Effect of oxygen at high pressure transmitter release spontaneous
on
JOEL S. COLTON AND CAROL A. COLTON Department of Physiology, School of Medical Sciences, University
COLTON,JOEL S., AND CAROL A. C~~~~~.Effictofo~ygen at high pressure on spontaneous transmitter release. Am. J. Physiol. 235(5): C233-C237, 1978 or Am. J. Physiol.: Cell Physiol. 4(3): C233-C237, 1978. -The effect of oxygen at high pressure (OHP) on resting membrane properties (effective membrane resistance (R,,) and membrane potential (Vm)) and the spontaneous release of excitatory transmitter were examined at the lobster neuromuscular junction. Pressurization with 100% oxygen to 150 pounds per square inch gauge pressure (psig) or with nitrogen to 150 psig (7,000 mmHg nitrogen and 135 mmHg oxygen) produced a decrease in Reff associated with a hyperpolarization of V,. These changes, however, returned to control values within 20-30 min after completion of pressurization. Spontaneous release of excitatory transmitter was shown to increase dramatically in the presence of 100% oxygen at 150 psig. The increase in miniature end-plate potential (MEPP) frequency persisted beyond the transient changes seen with Reff and V,. This effect was selective to oxygen, as pressurization with nitrogen did not produce an increase in MEPP frequency. No change in average MEPP amplitude was seen with either OHP or pressure alone. An OHP-induced increase in MEPP frequency was also seen at the frog neuromuscular junction. The results indicate that both glutamate-mediated and acetylcholine-mediated synaptic transmission are altered by OHP.
hyperbaric oxygen; plate potentials
neuromuscular
junction;
miniature
end-
OXYGEN IS ESSENTIAL for the production of energy and the survival of aerobic cells, it is also a universal cellular poison, especially at high pressure (10, 11, 15, 17). Th’is t oxic effect of oxygen at high pressure (OHP) produces symptoms in man ranging from dizziness, vomiting, and disorientation to muscle twitching and finally grand ma1 seizures that appear indistinguishable from those seen during epilepsy (9, 19). The seizure activity accompanying oxygen toxicity suggests that OHP modifies neuronal and/or synaptic function. Changes in synaptic function may be an important factor in seizure activity seen during OHP since seizures could result from: 1) a decrease in the release of inhibitory transmitter, 2) a failure of inhibitory transmitter to interact with pre- or postsynaptic receptors, 3) an increase in release of excitatory transmitter, or 4) a potentiation of the excitatory postsynaptic response (5, 18). Of these, a decrease in the level of the inhibitory transmitter y-aminobutyric acid (GABA) in ALTHOUGH
0363-6143/78/0000-OOOO$Ol. 25 Copyright
0
1978 the American
Physiological
of Nevada, Reno, Nevada 89557
intact brain has already been suggested as a factor in OHP-induced convulsions (22, 23, 24). However, inhibitory transmission at the junctional level during OHP on excitatory has yet to be examined. Information transmission during OHP is also lacking. To characterize the effects of OHP on the nonstimulated properties of the synapse, i.e., resting potential, membrane resistance, and spontaneous transmitter release, a series of experiments was carried out on the neuromuscular junctions of the lobster Homarus americanus and the frog Rana p&ens. The stretcher muscle from the walking leg of the lobster was chosen since it has been shown to have both inhibitory (GABA-mediated) and excitatory (glutamate-mediated) junctions (3, 14). The frog sartorius muscle was used to examine the effects of OHP on an acetylcholine-mediated synapse. METHODS
The stretcher muscle from the lobster walking leg was prepared as described previously (3, 4) and placed in a temperature-controlled, artificial-seawater bath. Using a single remote-controlled micromanipulator and a Narishge dual-microelectrode holder (model HMD-2), two closely spaced glass microelectrodes were aligned over a single surface muscle fiber. The microelectrodes used for voltage recordings and current passage were prepared by standard methods (3) and filled with either 3 M KC1 or 2 M NaCl. Once the microelectrodes were aligned, the entire preparation was moved into a hyperbaric chamber and the chamber closed. The microelectrodes were then lowered into a single surface cell by remote control (4). After stabilization of the preparation, control measurements of membrane potential (Vd, effective membrane resistance (R,,), and excitatory miniature endplate potential (MEPP) amplitude and frequency were performed. R eff was found from the slope of the linear region around the resting potential of a current-voltage relationship. Excitatory MEPP amplitude and frequency were recorded on a Grass strip chart recorder using a low pass filter set for 6 db down at 75 Hz. Experimental values were averaged over 2-3 min of recording, with each preparation serving as its own control. Miniature amplitudes were corrected for shifts in V, using the relationship described by Hubbard et al (12). A reversal potential of - 15 mV (3) was used in Society
Downloaded from www.physiology.org/journal/ajpcell at Glasgow Univ Lib (130.209.006.061) on February 14, 2019.
C233
C234
J.
the correction formula. Inhibitory miniature end-plate potentials were not measured since they were indistinguishable from background noise. Once control measurements were taken, the chamber was pressurized with the appropriate gas mixture and the measurements repeated at 5-lo-min intervals after completion of pressurization. For compression with oxygen, the chamber was completely flushed with 100% oxygen and pressurized to 150 psig (7,135 mmHg 0, total). This value of pressure was chosen to insure a more than adequate gradient for oxygen at the muscle fiber. For nitrogen control studies, the chamber was pressurized with nitrogen to produce a final atmospheric pressure of 7,000 mmHg nitrogen plus 135 mmHg oxygen. Similar experiments were carried out on the neuromuscular junction of the frog. Sartorius muscles were isolated as described by Fatt and Katz (7) and an end plate localized. To reduce muscle contraction, especially during OHP, some fibers were placed in frog Ringer solution made hypertonic by the addition of 95 mM sucrose. When sucrose was used, nitrogen controls were also carried out in sucrose Ringers. This enabled the comparison of oxygen at high pressure and pressure alone under the same experimental conditions. RESULTS
The effect of hyperbaric oxygen on the spontaneous release of excitatory transmitter is shown in Fig. 1. The recordings depict MEPP frequency under control conditions (ambient 0, and ambient pressure), immediately after completion of pressurization with 100% oxygen to 150 pounds per square inch gauge pressure (psig), after 12 min in 100% oxygen at 150 psig, and after 22 min in 100% oxygen at 150 psig. As can be seen, miniature activity steadily increased in the presence of OHP. At 22 min after pressurization the frequency increase was so great that a highly irregular wave form with periods of sustained depolarization rather than individual miniatures was recorded. Shortly after the sustained depolarizations were observed, contraction of
S. COLTON
AND
C. A.
COLTON
the muscle and the subsequent displacement and damage of the microelectrodes occurred. A similar response was seen in every fiber studied, although the time to onset of the large MEPP frequency increase varied (average time t SE = 26 t 3.33 min, n = 5). When nitrogen was used to pressurize to 150 psig instead of oxygen (Fig. Z), MEPP frequency was seen to increase on pressurization (from 0.46 t 0.15 under control conditions to 0.65 t 0.18 immediately after pressurization, n = 3) and then to decrease within 40 min to a value about 50% below the control frequency (0.24 t 0.06, n = 3). This new value of MEPP frequency was maintained for the remainder of the experiment (approximately 40 min) . Observation of average miniature amplitude (I&& during control conditions, immediately after pressurization with 100% oxygen to 150 psig, and at an intermediate time revealed no significant change in VMEPP. Because of the summation of individual miniatures, it was not possible to measure MEPP amplitude after the large frequency increase was seen. Examination of Vlll& amplitude during nitrogen pressurization to 150 psig also showed no significant change. dHP-induced changes in the resting membrane properties, i.e., Reff and V,, of the postsynaptic cell were evident in every fiber studied. As shown in Fig. 3, pressurization to 150 psig with 100% oxygen produced a decrease in R eff accompanied ‘by a hyperpolarization of the membrane potential (about 2 mV for this fiber). However, over the experimental period, membrane resistance and potential returned slowly toward their control values. Average values of Reff and V, are shown in Table 1. These effects were not specific to oxygen since pressurization with nitrogen to 150 psig produced a similar effect over the same time course. To determine if the change in resting membrane properties was caused by the physical injection of K+ ions inside the cell due to the increased atmospheric pressure on the KC1 microelectrodes, NaCl microelectrodes were used. A hynernolarization and decrease in resistance was again bbserved, indicating that the
FIG. 1. Strip chart recording from a typical muscle fiber of the lobster demonstrating effect of hyperbaric oxygen on spontaneous transmitter release. A : control conditions of ambient oxygen and ambient pressure; fMEPP = 1.08 s-l, VMErr = 0.07 mV. B: immediately after completion of pressurization with 100% oxygen to 150 psig (7,135 mmHg); f,,,, = 1.26 s-l, VMEPP = 0.07 mV. C: at 10 min after completion of pressurization; f,,,, = 4.10 s-l, VMEPr = 0.07 mV. D: at 22 min after completion of pressurization; determination of fMEPP and VMErP was not possible.
Downloaded from www.physiology.org/journal/ajpcell at Glasgow Univ Lib (130.209.006.061) on February 14, 2019.
EFFECT
OF
OHP
ON
8
~~~~+-khWdk~~~~~*,~
C
-p-wwewW~;v~
0
SPONTANEOUS
TRANSMITTER
C235
RELEASE
FIG. 2. Strip chart recording from a typical muscle fiber of the lobster demonstrating effect of pressure alone on spontaneous transmitter release. A: control conditions of ambient oxygen and ambient pressure; fhlEPP = 0.31 s-l, VMErP = 0.10 mV. B: immediately after completion of pressurization with nitrogen to a final pressure of 7,000 mmHg nitrogen plus 135 mmHg oxygen; fNIEPP = 0.53 s-l, VMEpp = 0.09 mV. C: at 36 min after completion of nitrogen pressurization; fhlEPP = 0.18 s-l, v MEPP = 0.10 mV. D: at 53 min after completion of nitrogen pressurization; fMEPP = 0.22 s-‘, VMEPP = 0.09 mV.
I ~-JJ+w%y./w
4.~w#j+‘~~fi&&$41u~
Ibroqremq 48’ ~~*y+‘y&+&-~‘~~~
L
ta%h&MY
0.2mV 1 I set TABLE 1. Effect of OHP on Reffand V, in lobster muscle fiber
Avm (m v
Control
Parameter
R eff
+5
7 x
lo5
:0
vrn,
d
mv
0
Values determined 29A).
X
0
I
4.5
+2.5
I
(IO-’ 0
Amps)
0
a
2.03 + 0.42 5 81.; 12.6 n=5
o;, 1500,“i”;g =
1.56 + 0.33* n=5 85.7 + 3.1** n = 5
of experiments. are means + SE; n = number using paired t test (Hewlett-Packard, * P < 0.01. ** P < 0.04.
y”
‘,“,“rgi$
1.76 + 0.46 n=5 81.7 k 4.1 n = 5 Significance STAT PAK
l-
and pressure to 11.2 t 2.7 s-l at 150 psig and 100% oxygen. Pressurization with nitrogen produced no consistent change in MEPP frequency. DISCUSSION
OOX X O
0
-- 5
0
X O
0
0 0
X 0
FIG. 3. Current-voltage relationship from a typical muscle fiber of the lobster during OHP. CZosed circles, ambient oxygen and ambient pressure; R eff = 3.3 x lo5 Q V, = - 76 mV. Open circles, immediately after completion of pressurization with 100% oxygen to 150 psig; R,ff = 2.5 x lo5 a, V, = -77.5 mV. Crosses, 20 min after completion of pressurization; R eff = 3.2 x lo5 a, V, = -75 mV.
effect of pressurization with oxygen or nitrogen was not an electrode-induced artifact. In experiments on the frog neuromuscular junction, pressurization with 100% oxygen to 150 psig also produced a large increase in MEPP frequency. The onset of this frequency increase, however, was more rapid than in the lobster and could be seen immediately after completion of pressurization (Fig. 4). Compared to control, the frequency immediately after pressurization increased more than six times, i.e., from an average of 1.8 t 0.1 s-l (mean t SE, n = 3) under ambient oxygen
These experiments indicate that the effect of OHP on the function of the nonstimulated neuromuscular junction is primarily presynaptic, although the resting membrane properties of the postsynaptic cell are changed during pressurization. The alteration of resting membrane properties induced by oxygen or nitrogen at high pressure appears to be transient, occurring primarily during the actual process of pressurization. Since the decrease in R eff is associated with a hyperpolarization rather than a depolarization of V,, nonspecific membrane damage is an unlikely event. In addition, studies using NaCl rather than KC1 microelectrodes suggest that the membrane changes are not due to a microelectrode artifact such as injection of ions into the cell. Rather, the changes in resting properties may be a response of the membrane to the mechanical stress of pressurization. The significance of this effect to OHP-induced seizures is not clear at this time. However, the direction and the small magnitude of the changes in R eff and V, plus their transient nature make it doubtful that they play any important role in the production of seizure activity. The dramatic increase in excitatory MEPP frequency during pressurization with 100% oxygen, which persists beyond the transient change seen with Reff and V,, suggests that a major action of OHP is on the nerve terminal. Since nitrogen at 150 psig did not produce a similar response, this effect cannot be due to pressure
Downloaded from www.physiology.org/journal/ajpcell at Glasgow Univ Lib (130.209.006.061) on February 14, 2019.
C236
J. S. COLTON
AND
C. A. COLTON
I
AA.
B*w’ /
C.
b
&;&@+f+$@/~q\&&*~p$&; A
0.2
FIG. 4. Strip chart recording of miniature endplate potentials from a typical frog muscle fiber. A: obtained under ambient oxygen and ambient pressure; fhlEPP = 1.7 s-l, VMEPP = 0.09 mV. B: obtained immediately after completion of pressurization to 150 psig with 100% oxygen; f,,,, = 9.9 s-l, VMEpp = 0.08 mV. C: obtained at 20 min after completion of pressurization with 100% oxygen; frequency and amplitude of MEPPs not discernible due to summation of individual miniatures.
mvl ISeC
alone. Furthermore, since both glutamate-mediated (lobster) and acetylcholine-mediated (frog) neuromuscular junctions are affected in a similar manner, the action of OHP appears to be on some general property of the release mechanism. It has been demonstrated that OHP, by oxidizing sulfhydryl-containing enzymes such as coenzyme A, succinate dehydrogenase, or glutamic acid decarboxylase, can decrease ATP production (10, 11, 21) and in turn interrupt the sodium-potassium pump. In fact, studies have shown that sodium transport across isolated frog skin is inhibited during OHP (6). By extrapolation of these observations it is possible to suggest that, at the axon terminal, blockage of the Na+-K+ pump by OHP could result in intracellular Na+ accumulation. Because of its small size, the axon terminal is more susceptible to this type of an effect than a larger fiber such as a muscle. In addition, Na+ accumulation in the presynaptic terminal has been shown to increase the frequency of spontaneous transmitter release (1, 2, 16). It is possible, however, that oxygen has a more direct effect on release by increasing the formation of disul-
fide bonds in the terminal membrane. Werman et al. (20) have shown that diamide, a thiol-oxidizing agent, produces a large increase in MEPP frequency with no change in VMEPP. They proposed that disulfide bond formation in the terminal membrane is a critical factor in the actual expulsion of transmitter from a vesicle (13). Agents that increase disulfide bonds, such as OHP, might then be expected to increase transmitter release. Since many of the experiments with OHP resulted in contraction of the muscle, the amount of excitatory transmitter released by the increased spontaneous activity appears to be quite large. Hence, activation of the postsynaptic cell by massive transmitter release could be an extremely important factor in the seizures seen during OHP. The authors thank Dr. George T. Smith for his help in obtaining equipment that allowed this project to proceed. This work was supported in part by Grant 7-l-228-5452-011 from the Research Advisory Board, University of Nevada, Reno and Grant 7-l-331-5452-073 from the Luke B. Hancock Foundation. Received
16 August
1977; accepted
in final
form
14 June
1978.
REFERENCES 1. ATWOOD, H. L., L. E. SWENARCHUK, AND C. R. GRUENWALD. Long term synaptic facilitation during sodium accumulation in nerve terminal. Bruin Res. 100: 198-204, 1975. 2. BIRKS, R. I., AND M. W. COHEN. The influence of internal sodium on the behavior of motor nerve endings. Proc. Roy. Sot. London Ser. B. 170: 401-421, 1968. 3. COLTON, C. A., AND A. R. FREEMAN. Dual response of lobster muscle fibers to L-glutamate. Comp. Biochem. Physiol. 51C: 275 284, 1975. 4. COLTON, J. S., AND A. R. FREEMAN. Intracellular measurements in a closed hyperbaric chamber. J. AppZ. Physiol. 35: 578-580, 1973. 5. ESPLIN, D. W., AND B. ZABLOCKA-ESPLIN. Mechanisms of actions of convulsants. In: Basic Mechanisms of the Epilepsies, edited by H. H. Jasper, A. N. Ward, and A. Pope. Boston: Little, Brown, 1969, p. 167-194. 6. FALSETTI, H. Effects of oxygen tension on sodium transport across isolated frog skin. Proc. Sot. ExptZ. BioZ. Med. 101: 721722, 1959. 7. FATT, P., AND B. KATZ. An analysis of the end plate potential recorded with an intracellular electrode. J. Physiol. London 115: 320-370, 1951. 8. GERSCHENFELD, H. M. Chemical transmission in invertebrate central nervous systems and neuromuscular junctions. Physiol. Rev. 53: 1-119, 1973. 9. HAREL, D., AND S. LAVEY. Changes in electrical activity of the brain as a criterion of hyperbaric oxygen toxicity in the central nervous system. Life Sci. 1: 11-114, 1971.
10. HAUGAARD, N. Cellular mechanisms of oxygen toxicity. Physiol. Rev. 48: 311-373, 1968. 11. HAUGAARD, N. The scope of chemical oxygen poisoning. In: Proceedings of the 4th Symposium on Underwater PhysioZogy, edited by C. J. Lambertsen. New York: Academic, 1971, p. l-8. 12. HUBBARD, J. I., R. LLINAS, AND D. M. J. QUASTAL. Electrophysiological analysis of synaptic transmission. London: Arnold, 1969. 13. KOSOWER, E. M., AND R. WERMAN. New step in transmitter release at the myoneural junction. Nature 233: 121-123, 1971. 14. KRAVITZ, E. A., S. W. KUFFLER, AND D.D. POTTER. Gamma aminobutyric acid and other blocking compounds in crustacea. III. Their relative concentrations in separated motor and inhibitory axons. J. Neurophysiol. 26: 739-751, 1963. 15. LAMBERTSEN, C. J. Effects of oxygen at high partial pressure. In: Handbook of Physiology. Respiration. Washington, D.C.: Am. Physiol. Sot., 1965, sect. 3, vol. II, p. 1027-1046. 16. LILEY, A. W. The effects of presynaptic polarization on the spontaneous activity at the mammalian neuromuscular junction. J. Physiol. London 134: 427-443, 1956. 17. MEIJNE, N. G. Hyperbaric Oxygen and Its Clinical Value, edited by I. N. Kugelmass. Chicago: Thomas, 1970. 18. SWANSON, P. D. Convulsive disorders. In: Biochemistry ofNeural Disease, edited by M. M. Cohen. New York: Harper and Row, 1975, p. 19-32. 19. TOWER, D. D. GABA and seizures: clinical correlates in man. In: GABA in Nervous System Function, Kroc Foundation Series, edited by E. Roberts, T. Chase, and D. Tower. New York: Raven Press, vol. 5, 1976, p. 461-478.
Downloaded from www.physiology.org/journal/ajpcell at Glasgow Univ Lib (130.209.006.061) on February 14, 2019.
RELEASE
C237
20. WERMAN, R., P. L. CARLEN, M. KUSHNIR, AND E. M. KOSOWER. Effect of the thiol-oxidizing agent diamide on acetylcholine release at the frog end plate. Nature 233: 120-121, 1971. 21. WILLIAMS, C. D., AND N. HAUGAARD. Toxic effects of oxygen on cerebral metabolism. J. Neurochem. 17: 709-720, 1970. 22. WOOD, J. D. Oxygen toxicity in neuronal elements. In: Proceedings of the 4th Symposium on Underwater Physiology, edited by C. J. Lambertsen. New York: Academic, 1971, p. 9-18.
23. WOOD, J. D., M. W. RADOMSKI, AND W. J. WATSON. A study of possible mechanisms involved in hyperbaric oxygen induced changes in cerebral gamma aminobutyric acid levels and accompanying seizures. Can. J. Biochem. 49: 543-547, 1971. 24. WOOD, J. D., W. J. WATSON, AND G. W. MURRAY. Correlation between decreases in brain gamma aminobutyric acid levels and susceptibility to convulsions induced by hyperbaric oxygen. J. Neurochem. 16: 281-287, 1969.
EFFECT
OF
OHP
ON
SPONTANEOUS
TRANSMITTER
Downloaded from www.physiology.org/journal/ajpcell at Glasgow Univ Lib (130.209.006.061) on February 14, 2019.
on
JOEL S. COLTON AND CAROL A. COLTON Department of Physiology, School of Medical Sciences, University
COLTON,JOEL S., AND CAROL A. C~~~~~.Effictofo~ygen at high pressure on spontaneous transmitter release. Am. J. Physiol. 235(5): C233-C237, 1978 or Am. J. Physiol.: Cell Physiol. 4(3): C233-C237, 1978. -The effect of oxygen at high pressure (OHP) on resting membrane properties (effective membrane resistance (R,,) and membrane potential (Vm)) and the spontaneous release of excitatory transmitter were examined at the lobster neuromuscular junction. Pressurization with 100% oxygen to 150 pounds per square inch gauge pressure (psig) or with nitrogen to 150 psig (7,000 mmHg nitrogen and 135 mmHg oxygen) produced a decrease in Reff associated with a hyperpolarization of V,. These changes, however, returned to control values within 20-30 min after completion of pressurization. Spontaneous release of excitatory transmitter was shown to increase dramatically in the presence of 100% oxygen at 150 psig. The increase in miniature end-plate potential (MEPP) frequency persisted beyond the transient changes seen with Reff and V,. This effect was selective to oxygen, as pressurization with nitrogen did not produce an increase in MEPP frequency. No change in average MEPP amplitude was seen with either OHP or pressure alone. An OHP-induced increase in MEPP frequency was also seen at the frog neuromuscular junction. The results indicate that both glutamate-mediated and acetylcholine-mediated synaptic transmission are altered by OHP.
hyperbaric oxygen; plate potentials
neuromuscular
junction;
miniature
end-
OXYGEN IS ESSENTIAL for the production of energy and the survival of aerobic cells, it is also a universal cellular poison, especially at high pressure (10, 11, 15, 17). Th’is t oxic effect of oxygen at high pressure (OHP) produces symptoms in man ranging from dizziness, vomiting, and disorientation to muscle twitching and finally grand ma1 seizures that appear indistinguishable from those seen during epilepsy (9, 19). The seizure activity accompanying oxygen toxicity suggests that OHP modifies neuronal and/or synaptic function. Changes in synaptic function may be an important factor in seizure activity seen during OHP since seizures could result from: 1) a decrease in the release of inhibitory transmitter, 2) a failure of inhibitory transmitter to interact with pre- or postsynaptic receptors, 3) an increase in release of excitatory transmitter, or 4) a potentiation of the excitatory postsynaptic response (5, 18). Of these, a decrease in the level of the inhibitory transmitter y-aminobutyric acid (GABA) in ALTHOUGH
0363-6143/78/0000-OOOO$Ol. 25 Copyright
0
1978 the American
Physiological
of Nevada, Reno, Nevada 89557
intact brain has already been suggested as a factor in OHP-induced convulsions (22, 23, 24). However, inhibitory transmission at the junctional level during OHP on excitatory has yet to be examined. Information transmission during OHP is also lacking. To characterize the effects of OHP on the nonstimulated properties of the synapse, i.e., resting potential, membrane resistance, and spontaneous transmitter release, a series of experiments was carried out on the neuromuscular junctions of the lobster Homarus americanus and the frog Rana p&ens. The stretcher muscle from the walking leg of the lobster was chosen since it has been shown to have both inhibitory (GABA-mediated) and excitatory (glutamate-mediated) junctions (3, 14). The frog sartorius muscle was used to examine the effects of OHP on an acetylcholine-mediated synapse. METHODS
The stretcher muscle from the lobster walking leg was prepared as described previously (3, 4) and placed in a temperature-controlled, artificial-seawater bath. Using a single remote-controlled micromanipulator and a Narishge dual-microelectrode holder (model HMD-2), two closely spaced glass microelectrodes were aligned over a single surface muscle fiber. The microelectrodes used for voltage recordings and current passage were prepared by standard methods (3) and filled with either 3 M KC1 or 2 M NaCl. Once the microelectrodes were aligned, the entire preparation was moved into a hyperbaric chamber and the chamber closed. The microelectrodes were then lowered into a single surface cell by remote control (4). After stabilization of the preparation, control measurements of membrane potential (Vd, effective membrane resistance (R,,), and excitatory miniature endplate potential (MEPP) amplitude and frequency were performed. R eff was found from the slope of the linear region around the resting potential of a current-voltage relationship. Excitatory MEPP amplitude and frequency were recorded on a Grass strip chart recorder using a low pass filter set for 6 db down at 75 Hz. Experimental values were averaged over 2-3 min of recording, with each preparation serving as its own control. Miniature amplitudes were corrected for shifts in V, using the relationship described by Hubbard et al (12). A reversal potential of - 15 mV (3) was used in Society
Downloaded from www.physiology.org/journal/ajpcell at Glasgow Univ Lib (130.209.006.061) on February 14, 2019.
C233
C234
J.
the correction formula. Inhibitory miniature end-plate potentials were not measured since they were indistinguishable from background noise. Once control measurements were taken, the chamber was pressurized with the appropriate gas mixture and the measurements repeated at 5-lo-min intervals after completion of pressurization. For compression with oxygen, the chamber was completely flushed with 100% oxygen and pressurized to 150 psig (7,135 mmHg 0, total). This value of pressure was chosen to insure a more than adequate gradient for oxygen at the muscle fiber. For nitrogen control studies, the chamber was pressurized with nitrogen to produce a final atmospheric pressure of 7,000 mmHg nitrogen plus 135 mmHg oxygen. Similar experiments were carried out on the neuromuscular junction of the frog. Sartorius muscles were isolated as described by Fatt and Katz (7) and an end plate localized. To reduce muscle contraction, especially during OHP, some fibers were placed in frog Ringer solution made hypertonic by the addition of 95 mM sucrose. When sucrose was used, nitrogen controls were also carried out in sucrose Ringers. This enabled the comparison of oxygen at high pressure and pressure alone under the same experimental conditions. RESULTS
The effect of hyperbaric oxygen on the spontaneous release of excitatory transmitter is shown in Fig. 1. The recordings depict MEPP frequency under control conditions (ambient 0, and ambient pressure), immediately after completion of pressurization with 100% oxygen to 150 pounds per square inch gauge pressure (psig), after 12 min in 100% oxygen at 150 psig, and after 22 min in 100% oxygen at 150 psig. As can be seen, miniature activity steadily increased in the presence of OHP. At 22 min after pressurization the frequency increase was so great that a highly irregular wave form with periods of sustained depolarization rather than individual miniatures was recorded. Shortly after the sustained depolarizations were observed, contraction of
S. COLTON
AND
C. A.
COLTON
the muscle and the subsequent displacement and damage of the microelectrodes occurred. A similar response was seen in every fiber studied, although the time to onset of the large MEPP frequency increase varied (average time t SE = 26 t 3.33 min, n = 5). When nitrogen was used to pressurize to 150 psig instead of oxygen (Fig. Z), MEPP frequency was seen to increase on pressurization (from 0.46 t 0.15 under control conditions to 0.65 t 0.18 immediately after pressurization, n = 3) and then to decrease within 40 min to a value about 50% below the control frequency (0.24 t 0.06, n = 3). This new value of MEPP frequency was maintained for the remainder of the experiment (approximately 40 min) . Observation of average miniature amplitude (I&& during control conditions, immediately after pressurization with 100% oxygen to 150 psig, and at an intermediate time revealed no significant change in VMEPP. Because of the summation of individual miniatures, it was not possible to measure MEPP amplitude after the large frequency increase was seen. Examination of Vlll& amplitude during nitrogen pressurization to 150 psig also showed no significant change. dHP-induced changes in the resting membrane properties, i.e., Reff and V,, of the postsynaptic cell were evident in every fiber studied. As shown in Fig. 3, pressurization to 150 psig with 100% oxygen produced a decrease in R eff accompanied ‘by a hyperpolarization of the membrane potential (about 2 mV for this fiber). However, over the experimental period, membrane resistance and potential returned slowly toward their control values. Average values of Reff and V, are shown in Table 1. These effects were not specific to oxygen since pressurization with nitrogen to 150 psig produced a similar effect over the same time course. To determine if the change in resting membrane properties was caused by the physical injection of K+ ions inside the cell due to the increased atmospheric pressure on the KC1 microelectrodes, NaCl microelectrodes were used. A hynernolarization and decrease in resistance was again bbserved, indicating that the
FIG. 1. Strip chart recording from a typical muscle fiber of the lobster demonstrating effect of hyperbaric oxygen on spontaneous transmitter release. A : control conditions of ambient oxygen and ambient pressure; fMEPP = 1.08 s-l, VMErr = 0.07 mV. B: immediately after completion of pressurization with 100% oxygen to 150 psig (7,135 mmHg); f,,,, = 1.26 s-l, VMEPP = 0.07 mV. C: at 10 min after completion of pressurization; f,,,, = 4.10 s-l, VMEPr = 0.07 mV. D: at 22 min after completion of pressurization; determination of fMEPP and VMErP was not possible.
Downloaded from www.physiology.org/journal/ajpcell at Glasgow Univ Lib (130.209.006.061) on February 14, 2019.
EFFECT
OF
OHP
ON
8
~~~~+-khWdk~~~~~*,~
C
-p-wwewW~;v~
0
SPONTANEOUS
TRANSMITTER
C235
RELEASE
FIG. 2. Strip chart recording from a typical muscle fiber of the lobster demonstrating effect of pressure alone on spontaneous transmitter release. A: control conditions of ambient oxygen and ambient pressure; fhlEPP = 0.31 s-l, VMErP = 0.10 mV. B: immediately after completion of pressurization with nitrogen to a final pressure of 7,000 mmHg nitrogen plus 135 mmHg oxygen; fNIEPP = 0.53 s-l, VMEpp = 0.09 mV. C: at 36 min after completion of nitrogen pressurization; fhlEPP = 0.18 s-l, v MEPP = 0.10 mV. D: at 53 min after completion of nitrogen pressurization; fMEPP = 0.22 s-‘, VMEPP = 0.09 mV.
I ~-JJ+w%y./w
4.~w#j+‘~~fi&&$41u~
Ibroqremq 48’ ~~*y+‘y&+&-~‘~~~
L
ta%h&MY
0.2mV 1 I set TABLE 1. Effect of OHP on Reffand V, in lobster muscle fiber
Avm (m v
Control
Parameter
R eff
+5
7 x
lo5
:0
vrn,
d
mv
0
Values determined 29A).
X
0
I
4.5
+2.5
I
(IO-’ 0
Amps)
0
a
2.03 + 0.42 5 81.; 12.6 n=5
o;, 1500,“i”;g =
1.56 + 0.33* n=5 85.7 + 3.1** n = 5
of experiments. are means + SE; n = number using paired t test (Hewlett-Packard, * P < 0.01. ** P < 0.04.
y”
‘,“,“rgi$
1.76 + 0.46 n=5 81.7 k 4.1 n = 5 Significance STAT PAK
l-
and pressure to 11.2 t 2.7 s-l at 150 psig and 100% oxygen. Pressurization with nitrogen produced no consistent change in MEPP frequency. DISCUSSION
OOX X O
0
-- 5
0
X O
0
0 0
X 0
FIG. 3. Current-voltage relationship from a typical muscle fiber of the lobster during OHP. CZosed circles, ambient oxygen and ambient pressure; R eff = 3.3 x lo5 Q V, = - 76 mV. Open circles, immediately after completion of pressurization with 100% oxygen to 150 psig; R,ff = 2.5 x lo5 a, V, = -77.5 mV. Crosses, 20 min after completion of pressurization; R eff = 3.2 x lo5 a, V, = -75 mV.
effect of pressurization with oxygen or nitrogen was not an electrode-induced artifact. In experiments on the frog neuromuscular junction, pressurization with 100% oxygen to 150 psig also produced a large increase in MEPP frequency. The onset of this frequency increase, however, was more rapid than in the lobster and could be seen immediately after completion of pressurization (Fig. 4). Compared to control, the frequency immediately after pressurization increased more than six times, i.e., from an average of 1.8 t 0.1 s-l (mean t SE, n = 3) under ambient oxygen
These experiments indicate that the effect of OHP on the function of the nonstimulated neuromuscular junction is primarily presynaptic, although the resting membrane properties of the postsynaptic cell are changed during pressurization. The alteration of resting membrane properties induced by oxygen or nitrogen at high pressure appears to be transient, occurring primarily during the actual process of pressurization. Since the decrease in R eff is associated with a hyperpolarization rather than a depolarization of V,, nonspecific membrane damage is an unlikely event. In addition, studies using NaCl rather than KC1 microelectrodes suggest that the membrane changes are not due to a microelectrode artifact such as injection of ions into the cell. Rather, the changes in resting properties may be a response of the membrane to the mechanical stress of pressurization. The significance of this effect to OHP-induced seizures is not clear at this time. However, the direction and the small magnitude of the changes in R eff and V, plus their transient nature make it doubtful that they play any important role in the production of seizure activity. The dramatic increase in excitatory MEPP frequency during pressurization with 100% oxygen, which persists beyond the transient change seen with Reff and V,, suggests that a major action of OHP is on the nerve terminal. Since nitrogen at 150 psig did not produce a similar response, this effect cannot be due to pressure
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C236
J. S. COLTON
AND
C. A. COLTON
I
AA.
B*w’ /
C.
b
&;&@+f+$@/~q\&&*~p$&; A
0.2
FIG. 4. Strip chart recording of miniature endplate potentials from a typical frog muscle fiber. A: obtained under ambient oxygen and ambient pressure; fhlEPP = 1.7 s-l, VMEPP = 0.09 mV. B: obtained immediately after completion of pressurization to 150 psig with 100% oxygen; f,,,, = 9.9 s-l, VMEpp = 0.08 mV. C: obtained at 20 min after completion of pressurization with 100% oxygen; frequency and amplitude of MEPPs not discernible due to summation of individual miniatures.
mvl ISeC
alone. Furthermore, since both glutamate-mediated (lobster) and acetylcholine-mediated (frog) neuromuscular junctions are affected in a similar manner, the action of OHP appears to be on some general property of the release mechanism. It has been demonstrated that OHP, by oxidizing sulfhydryl-containing enzymes such as coenzyme A, succinate dehydrogenase, or glutamic acid decarboxylase, can decrease ATP production (10, 11, 21) and in turn interrupt the sodium-potassium pump. In fact, studies have shown that sodium transport across isolated frog skin is inhibited during OHP (6). By extrapolation of these observations it is possible to suggest that, at the axon terminal, blockage of the Na+-K+ pump by OHP could result in intracellular Na+ accumulation. Because of its small size, the axon terminal is more susceptible to this type of an effect than a larger fiber such as a muscle. In addition, Na+ accumulation in the presynaptic terminal has been shown to increase the frequency of spontaneous transmitter release (1, 2, 16). It is possible, however, that oxygen has a more direct effect on release by increasing the formation of disul-
fide bonds in the terminal membrane. Werman et al. (20) have shown that diamide, a thiol-oxidizing agent, produces a large increase in MEPP frequency with no change in VMEPP. They proposed that disulfide bond formation in the terminal membrane is a critical factor in the actual expulsion of transmitter from a vesicle (13). Agents that increase disulfide bonds, such as OHP, might then be expected to increase transmitter release. Since many of the experiments with OHP resulted in contraction of the muscle, the amount of excitatory transmitter released by the increased spontaneous activity appears to be quite large. Hence, activation of the postsynaptic cell by massive transmitter release could be an extremely important factor in the seizures seen during OHP. The authors thank Dr. George T. Smith for his help in obtaining equipment that allowed this project to proceed. This work was supported in part by Grant 7-l-228-5452-011 from the Research Advisory Board, University of Nevada, Reno and Grant 7-l-331-5452-073 from the Luke B. Hancock Foundation. Received
16 August
1977; accepted
in final
form
14 June
1978.
REFERENCES 1. ATWOOD, H. L., L. E. SWENARCHUK, AND C. R. GRUENWALD. Long term synaptic facilitation during sodium accumulation in nerve terminal. Bruin Res. 100: 198-204, 1975. 2. BIRKS, R. I., AND M. W. COHEN. The influence of internal sodium on the behavior of motor nerve endings. Proc. Roy. Sot. London Ser. B. 170: 401-421, 1968. 3. COLTON, C. A., AND A. R. FREEMAN. Dual response of lobster muscle fibers to L-glutamate. Comp. Biochem. Physiol. 51C: 275 284, 1975. 4. COLTON, J. S., AND A. R. FREEMAN. Intracellular measurements in a closed hyperbaric chamber. J. AppZ. Physiol. 35: 578-580, 1973. 5. ESPLIN, D. W., AND B. ZABLOCKA-ESPLIN. Mechanisms of actions of convulsants. In: Basic Mechanisms of the Epilepsies, edited by H. H. Jasper, A. N. Ward, and A. Pope. Boston: Little, Brown, 1969, p. 167-194. 6. FALSETTI, H. Effects of oxygen tension on sodium transport across isolated frog skin. Proc. Sot. ExptZ. BioZ. Med. 101: 721722, 1959. 7. FATT, P., AND B. KATZ. An analysis of the end plate potential recorded with an intracellular electrode. J. Physiol. London 115: 320-370, 1951. 8. GERSCHENFELD, H. M. Chemical transmission in invertebrate central nervous systems and neuromuscular junctions. Physiol. Rev. 53: 1-119, 1973. 9. HAREL, D., AND S. LAVEY. Changes in electrical activity of the brain as a criterion of hyperbaric oxygen toxicity in the central nervous system. Life Sci. 1: 11-114, 1971.
10. HAUGAARD, N. Cellular mechanisms of oxygen toxicity. Physiol. Rev. 48: 311-373, 1968. 11. HAUGAARD, N. The scope of chemical oxygen poisoning. In: Proceedings of the 4th Symposium on Underwater PhysioZogy, edited by C. J. Lambertsen. New York: Academic, 1971, p. l-8. 12. HUBBARD, J. I., R. LLINAS, AND D. M. J. QUASTAL. Electrophysiological analysis of synaptic transmission. London: Arnold, 1969. 13. KOSOWER, E. M., AND R. WERMAN. New step in transmitter release at the myoneural junction. Nature 233: 121-123, 1971. 14. KRAVITZ, E. A., S. W. KUFFLER, AND D.D. POTTER. Gamma aminobutyric acid and other blocking compounds in crustacea. III. Their relative concentrations in separated motor and inhibitory axons. J. Neurophysiol. 26: 739-751, 1963. 15. LAMBERTSEN, C. J. Effects of oxygen at high partial pressure. In: Handbook of Physiology. Respiration. Washington, D.C.: Am. Physiol. Sot., 1965, sect. 3, vol. II, p. 1027-1046. 16. LILEY, A. W. The effects of presynaptic polarization on the spontaneous activity at the mammalian neuromuscular junction. J. Physiol. London 134: 427-443, 1956. 17. MEIJNE, N. G. Hyperbaric Oxygen and Its Clinical Value, edited by I. N. Kugelmass. Chicago: Thomas, 1970. 18. SWANSON, P. D. Convulsive disorders. In: Biochemistry ofNeural Disease, edited by M. M. Cohen. New York: Harper and Row, 1975, p. 19-32. 19. TOWER, D. D. GABA and seizures: clinical correlates in man. In: GABA in Nervous System Function, Kroc Foundation Series, edited by E. Roberts, T. Chase, and D. Tower. New York: Raven Press, vol. 5, 1976, p. 461-478.
Downloaded from www.physiology.org/journal/ajpcell at Glasgow Univ Lib (130.209.006.061) on February 14, 2019.
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20. WERMAN, R., P. L. CARLEN, M. KUSHNIR, AND E. M. KOSOWER. Effect of the thiol-oxidizing agent diamide on acetylcholine release at the frog end plate. Nature 233: 120-121, 1971. 21. WILLIAMS, C. D., AND N. HAUGAARD. Toxic effects of oxygen on cerebral metabolism. J. Neurochem. 17: 709-720, 1970. 22. WOOD, J. D. Oxygen toxicity in neuronal elements. In: Proceedings of the 4th Symposium on Underwater Physiology, edited by C. J. Lambertsen. New York: Academic, 1971, p. 9-18.
23. WOOD, J. D., M. W. RADOMSKI, AND W. J. WATSON. A study of possible mechanisms involved in hyperbaric oxygen induced changes in cerebral gamma aminobutyric acid levels and accompanying seizures. Can. J. Biochem. 49: 543-547, 1971. 24. WOOD, J. D., W. J. WATSON, AND G. W. MURRAY. Correlation between decreases in brain gamma aminobutyric acid levels and susceptibility to convulsions induced by hyperbaric oxygen. J. Neurochem. 16: 281-287, 1969.
EFFECT
OF
OHP
ON
SPONTANEOUS
TRANSMITTER
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