.V~~~~ropl~or~nucologp 1975. 14, 537-545. Pergamon Press. Prmted in Gt. Bntain
FACILITATION OF LEAO’S SPREADING DEPRESSION BY A PYRROLOPYRIMIDINE DERIVATIVE B. DELUCA*, M. SHIBATA?.G. BRO~EKand J. BURES Institute of Physiology, Czechoslovak Academy of Sciences, Prague (Accepted 14 Novembrr
1974)
Summary-The facilitatory effect of the drug BW 58-271 (2-methyl 4-benzyl-aminopyrrolo/2,3d/pyrimidine) on LeBo’s spreading depression was examined in 67 adult rats. anaesthetized with Allobarbital or Nembutal. Intraperitoneal injection of BW 58-271 (10 mg/kg) increased the cortical spreading depression susceptibility, increased the amplitude of the slow potential change by 60% and the propagation rate by 28%. The drug abruptly increased the percentage of cortical spreading depression waves penetrating into the caudate nucleus from 34% to 100% and reduced the corticocaudate or caudato-cortical conduction time from 5.6 to 3.1 min. All these effects were maximal 20 min after BW 58-271 injection and decayed over the following 2 hr. Measurements with potassium selective microelectrodes showed that BW 58-271 did not change the resting level of extracellular potassium in the cortex but increased the peak extracellular potassium level attained during spreading depression from 65 to 85 mM. Reverberation of cortical spreading depression around a circular lesion (5.2mm in diameter) in the frontal cortex was enhanced by BW 58-271; the number of completed cycles increasing from 12 in control to 53 in treated rats. The changes of the spreading depression properties indicate that the drug increases potassium permeability of the neural membrance without interfering with the mechanism of the active transport.
Analysis of spreading depression often requires the use of procedures which increase the reliability of the phenomenon or accelerate its recovery cycle. Anoxia and hypoglycemia (BURESand BuRESOVA, 1960) reduce the threshold but prolong the refractory period of spreading depression. Systemic administration of metabolic poisons has analogous effects (BuRES, 1957; GOLDRING, O’LEARY and LAM, 1953). A pyrrolopyrimidine drug, BW 58-271 (2-methyl 4-benzyl-aminopyrrolo/2,3-d/pyrimidine) was shown to evoke marked suppression of EEG activity in cats (NORTON and JEWETT, 1966) and in rats (NORTON and JEWETT, 1967). The presence of the characteristic slow potential changes suggested that the drug induces repeated waves of spreading depression. The purpose of the present paper was to examine the effect of BW 58-271 in more detail, to establish the dynamics of its action and to employ it for facilitation of the reverberatory cortical spreading depression and of cortico-caudate spreading depression propagation. METHODS
Male hooded rats (Druckrey strain) aged 2-3 months were used throughout. In animals anaesthetized with Allobarbital (Spofa, 30mg/kg) or with Nembutal (45mg/kg), trephine openings were made according to the scheme shown in Figures 1 and 3. Spreading depression was elicited by microinjection of 10% KC1 into the cerebral tissue or by application of filter papers soaked with 0.6% or 0.8% KC1 onto the dura covered cortex exposed by a 4mm trephine opening. Special care was taken to avoid mechanical stimulation when applying the filter papers. The slow potential changes were led off with wick calomel cell electrodes applied on the exposed cortical surface or by contacting the saline in the capillary electrode inserted into the caudate nucleus. The reference electrode was placed on the frontal bone or on the contralateral hemisphere. The electrodes were connected to high impedance inputs of a 3 channel recording millivoltmeter. Extracellular potassium activity was measured with the liquid membrane potassium selective microelectrodes of WALKER (1971), using the technique described elsewhere * Visiting scientist from Istituto di Fisiolgia Umana, Naples, Italy. t Visiting scientist from Gifu University, Gifu, Japan. \I’ I-ls \
537
B. DEL~JCAet a/
538
(VYSKOCIL,KRiZ and BURES,1972). The potassium electrode and the reference capillary electrode were mounted in such a way that their tips were parallel and not more than 50pm apart. Chlorinated silver wires contacting the fluids in the electrode vessels (0.5 M KC1 and saline, respectively) were connected through a high impedance operational amplifier to the recording millivoltmeter. The system was calibrated with solutions of different KC1 concentrations (3 to 1OOmM)and constant NaCl (150m~) content. The response to a tenfold increase of KC1 concentration from 4rn~ to 40m~ was typically 50 mV. Reverberating cortical spreading depression was elicited by the technique described by SHIBATAand BURES (1974). A circular lesion, 5 mm in diameter and 3 mm deep, was produced by thermocoagulation in the frontal cortex (see Fig. 6) in such a way that it was surrounded on all sides by at least 2mm wide bridges of intact tissue. Recording electrodes (1 to 3) were placed into small trephine openings surrounding the lesion at a distance of 1.5 to 2.0mm. Two other small openings (A and B) served for intracortical microinjection of 10% KCl. Reverberation was started by two appropriately timed injections of 0.5~1 KC1 into the trephine openings A and B. The drug BW 58-271 was dissolved in distilled water with added acetic acid. The lo/, solution of the drug had a pH of 5-O and was injected intraperitoneally (lOmg/kg). In some experiments, additional quantities of the drug (5mg/kg) were injected after 30 min or later. Drug administration was always combined with barbiturate anaesthesia.
RESULTS Cortical
spreading
depression
susceptibility
(Experiment
1)
In 14 rats, cortical spreading depression susceptibility was established by repeated application of filter papers (3mm in diameter) soaked with 0.6 or 0.8% KC1 solution onto the exposed parietal cortical surface covered by dura. The effect of the applied KCl solutions was checked by slow potential change recording from a point in the frontal cortex, 3 mm rostra1 from the treated area. Irrespective of whether or not cortical spreading depression was evoked, the filter paper was removed after 20 min, excess fluid was dried up and a new filter paper soaked with the same KC1 solution was applied. Under control conditions, repeated applications of 0.6% KC1 did not elicit cortical spreading depression (0 in 14 applications). Forty to 100 min after pyrrolopyrimidine injection, 0.6% KC1 elicited cortical spreading depression 9 times in 28 applications, and O*S’AKC1 17 times in 28 applications. Student’s t-test for binomial data shows that pyrrolopyrimidine significantly increased the incidence of cortical spreading depression to 0.6% KC1 (P < 0.05) but not to 0.8% KC1 (P > 0.05, t = 1.65). When results obtained with both concentrations were pooled (5/28 in controls and 26/56 in the experimental animals) the significance increased to P < 0.01. Cortical
spreading
depression
propagation
rate (Experiment
2)
In six rats, three trephine openings were made in the parasagittal plane L 3 according to the scheme in Figure 1. In order to increase the distance available for measurement of cortical spreading depression propagation rate, the triggering stimulus was applied through the small rostra1 opening. The injection of 0.5 ~1 of 10% KC1 applied 1 mm below the surface elicited cortical spreading depression in all cases. The slow potential change was recorded from openings 1 and 2 (distance 7mm). The cortical spreading depression waves were elicited before BW 58-271 application and at 15 min intervals starting 5 to 10 min after injection. Example of a typical experiment is shown in Figure 1 and all results are summarised in Figure 2. Conduction time measured between the negative peaks of the slow potential changes at electrodes 1 and 2 was an average of 2.3 k 0.1 min and was quite stable before BW 58-271 administration. Approximately 20 min after drug injection, spreading depression propagation between the two cortical electrodes was accelerated; the conduction time dropping to 1% + 0.1 min (P < 0.01).
Leao’s spreading depression
539
4.n mm/min .r
3.33mmjmin 3,25mm/min
-
r
SmV I
12Omin
C
Fig. I. The effect of pyrrolopyrimidine on cortical spreading depression. Brain scheme indicates the location of electrodes (1,2) and the injection site (KCl Co.). Slow potential recordings before (C) and 20 or 120 min after injection of BW 58-271 (10 mg/kg). Injection of KCl indicated by arrow. Spreading rates corresponding to the electrode distance and peak conduction time are shown above the record. Calibration: 5 mV, 2 min. Negativity of the active electrode upward. Note increase of slow potential change amplitude and propagation rate and decrease of slow potential change duration 20 min after drug application.
After the same time, amplitude of the slow potential change increased from the control level of 7.5 to 12.2mV. Both effects gradually decayed over the following 2 hours to the predrug value. Twenty minutes after drug injection, duration of the negative wave decreased from I.0 to 0.8 min. but the latter effect was only shortlasting and slow potential changes were prolonged after 1 hour. min 2.51
0 t
20
40
60
80
100
120 min
Fig. 2. Pyrrolopyrimidine induced changes of conduction time (above) slow potential change amplitude (middle) and slow potential change duration at 500/, of negative maximum (below). Abscissa: time after pyrrolopyrimidine injection in min. Vertical bars denote f S.E.M. The curves are based on 45 measurements each (4 to 6 measurements per point).
540
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DELUCA et nl.
Propagation of spreading depression between caudate nucleus and neocortex (Experiment 3)
In agreement with the earlier reports (FIFKOVP;and BURES, 1964; FIFKOVP;and SYKA, 1964), spreading depression propagation from the neocortex to the caudate nucleus and vice versa was irregular. Of six control animals, two displayed at least one spreading depression transition in both directions. In one rat, spreading depression penetrated from the caudate nucleus to neocortex and no transition was observed in the remaining three rats. With repeated testing, transition often failed. Out of 17 cortical spreading depression waves, 5 reached the caudate nucleus and out of 21 caudate spreading depression waves, 8 reached the parietal cortex. The overall incidence of transition in both directions was 34%. Propagation rate was the same in both directions (Fig.3A), the conduction time being 5.7 ) 0.7 and 5.6 t_ 0.6 min for the cortico-caudate and caudatecortical transmission, respectively. Due to the rare incidence of bidirectional propagation under control conditions, pyrrolopyrimidine-induced facilitation of cortico-caudate transmission could be seldom demonstrated in the same animal. In the example shown in Figure 3, the predrug values of the cortico-caudate and caudate-cortical conduction times were 5.8 and 6.3 min and decreased 60 and 90 min after injection to 4.0 and 5.3 min, respectively. A serious disadvantage of such experiments was that pyrrolopyrimidine application followed after prolonged testing (2 to 3 hours). In order to eliminate the potential influence of repeated spreading depression waves on the pyrrolopyrimidine effect, in another group of 7 rats measurements were started only after drug administration. Statistical comparisons between the control and experimental groups were made with Student’s t-test for unpaired data. Ten to 20 min after injection, spreading depression spread regularly between
I
5mV
t KCI
I
4
SmV
Zmm
-I
SmV
Leio’s spreading depression
541
30min
5mpgg[
J--J@
20min
c
Fig. 4. Effect of pyrrolopyrimidine on the transition of spreading depression from caudate to neocortex. Transition failed before drug application (C), was rapid 20 min and slow 100 min after injection. Other description as in Fig. 3.
the two structures (Fig. 4). Propagation in both directions was similarly affected so that it was possible to pool the data obtained with cortico-caudate and caudatecortical spread. In total, 36 spreading depression waves were evoked at various intervals after drug application. Probability of transition increased to 100% and the conduction time decreased to 3.1 + 0.3 min during the first 20 min after injection and was maintained at this level for an hour before it started to return to the control value. The time course of this change was similar to that in the cerebral cortex, but the maximum effect was more pronounced (45% instead of 22% reduction, P < 0.01). On the other hand, changes of amplitude and duration of caudate slow potential changes were not significant. Extracellular
potassium
shifts accompanying
cortical
spreading
depression
(Experiment
4)
In an attempt to analyse the mechanism of facilitation of spreading depression induced by BW 58-271, the resting level of extracellular potassium [Kc’] was measured 1 mm below the cortical surface in 5 rats before and after injection of the drug (lOmg/kg). A typical experiment is illustrated in Figure 5, in which the resting level of 4*0mM (mean 3.0 + 05m~ for the 5 rats) was not raised during 2 hours after BW 58-271 administration. During spreading depression, the extracellular potassium abruptly rose to 70m~ (average for all animals was 64 + 5 mM) and after 30 to 40 set started to return more slowly to or even below the resting level (Fig. 5C). Pyrrolopyrimidine treatment did not significantly change the slope of the potassium shifts, but the maximum concentration was increased to 90m~, as shown in Figure 5P (average 85 + 7 mu, P < 0.05). Since the higher [K,‘] level was attained during the same time as before injection, the rate of [K,‘] increase was slightly faster. The peak [Kc:] was sometimes maintained for several tens of seconds (Fig. 5P). Reabsorption rate was not affected mHK+ loo80-
C
P
60SO403020-
10-
543-
-
J I
I
542
B.
DELUCA et al.
by drug administration. During the steep part of the recovery phase, 50% decrease of [K:] took 4.5 f 0.4 set and 4.2 f 07 set before and after pyrrolopyrimidine application respectively. When a train of slow potential waves was evoked by application of loo/, KC1 on a remote cortical region, the potassium waves generated in rapid succession were of equal amplitude and displayed no decrement, which is commonly seen under similar conditions in untreated animals. The interwave interval decreased from 3.8 + 0.2 min before to 2.5 f 0.3 min after pyrrolopyrimidine injection. Reverberating spreading depression (Experiment
5)
The above findings suggest that BW 58-271 lowers the spreading depression threshold, increases the slow potential change amplitude and accelerates the propagation rate of spreading depression, without interfering with the recovery of the process. Since all these changes may favour reverberation of cortical spreading depression around a cortical lesion (SHIBATAand BURES,1972), the effect of BW 58-271 on reverberating cortical spreading depression was examined in 11 rats. Reverberation was elicited by the double injection method (Fig. 6) described in detail elsewhere (SHIBATAand BURES,1972, 1974). After the first spreading depression wave elicited from the trephine opening A passed under electrode 2, a second spreading depression wave was triggered by KCI injection into the point B. Since the cat&l parts of the hemisphere were occupied by the first spreading depression, the new wave could only spread into the recovered cortical region
Fig. 6. Facilitation of reverberating cortical spreading depression by pyrrolopyrimidine. In the brain scheme, numbers 1 to 3 indicate the position of the electrodes, letters A and B the injection sites and shading the thermocoagulated area. The slow potential change recordings from electrodes I
LeBo’s spreading
depression
543
between the lesion and the medial surface of the hemisphere. The new wave reached electrode 1 and started to propagate around the lesion. Since the first spreading depression had expired in the meantime at the boundaries of the cortex, nothing hindered the spread of the incipient circle wave which reached the caudal entrance to the medial cortical segment, completed the first cycle and continued to circulate around the lesion. In the experiment illustrated by Figure 6, reverberation lasted for 12.7 hours and stopped spontaneously after 107 cycles, when a slowly developing block in the medial segment prevented transition from electrode 2 to electrode 1. Comparison of the average number of completed cycles of rever~rating cortical spreading depression in 19 control animals (Allobarbital anaesthesia only) and in 11 rats treated with BW 58-271, showed an increment from 12.0 ) 2.0 to 52.9 + 9.2 (P < @Ol) cycles. More than 20 and 25 cycles were completed by only 30% and 15% of untreated rats, respectively, but by BOY//of pyrrolopyrimidine injected rats. The effect of BW 58-27 1 was also manifested by the increased amplitude of the cortical slow potential, the average amplitude of the first three reverberating cortical spreading depression waves being almost twice the height in treated than in untreated rats. Whereas in control animals the amplitude smoothly decreased from the first to the last wave, in pyrrolopyrimidine injected animals the amplitude was maintained during the first 10 cycles and started to decline approximately 2 hours after reverberation onset. The reverberation time was unaffected by pyrrolopyrimidine, if the comparison was based on the average duration of the first three cycles. It must be pointed out, however, that comparisons made on an absolute time scale showed faster rever~ration in the pyrrolopyrimidine treated rats; the average cycle duration after one hour of reverberation being 62 + 01 min (n = 35) for the experimental and 65 + 0.1 min (n = 78) for the control animals (P < 0.05). In the last 3 cycles, reverberation was significantly slower in the pyrrolopyrimidine animals, but this was probably due to exhaustion caused by the preceding several hours of continuous spreading depression. DISCUSSION
The present paper confirms earlier reports by NORTONand JEWET~(1966, 1967) that systemic application of BW 58-271 evokes cortical spreading depression, and describes the effects of a subthreshold dosage which, after a short latency, caused marked facilitation of spreading depression. According to BURES,BuRESOVP;and KRIVANEK(1960), KC1 threshold (EDso) is 0.6% in rats anaesthetised with Allobarbital, when the filter paper is applied into a 5mm trephine opening. As the threshold concentration is indirectly proportional to the area of the exposed surface, the predrug effects of the @6 and O*S% KC1 solutions applied into a 4mm trephine opening (Experiment 1) are within the expected limits. The increase of susceptibility of cortical spreading depression caused by lOmg/kg of BW 58-271 is significant but small in comparison with the facilitation due, for instance, to hypoglycemia. As shown by BURESand BURES~VA(1960), one hour after intraperitoneal injection of 0.1 IU insulin, KC1 threshold dropped to 0.22%. Similar effects were reported after 1 min anoxia (BURES and BuRESOVA,1960) and after systemic application of metabolic poisons (BuRES, 1957). Con~asting with the small increase of cortical spreading depression su~ptibility, is the marked increase of propagation rate. According to the mathemati~l model of spreading depression proposed by A. L. Hodgkin (cited according to BuR&, B~RESOVA. and KKIVANEK,1974), conduction rate u can be expressed by the equation u = + Y;,- 2~0 D Y., J 2T where D is the diffusion coefficient of K+ in brain tissue, y;, is the maximum extracellular potassium level reached at the peak of the negative slow potential, y,, is the threshold concentration of extracellular K,? causing depolarization of adjacent elements and T
544
B. DELUCA et al
is the time constant with which the extracellular potassium concentration reaches maximum. When the extracellular potassium level was measured before and after administration of pyrrolopyrimidine, the drug increased y:, approximately by 30% (Experiment 4). The 60% increase of cortical slow potential change amplitude is also compatible with raised y;,, but the two values are not directly proportional, since slow potential change depends not only on the membrane potential but also on the geometry of the dipoles and on specific cortical impedence. After pyrrolopyrimidine, depolarization seemed to reach deeper cortical layers. Higher peak values of [K,‘] indicate a greater reduction of extracellular volume and a corresponding increase of impedance. All these changes account for the observed increment of slow potential change amplitudes. A 20% reduction of y,, can be inferred from Experiment 1. Since the y:, and y0 values established in control experiments (VYSKO&Let al., 1972) are 65 and 11 mM respectively, the pyrrolopyrimidine induced changes of y;, and y0 would account for a 20% increase of conduction rate. Further acceleration of spreading could be due to a faster rise time of the extracellular [K:], suggested by the results of Experiment 4. Decrease of T by lo%, together with the changes of y., and yO, would increase the propagation rate to the value 128% observed in Experiment 2. even when it is assumed that the value of D remains unchanged. It must be pointed out that only few interventions can accelerate spreading depression propagation. Local treatment of the cerebral cortex with 0.2% KC1 (BuRES, 1962), with isotonic sodium acetate or hypotonic NaCl (30 to 40mM) (Ltio, 1963), increased body temperature (BuRES, BURESOVA and ZACHAROVA,1957). Other agents which reduce the cortical spreading depression threshold decrease the spreading rate (for instance anoxia, BURES,1957) and can thus be distinguished from the pyrrolopyrimidine effect. The possibility of facilitating spreading depression penetration into refractory regions was studied by MARSHALL,ESSIG and DUBROFF( 1951), VAN HARREVELDand BOGEN (1956), LGo and MARTINS-FERREIRA (1956, 1961). Local application of subthreshold concentrations of KC1 or of metabolic inhibitors used in these studies would be impossible in the case of cortico-caudate transmission. According to FIFKOVAand BURES(1964) and FIF~ovli and SYKA (1964) the critical regions are the boundaries between neocortex and amygdala and nucleus accumbens, i.e., points which cannot be influenced from the exposed brain surface. Since faster spreading depression propagation would account only for a decrease in the cortico-caudate conduction time to 78% but not to 55% of the control level, the possibility must be taken into account that the cortico-caudate propagation follows a shorter pathway in pyrrolopyrimidine-treated animals the transition proceeding across the thin layer of subcortical white matter separating temporal cortex from the caudate nucleus. Another feature of the pyrrolopyrimidine effect is the decreased spreading depression refractoriness which is manifest by the undiminished K+ shifts accompanying a rapid succession of spreading depression waves, This explains the striking facilitation of reverberating cortical spreading depression. Since the relative refractory period of spreading depression in rat is approximately 15 min (ZACHARand ZACHAROVA,1961), with the average reverberation time of 6 min, the reverberating cortical spreading depression moves continuously over a partly refractory tissue. This circumstance decreases the safety factor of spreading and increases the probability of random interference. Although the relative refractory period after pyrrolopyrimidine application was not exactly estimated, comparison of the rate and amplitude of the slow potential changes and potassium shifts generated by application of equally strong suprathreshold stimuli indicates that the shortest interval compatible with generation of undiminished successive waves decreases from 3.8 min to 2.5 min. Reverberation cqntinues for an average of 12 cycles in control animals, which indicates that the probability of continuing reverberation decreases with each cycle by approximately 0.94. After BW 58-271 application, this coefficient increases to 0.99. Although the exact mechanism of the pyrrolopyrimidine effect remains obscure, reduced KC1 threshold and raised peak concentration of extracellular potassium could
Leao’s spreading depression
545
be due to increased potassium permeability of the nerve cell membrane. At the same time, a faster recovery cycle of spreading depression indicates that the drug does not interfere with the mechanism of active transport. Acknowledgement-The ing the BW 58-271.
authors wish to thank Burroughs-Wellcome and Co., Tuckahoe, New York, for supply-
REFERENCES BURE$ J. (1957). The effect of anoxia and asphyxia on spreading EEG depression. Physiologia bohemoslou. 6: 444-453.
BURES,J. (1962). Spreading EEG Depression, ifs Mechanism and Application. (in Czech). SZN, Prague. BURES,J. and BuRESOVA,0. (1960). Activation of latent foci of spreading cortical depression in rats. J. Neurophysiol. 23: 225236.
BuRES,J., BuRESOVA,0. and KRIV~NEK,J. (1960). Some metabolic aspects of Leao’s spreading cortical depression. In: Structure and Function of the Cerebral Cortex (TOWER,D. B. and SCHAD~,J. P., Eds.), pp. 257-265. Elsevier, Amsterdam. BuRES,J., BuRE~v.&, 0. and KRIVANEK,J. (1974). The Mechanism and Applications of Ledo’s Spreading Depression of Electroencephalographic Activity. Academia, Prague. BuRES, J., BuRESOV.&0. and ZACHARO~P;,D. (1957). E&t of changes in body temperature on spreading EEG demession. Phvsioloaia bohemoslov. 6: 454461. FIFKOVA,E: and BuREQ,~J. (1964). Spreading depression in the mammalian striatum. Archs int. Physiol. Biochim. 12: 171-179. FIFKOV.&,E. and SYKA, J. (1964). Relationships between cortical and striatal spreading depression in rat. Expl Neural. 9: 355366.
GOLDRING,S., O’LEARY,J. L. and LAM, R. (1953). Effect of malonitrile upon the electrocorticogram of the rabbit. Electroenceph. clin. Neurophysiol. 5: 39S400. LEO, A. A. P. (1963). On the spread of spreading depression. In: Brain Function, Vol. 1 (BRAZIER,M. A. B.. Ed.), pp. 78-85. University of California Press, Berkeley. Ldo, A. A. P. and MARTINS-FERREIRA, H. M. (1956). Condicbes necesirias para a ocor&cia da depreslo alastrante. An. Acad. Brasil. Ci. 28: XV. Ltio, A. A. P. and MARTINS-FERREIRA, H. M. (1961). Nota adrca da depress%0 alastrante no cerebelo, tuberculo, quadrigsmino anterior e bulbo olfativo. An. Acad. Emil. Ci. 33: 3950. MARSHALL,W. H., ESSIG, C. F. and DUBROFF,S. J. (1951). Relation of temperature of cerebral cortex to spreading depression of Lelo. J. Neurophysiol. 14: 15>166. NORTON,S. and JEWETT,R. E. (1966). A pyrrolopyrimidine with depressant action on the CNS. J. Pharmac. exp. Ther. lJ4: 152-160. NORTON,S. and JEWETT,R. E. (1967). Effect of prolonged EEG supression by a pyrrolopyrimidine on learned behavior. Physiol. Behau. 2: 317-322. SHIBATA,M. and BURES,J. (1972). Reverberation of cortical spreading depression along closed-loop pathways in rat cerebral cortex. J. Neurophysiol. 35: 381-388. SHIBATA,M. and BURES,J. (1974). Optimum topographical conditions for reverberating cortical spreading depression in rats. J. Neurobiol. 5: 107-l 18. VAN HARREVELD, A. and BOGEN, J. E. (1956). Regional differences in propagation of spreading cortical depression in the rabbit. Proc. Sot. exp. Biol. Med. 91: 2977302. VYSK&IL, F.. KRiZ. N. and BURES,J. (1972). Potassium-selective microelectrodes used for measuring the extracellular brain potassium during spreading depression and anoxic depolarization in rats. Brain Res. 39: 255259. WALKER,J. L. JR. (1971). Ion-specific liquid ion-exchanger microelectrodes. Analyt. Chem. 43: 89A-92A. ZACHAR,J. and ZACHAROV.~, D. (1961). The refractory phase of Leao’s spreading cortical depression. Physiologia bohernoslov. 10: 341-348.
FACILITATION OF LEAO’S SPREADING DEPRESSION BY A PYRROLOPYRIMIDINE DERIVATIVE B. DELUCA*, M. SHIBATA?.G. BRO~EKand J. BURES Institute of Physiology, Czechoslovak Academy of Sciences, Prague (Accepted 14 Novembrr
1974)
Summary-The facilitatory effect of the drug BW 58-271 (2-methyl 4-benzyl-aminopyrrolo/2,3d/pyrimidine) on LeBo’s spreading depression was examined in 67 adult rats. anaesthetized with Allobarbital or Nembutal. Intraperitoneal injection of BW 58-271 (10 mg/kg) increased the cortical spreading depression susceptibility, increased the amplitude of the slow potential change by 60% and the propagation rate by 28%. The drug abruptly increased the percentage of cortical spreading depression waves penetrating into the caudate nucleus from 34% to 100% and reduced the corticocaudate or caudato-cortical conduction time from 5.6 to 3.1 min. All these effects were maximal 20 min after BW 58-271 injection and decayed over the following 2 hr. Measurements with potassium selective microelectrodes showed that BW 58-271 did not change the resting level of extracellular potassium in the cortex but increased the peak extracellular potassium level attained during spreading depression from 65 to 85 mM. Reverberation of cortical spreading depression around a circular lesion (5.2mm in diameter) in the frontal cortex was enhanced by BW 58-271; the number of completed cycles increasing from 12 in control to 53 in treated rats. The changes of the spreading depression properties indicate that the drug increases potassium permeability of the neural membrance without interfering with the mechanism of the active transport.
Analysis of spreading depression often requires the use of procedures which increase the reliability of the phenomenon or accelerate its recovery cycle. Anoxia and hypoglycemia (BURESand BuRESOVA, 1960) reduce the threshold but prolong the refractory period of spreading depression. Systemic administration of metabolic poisons has analogous effects (BuRES, 1957; GOLDRING, O’LEARY and LAM, 1953). A pyrrolopyrimidine drug, BW 58-271 (2-methyl 4-benzyl-aminopyrrolo/2,3-d/pyrimidine) was shown to evoke marked suppression of EEG activity in cats (NORTON and JEWETT, 1966) and in rats (NORTON and JEWETT, 1967). The presence of the characteristic slow potential changes suggested that the drug induces repeated waves of spreading depression. The purpose of the present paper was to examine the effect of BW 58-271 in more detail, to establish the dynamics of its action and to employ it for facilitation of the reverberatory cortical spreading depression and of cortico-caudate spreading depression propagation. METHODS
Male hooded rats (Druckrey strain) aged 2-3 months were used throughout. In animals anaesthetized with Allobarbital (Spofa, 30mg/kg) or with Nembutal (45mg/kg), trephine openings were made according to the scheme shown in Figures 1 and 3. Spreading depression was elicited by microinjection of 10% KC1 into the cerebral tissue or by application of filter papers soaked with 0.6% or 0.8% KC1 onto the dura covered cortex exposed by a 4mm trephine opening. Special care was taken to avoid mechanical stimulation when applying the filter papers. The slow potential changes were led off with wick calomel cell electrodes applied on the exposed cortical surface or by contacting the saline in the capillary electrode inserted into the caudate nucleus. The reference electrode was placed on the frontal bone or on the contralateral hemisphere. The electrodes were connected to high impedance inputs of a 3 channel recording millivoltmeter. Extracellular potassium activity was measured with the liquid membrane potassium selective microelectrodes of WALKER (1971), using the technique described elsewhere * Visiting scientist from Istituto di Fisiolgia Umana, Naples, Italy. t Visiting scientist from Gifu University, Gifu, Japan. \I’ I-ls \
537
B. DEL~JCAet a/
538
(VYSKOCIL,KRiZ and BURES,1972). The potassium electrode and the reference capillary electrode were mounted in such a way that their tips were parallel and not more than 50pm apart. Chlorinated silver wires contacting the fluids in the electrode vessels (0.5 M KC1 and saline, respectively) were connected through a high impedance operational amplifier to the recording millivoltmeter. The system was calibrated with solutions of different KC1 concentrations (3 to 1OOmM)and constant NaCl (150m~) content. The response to a tenfold increase of KC1 concentration from 4rn~ to 40m~ was typically 50 mV. Reverberating cortical spreading depression was elicited by the technique described by SHIBATAand BURES (1974). A circular lesion, 5 mm in diameter and 3 mm deep, was produced by thermocoagulation in the frontal cortex (see Fig. 6) in such a way that it was surrounded on all sides by at least 2mm wide bridges of intact tissue. Recording electrodes (1 to 3) were placed into small trephine openings surrounding the lesion at a distance of 1.5 to 2.0mm. Two other small openings (A and B) served for intracortical microinjection of 10% KCl. Reverberation was started by two appropriately timed injections of 0.5~1 KC1 into the trephine openings A and B. The drug BW 58-271 was dissolved in distilled water with added acetic acid. The lo/, solution of the drug had a pH of 5-O and was injected intraperitoneally (lOmg/kg). In some experiments, additional quantities of the drug (5mg/kg) were injected after 30 min or later. Drug administration was always combined with barbiturate anaesthesia.
RESULTS Cortical
spreading
depression
susceptibility
(Experiment
1)
In 14 rats, cortical spreading depression susceptibility was established by repeated application of filter papers (3mm in diameter) soaked with 0.6 or 0.8% KC1 solution onto the exposed parietal cortical surface covered by dura. The effect of the applied KCl solutions was checked by slow potential change recording from a point in the frontal cortex, 3 mm rostra1 from the treated area. Irrespective of whether or not cortical spreading depression was evoked, the filter paper was removed after 20 min, excess fluid was dried up and a new filter paper soaked with the same KC1 solution was applied. Under control conditions, repeated applications of 0.6% KC1 did not elicit cortical spreading depression (0 in 14 applications). Forty to 100 min after pyrrolopyrimidine injection, 0.6% KC1 elicited cortical spreading depression 9 times in 28 applications, and O*S’AKC1 17 times in 28 applications. Student’s t-test for binomial data shows that pyrrolopyrimidine significantly increased the incidence of cortical spreading depression to 0.6% KC1 (P < 0.05) but not to 0.8% KC1 (P > 0.05, t = 1.65). When results obtained with both concentrations were pooled (5/28 in controls and 26/56 in the experimental animals) the significance increased to P < 0.01. Cortical
spreading
depression
propagation
rate (Experiment
2)
In six rats, three trephine openings were made in the parasagittal plane L 3 according to the scheme in Figure 1. In order to increase the distance available for measurement of cortical spreading depression propagation rate, the triggering stimulus was applied through the small rostra1 opening. The injection of 0.5 ~1 of 10% KC1 applied 1 mm below the surface elicited cortical spreading depression in all cases. The slow potential change was recorded from openings 1 and 2 (distance 7mm). The cortical spreading depression waves were elicited before BW 58-271 application and at 15 min intervals starting 5 to 10 min after injection. Example of a typical experiment is shown in Figure 1 and all results are summarised in Figure 2. Conduction time measured between the negative peaks of the slow potential changes at electrodes 1 and 2 was an average of 2.3 k 0.1 min and was quite stable before BW 58-271 administration. Approximately 20 min after drug injection, spreading depression propagation between the two cortical electrodes was accelerated; the conduction time dropping to 1% + 0.1 min (P < 0.01).
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Fig. I. The effect of pyrrolopyrimidine on cortical spreading depression. Brain scheme indicates the location of electrodes (1,2) and the injection site (KCl Co.). Slow potential recordings before (C) and 20 or 120 min after injection of BW 58-271 (10 mg/kg). Injection of KCl indicated by arrow. Spreading rates corresponding to the electrode distance and peak conduction time are shown above the record. Calibration: 5 mV, 2 min. Negativity of the active electrode upward. Note increase of slow potential change amplitude and propagation rate and decrease of slow potential change duration 20 min after drug application.
After the same time, amplitude of the slow potential change increased from the control level of 7.5 to 12.2mV. Both effects gradually decayed over the following 2 hours to the predrug value. Twenty minutes after drug injection, duration of the negative wave decreased from I.0 to 0.8 min. but the latter effect was only shortlasting and slow potential changes were prolonged after 1 hour. min 2.51
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Fig. 2. Pyrrolopyrimidine induced changes of conduction time (above) slow potential change amplitude (middle) and slow potential change duration at 500/, of negative maximum (below). Abscissa: time after pyrrolopyrimidine injection in min. Vertical bars denote f S.E.M. The curves are based on 45 measurements each (4 to 6 measurements per point).
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Propagation of spreading depression between caudate nucleus and neocortex (Experiment 3)
In agreement with the earlier reports (FIFKOVP;and BURES, 1964; FIFKOVP;and SYKA, 1964), spreading depression propagation from the neocortex to the caudate nucleus and vice versa was irregular. Of six control animals, two displayed at least one spreading depression transition in both directions. In one rat, spreading depression penetrated from the caudate nucleus to neocortex and no transition was observed in the remaining three rats. With repeated testing, transition often failed. Out of 17 cortical spreading depression waves, 5 reached the caudate nucleus and out of 21 caudate spreading depression waves, 8 reached the parietal cortex. The overall incidence of transition in both directions was 34%. Propagation rate was the same in both directions (Fig.3A), the conduction time being 5.7 ) 0.7 and 5.6 t_ 0.6 min for the cortico-caudate and caudatecortical transmission, respectively. Due to the rare incidence of bidirectional propagation under control conditions, pyrrolopyrimidine-induced facilitation of cortico-caudate transmission could be seldom demonstrated in the same animal. In the example shown in Figure 3, the predrug values of the cortico-caudate and caudate-cortical conduction times were 5.8 and 6.3 min and decreased 60 and 90 min after injection to 4.0 and 5.3 min, respectively. A serious disadvantage of such experiments was that pyrrolopyrimidine application followed after prolonged testing (2 to 3 hours). In order to eliminate the potential influence of repeated spreading depression waves on the pyrrolopyrimidine effect, in another group of 7 rats measurements were started only after drug administration. Statistical comparisons between the control and experimental groups were made with Student’s t-test for unpaired data. Ten to 20 min after injection, spreading depression spread regularly between
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t KCI
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Fig. 4. Effect of pyrrolopyrimidine on the transition of spreading depression from caudate to neocortex. Transition failed before drug application (C), was rapid 20 min and slow 100 min after injection. Other description as in Fig. 3.
the two structures (Fig. 4). Propagation in both directions was similarly affected so that it was possible to pool the data obtained with cortico-caudate and caudatecortical spread. In total, 36 spreading depression waves were evoked at various intervals after drug application. Probability of transition increased to 100% and the conduction time decreased to 3.1 + 0.3 min during the first 20 min after injection and was maintained at this level for an hour before it started to return to the control value. The time course of this change was similar to that in the cerebral cortex, but the maximum effect was more pronounced (45% instead of 22% reduction, P < 0.01). On the other hand, changes of amplitude and duration of caudate slow potential changes were not significant. Extracellular
potassium
shifts accompanying
cortical
spreading
depression
(Experiment
4)
In an attempt to analyse the mechanism of facilitation of spreading depression induced by BW 58-271, the resting level of extracellular potassium [Kc’] was measured 1 mm below the cortical surface in 5 rats before and after injection of the drug (lOmg/kg). A typical experiment is illustrated in Figure 5, in which the resting level of 4*0mM (mean 3.0 + 05m~ for the 5 rats) was not raised during 2 hours after BW 58-271 administration. During spreading depression, the extracellular potassium abruptly rose to 70m~ (average for all animals was 64 + 5 mM) and after 30 to 40 set started to return more slowly to or even below the resting level (Fig. 5C). Pyrrolopyrimidine treatment did not significantly change the slope of the potassium shifts, but the maximum concentration was increased to 90m~, as shown in Figure 5P (average 85 + 7 mu, P < 0.05). Since the higher [K,‘] level was attained during the same time as before injection, the rate of [K,‘] increase was slightly faster. The peak [Kc:] was sometimes maintained for several tens of seconds (Fig. 5P). Reabsorption rate was not affected mHK+ loo80-
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by drug administration. During the steep part of the recovery phase, 50% decrease of [K:] took 4.5 f 0.4 set and 4.2 f 07 set before and after pyrrolopyrimidine application respectively. When a train of slow potential waves was evoked by application of loo/, KC1 on a remote cortical region, the potassium waves generated in rapid succession were of equal amplitude and displayed no decrement, which is commonly seen under similar conditions in untreated animals. The interwave interval decreased from 3.8 + 0.2 min before to 2.5 f 0.3 min after pyrrolopyrimidine injection. Reverberating spreading depression (Experiment
5)
The above findings suggest that BW 58-271 lowers the spreading depression threshold, increases the slow potential change amplitude and accelerates the propagation rate of spreading depression, without interfering with the recovery of the process. Since all these changes may favour reverberation of cortical spreading depression around a cortical lesion (SHIBATAand BURES,1972), the effect of BW 58-271 on reverberating cortical spreading depression was examined in 11 rats. Reverberation was elicited by the double injection method (Fig. 6) described in detail elsewhere (SHIBATAand BURES,1972, 1974). After the first spreading depression wave elicited from the trephine opening A passed under electrode 2, a second spreading depression wave was triggered by KCI injection into the point B. Since the cat&l parts of the hemisphere were occupied by the first spreading depression, the new wave could only spread into the recovered cortical region
Fig. 6. Facilitation of reverberating cortical spreading depression by pyrrolopyrimidine. In the brain scheme, numbers 1 to 3 indicate the position of the electrodes, letters A and B the injection sites and shading the thermocoagulated area. The slow potential change recordings from electrodes I
LeBo’s spreading
depression
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between the lesion and the medial surface of the hemisphere. The new wave reached electrode 1 and started to propagate around the lesion. Since the first spreading depression had expired in the meantime at the boundaries of the cortex, nothing hindered the spread of the incipient circle wave which reached the caudal entrance to the medial cortical segment, completed the first cycle and continued to circulate around the lesion. In the experiment illustrated by Figure 6, reverberation lasted for 12.7 hours and stopped spontaneously after 107 cycles, when a slowly developing block in the medial segment prevented transition from electrode 2 to electrode 1. Comparison of the average number of completed cycles of rever~rating cortical spreading depression in 19 control animals (Allobarbital anaesthesia only) and in 11 rats treated with BW 58-271, showed an increment from 12.0 ) 2.0 to 52.9 + 9.2 (P < @Ol) cycles. More than 20 and 25 cycles were completed by only 30% and 15% of untreated rats, respectively, but by BOY//of pyrrolopyrimidine injected rats. The effect of BW 58-27 1 was also manifested by the increased amplitude of the cortical slow potential, the average amplitude of the first three reverberating cortical spreading depression waves being almost twice the height in treated than in untreated rats. Whereas in control animals the amplitude smoothly decreased from the first to the last wave, in pyrrolopyrimidine injected animals the amplitude was maintained during the first 10 cycles and started to decline approximately 2 hours after reverberation onset. The reverberation time was unaffected by pyrrolopyrimidine, if the comparison was based on the average duration of the first three cycles. It must be pointed out, however, that comparisons made on an absolute time scale showed faster rever~ration in the pyrrolopyrimidine treated rats; the average cycle duration after one hour of reverberation being 62 + 01 min (n = 35) for the experimental and 65 + 0.1 min (n = 78) for the control animals (P < 0.05). In the last 3 cycles, reverberation was significantly slower in the pyrrolopyrimidine animals, but this was probably due to exhaustion caused by the preceding several hours of continuous spreading depression. DISCUSSION
The present paper confirms earlier reports by NORTONand JEWET~(1966, 1967) that systemic application of BW 58-271 evokes cortical spreading depression, and describes the effects of a subthreshold dosage which, after a short latency, caused marked facilitation of spreading depression. According to BURES,BuRESOVP;and KRIVANEK(1960), KC1 threshold (EDso) is 0.6% in rats anaesthetised with Allobarbital, when the filter paper is applied into a 5mm trephine opening. As the threshold concentration is indirectly proportional to the area of the exposed surface, the predrug effects of the @6 and O*S% KC1 solutions applied into a 4mm trephine opening (Experiment 1) are within the expected limits. The increase of susceptibility of cortical spreading depression caused by lOmg/kg of BW 58-271 is significant but small in comparison with the facilitation due, for instance, to hypoglycemia. As shown by BURESand BURES~VA(1960), one hour after intraperitoneal injection of 0.1 IU insulin, KC1 threshold dropped to 0.22%. Similar effects were reported after 1 min anoxia (BURES and BuRESOVA,1960) and after systemic application of metabolic poisons (BuRES, 1957). Con~asting with the small increase of cortical spreading depression su~ptibility, is the marked increase of propagation rate. According to the mathemati~l model of spreading depression proposed by A. L. Hodgkin (cited according to BuR&, B~RESOVA. and KKIVANEK,1974), conduction rate u can be expressed by the equation u = + Y;,- 2~0 D Y., J 2T where D is the diffusion coefficient of K+ in brain tissue, y;, is the maximum extracellular potassium level reached at the peak of the negative slow potential, y,, is the threshold concentration of extracellular K,? causing depolarization of adjacent elements and T
544
B. DELUCA et al
is the time constant with which the extracellular potassium concentration reaches maximum. When the extracellular potassium level was measured before and after administration of pyrrolopyrimidine, the drug increased y:, approximately by 30% (Experiment 4). The 60% increase of cortical slow potential change amplitude is also compatible with raised y;,, but the two values are not directly proportional, since slow potential change depends not only on the membrane potential but also on the geometry of the dipoles and on specific cortical impedence. After pyrrolopyrimidine, depolarization seemed to reach deeper cortical layers. Higher peak values of [K,‘] indicate a greater reduction of extracellular volume and a corresponding increase of impedance. All these changes account for the observed increment of slow potential change amplitudes. A 20% reduction of y,, can be inferred from Experiment 1. Since the y:, and y0 values established in control experiments (VYSKO&Let al., 1972) are 65 and 11 mM respectively, the pyrrolopyrimidine induced changes of y;, and y0 would account for a 20% increase of conduction rate. Further acceleration of spreading could be due to a faster rise time of the extracellular [K:], suggested by the results of Experiment 4. Decrease of T by lo%, together with the changes of y., and yO, would increase the propagation rate to the value 128% observed in Experiment 2. even when it is assumed that the value of D remains unchanged. It must be pointed out that only few interventions can accelerate spreading depression propagation. Local treatment of the cerebral cortex with 0.2% KC1 (BuRES, 1962), with isotonic sodium acetate or hypotonic NaCl (30 to 40mM) (Ltio, 1963), increased body temperature (BuRES, BURESOVA and ZACHAROVA,1957). Other agents which reduce the cortical spreading depression threshold decrease the spreading rate (for instance anoxia, BURES,1957) and can thus be distinguished from the pyrrolopyrimidine effect. The possibility of facilitating spreading depression penetration into refractory regions was studied by MARSHALL,ESSIG and DUBROFF( 1951), VAN HARREVELDand BOGEN (1956), LGo and MARTINS-FERREIRA (1956, 1961). Local application of subthreshold concentrations of KC1 or of metabolic inhibitors used in these studies would be impossible in the case of cortico-caudate transmission. According to FIFKOVAand BURES(1964) and FIF~ovli and SYKA (1964) the critical regions are the boundaries between neocortex and amygdala and nucleus accumbens, i.e., points which cannot be influenced from the exposed brain surface. Since faster spreading depression propagation would account only for a decrease in the cortico-caudate conduction time to 78% but not to 55% of the control level, the possibility must be taken into account that the cortico-caudate propagation follows a shorter pathway in pyrrolopyrimidine-treated animals the transition proceeding across the thin layer of subcortical white matter separating temporal cortex from the caudate nucleus. Another feature of the pyrrolopyrimidine effect is the decreased spreading depression refractoriness which is manifest by the undiminished K+ shifts accompanying a rapid succession of spreading depression waves, This explains the striking facilitation of reverberating cortical spreading depression. Since the relative refractory period of spreading depression in rat is approximately 15 min (ZACHARand ZACHAROVA,1961), with the average reverberation time of 6 min, the reverberating cortical spreading depression moves continuously over a partly refractory tissue. This circumstance decreases the safety factor of spreading and increases the probability of random interference. Although the relative refractory period after pyrrolopyrimidine application was not exactly estimated, comparison of the rate and amplitude of the slow potential changes and potassium shifts generated by application of equally strong suprathreshold stimuli indicates that the shortest interval compatible with generation of undiminished successive waves decreases from 3.8 min to 2.5 min. Reverberation cqntinues for an average of 12 cycles in control animals, which indicates that the probability of continuing reverberation decreases with each cycle by approximately 0.94. After BW 58-271 application, this coefficient increases to 0.99. Although the exact mechanism of the pyrrolopyrimidine effect remains obscure, reduced KC1 threshold and raised peak concentration of extracellular potassium could
Leao’s spreading depression
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be due to increased potassium permeability of the nerve cell membrane. At the same time, a faster recovery cycle of spreading depression indicates that the drug does not interfere with the mechanism of active transport. Acknowledgement-The ing the BW 58-271.
authors wish to thank Burroughs-Wellcome and Co., Tuckahoe, New York, for supply-
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BURES,J. (1962). Spreading EEG Depression, ifs Mechanism and Application. (in Czech). SZN, Prague. BURES,J. and BuRESOVA,0. (1960). Activation of latent foci of spreading cortical depression in rats. J. Neurophysiol. 23: 225236.
BuRES,J., BuRESOVA,0. and KRIV~NEK,J. (1960). Some metabolic aspects of Leao’s spreading cortical depression. In: Structure and Function of the Cerebral Cortex (TOWER,D. B. and SCHAD~,J. P., Eds.), pp. 257-265. Elsevier, Amsterdam. BuRES,J., BuRE~v.&, 0. and KRIVANEK,J. (1974). The Mechanism and Applications of Ledo’s Spreading Depression of Electroencephalographic Activity. Academia, Prague. BuRES, J., BuRESOV.&0. and ZACHARO~P;,D. (1957). E&t of changes in body temperature on spreading EEG demession. Phvsioloaia bohemoslov. 6: 454461. FIFKOVA,E: and BuREQ,~J. (1964). Spreading depression in the mammalian striatum. Archs int. Physiol. Biochim. 12: 171-179. FIFKOV.&,E. and SYKA, J. (1964). Relationships between cortical and striatal spreading depression in rat. Expl Neural. 9: 355366.
GOLDRING,S., O’LEARY,J. L. and LAM, R. (1953). Effect of malonitrile upon the electrocorticogram of the rabbit. Electroenceph. clin. Neurophysiol. 5: 39S400. LEO, A. A. P. (1963). On the spread of spreading depression. In: Brain Function, Vol. 1 (BRAZIER,M. A. B.. Ed.), pp. 78-85. University of California Press, Berkeley. Ldo, A. A. P. and MARTINS-FERREIRA, H. M. (1956). Condicbes necesirias para a ocor&cia da depreslo alastrante. An. Acad. Brasil. Ci. 28: XV. Ltio, A. A. P. and MARTINS-FERREIRA, H. M. (1961). Nota adrca da depress%0 alastrante no cerebelo, tuberculo, quadrigsmino anterior e bulbo olfativo. An. Acad. Emil. Ci. 33: 3950. MARSHALL,W. H., ESSIG, C. F. and DUBROFF,S. J. (1951). Relation of temperature of cerebral cortex to spreading depression of Lelo. J. Neurophysiol. 14: 15>166. NORTON,S. and JEWETT,R. E. (1966). A pyrrolopyrimidine with depressant action on the CNS. J. Pharmac. exp. Ther. lJ4: 152-160. NORTON,S. and JEWETT,R. E. (1967). Effect of prolonged EEG supression by a pyrrolopyrimidine on learned behavior. Physiol. Behau. 2: 317-322. SHIBATA,M. and BURES,J. (1972). Reverberation of cortical spreading depression along closed-loop pathways in rat cerebral cortex. J. Neurophysiol. 35: 381-388. SHIBATA,M. and BURES,J. (1974). Optimum topographical conditions for reverberating cortical spreading depression in rats. J. Neurobiol. 5: 107-l 18. VAN HARREVELD, A. and BOGEN, J. E. (1956). Regional differences in propagation of spreading cortical depression in the rabbit. Proc. Sot. exp. Biol. Med. 91: 2977302. VYSK&IL, F.. KRiZ. N. and BURES,J. (1972). Potassium-selective microelectrodes used for measuring the extracellular brain potassium during spreading depression and anoxic depolarization in rats. Brain Res. 39: 255259. WALKER,J. L. JR. (1971). Ion-specific liquid ion-exchanger microelectrodes. Analyt. Chem. 43: 89A-92A. ZACHAR,J. and ZACHAROV.~, D. (1961). The refractory phase of Leao’s spreading cortical depression. Physiologia bohernoslov. 10: 341-348.
