. and O'Donnell, J.M., Antagonism of the responses to isoproterenol in the rat hippocampal slice with subtype-selective antagonists, Eur. J. Pharmacol., 153 (1988) 105-110. 11 Fredholm, B.B., Dunwiddie, T.V., Bergman, B. and Lindstrom, K. Levels of adenosine and adenine nucleotides in slices of rat hippocampus, Brain Research, 295 (1984) 127-136. 12 Fujii, T., Baumgartl, H. and Lubbers, D.W., Limiting section thickness of guinea pig olfactory cortical slices
384 studied from tissue pO 2 values and electrical activities, Pflfigers Arch, 393 (1982) 83-87. 13 Fujiwara, N., Higshi, H., Shimoji, K. and Yoshimura, M., Effects of hypoxia on rat hippocampal neurones in vitro, J. Physiol. (Lond.), 384 (1987) 131-151. 14 Greene, R.W. and Haas, H.L., Adenosine actions on CA1 pyramidal neurones in rat hippocampal slices, J. Physiol. (Lond.), 366 (1985) 119-127. 15 Haas, H.L. and Greene, R.W., Adenosine enhances afterhyperpolarization and accommodation in hippoeampal pyramidal cells, Pfliigers Arch., 420 (1984) 244-247. 16 Haas, H.L. and Greene, R.W., Endogenous adenosine inhibits hippocampal CAI neurones: further evidence from extra- and intracellular recording, Naunyn-Schmiedeberg's Arch. Pharmacol., 337 (1988) 561-565. 17 Halliwell, J.V. and Scholfield, C.N., Somatically recorded Ca-currents in guinea pig hippocampal and olfactory cortex neurones are resistant to adenosine action, Neurosci. Lett., 50 (1984) 13-18. 18 Hansen, A.J., Hounsgaard, J. and Jahnsen, H., Anoxia increases potassium conductance in hippocampal nerve cells, Acta Physiol. Scand., 115 (1982) 301-310. 19 Hollins, C. and Stone, T.W., Characteristics of the release of adenosine from slices of rat cerebral cortex, J. Physiol. (Lond.), 303 (1980) 73-82. 20 Jefferys, J.G.R. and Haas, H.L., Synchronized bursting of CA1 hippocampal pyramidal cells in the absence of synaptic transmission, Nature, 300 (1982) 448-450. 21 Jonzon, B. and Fredholm, B.B., Release of purines, noradrenaline, and GABA from rat hippocampal slices by field stimulation, J. Neurochem., 44 (1985) 217-224. 22 Katz, A.M., Repke, D.I. and Hasselback, W., Dependence of iontophore- and caffeine-induced calcium release from sarcoplasmic reticulum vesicles on external and internal calcium ion concentrations, J. Biol. Chem., 252 (1977) 1938-1949. 23 Lee, K.S., Schubert, P. and Heinemann, U., The anticonvulsant action of adenosine: a postsynaptic, dendritic action by a possible endogenous anticonvuslant., Brain Research, 321 (1984) 160-164. 24 Lipton, P. and Robacker, K., Adenosine may cause early inhibition of synaptic transmission during anoxia, Soc. Neurosci. Abstr., 8 (1982) 993. 25 Lipton, P. and Whittingham, T.S., Reduced ATP concentration as a basis for synaptic transmission failure during hypoxia in the in vitro guinea pig, J. Physiol. (Lond.), 325 (1982) 51-65. 26 Lloyd, H.G.E., Spence, I. and Johnston, G.A.R., Involvement of adenosine in synaptic depression induced by a brief period of hypoxia in isolated spinal cord of neonatal rat, Brain Research, 462 (1988) 391-395. 27 Michaelis, M.L., Johe, K.K., Moghadam, B. and Adams, R.N.. Studies on the ionic mechanism for neuromodulatory actions of adenosine in the brain, Brain Research, 473 (1988) 249-260. 28 Misgeld, U. and Frotscher, M., Dependence of the viability of neurons in hippocampal slices on oxygen supply, Brain Res. Bull., 8 (1982) 95-100. 29 Novelli, A., Railly, J.A., Lysko, P.G. and Henneberry, R.C., Glutamate becomes neurotoxic via the N-methylD-aspartate receptor when intracellular energy levels are reduced, Brain Research, 451 (1988) 205-212. 30 Phillis, J.W., Edstr6m, J.P., Kostopoulos, G.K. and
Kirkpatrick, J.R., Effects of adenosine and adenine nucleotides on synaptic transmission in the cerebral cortex, Can. J. Physiol. Pharmacol., 57 (1979) 1289-1313. 31 Phillis, J.W. and Kostopoulos, G.K., Adenosine as a putative transmitter in the cerebral cortex. Studies with potentiators and antagonists, Life Sci., 17 (1975) 10851094. 32 Phillis, J.W., Walter, G.A., O'Regan, M.H. and Stair, R.E., Increases in cerebral cortical perfusate adenosine and inosine concentrations during hypoxia and ischemia, J. Cereb. Blood Flow Metabol., 7 (1987) 679-686. 33 Proctor, W.R. and Dunwiddie, T.V., Adenosine inhibits calcium spikes in hippocampal pyramidal neurons in vitro, Neurosci. Lett., 35 (1983) 197-201. 34 Reddington, M., Lee, K. and Schubert, P., An A 1adenosine receptor characterized by [3H]cyclohexyladenosine binding, mediates the depression of evoked potentials in a rat hippocampal slice preparation, Neurosci. Lett., 28 (1982) 275-279. 35 Rudolphi, K.A., Keil, M. and Westhofer, U., Effect of theophylline in ischemically induced hippocampal damage in Mongolian gerbils. In Krieglstein J. (Ed.), Pharmacology of Cerebral lschemia, Elsevier Amsterdam, 1986, pp. 358-362. 36 Schiff, S.J. and Somjen, G.G., Hyperexcitability following moderate hypoxia in hippocampal tissue slices, Brain Research, 337 (1985) 337-340. 37 Schiff, S.J. and Somjen, G.G., The effect of graded hypoxia on the hippocampal slice: an in vitro model of the ischemic penumbra, Stroke, 18 (1987) 30-37. 38 Schubert, P. and Lee, K.S., Non-synaptic modulation of repetitive firing by adenosine is antagonized by 4-aminopyridine in a rat hippocampal slice, Neurosci. Lett., 67 (1986) 334-338. 39 Schurr, A., Reid, K.H., Tseng, M.T., Edmonds Jr., H.L. and Rigor, B.M., A dual chamber for comparative studies using the brain slice preparation, Comp. Biochem. Physiol., 82 (1985) 701-704. 40 Schurr, A., Reid, K.H., Tseng, M.T., West, C. and Rigor, B.M.K., Adaptation of adult brain tissue to anoxia and hypoxia in vitro,, Brain Research, 374 (1986) 244-248. 41 Segal, M., Intracellular analysis of a postsynaptic action of adenosine in the rat hippocampus, Eur. J. Pharmacol., 79 (1982) 193-199. 42 Sick, T.J., Solow, E.L. and Roberts Jr., E.L., Extracellular potassium ion activity and electrophysiology in the hippocampal slice: paradoxical recovery of synaptic transmission during anoxia, Brain Research, 418 (1987) 227-234. 43 Taylor, C.P. and Dudek, F.E., Synchronous neural afterdischarges in rat hippocampal slices without active chemical synapses, Science, 218 (1982) 810-812. 44 Taylor, M.D., Mellert, T.K., Parmentier, J.L. and Eddy, L.J., Pharmacological protection of reoxygenation damage to in vitro brain slice tissue, Brain Research, 347 (1985) 268-273. 45 Thurston, J.H., Hauhart, R.E. and Dirgo, J.A., Aminophylline increases cerebral metabolic rate and decreases anoxic survival in young mice, Science, 201 (1978) 649-651. 46 Zetterstrom, T., Vernet, L.. Understedt, U., Tossman, U., Jonzon, B. and Fredholm, B.B., Purine levels in the intact rat brain. Studies with an implanted perfused hollow fibre, Neurosci. Lett., 29 (1982) 111-115.
Brain Research, 490 (1989) 378 384
Elsevicr BRE 23578
Adenosine antagonists delay hypoxia-induced depression of neuronal activity in hippocampal brain slice John C. Fowler Physiology Group, LS-1, Life Sciences Division, Los Alarnos National Laboratory, Los Alamos, NM 87545 (U.S.A.)
(Accepted 13 March 1989) Key words: Adenosine; Hippocampus; Hypoxia; Theophylline; 8-Phenyltheophylline
Submerged rat hippocampal slices were exposed to hypoxic medium prepared with 95% N2/5% CO 2. The population spikes recorded from CA1 cell layer were completely blocked within a range of 5-10 min. The adenosine antagonist theophylline (100/~M) delayed and partially prevented the hypoxia-induced depression. Increasing concentrations of the more potent adenosine antagonist 8-phenyltheophylline (8-PT; 0.1, 1, 10/~M) resulted in progressively less hypoxia-induced depression. The antidromically elicited afterpotentials recorded in the absence of synaptic transmission in low calcium, high magnesium medium were blocked within 8 rain of hypoxia. Theophylline (100/~M) and 8-PT (10/~M) delayed to a similar extent the hypoxia-induced depression of the first afterpotential but did not prevent its complete depression.
Adenosine is a potent neuroactive substance present in the mammalian central nervous system in sufficient concentrations to generate a tonic inhibitory tone 46. This purine and its analogs inhibit both spontaneous and evoked neuronal firing 3°'31. There also exists an inhibitory adenosinergic tone in the isolated hippocampal slice preparation. Thus, competitive adenosine antagonists, such as theophylline and 8-phenyltheophylline (8-PT), increase pyramidal cell excitability and the amplitude of synaptic responses 4-6. Adenosine's inhibitory actions at the cellular level, most completely demonstrated in the hippocampal slice, may be explained by a variety of actions including a decrease in calcium influx (although see refs. 17, 27), a membrane hyperpolarization and a decrease in membrane resistance attributed to an increase in potassium conductance 14" 15,33,41. Adenosine's depressant action in the hippocampus appears to be due to activation of A 1 subtype receptors 34. Adenosine is considered a neuromodulator because its regulation is not directly contingent on calcium-dependent, stimulus-secretion coupling. Thus, unlike the more classical neurotransmitters,
the bulk of purine release in brain slices occurs after the termination of electrical or potassium stimulation 19'21. Even when synaptic transmission is blocked there is sufficient adenosine present in the hippocampal slice to depress neuronal excitability 9' 16 Adenosine levels in the brain are thought to be regulated by the balance between energy supply and demand of the tissue (for review, see ref. 7). For example, hypoxia results in a rapid increase in adenosine levels 32'46. Hypoxia induces a depression of neuronal activity 13'25'28'36'37'4°'42'44. In a manner reminiscent of the actions of adenosine, hypoxiainduced depression of electrical activity is associated with an initial hyperpolarization and a decreased membrane resistance due to an increase in potassium
conductancel3,18,28. The orthodromically evoked population spike recorded from the CA1 stratum pyramidale region of the submerged hippocampal slice is reversibly depressed within minutes of exposure to physiological medium equilibrated with 95% N2/5% CO236'37. Experiments were designed to determine, in the rat hippocampal slice, the contribution of endogenous
Correspondence: J.C. Fowler, Physiology Group, LS-1, MS M-882, Life Sciences Division, Los Alamos National Laboratory, Los Alamos, NM 87545, U.S.A.
0006-8993/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
379 adenosine to this hypoxia-induced depression of neuronal activity. In addition, experiments were performed to determine if, in the absence of synaptic transmission, hypoxia could alter adenosine levels sufficiently to depress neuronal excitability. Transverse hippocampal slices (400 /~m) were prepared from Sprague-Dawley rats (35-45 days old) in a manner similar to that previously described 9'm. Slices were incubated in a static bath containing physiological medium (in mM): NaC1 124, KC! 5.9, NaH2PO 4 1.2, MgSO 4 1.3, CaCI 2 3.1, N a H C O 3 25.6, glucose 10. Slices used to examine the effect of hypoxia in the absence of synaptic transmission were incubated for at least two hours in a modified physiological medium substituted with (in mM): MgSO 4 4.0, CaCI 2 0.24. This medium has a relatively low calcium, high magnesium concentration that blocks synaptic transmission and prevents spontaneous burst discharges in the hippocampal slice 20.3s.43.
Before recording, slices were transferred to a recording chamber where they were submerged and superfused at 2 ml/min with the appropriate medium at a temperature of 33-34 °C. For normoxic conditions the medium was equilibrated with a 95% 02/5% CO 2 gas mixture. Hypoxia was induced by switching to a medium equilibrated with 95% N2/5% C O 2.
Bath pO 2 was measured, using a blood-gas machine, in samples of medium withdrawn from the recording chamber while perfusing with normoxic medium and after 10 min of perfusion with the nominally anoxic medium equilibrated with 95% N2/5% CO 2. Normoxic p O 2 values ranged from 400 to 500 mm Hg. Five measurements of bath p O 2 during hypoxic conditions resulted in a m e a n p O 2 of 155 __+ 17 mm Hg (mean + S.E.M.). This elevated pO 2 value is ascribed to the gain of oxygen from atmosphere through plastic tubing 36"37. Because of the steep diffusion gradient, this bath pO 2 is expected to produce an estimated pO 2 near the center of the 400-/~m slice of 0-20 mm Hg 12. pH in both normoxic and hypoxic medium was 7.3-7.4. In normal medium the monosynaptic population spike recorded in the pyramidal cell layer of CA1 was elicited by stimulating the stratum radiatum. In modified medium stimulation was in the alveus containing the axonal projections from the pyrami-
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Fig. 1. Response of population spikes to hypoxia (open squares), to hypoxia + theophylline (100/aM) (filled circles), and after wash-out of theophylline (filled squares). Amplitude of population spike is expressed as percent of control spike measured at time = 0 min. Error bars are mean + S.E.M. Means are of 7 slices exposed to all 3 conditions.
dal cells. In both cases stimulation was suprathreshold at a frequency of 1 every 30 s. The amplitude of the population spike was measured as described before 1°. In modified medium a stimulation pulse applied to the alvear fibers produced an extracellularly recorded antidromic action potential followed by 2-4 afterpotentials recorded from the CA1 pyramidal cell layer. The amplitudes of the action potential or first spike and the first afterpotential were measured as described previously 9. Theophylline (Sigma, St. Louis, MO) was made up in distilled water at 500 times the desired final concentration and added to the perfusion reservoir to a final concentration of 100/tM. A stock solution of 8-phenyltheophylline (Research Biochemicals Inc., Natick, MA) was prepared in dimethyl sulfoxide and added to the reservoir at dilutions of 10 -4 for 0.1 ~tM and 10 -3 for final concentrations of 1 and 10 ktM. Dimethyl sulfoxide at 10- 3 alone did not affect the time course of the hypoxia-induced depression. Exposing the slice to hypoxic medium completely blocked the population spikes over a range of 5-10 min (Fig. 1). This depression was reversible, with an average maximum population spike amplitude of 5.29 ___ 0.44 mV (mean + S.E.M.; n = 7) before hypoxia and 5.24 + 0.51 mV following reoxygenation. To determine the possible role of endogenous
380 adenosine in hypoxia-induced depression, slices were exposed to the competitive adenosine antagonist, theophylline (100/~M), for at least two min before and then continuously throughout the subsequent hypoxic period. In the presence of this antagonist, the population spike amplitude declined at a slower rate and persisted even after 12 min of exposure to hypoxia as illustrated in Fig. 1. Following 12 min of exposure to hypoxia the mean population spike amplitude measured in 7 slices was 33 _+ 14% of the prehypoxic value. Variance between slices was high: the population spikes in 3 out of 7 of these slices in the presence of theophylline were completely depressed within 8-10 min of hypoxia. A paired comparison between slices exposed to hypoxia in the absence and presence of theophyUine revealed that in each case the time to complete block of the population spike was extended in the presence of the antagonist. After wash-out of theophylline in normoxic medium the same slices were re-exposed to hypoxic medium. As in the predrug hypoxic period, the population spike was completely blocked in all slices within 5-10 min (Fig. 1). The partial protection against hypoxia-induced depression offered by 100/~M theophylline may be due to increasing adenosine levels overcoming theophylline's antagonism. Theophylline above 200 /~M begins to exert significant effects on phosphodiesterase activity and on intracellularly bound calcium 2'22. For this reason the effect of the more selective adenosine antagonist 8-PT6 on hypoxiainduced depression was investigated• Slices were either used once only at a single concentration of the drug in the presence of hypoxia or were serially exposed to hypoxic conditions, first in the absence of drug and then in successively higher concentrations of 0.1, 1, and 10/~M 8-PT. Following each hypoxic period, population spikes recovered under normoxic conditions. Cumulative dose-response exposures were performed as the 8-PT effect persisted after wash-out periods as long as 50 min. The response to 8-PT in an individual slice is illustrated in Fig. 2. Hypoxia completely blocked both the population spike and the synaptic component of the evoked response within 7 min (Fig. 2A, bottom). The slice had been prepared so as to preserve the presynaptic fiber potential (at arrow).
This field potential was unaffected by hypoxia (Fig. 2A, bottom). During reoxygenation the population spike returned to 77% of prehypoxic amplitude (Fig. 2B, top). In the presence of 10 /~M 8-PT, the amplitude of the evoked response was completely preserved even after 15 min of re-exposure to hypoxia (Fig. 2B, bottom). Increasing concentrations of 8-PT resulted in progressively less of an hypoxia-induced depression of the population spike as seen in Fig. 2C. Thus, at the end of 15 min of hypoxia, population spike amplitude was completely blocked in the absence of drug. Spike amplitude was 19 _ 13% (n = 4) of prehypoxic amplitude in the presence of 0.1 ktM
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Time (rain) Fig. 2. Response of population spike to hypoxia and to hypoxia + 8-phenyltheophylline (8-PT). A: population spike recorded under normoxic conditions (top) and 7 min after exposure to hypoxia (bottom). The population spike was completely blocked within 7 min. Note fiber potential is still present. B: population spike recorded from the same slice after return to normoxic conditions (top) and after 15 min in hypoxia + 8-PT (10/~M) (bottom). C: response of population spike to hypoxia alone (open squares, n = 7), to hypoxia + 0.1 /~M 8-PT (filled squares, n = 4), to hypoxia + 1 /~M 8-PT (Open circles, n = 6), and to hypoxia + 10 # M 8-PT (filled circles, n = 8). Population spike amplitudes expressed as percent of control spike at time = 0 min. Scale: 5 mV, 2 ms. Some error bars are removed for clarity.
381 8-PT, 60 + 14% (n = 6) in 1 BM 8-PT, and 85 + 9% (n = 8) in the presence of 10/zM 8-PT. Two aspects of this experimental design need to be addressed. First, it is well known that adenosine antagonists often increase the amplitudes of the synaptic responses 4'5'16. Thus, a slowing of the time course of the hypoxia-induced depression of the population spike could be due to an essentially independent coincident decrease in inhibitory adenosinergic tone. To investigate this possibility the effect of 10/tM 8-PT added to normoxic medium on the population spike evoked with suprathreshold stimulation was determined. Four slices were exposed to 8-PT for 15 min and the population spike was measured at 5, 10, and 15 min. Spike amplitude was 102 + 5%, 102 + 8%, and 92 + 3% of predrug amplitude at 5, 10, and 15 min, respectively. These values are not significantly different from the predrug values. The conclusion is that 8-PT does not affect the amplitude of the suprathreshold-evoked population spike during the 15-min hypoxic periods used to collect the data for Fig. 2. A second concern in the interpretation of these results involves the observation that a brief exposure of hippocampal slices in an interface chamber to anoxia significantly attenuates the depression of the population spike during a subsequent hypoxic exposure 4°. Thus, in the present experiments, the protection provided by adenosine antagonists in submerged slices previously exposed to a hypoxic period could in fact be due to this anoxic adaptation effect. This possibility was investigated in a number of ways. First, the time course of hypoxia-induced depression was found to essentially overlap in two slices exposed to repeated hypoxic periods interrupted by periods of recovery in normoxic medium. Second, 3 slices were exposed to their first hypoxic episode in the presence of 10/~M 8-PT. After 15 min of hypoxia, the population spike amplitudes recorded in these slices was 99 _+ 15% of the prehypoxic amplitude. This value is not significantly different from that observed in slices exposed to hypoxia and 10/xM 8-PT after preceding hypoxic exposures. Thus, the adenosine antagonist prevents hypoxia-induced depression of the population spike during the first exposure to hypoxia. Finally, supportive evidence against an adaptive effect of repeated hypoxic episodes is provided by the data
presented in Fig. 1. These data indicate that after the rinse-out of theophylline, the time course of hypoxia-induced depression during a third exposure to hypoxia does not significantly differ from the first response to hypoxia observed in the absence of the drug. Sufficient endogenous adenosine exists in the absence of synaptic transmission to depress neuronal excitability9. However, it is not clear whether hypoxia can elevate adenosine levels when synaptic transmission is blocked. In modified (low calcium, high magnesium) medium, that blocks synaptic transmission, the antidromically elicited response consists of an extracellularly recorded action potential or first spike followed by a series of afterpotentials recorded in the CA1 pyramidal cell layer9'23. Exogenous adenosine has been shown to block the afterpotentials with little effect on the first spike 23. Hypoxia alone completely blocked the amplitude of the first afterpotential after approximately 8 min (Fig. 3A). The first spike was not consistently affected at this time by the exposure to hypoxia (Fig. 3B). As in the case of the orthodromically evoked population spike, the hypoxic-induced depression was reversible and repeatable. Theophylline (100 /~M) delayed the depression of the first afterpotential but did not prevent its complete block (Fig. 3A). The amplitudes of the first afterpotential were significantly greater in the presence of theophylline 4 min after the start of hypoxia (P < 0.005, unpaired Student's t-test). The more potent antagonist, 8-PT (10/xM), essentially mimicked the effect of theophylline in delaying the loss of the afterpotential during hypoxia (Fig. 3A). The main finding of this study is that adenosine antagonists greatly delay the development of hypoxia-induced depression of neuronal activity in the brain slice. These results are consistent with the interpretation that adenosine plays a central role in the depression of neuronal activity during hypoxia. At a concentration of 10/~M, the adenosine antagonist 8-PT prevents the depression of the population spike observed during early exposure to hypoxia. An estimate of 50/~M for the concentration of adenosine associated with hypoxia is consistent with a potency series done in the hippocampal slice indicating that 5/~M 8-PT completely suppresses the electrophysiological effect of 50 /xM adenosine 6. The greater
382 potency of 8-PT when compared to that of theophylline is apparent from the present study. 10 ~M 8-PT preserved the population spike to a greater extent than did 100 #M theophylline. Previous reports investigating the role of adenosine in hypoxia-induced depression in the hippocampal slice have suggested at least a partial contribution. The adenosine antagonist, 3-isobutyl methylxanthine, was found to slightly delay the loss of the population spike recorded from guinea pig dentate granule cell layer 24. 8-PT (10-20 ~tM) somewhat delayed the fall of the evoked response measured in rat CA1 region during hypoxia ~. This occurred despite the observation that adenosine levels were not appreciably increased until after 14 min of hypoxia. The present results closely resemble those ob-
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Time (min) Fig. 3. Response of first afterpotential and first spike to hypoxia alone and in the presence of two adenosine antagonists. A: first afterpotential expressed as percent of control
afterpotential at time = 0 min was depressed in hypoxia alone (open squares, n = 8). The rate of decline during hypoxia was slowed by the addition of either theophylline (100/~M; closed circles, n = 6) or 8-PT (10 #M; open circles, n = 10). B: the amplitude of the first spike is expressed as percent of control at time = 0 min. The first spike was not consistently affected during hypoxia alone (open squares), or in the presence of either theophylline (100 #M, closed circles) or 8-PT (10 #M, open circles).
served in the isolated, superfused spinal cord. The adenosine antagonist, 8-cyclopentyltheophylline, for the most part prevented the hypoxia-induced depression of the monosynaptic reflex during early hypoxia 2~. The present results also indicate that adenosine levels may be altered by hypoxia in the absence of synaptic transmission. Both 8-PT and theophyUine slow the development of hypoxia-induced depression. However, it is not clear why no potency difference is observed between these two antagonists as both theophylline (100 #M) and 8-PT (10 #M) prolonged the time course of depression to a similar degree. One possible explanation is that somewhat less adenosine is released in the modified medium containing low calcium, high magnesium. Thus, 100 uM theophylline is sufficient to completely block the response to this increase in adenosine concentration. Other as yet unidentified processes must underlie the remaining depression that occurs fairly early during hypoxia. The present results suggest that adenosine may play a protective role during periods of metabolic stress by inhibiting neuronal activity and thus conserving cerebral energy supplies. The stable adenosine analogue, 2-chloroadenosine, protects against ischemic hippocampal CA1 cell loss s while theophylline exacerbates CAI damage 35. Adenosine influences cerebral energy consumption. In mice administration of the adenosine antagonist, aminophyiline, is associated with an increase in cerebral metabolic rate and a decrease in anoxic survival in vivo 45. The protective action of adenosine extends to its ability to depress the release of excitatory neurotransmitters such as glutamate 3. During conditions of metabolic stress glutamate exerts a neurotoxic effect and the neurotoxicity of excitatory transmitters is particularly evident when intracellular energy levels are reduced 29. The role of adenosine is of particular interest because, at least under the present conditions, adenosine inhibits neuronal function during the metabolic stress of hypoxia. The inhibition of central nervous function occurs before such activity is limited by the more severe pathological consequences of cerebral energy failure. It is worth noting that, in the present experiments, the population spike recorded in the submerged hippocampal slice did not experience an increased
383 It has been suggested that anoxic adaptation may be due to elevated glycolysis providing the necessary energy during the subsequent hypoxic period 4°. In the present case, it is certainly of interest to determine the source of energy consumed during the hypoxic period when adenosine antagonists are blocking the hypoxia-induced depression of the population spike. The initial metabolic state of the submerged slice and the slice with a surface directly exposed to oxygenated gas do differ. This difference is evident from the observation that the exposed slice undergoes a decline in energy charge when submerged ~. It is probable that the differences exhibited by submerged slices and by slices in an interface chamber exposed to hypoxia/anoxia represent a physiological response continuum to this form of metabolic stress. An understanding of these differences and of the role of possible endogenous modulators in these responses will further our understanding of the neuronal response to hypoxia and ischemia.
tolerance to hypoxia as a result of repeated exposure to hypoxia. This observation contrasts with that made in slices in an interface chamber. These slices exhibited an anoxic adaptation expressed as an increased resistance to hypoxia-induced depression of the population spike following a brief conditioning exposure to anoxia 4°. The reason for this difference is not clear but may involve the experimental dissimilarities between the submerged and the interface conditions. Slices in an interface chamber, where the humidified gas is passed directly over the exposed surface of the slice, are exposed to very nearly true anoxic conditions (defined as the complete lack of oxygen) in the presence of 95% N J 5 % C O 2, whereas the submerged slice exposed to nominally anoxic medium equilibrated with 95% N2/5% CO2 is actually exposed to hypoxic conditions with bath pO 2 near that of atmospheric pO237. Perhaps reflective of this difference in severity of hypoxic/anoxic conditions is the observation that electrical .activity recorded in slices in an interface chamber is irreversibly abolished after roughly 10 min of exposure to 95% N2/5% CO21'39"44, while submerged slices exhibit recovery after much longer periods of hypoxia 37. Submerged slices recover electrical activity after hypoxic periods as long as 30-45 min 36. The increased sensitivity of slices in an interface chamber may be due to the development of spreading depression which is not seen in submerged slices I .
The review of an earlier manuscript by Drs. John E McEiroy and Donal G. Sinex is appreciated. This work was supported by institutionally supported research funds of the Los Alamos National Laboratory under the auspices of the Department of Energy.
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