Brain Research, 88 (1975) 145-149
145
~ Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
Effect of ouabain and phenobarbital on oxidative metabolic activity associated with spreading cortical depression in cats
JOSEPH C. LAMANNA AND M Y R O N R O S E N T H A L
Department of Physiology and Pharmacology, Duke University Medical School, Durham, N.C. 27710
(u.s.A.) (Accepted January 14th, 1975)
Spreading cortical depression (SD) is accompanied by large changes in the oxidative metabolic activity of the cerebral cortex of cats 11 and rats 6 recorded fluorometrically. SD is characterized by depression of the E C o G activity together with a negative shift of the cortical steady potential (SP) and an increase in extracellular potassium activity4-L Prior to this SP shift, no changes in tissue fluorescence occur, but during the period of depression, this signal declines and later begins to return to baseline after SP and E C o G approximate full recovery. The fluorometric changes were interpreted as due to an oxidation of intramitochondrial nicotinamide adenine dinucleotide ( N A D H ) to N A D + followed by a reduction of the N A D + to original N A D H levels, a sequence of events associated with transiently increased tissue activity1, 3,s. This occurs because reduced N A D H fluoresces while the oxidized form, N A D ~', does not. In this study, we examine the effects of small intracortical injections of ouabain and of systemically administered phenobarbital on the fluorescence changes that occur due to the production of spreading depression. Recently we reported the effects of these two drugs administered in the same way on the fluorescence changes that occur on the cortical surface when potentials are evoked 9. In that study, up to 600 pmoles of 0.1 m M ouabain slowed the rate of fluorescence decrease ( N A D H oxidation or ' o n ' kinetics) caused by direct cortically stimulated evoked potentials but had no effect on the rate of recovery of fluorescence to baseline levels ('off' kinetics). Higher ouabain doses abolished both electrical and metabolic events. Phenobarbital slowed the 'off' or recovery rate but had no effect on the initial fluorescence decrease. Similar to the results caused by evoked potentials, we report here that ouabain changes the rate of the larger decrease in fluorescence caused by SD and has no effect on the rate of recovery of fluorescence. In contrast, phenobarbital slows the return of fluorescence with no effect on the initial decrease of the signal. These results, combined with those obtained previously, support a hypothesis that the rate of oxidation of N A D H is controlled by the rising A D P concentration due to the breakdown of ATP by the ouabain-sensitive N a - K ATPase membrane transport system while the recovery
146 rate of N A D H from N A D is a function of the respiratory chain itself. A preliminar> report of these investigations has been presented 1°. The trachea and a l'emoral vein were cannulated in cats that were anesthetized with ether. The cisterna was drained of CSF, then a portion of tile calvarium w~ts excised, the dura removed and the brain stem was transected with an electrolytic lesion at the A-P 0.0 coordinates between the colliculi, as in the classical cerveau isold preparation. Ether was discontinued and the cats were allowed to rest for at least 3 h. The cats were heated with feedback thermal controls, expiratory CO2 levels were monitored and the cortex was kept moist with saline or artificial CSF. Up to t4 ,ul of ouabain was injected through a 30-gauge stainless steel needle with its delivery tip implanted 0.5 mm under the cortical surface within the suprasylvian gyrus. The solution was forced into the center of the optical field formed by the epi-illumination optics of the microscope system used for light delivery and monitoring 9. Field diameters of 3.2 mm and 1 mm were used. Microliter quantities of ouabain solutions (0. I raM) in distilled water or saline solution (0.9 ~,,; NaCI) could be administered during the period of optical and electrical monitoring. Effects of volume changes on electrical potentials or fluorescence changes were not observed with up to 10 #1 of water or saline. Phenobarbital was injected i.v. The fluorometric monitoring technique has been described previously 3,,q. The fluorescence of N A D H at approximately 450 nm is recorded with reflected excitation light at 366 rim. Reflectance is then subtracted from fluorescence to produce the 'metabolic signal' (F-R). This signal is corrected for changes due to blood volume, light scattering, volumetric or other geometry changes which appear in both the F and R channels. Spreading depression was produced by suprathreshold intensities of direct cortical stimulation, usually 1 sec trains of 20 Hz pulses of 0.5 msec duration. Stimulation intensities were kept constant through an experiment. Fig. 1 shows that increasing ouabain doses slow the decrease in the metabolic signal during SD. The drug, however, has no effect on the subsequent rate of recovery of this signal to baseline levels. In fact, ouabain has three apparent effects on the optical signal. Most obvious is the retardation of the 'on' kinetics. The drug also decreases the signal amplitude and finally, it was consistently observed that after ouabain administration the decrease of fluorescence becomes more complex and a plateau effect is apparent. This effect is more obvious in a 1 mm diameter field although the retardation of 'on' kinetics is also seen in a 3.2 mm field. Phenobarbital has no effect on 'on' kinetics. However, the barbiturate decreases the amplitude of the metabolic signal and greatly prolongs the time of the return to baseline levels (Fig. 2). This prolongation of the decreased fluorescence is due to the fact that recovery after I-2 rain is reduced progressively with increasing phenobarbital doses, i.e., the lower data set indicates a 500 }; increase in the time required for half of the fluorescence to return to baseline after a dose of 80 mg/kg of phenobarbital. Phenobarbital at 20 mg/kg shows a smaller but still apparent inhibitory effect on "off' kinetics. Similar results were obtained with amobarbital. The effects of both ouabain and phenobarbital were consistent and irreversible
147
B
C
9
2 0 sec Fig. 1. Ouabain effects on metabolism in spreading depression. Ouabain was microinjected 0.5 mm below the surface at the center of the 1 mm diameter optical field. A is the control metabolic signal (F-R) caused by SD; B is response after 4 ffl of 0.1 mM ouabain; C is response after 8 ffl; and D is response after 12 ffl. In this and subsequent trace, a decrease in the optical signal (F-R) denotes an oxidation of NADH. This decrease is expressed as a per cent with the 'resting' F-R level set at 100 relative to a dark field.
u p o n r e p e a t e d S D episodes in the same area over the e x p e r i m e n t a l p e r i o d which was up to a p p r o x i m a t e l y 4 h. The o u a b a i n a n d p h e n o b a r b i t a l i n h i b i t i o n s o f the m e t a b o l i c response to S D are similar to those o b s e r v e d when e v o k e d p o t e n t i a l s were m e a s u r e d 9. These results, however, are different f r o m those o b s e r v e d in studies using e v o k e d p o t e n t i a l s in 3 respects : (1) S D responses were present with o u a b a i n doses larger t h a n those which consistently e l i m i n a t e d e v o k e d p o t e n t i a l s ( e v o k e d p o t e n t i a l s were virtually e l i m i n a t e d by 4 #1 o f o u a b a i n a n d t o t a l l y e l i m i n a t e d at 6 ffl); (2) at low o u a b a i n doses, e v o k e d p o t e n t i a l o p t i c a l responses were a t t e n u a t e d in a 3 m m d i a m e t e r field with no kinetic changes. In a 1 m m field, these same doses a t t e n u a t e d signal a n d i n h i b i t e d ' o n ' kinetics. In contrast, the effect o f o u a b a i n on S D kinetics was seen in b o t h 1 m m a n d 3 m m fields; (3) o u a b a i n p r o d u c e d p l a t e a u s o f the ' o n ' kinetics o f the S D response. Since e v o k e d p o t e n t i a l s are e l i m i n a t e d at doses o f o u a b a i n which do not prevent SD, it is a p p a r e n t t h a t the ability to p r o d u c e e v o k e d p o t e n t i a l s is not necessary to
148
A
?
irnir
B
cL Fig. 2. Phenobarbital effects on spreading depression. A is the control (F-R) response; B is the response after 20 mg/kg phenobarbital administered i.v., and C is the response after 80 rng/kg phenobarbital.
produce SD by electrical stimulation. Ouabain causes cell swelling in CNS tissue lz, and this may decrease the integrity of individual CNS cells or the integrity of neuronal interconnections. It appears that a higher degree o f integrity of the nervous system is required for evoked potentials than for SD. The disruption of intercellular communication would be expected to inhibit evoked potentials more than SD since the latter is presumed to be communicated by elevated extracellular potassium 2 and may not depend upon connections between neurons or between glial elements. Furthermore, if the spread of an evoked potential involves interneuronal contacts and these are inhibited by ouabain, then less cells at the periphery would be involved. It might be that the cells at the periphery of a 3 m m field are relatively unaffected by ouabain and respond normally to electrical stimuli although fewer peripheral cells receive stimulation. This might explain the fact that in the presence of ouabain, evoked potential associated optical responses are attenuated but have normal kinetics when recorded in a 3 m m field. In a smaller field, however, the number of cells
149 r e s p o n d i n g to t r a n s s y n a p t i c influences m a y also be lessened b u t o u a b a i n affected ceils m a y also be excited by the electrical pulses directly or by increased extracellular potassium. In SD, passive p o t a s s i u m diffusion could m e a n t h a t m o r e o u a b a i n affected cells are p a r t i c i p a t i n g in the reactions. The p a r t i c i p a t i o n o f these cells m a y result in the effects o f o u a b a i n on signal a m p l i t u d e a n d on kinetics in a 3 m m field as well as in 1 mm fields. A n alternate possibility, however, is t h a t the m u c h larger extracellular p o t a s s i u m levels in SD4,5, 7 stimulate t r a n s p o r t a n d recovery systems o t h e r t h a n those a c t i v a t e d by e v o k e d potentials. Finally, the p l a t e a u effect o f o u a b a i n indicates the p o s s i b i l i t y that in S D multiple t r a n s p o r t systems might be involved, each with a variable t i m e course a n d sensitivity to o u a b a i n inhibition. Analysis a n d c o r r e l a t i o n o f the time courses o f the steady p o t e n t i a l shifts a n d the rise a n d fall o f extracellular p o t a s s i u m activity with simultaneous fluorescence m o n i t o r i n g m a y resolve this question. W e wish to express o u r a p p r e c i a t i o n to Dr. F. F. J6bsis for his advice a n d e n c o u r a g e m e n t t h r o u g h o u t these investigations. This w o r k was s u p p o r t e d b y PHS G r a n t s NS-10385, NS-06233 a n d MH-12333.
1 CHAPMAN, J. B., Fluorometric studies of oxidative metabolism in isolated papillary muscle of the rabbit, J. gen. Physiol., 59 (1972) 135-154. 2 GRAFSTEIN,B., Mechanism of spreading cortical depression, J. Neurophysiol., 19 (1956) 154-171. 3 J/3BSlS,F. F., O'CONNOR, M., VITALE, A., AND VREMAN,H., lntracellular redox changes in functioning cerebral cortex. I. Metabolic effects of epileptiform activity, J. Neurophysiol., 34 (1971) 735-749. 4 J(~BSIS,F. F., ROSENTHAL,M., LAMANNA,J. C., LOTHMAN,E., CORDINGLEY,G., AND SOMJEN, G., Metabolic activity in epileptic seizures. In D. INGVARAND N. LASSEN (Eds.), Benzon S),mposium on the Working Brahl, Munksgaard, Copenhagen, in press. 5 LOTHMAN,E., LAMANNA,J. C., CORDINGLEY,G., ROSENTHAL,M., AND SOMJEN,G., Responses of electrical potential, potassium levels, and oxidative metabolic activity of the cerebral neocortex of cats, Brain Research, 88 (1975) 15-36. 6 MAYEVSKY,A., AND CHANCE,B., Repetitive patterns of metabolic changes during cortical spreading depression of the awake rat, Brain Research, 65 (1974) 529-533. 7 MAYEVSKY,A., ZEUTHEN, T., AND CHANCE, B., Measurements of extracellular potassium, ECoG and pyridine nucleotide levels during cortical spreading depression in rats, Brain Research, 76 (1974) 347-349. 8 ROSENTHAL,M., AND J~)BSIS,F. F., Intracellular redox changes in functioning cerebral cortex. 11. Effects of direct cortical stimulation, J. Neurophysiol., 34 (1971) 750-761. 9 ROSENTHAL, M., AND LAMANNA, J. C., Effect of ouabain and phenobarbital on the kinetics of cortical metabolic transients associated with evoked potentials, J. Neurochem., 24 (1975) 111-116. 10 ROSENTHAL,M., LAMANNA,J. C., AND J/3BSIS, F. F., Resolution of the metabolic response to spreading depression, Fed. Proe., 33 (1974) 399. 11 ROSENTHAL,M., AND SOMJEN,G., Spreading depression, sustained potential shifts and metabolic activity of cerebral cortex of cats, J. Neurophysioi., 36 (1973) 739-749. 12 VENTURIN1, G., AND PALLADINI, G., ATPase activity, sodium and potassium content in guinea pig cortex after ouabain treatment in vivo, J. Neurochem., 20 (1973) 237-239.
145
~ Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
Effect of ouabain and phenobarbital on oxidative metabolic activity associated with spreading cortical depression in cats
JOSEPH C. LAMANNA AND M Y R O N R O S E N T H A L
Department of Physiology and Pharmacology, Duke University Medical School, Durham, N.C. 27710
(u.s.A.) (Accepted January 14th, 1975)
Spreading cortical depression (SD) is accompanied by large changes in the oxidative metabolic activity of the cerebral cortex of cats 11 and rats 6 recorded fluorometrically. SD is characterized by depression of the E C o G activity together with a negative shift of the cortical steady potential (SP) and an increase in extracellular potassium activity4-L Prior to this SP shift, no changes in tissue fluorescence occur, but during the period of depression, this signal declines and later begins to return to baseline after SP and E C o G approximate full recovery. The fluorometric changes were interpreted as due to an oxidation of intramitochondrial nicotinamide adenine dinucleotide ( N A D H ) to N A D + followed by a reduction of the N A D + to original N A D H levels, a sequence of events associated with transiently increased tissue activity1, 3,s. This occurs because reduced N A D H fluoresces while the oxidized form, N A D ~', does not. In this study, we examine the effects of small intracortical injections of ouabain and of systemically administered phenobarbital on the fluorescence changes that occur due to the production of spreading depression. Recently we reported the effects of these two drugs administered in the same way on the fluorescence changes that occur on the cortical surface when potentials are evoked 9. In that study, up to 600 pmoles of 0.1 m M ouabain slowed the rate of fluorescence decrease ( N A D H oxidation or ' o n ' kinetics) caused by direct cortically stimulated evoked potentials but had no effect on the rate of recovery of fluorescence to baseline levels ('off' kinetics). Higher ouabain doses abolished both electrical and metabolic events. Phenobarbital slowed the 'off' or recovery rate but had no effect on the initial fluorescence decrease. Similar to the results caused by evoked potentials, we report here that ouabain changes the rate of the larger decrease in fluorescence caused by SD and has no effect on the rate of recovery of fluorescence. In contrast, phenobarbital slows the return of fluorescence with no effect on the initial decrease of the signal. These results, combined with those obtained previously, support a hypothesis that the rate of oxidation of N A D H is controlled by the rising A D P concentration due to the breakdown of ATP by the ouabain-sensitive N a - K ATPase membrane transport system while the recovery
146 rate of N A D H from N A D is a function of the respiratory chain itself. A preliminar> report of these investigations has been presented 1°. The trachea and a l'emoral vein were cannulated in cats that were anesthetized with ether. The cisterna was drained of CSF, then a portion of tile calvarium w~ts excised, the dura removed and the brain stem was transected with an electrolytic lesion at the A-P 0.0 coordinates between the colliculi, as in the classical cerveau isold preparation. Ether was discontinued and the cats were allowed to rest for at least 3 h. The cats were heated with feedback thermal controls, expiratory CO2 levels were monitored and the cortex was kept moist with saline or artificial CSF. Up to t4 ,ul of ouabain was injected through a 30-gauge stainless steel needle with its delivery tip implanted 0.5 mm under the cortical surface within the suprasylvian gyrus. The solution was forced into the center of the optical field formed by the epi-illumination optics of the microscope system used for light delivery and monitoring 9. Field diameters of 3.2 mm and 1 mm were used. Microliter quantities of ouabain solutions (0. I raM) in distilled water or saline solution (0.9 ~,,; NaCI) could be administered during the period of optical and electrical monitoring. Effects of volume changes on electrical potentials or fluorescence changes were not observed with up to 10 #1 of water or saline. Phenobarbital was injected i.v. The fluorometric monitoring technique has been described previously 3,,q. The fluorescence of N A D H at approximately 450 nm is recorded with reflected excitation light at 366 rim. Reflectance is then subtracted from fluorescence to produce the 'metabolic signal' (F-R). This signal is corrected for changes due to blood volume, light scattering, volumetric or other geometry changes which appear in both the F and R channels. Spreading depression was produced by suprathreshold intensities of direct cortical stimulation, usually 1 sec trains of 20 Hz pulses of 0.5 msec duration. Stimulation intensities were kept constant through an experiment. Fig. 1 shows that increasing ouabain doses slow the decrease in the metabolic signal during SD. The drug, however, has no effect on the subsequent rate of recovery of this signal to baseline levels. In fact, ouabain has three apparent effects on the optical signal. Most obvious is the retardation of the 'on' kinetics. The drug also decreases the signal amplitude and finally, it was consistently observed that after ouabain administration the decrease of fluorescence becomes more complex and a plateau effect is apparent. This effect is more obvious in a 1 mm diameter field although the retardation of 'on' kinetics is also seen in a 3.2 mm field. Phenobarbital has no effect on 'on' kinetics. However, the barbiturate decreases the amplitude of the metabolic signal and greatly prolongs the time of the return to baseline levels (Fig. 2). This prolongation of the decreased fluorescence is due to the fact that recovery after I-2 rain is reduced progressively with increasing phenobarbital doses, i.e., the lower data set indicates a 500 }; increase in the time required for half of the fluorescence to return to baseline after a dose of 80 mg/kg of phenobarbital. Phenobarbital at 20 mg/kg shows a smaller but still apparent inhibitory effect on "off' kinetics. Similar results were obtained with amobarbital. The effects of both ouabain and phenobarbital were consistent and irreversible
147
B
C
9
2 0 sec Fig. 1. Ouabain effects on metabolism in spreading depression. Ouabain was microinjected 0.5 mm below the surface at the center of the 1 mm diameter optical field. A is the control metabolic signal (F-R) caused by SD; B is response after 4 ffl of 0.1 mM ouabain; C is response after 8 ffl; and D is response after 12 ffl. In this and subsequent trace, a decrease in the optical signal (F-R) denotes an oxidation of NADH. This decrease is expressed as a per cent with the 'resting' F-R level set at 100 relative to a dark field.
u p o n r e p e a t e d S D episodes in the same area over the e x p e r i m e n t a l p e r i o d which was up to a p p r o x i m a t e l y 4 h. The o u a b a i n a n d p h e n o b a r b i t a l i n h i b i t i o n s o f the m e t a b o l i c response to S D are similar to those o b s e r v e d when e v o k e d p o t e n t i a l s were m e a s u r e d 9. These results, however, are different f r o m those o b s e r v e d in studies using e v o k e d p o t e n t i a l s in 3 respects : (1) S D responses were present with o u a b a i n doses larger t h a n those which consistently e l i m i n a t e d e v o k e d p o t e n t i a l s ( e v o k e d p o t e n t i a l s were virtually e l i m i n a t e d by 4 #1 o f o u a b a i n a n d t o t a l l y e l i m i n a t e d at 6 ffl); (2) at low o u a b a i n doses, e v o k e d p o t e n t i a l o p t i c a l responses were a t t e n u a t e d in a 3 m m d i a m e t e r field with no kinetic changes. In a 1 m m field, these same doses a t t e n u a t e d signal a n d i n h i b i t e d ' o n ' kinetics. In contrast, the effect o f o u a b a i n on S D kinetics was seen in b o t h 1 m m a n d 3 m m fields; (3) o u a b a i n p r o d u c e d p l a t e a u s o f the ' o n ' kinetics o f the S D response. Since e v o k e d p o t e n t i a l s are e l i m i n a t e d at doses o f o u a b a i n which do not prevent SD, it is a p p a r e n t t h a t the ability to p r o d u c e e v o k e d p o t e n t i a l s is not necessary to
148
A
?
irnir
B
cL Fig. 2. Phenobarbital effects on spreading depression. A is the control (F-R) response; B is the response after 20 mg/kg phenobarbital administered i.v., and C is the response after 80 rng/kg phenobarbital.
produce SD by electrical stimulation. Ouabain causes cell swelling in CNS tissue lz, and this may decrease the integrity of individual CNS cells or the integrity of neuronal interconnections. It appears that a higher degree o f integrity of the nervous system is required for evoked potentials than for SD. The disruption of intercellular communication would be expected to inhibit evoked potentials more than SD since the latter is presumed to be communicated by elevated extracellular potassium 2 and may not depend upon connections between neurons or between glial elements. Furthermore, if the spread of an evoked potential involves interneuronal contacts and these are inhibited by ouabain, then less cells at the periphery would be involved. It might be that the cells at the periphery of a 3 m m field are relatively unaffected by ouabain and respond normally to electrical stimuli although fewer peripheral cells receive stimulation. This might explain the fact that in the presence of ouabain, evoked potential associated optical responses are attenuated but have normal kinetics when recorded in a 3 m m field. In a smaller field, however, the number of cells
149 r e s p o n d i n g to t r a n s s y n a p t i c influences m a y also be lessened b u t o u a b a i n affected ceils m a y also be excited by the electrical pulses directly or by increased extracellular potassium. In SD, passive p o t a s s i u m diffusion could m e a n t h a t m o r e o u a b a i n affected cells are p a r t i c i p a t i n g in the reactions. The p a r t i c i p a t i o n o f these cells m a y result in the effects o f o u a b a i n on signal a m p l i t u d e a n d on kinetics in a 3 m m field as well as in 1 mm fields. A n alternate possibility, however, is t h a t the m u c h larger extracellular p o t a s s i u m levels in SD4,5, 7 stimulate t r a n s p o r t a n d recovery systems o t h e r t h a n those a c t i v a t e d by e v o k e d potentials. Finally, the p l a t e a u effect o f o u a b a i n indicates the p o s s i b i l i t y that in S D multiple t r a n s p o r t systems might be involved, each with a variable t i m e course a n d sensitivity to o u a b a i n inhibition. Analysis a n d c o r r e l a t i o n o f the time courses o f the steady p o t e n t i a l shifts a n d the rise a n d fall o f extracellular p o t a s s i u m activity with simultaneous fluorescence m o n i t o r i n g m a y resolve this question. W e wish to express o u r a p p r e c i a t i o n to Dr. F. F. J6bsis for his advice a n d e n c o u r a g e m e n t t h r o u g h o u t these investigations. This w o r k was s u p p o r t e d b y PHS G r a n t s NS-10385, NS-06233 a n d MH-12333.
1 CHAPMAN, J. B., Fluorometric studies of oxidative metabolism in isolated papillary muscle of the rabbit, J. gen. Physiol., 59 (1972) 135-154. 2 GRAFSTEIN,B., Mechanism of spreading cortical depression, J. Neurophysiol., 19 (1956) 154-171. 3 J/3BSlS,F. F., O'CONNOR, M., VITALE, A., AND VREMAN,H., lntracellular redox changes in functioning cerebral cortex. I. Metabolic effects of epileptiform activity, J. Neurophysiol., 34 (1971) 735-749. 4 J(~BSIS,F. F., ROSENTHAL,M., LAMANNA,J. C., LOTHMAN,E., CORDINGLEY,G., AND SOMJEN, G., Metabolic activity in epileptic seizures. In D. INGVARAND N. LASSEN (Eds.), Benzon S),mposium on the Working Brahl, Munksgaard, Copenhagen, in press. 5 LOTHMAN,E., LAMANNA,J. C., CORDINGLEY,G., ROSENTHAL,M., AND SOMJEN,G., Responses of electrical potential, potassium levels, and oxidative metabolic activity of the cerebral neocortex of cats, Brain Research, 88 (1975) 15-36. 6 MAYEVSKY,A., AND CHANCE,B., Repetitive patterns of metabolic changes during cortical spreading depression of the awake rat, Brain Research, 65 (1974) 529-533. 7 MAYEVSKY,A., ZEUTHEN, T., AND CHANCE, B., Measurements of extracellular potassium, ECoG and pyridine nucleotide levels during cortical spreading depression in rats, Brain Research, 76 (1974) 347-349. 8 ROSENTHAL,M., AND J~)BSIS,F. F., Intracellular redox changes in functioning cerebral cortex. 11. Effects of direct cortical stimulation, J. Neurophysiol., 34 (1971) 750-761. 9 ROSENTHAL, M., AND LAMANNA, J. C., Effect of ouabain and phenobarbital on the kinetics of cortical metabolic transients associated with evoked potentials, J. Neurochem., 24 (1975) 111-116. 10 ROSENTHAL,M., LAMANNA,J. C., AND J/3BSIS, F. F., Resolution of the metabolic response to spreading depression, Fed. Proe., 33 (1974) 399. 11 ROSENTHAL,M., AND SOMJEN,G., Spreading depression, sustained potential shifts and metabolic activity of cerebral cortex of cats, J. Neurophysioi., 36 (1973) 739-749. 12 VENTURIN1, G., AND PALLADINI, G., ATPase activity, sodium and potassium content in guinea pig cortex after ouabain treatment in vivo, J. Neurochem., 20 (1973) 237-239.
