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Functional and metabolic correlates of long series of cortical spreading depression waves in rats
Brain Research, 404 (1987) 379 38 l Elsevier
379
BRE 22066
Functional and metabolic correlates of long series of cortical spreading depression waves in rats Natalia A. Gorelova 1, Jif/Kfivfinek
2 and Jan Bureg 2
lblstitule qf Higher Nervous Activi(v and Neurot)hysiology, Academy of Sciences, Moscow (U.S.S.R.) and 2Institute qf Physiologv. Czechoslovak Academy of Sciences. Prague (Czechoslovakia) (Accepted 21 October 1986)
Key words'." Spreading depression (SD); Slow potential change; Cyclic adenosine monophosphate (cAMP): Cerebral cortex: Rat
Repetitive generation of spreading depression (SD) waves may induce metabolic and functional disturbances in the invaded brain regions. In order to better characterize this state, up to 60 SD waves were elicited in the cerebral cortex of anesthetized rats by intracortical injections of 2 tll of isotonic K-acetate repeated at regular 5-rain intervals. Propagation rate of SD between capillary electrodes located 1 mm and 7 mm from the injection site gradually decreased and after 35-45 waves became irregular. In another series of experiments SD-induced changes of cyclic adenosine monophosphate (cAMP) level were examined after 24 SD waves. Although no irregularities of SD propagation were observed, maximum level of cAMP in the SD-invaded cortex decreased from 270% in controls to 170% after 24 waves. Also, the recovery of the nucleotide was slowed down. Metabolic alterations thus preceded irregularities in SD propagation. Leao's spreading depression (SD) continues to at-
M) applied intracortically with an automatic microin-
tract attention of neurobiologists (see recent reviews 2't°) as a model of deep disturbance of the homeostasis of brain microenvironment. Some biochemical as well as behavioral studies require longlasting generation of SD waves, usually achieved by repeated application of concentrated KC1 solutions onto the exposed cortical surface 3'5. A major disadvantage of this technique is that SD generation and
jector. The slow potential change (SPC) was monitored with two saline-filled capillaries (5 u m tip) connected to calomel cell electrodes. The micropipettes were placed 1 mm and 7 mm from the injection site and inserted 1 m m deep into cortex. Additional pentobarbital was applied when necessary and colonic temperature was maintained between 35 and 33 °C with a heating pad. The following parameters of the
propagation soon become irregular. U n d e r these conditions estimation of various parameters of the SD waves and of their dynamic changes is rather difficult. A n o t h e r complicating factor is serious cell damage in the areas treated with the highly hypertonic KCl solutions ~t. To avoid these drawbacks, SD waves were elicited by microinjections of isotonic potassium acetate which elicits SD more reliably than KCI 4.
individual SD waves were monitored: m a x i m u m amplitude, duration (defined as the time interval between the ascending and descending limbs of SPC at 50% of its maximum amplitude), velocity of spreading (defined as the time elapsed between appearance of the wave under near and far electrodes, respectively). For determination of c A M P level during various phases of SD development, the animals were killed by immersion into liquid nitrogen one rain after onset of the SD wave at the near or at the far electrode. Thus in the first case, cAMP level was determined at return of SPC to zero at the near electrode and after 3.5 rain recovery at the far electrode. In the second case the brain was frozen during SPC return
Male albino rats (Wistar strain) anesthetized with pentobarbital were used throughout. Two trephine openings 4 m m in diameter were made over both hemispheres. Single SD waves were evoked at regular 5-min intervals by potassium acetate (2/A, 0.15
Correspondence: Jiff Kfiwinek, Institute of Physiology, Czechoslovak Academy of Sciences, Vfdefisk~i 1083, CS - 142 20, Prague 4Kr~, Czechoslovakia. 0006-8993/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
380
0-2
~
~
17-20
_ I-5
27- 31
% _: ~___,
i
;
i
i
i
I
i r>'
____/L
\
120mv
4 0 - 48
l.
5rain Fig. 1. Changes of SPC characteristics of long series of SD waves. Recordings are identified by ordinal numbers of the SD waves. Arrows indicate injection of2td of 0.15 M K-acetate. 1, the near electrode located 1 mm from the potassium acetate (K-Ac) injection: 2, the far electrode, 6 mm from the near one; 0-1,0-2, recordings of the slow potential change (SPC) at the electrode I and 2, respectiveIv,
to zero at the far electrode
andafter 1.5 min recovery
at the near electrode. In this way cAMP was measured immediately after return of SPC to zero (i.e. during maximal increase of cAMP 7) as well as 1.5 rain or 3.5 min later. Tissue preparation and c A M P have been already described 7. Briefly, about 1 into ~ samples dissected at - 2 0 °C from the cortex exposed by trephine openings were transferred to precooled 1 0 ~ (w/v) trichloracetic acid (TCA) and kept frozen until homogenized in a cold room. Cyclic AMP was determined in the T C A extracts following removal of T C A by diethylether. The protein binding method was used ~. Statistical evaluation of the results was performed with the Student's t-test.
assay
In the first series of experiments individual SD waves were elicited for 5 h (60 waves). Fig. 1 shows that the first 5 waves have the usual characteristics. After 90 rain (17-20 waves), duration of SD waves
increased, spreading rate decreased and amplitude remained unchanged. After another hour (27-30 waves), the above effect was still more expressed and some waves appearing at the near electrode failed to reach the far electrode. With continued K-acetate application (40-48 waves), the percentage of abortive SD waves progressively increased. SD is accompanied by remarkable changes of various chemical constituents of brain tissue including glucose, lactic acid, glycogen, cAMP and others ~' s. It is conceivable that a sort of metabolic 'exhaustion' precedes the failure of regular SD propagation. In this case, some quantitative alteration of the biochemical response(s) should precede the appearance of the anomalous SD waves. Changes of cAMP level seemed particularly well suited for this purpose because SD effects on cAMP metabolism had been studied in detail ?'s.
381 - 30
-20 >E -10
~10 300 ~" 250"
t ~
200"
100
•
-1
0
1
2
3
4rnin
Fig. 2. Cyclic AMP levels accompanying passage of the second (control; closed circles) and 24th SPC waves (open circles). Results are expressed in percentage of controls (values from the contralateral, intact hemicortices). Zero of the time axis denotes return of SPC to the zero value (upper part of the figure). Individual SD waves were elicited during 2 h (24 waves). While SD p r o p a g a t i o n was still regular and SPC amplitude r e m a i n e d unchanged under these conditions, duration of the SPC waves was p r o l o n g e d by 58% and 26% at the near and far electrodes, re1 Brown, B.L., Albano, J.D., Ekins, R.P. and Scherzi, A.M., A simple and sensitive saturation assay method for the measurement of adenosine Y,5'-cyclic monophosphate, Biochem. J., 121 (1971) 561-562. 2 Bureg, J., Buregov& O. and K~iv~inek,J., The Mechanism and Applications of Leao's Spreading Depression of Electroencephalographic Activity, Academia, Prague, 1974. 3 Buregov£ O. and Bureg, J., The effect of prolonged cortical spreading depression on learning and memory in rats, J. Neurobiol., 1 (1969) 135-146. 4 Gorelova, N.A., Koroleva, V.I., Amemori, T., Pavh'k, V. and Bureg, J., Ketamine blockade of cortical spreading depression in rats, EEG Clin. Neurophysiol., in press. 5 Kfivfinek, J., Changes in protein, electrolyte and water metabolism caused by prolonged spreading cortical depression in rats, J. Neurobiol., 1 (1969) 147-154.
spectively, and velocity of spreading was r e d u c e d by 27%. The response of c A M P is depicted in Fig. 2. A t the time of return of the 24th SPC to zero, the c A M P level was only 170% of that in the control, contralateral hemicortex (21.8 + 0.9 p m o l . m g prot. q vs 12.8 + 0.4 p m o l . m g prot.-a). In the control animals (2 SD waves), c A M P level reached 270% of the normal value (34.3 _+ 0.9 vs 12.7 _+ 0.5). The rate of metabolic change (pmol r e m o v e d p e r rain) was lower in the cortex through which a long series of waves had passed. F o r 1.5 min recovery this rate is 3.1 pmol-min -a and 8.0 pmol-min -a in e x p e r i m e n t a l and control (2 waves) hemicortex, respectively. The corresponding values for 3.5 min recovery are 1.6 and 2.5 pmol-min -1 in control and experimental hemicortex, respectively. It appears that the onset of irregular SD propagation is h e r a l d e d by changes in some biochemical events, an example of which is d e m o n s t r a t e d by the present experiments. The exact nature of the c A M P changes in the course of SD is known neither for individual nor for r e p e a t e d SD waves. The discussion of the mechanism involved is b e y o n d the scope of this report. A b s o l u t e SD refractory p e r i o d terminates at a time when the recovery of many chemical constituents of the SD-invaded brain tissue is still incomplete 2'6'8. With repeated SD waves this ' m e t a b o l i c deficit' may increase and eventually lead to failure of the autoregenerative mechanisms of SD propagation. This effect is first seen at the far electrode, where the intensity of SD-eliciting stimulus (endogenously increased concentration of extracellular K + and of depolarizing neurotransmitters) is obviously w e a k e r then in the region directly affected by K-acetate application.
6 Kf'ivfinek, J., Some metabolic changes accompanying Leao's spreading cortical depression in the rat, J. Neurochem., 6 (1961) 183-189. 7 K~ivfinek, J., Brain cyclic adenosine Y,5'-monophosphate during depolarization of the cerebral cortical cells in vivo, Brain Research, 120 (1977) 493-505. 8 K~ivfinek, J., Ion Shifts and Metabolism of the Brain, (in Czech), Academia, Prague, 1983. 9 Leao, A.A.P., Spreading depression of activity in the cerebral cortex, J. Neurophysiol., 7 (1944) 359-390. 10 Martins Ferreira, H., (Ed.), Current Views on Leao's Spreading Depression, Am. Acad. Bras. Cienc., 56 (1984) no. 4. 11 Rug~kovfi, D., Effect of potassium ions on the morphological picture of cells of the cerebral cortex, Physiol. Bohemoslov., 13 (1964) 161-166.
379
BRE 22066
Functional and metabolic correlates of long series of cortical spreading depression waves in rats Natalia A. Gorelova 1, Jif/Kfivfinek
2 and Jan Bureg 2
lblstitule qf Higher Nervous Activi(v and Neurot)hysiology, Academy of Sciences, Moscow (U.S.S.R.) and 2Institute qf Physiologv. Czechoslovak Academy of Sciences. Prague (Czechoslovakia) (Accepted 21 October 1986)
Key words'." Spreading depression (SD); Slow potential change; Cyclic adenosine monophosphate (cAMP): Cerebral cortex: Rat
Repetitive generation of spreading depression (SD) waves may induce metabolic and functional disturbances in the invaded brain regions. In order to better characterize this state, up to 60 SD waves were elicited in the cerebral cortex of anesthetized rats by intracortical injections of 2 tll of isotonic K-acetate repeated at regular 5-rain intervals. Propagation rate of SD between capillary electrodes located 1 mm and 7 mm from the injection site gradually decreased and after 35-45 waves became irregular. In another series of experiments SD-induced changes of cyclic adenosine monophosphate (cAMP) level were examined after 24 SD waves. Although no irregularities of SD propagation were observed, maximum level of cAMP in the SD-invaded cortex decreased from 270% in controls to 170% after 24 waves. Also, the recovery of the nucleotide was slowed down. Metabolic alterations thus preceded irregularities in SD propagation. Leao's spreading depression (SD) continues to at-
M) applied intracortically with an automatic microin-
tract attention of neurobiologists (see recent reviews 2't°) as a model of deep disturbance of the homeostasis of brain microenvironment. Some biochemical as well as behavioral studies require longlasting generation of SD waves, usually achieved by repeated application of concentrated KC1 solutions onto the exposed cortical surface 3'5. A major disadvantage of this technique is that SD generation and
jector. The slow potential change (SPC) was monitored with two saline-filled capillaries (5 u m tip) connected to calomel cell electrodes. The micropipettes were placed 1 mm and 7 mm from the injection site and inserted 1 m m deep into cortex. Additional pentobarbital was applied when necessary and colonic temperature was maintained between 35 and 33 °C with a heating pad. The following parameters of the
propagation soon become irregular. U n d e r these conditions estimation of various parameters of the SD waves and of their dynamic changes is rather difficult. A n o t h e r complicating factor is serious cell damage in the areas treated with the highly hypertonic KCl solutions ~t. To avoid these drawbacks, SD waves were elicited by microinjections of isotonic potassium acetate which elicits SD more reliably than KCI 4.
individual SD waves were monitored: m a x i m u m amplitude, duration (defined as the time interval between the ascending and descending limbs of SPC at 50% of its maximum amplitude), velocity of spreading (defined as the time elapsed between appearance of the wave under near and far electrodes, respectively). For determination of c A M P level during various phases of SD development, the animals were killed by immersion into liquid nitrogen one rain after onset of the SD wave at the near or at the far electrode. Thus in the first case, cAMP level was determined at return of SPC to zero at the near electrode and after 3.5 rain recovery at the far electrode. In the second case the brain was frozen during SPC return
Male albino rats (Wistar strain) anesthetized with pentobarbital were used throughout. Two trephine openings 4 m m in diameter were made over both hemispheres. Single SD waves were evoked at regular 5-min intervals by potassium acetate (2/A, 0.15
Correspondence: Jiff Kfiwinek, Institute of Physiology, Czechoslovak Academy of Sciences, Vfdefisk~i 1083, CS - 142 20, Prague 4Kr~, Czechoslovakia. 0006-8993/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
380
0-2
~
~
17-20
_ I-5
27- 31
% _: ~___,
i
;
i
i
i
I
i r>'
____/L
\
120mv
4 0 - 48
l.
5rain Fig. 1. Changes of SPC characteristics of long series of SD waves. Recordings are identified by ordinal numbers of the SD waves. Arrows indicate injection of2td of 0.15 M K-acetate. 1, the near electrode located 1 mm from the potassium acetate (K-Ac) injection: 2, the far electrode, 6 mm from the near one; 0-1,0-2, recordings of the slow potential change (SPC) at the electrode I and 2, respectiveIv,
to zero at the far electrode
andafter 1.5 min recovery
at the near electrode. In this way cAMP was measured immediately after return of SPC to zero (i.e. during maximal increase of cAMP 7) as well as 1.5 rain or 3.5 min later. Tissue preparation and c A M P have been already described 7. Briefly, about 1 into ~ samples dissected at - 2 0 °C from the cortex exposed by trephine openings were transferred to precooled 1 0 ~ (w/v) trichloracetic acid (TCA) and kept frozen until homogenized in a cold room. Cyclic AMP was determined in the T C A extracts following removal of T C A by diethylether. The protein binding method was used ~. Statistical evaluation of the results was performed with the Student's t-test.
assay
In the first series of experiments individual SD waves were elicited for 5 h (60 waves). Fig. 1 shows that the first 5 waves have the usual characteristics. After 90 rain (17-20 waves), duration of SD waves
increased, spreading rate decreased and amplitude remained unchanged. After another hour (27-30 waves), the above effect was still more expressed and some waves appearing at the near electrode failed to reach the far electrode. With continued K-acetate application (40-48 waves), the percentage of abortive SD waves progressively increased. SD is accompanied by remarkable changes of various chemical constituents of brain tissue including glucose, lactic acid, glycogen, cAMP and others ~' s. It is conceivable that a sort of metabolic 'exhaustion' precedes the failure of regular SD propagation. In this case, some quantitative alteration of the biochemical response(s) should precede the appearance of the anomalous SD waves. Changes of cAMP level seemed particularly well suited for this purpose because SD effects on cAMP metabolism had been studied in detail ?'s.
381 - 30
-20 >E -10
~10 300 ~" 250"
t ~
200"
100
•
-1
0
1
2
3
4rnin
Fig. 2. Cyclic AMP levels accompanying passage of the second (control; closed circles) and 24th SPC waves (open circles). Results are expressed in percentage of controls (values from the contralateral, intact hemicortices). Zero of the time axis denotes return of SPC to the zero value (upper part of the figure). Individual SD waves were elicited during 2 h (24 waves). While SD p r o p a g a t i o n was still regular and SPC amplitude r e m a i n e d unchanged under these conditions, duration of the SPC waves was p r o l o n g e d by 58% and 26% at the near and far electrodes, re1 Brown, B.L., Albano, J.D., Ekins, R.P. and Scherzi, A.M., A simple and sensitive saturation assay method for the measurement of adenosine Y,5'-cyclic monophosphate, Biochem. J., 121 (1971) 561-562. 2 Bureg, J., Buregov& O. and K~iv~inek,J., The Mechanism and Applications of Leao's Spreading Depression of Electroencephalographic Activity, Academia, Prague, 1974. 3 Buregov£ O. and Bureg, J., The effect of prolonged cortical spreading depression on learning and memory in rats, J. Neurobiol., 1 (1969) 135-146. 4 Gorelova, N.A., Koroleva, V.I., Amemori, T., Pavh'k, V. and Bureg, J., Ketamine blockade of cortical spreading depression in rats, EEG Clin. Neurophysiol., in press. 5 Kfivfinek, J., Changes in protein, electrolyte and water metabolism caused by prolonged spreading cortical depression in rats, J. Neurobiol., 1 (1969) 147-154.
spectively, and velocity of spreading was r e d u c e d by 27%. The response of c A M P is depicted in Fig. 2. A t the time of return of the 24th SPC to zero, the c A M P level was only 170% of that in the control, contralateral hemicortex (21.8 + 0.9 p m o l . m g prot. q vs 12.8 + 0.4 p m o l . m g prot.-a). In the control animals (2 SD waves), c A M P level reached 270% of the normal value (34.3 _+ 0.9 vs 12.7 _+ 0.5). The rate of metabolic change (pmol r e m o v e d p e r rain) was lower in the cortex through which a long series of waves had passed. F o r 1.5 min recovery this rate is 3.1 pmol-min -a and 8.0 pmol-min -a in e x p e r i m e n t a l and control (2 waves) hemicortex, respectively. The corresponding values for 3.5 min recovery are 1.6 and 2.5 pmol-min -1 in control and experimental hemicortex, respectively. It appears that the onset of irregular SD propagation is h e r a l d e d by changes in some biochemical events, an example of which is d e m o n s t r a t e d by the present experiments. The exact nature of the c A M P changes in the course of SD is known neither for individual nor for r e p e a t e d SD waves. The discussion of the mechanism involved is b e y o n d the scope of this report. A b s o l u t e SD refractory p e r i o d terminates at a time when the recovery of many chemical constituents of the SD-invaded brain tissue is still incomplete 2'6'8. With repeated SD waves this ' m e t a b o l i c deficit' may increase and eventually lead to failure of the autoregenerative mechanisms of SD propagation. This effect is first seen at the far electrode, where the intensity of SD-eliciting stimulus (endogenously increased concentration of extracellular K + and of depolarizing neurotransmitters) is obviously w e a k e r then in the region directly affected by K-acetate application.
6 Kf'ivfinek, J., Some metabolic changes accompanying Leao's spreading cortical depression in the rat, J. Neurochem., 6 (1961) 183-189. 7 K~ivfinek, J., Brain cyclic adenosine Y,5'-monophosphate during depolarization of the cerebral cortical cells in vivo, Brain Research, 120 (1977) 493-505. 8 K~ivfinek, J., Ion Shifts and Metabolism of the Brain, (in Czech), Academia, Prague, 1983. 9 Leao, A.A.P., Spreading depression of activity in the cerebral cortex, J. Neurophysiol., 7 (1944) 359-390. 10 Martins Ferreira, H., (Ed.), Current Views on Leao's Spreading Depression, Am. Acad. Bras. Cienc., 56 (1984) no. 4. 11 Rug~kovfi, D., Effect of potassium ions on the morphological picture of cells of the cerebral cortex, Physiol. Bohemoslov., 13 (1964) 161-166.