Seizure activity and cortical spreading depression monitored by an extrinsic potential-sensitive molecular probe

350 Brain Research, 409 (1987) 350-357 Elsevier

BRE 22182

Short Communications

Seizure activity and cortical spreading depression monitored by an extrinsic potential-sensitive molecular probe

Diane Evans and J.C. Smith Department of Chemistry and Laboratory for Microbial and Biochemical Sciences, Georgia State University,

Atlanta, GA 30303 (U.S.A.)

(Accepted 23 December 1986)

Key words: Molecular probe; Spreading depression; Seizure activity; Anoxia; Mitochondrion; Bicuculline; Picrotoxin

Using surface fluorescence and reflectance measurements from the exposed cerebral cortex, several potential-sensitive molecular probes, primarily oxonol V, have been evaluated as indicators of electrical activity changes developing during seizure activity in the mongolian gerbil. Intraventricular injection of bicuculline, picrotoxin, or KCI produced characteristic cyclic molecular probe fluorescence intensity descreases that could be abolished by the uncoupler CCCP or the mitochondrial electron transport inhibitor rotenone. Smaller fluorophore signals were observed when KCI was applied topically to the exposed cortex. In some cases, as the animals were recovering from anoxia induced by nitrogen inhalation, oscillations similar to those due to spreading depression were observed in both the oxonol V and pyridine nucleotide signals. In all experiments, the molecular probe signals closely followed those of the intrinsic pyridine nucleotides. The drug-induced oxonol V signal alterations have been provisionally interpreted as due at least in part to the reduction of the mitochondrial membrane potential that accompanies a state 4 to state 3 transition. Measurements at the oxonol V fluorescence excitation wavelength indicated that only small changes in the reflectance signal occurred during seizure activity suggesting minimal blood volume change contributions to the extrinsic probe signal.

Numerous investigations have indicated that epi-

leptiform seizures result in an increase in oxygen con-

sumption in the cerebral cortex. This increase is de-

tectable by direct polarographic measurements on

the cortex surface or from A - V blood pO 2 differences

that are coupled to enhanced blood flow in the cortex during seizures 11'22'24'25. The increased oxygen con-

sumption has been shown to be manifested as a re-

duction in the fluorescence intensity from the cortical

pyridine nucleotides during spreading depression ep-

isodes 15 by the methods deve loped by Chance and as-

sociates 7-9 and subsequently employed in a number of studies using cat a2, conscious rat 18'19 or mongolian

gerbil models z°. More recently, Mayevsky, et al. 2t

have explored the relat ionship between the metabol-

ic responses to ischemia and seizure propensi ty in

both seizure-prone and non-seizure-prone gerbils

using both in vivo measurements and scanning sur-

face fluorescence measurements on funnel-frozen ce-

rebral cortex tissue. The pyridine nucleotide fluores-

cence signal from the cerebral cortex appears to orig-

inate virtually entirely from the mitochondrial matrix

since the quantum yield of the f luorophore in this re-

gion is much higher than that of cytosolic pyridine nu-

cleotides, and there appears to be only small changes

in the lower concentrat ion of N A D P H during convul-

sive activity episodes le. The cyclic pyridine nucleo-

tide fluorescence intensity decreases observed in the

rat brain and re la ted prepara t ions during spreading

depression episodes have been in terpre ted as due to

a mitochondrial state 4 to state 3 transit ion and hence

imply act ivated aerobic energy metabol ism during

seizure activity ~8. This communicat ion contains re-

sults from our initial applicat ions of fluorescence

molecular probes , pr imari ly oxonol V, to the detec-

tion and monitoring of electrical activity changes that

develop after the topical applicat ion or intraventricu-

lar injection of seizure-inducing agents. The probes

Correspondence: J.C. Smith, Department of Chemistry, Georgia State University, Atlanta, GA 30303, U.S.A.

0006-8993/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)

used in this work are known to be specifically sensitive to membrane potentials in isolated mitochondria and in preparations derived therefrom 4,27. Since both the origin and nature of the pyridine nucleotide fluorescence signal present during spreading depression episodes has been well establised, in many but not all of the experiments described herein, the behavior of the fluorescence intensity signal associated with extrinsic probes applied topically to the cortex has been compared with that from the pyridine nucleotides using the technique of surface fluorescence. Using vi- deo and image reconstitution techniques, Blasdel and Salama 5 have recently employed the merocya- nine oxazolone dye, NK2367, in an elegant investiga- tion of modular organization in the monkey striate cortex whereas Orbach, et al. 23 have employed pho-

todiode array methodology in investigations of the rat somatosensory and visual cortex primarily using the styryl probe RH414. Related applications of potential-sensitive probes to a variety of biological sys- tems have been described in reviews by Waggon- er 28'29, Cohen and Salzberg 1°, Bashford and Smith 3, Bashford 1 and Smith 26.

Mongolian gerbils (Meriones unguiculatis) obtained from Tumblebrook Farms, West Brookfield, MA, 50-75 g weight, of either sex or animals derived from inbread seizure susceptible stock from this source were employed in these investigations. The animals were anesthesized with sodium pentobarbital (10 mg/kg b. wt.) by i.p. injection. The latter do- sage was adequate to anesthesize the animal during the initial stages of the optical measurements; a minimal amount of anesthetic was employed in this work in order to avoid the possibility of depressing the development of electrical activity changes upon the administration of seizure-inducing drugs. Additional anesthetic was injected as necessary over the course of the experiments, which varied greatly in length, in order to keep the animal anesthesized. For work in- volving normoxic/anoxic transitions, the trachea of the animal was supplied with a 2 mm diameter cannula; nitrogen was administered from a cylinder by means of a length of tubing loosely fitted over this cannula. In all experiments, the gerbils were placed in either a plexiglass holder and secured with a brass headvice or a Kopf small animal stereotaxic appara- tus equipped with a microinjection unit. The skull was then removed with a dental drill over an approxi-

351

mately 2 mm diameter area to the level of the dura; in experiments requiring the topical application or in-

jection of various drugs, a second nominally 1.5 mm diameter opening located rostrally to the previous one and on the midline was generated in the same manner at a distance of approximately 3 mm from the 2 mm diameter opening used for optical measurements. The location of these two skull openings and the intraventricular injection site are indicated dia- grammatically in the insert to Fig. 1.

Dye solutions at 50-250 #M concentration were prepared in physiological saline" (0.9%) with a mini- mum amount of ethanol present to keep the probes in solution. Bicuculline, rotenone and CCCP (carbonyl cyanide m-chlorophenyl hydrazone) solutions were prepared in ethanol due to the low solubility of these materials in aqueous media. The probe solutions were applied topically to the 2 mm skull opening and allowed to stain the cortex over time periods varying from 30 s to 30 min; in some cases, the excess dye was removed by gently washing the exposed area with saline or by the capillary action of a tissue paper applied at the edge of the opening. As judged from fluorescence measurements of frozen oxonol V-stained gerbil cortex tissue, the probe penetrates this tissue to a depth of approximately 3 mm (ref. 2). After the dye staining procedures were complete, the animal in the headvice or stereotaxic assembly was positioned under a bifurcated quartz fiber optics light guide for optical measurements.

Surface fluorescence and reflectance measurements were performed using a Johnson Foundation 3 mm diameter or Welch Allyn (Skaneatelis Falls, NY) 1 or 2 mm diameter branching fiber optics light guides positioned over the 2 mm skull opening using a micromanipulator. Optical signals were monitored with a Johnson Foundation MB2 spinning disk (filter) spectrometer fitted with a 100 W mercury arc lamp and configured either as a dual fluorimeter or as a combination fluorimeter/reflectometer. Pyridine nucleotide emission was excited at 365 nm and the emission detected by means of a Corning 3-73 cutoff filter. The blue oxonol and cyanine probes were excited at 580 nm and the emission monitored with a Coming 2-60 cutoff filter. The overlap of the two fluorophore signals was eliminated by the time sharing feature of the spectrometer. Details are provided in the figure captions. The output from the spectrome-

INTRAVENTRICULAR INJECTION

TOP VIEW OF CRANIUM onterior

PTICS LIGHT HOLE

posterior

T LIJ t,/) ( { I mM BICUCULLINE INJECTION

352

I

0

I

2

5

4

5

t u rn 6

CROSS-SECTION AT BREGMA I , , , l l , , l , l , , j , I . . . . l ~ . . , I . . . . 1 . . . . I . . . . I . . . . I . . . . 1 . . . . I . . . . I

6 5 4 5 2 I 0 I 2 3 4 5 6

/ /2 FzQ CCCP INJECTION / ( 2 ) ( (hi (2) AF/F; 45.6 % (PN)

o . (,, j

LL I M I N U T E

Fig. 1. Seizure activity induced by the injection of 10/A of a 1 mM stock bicuculline solution (or 0.01 #tool of the drug) into the lateral ventricle. Traces (1) and (2) are the oxonol V and pyridine nucleotide fluorescence signals, respectively. The injection of 2/~g of the uncoupler CCCP into the lateral ventricle completely eliminates the spreading depression signal. The cortex tissue was stained for approximately 30 s with a 0.1 mM oxonol V solution in physiological saline. The dye and pyridine nucleotide emission were excited at 580 and 365 nm, respectively; the emission signals were monitored using Corning 2-60 and 3-73 cutoff filters, respectively. The instrument RC time constant was 600 ms. Light guide diameter: 2 mm.

ter was fed to a Linseis type 2045 strip chart recorder

equipped with G8405 amplifiers that afford a maxi-

mum 200 % background offset capability. Changes in the probe emission signal due to drug- or anoxia-in-

duced cerebral cortex electrical activity alterations

were expressed as a percentage of the background fluorescence signal, AF/F x 100; reflectance change

measurements were normalized to the background reflectance level using the same calculation procedure.

Coordinates specifying the lateral ventricle location were taken from the gerbil atlas compiled by Loskota, et a lJ 6. Histological studies were performed on the excised brain to insure that the seizure-inducing agents were being injected into this ventricle by substituting the readily visualized oxonol

V dye for the drugs. The oxonol V and VI probes were synthesized by

the methods described by Smith, et al.27; the cyanine

diS-C3-(5 ) was the generous gift of Prof. A. Waggon-

er or was purchased from Molecular Probes, Eugene,

OR. Reagent grade KCI and ethanol were supplied by Fisher Scientific, and sodium pentobarbital was purchased from Veterinary Laboratories, Lenexa,

KS. All other biochemicals were supplied by Sigma

Chemicals, St. Louis, MO. Since the behavior of the pyridine nucleotide sig-

nal during spreading depression episodes has been well studied and arises primarily from mitochondrial N A D H 12'is, the latter signal has been used as an in-

ternal marker with which to compare the behavior of the oxonol extrinsic probe using the dual fluorimetry instrument configuration described in the previous section. Bicuculline, picrotoxin, as well as KC1 when injected into the lateral ventricle induced characteristic periodic alterations in both the pyridine nucleo-

tide and molecular probe signals that are described in

353

AF /F - 5 . 8 5 % (OX-V)

T,,, , , ,

(2)

u-J (2) A F / F - 17.27 % {PN)

AF/F- 0 .62 % (OX-V)

I mM BICUCULLINE \ (I) j I mM BICUCULLINE

\ I mM ROTENONE .L ~ / i w ~ . . (!). p,,,_IIA,, ~ (I)

12) I MINUTE

AF/F- 3 , 6 4 % (PN) I q

Fig. 2. Seizure activity induced by the injection of I nmol bicuculline into the lateral ventricle. Traces (1) and (2) are the oxonol V and pyridine nucleotide signals, respectively. The injection of 10 nmol rotenone into the ventricle eliminated the drug-dependent signal; subsequent injections of 0.5 and I nmol bicuculline would not re-initiate seizure activity. The cortex was stained for 30 s with 0.1 mM oxonol V in physiological saline. Fluorescence excitation and emission parameters were the same as those given in Fig. 1. This experiment ran for approximately 33 min. Light guide diameter: I mm.

turn in this section. Fig. 1 illustrates the progressive

formation of the seizure-dependent signal character-

ized by a cyclic, short-lived decrease in both the pyri-

dine nucleotide and oxonol V fluorescence intensity

upon injection of 10/tl of a I mM bicuculline solution

into the lateral ventricle. Maximal signals of A F / F =

40% were observed from both fluorophores in these

experiments. The injection of the oxidative phospho-

rylation uncoupler CCCP into the ventricle com-

pletely eliminated the bicucuUine-induced signal within nominally 1 min after the injection. The animal was alive and breathing normally at the end of

the record shown in this figure as well as in those illustrated in Figs. 2-5 . Appropria te control injections

were made in a series of separate experiments on sev-

eral animals to ensure that the ethanol in which the

bicuculline and CCCP were dissolved did not cause a

cyclic reduction in the f luorophore signals.

Fig. 2 contains optical data on a somewhat more

compressed scale in which the decrease of both pyri-

dine nucleotide and oxonol V fluorescence intensity

caused by bicuculline injection is readily apparent. The characteristic cyclic signal from both fluoro-

phores could be maintained for at least a half hour in

this experiment. The injection of rotenone abolished the oscillatory signal which could not be re-initiated

by repeated injections of bicuculline. Similar oxonol V and pyridine nucleotide fluorescence intensity al-

terations were associated with seizure activity generated by the injection of picrotoxin and 1 M KCI into

the lateral ventricle. Representative portions of

records obtained using the latter agent are shown i n

PICROTOXIN

l ,2,

L~ ( I , ~F/F • 15.6 N (OX-V) Z 6 F / F . 15 6%{OX-V) to ' ~F/F - 189 % (OX-V)

AF/F- I Z 9 % (OX-V)

( I )

I MINUTE

Fig. 3. The effect on the oxonol V [trace (1)] and the pyridine nucleotide [trace (2)] caused by the injection of 10 pmol picrotoxin dissolved in physiological saline into the lateral ventricle. The cortex was stained with a 0.5 mM oxonol V solution for 30 min. The signals persisted for over 1.5 h. The fluorescence excitation and emission parameters were the same as those given in Fig. 1. Light guide diameter: I mm.

354

KCI

AF/F = 10.3% (OX-V) AF/F= 10.9 % (OX-V}

AF/F: 7 ,3% (OX-V)

(J)

T AF/F= 9'I % (OX'V)(I ' "Jr" I" ' #CA//"

AF/F= 12.1% (PN) AF/F =11.4 % (PN) (2) ~,FIF • I LO % (PN)

o (21 AFIF- 9.0 % (PN)

u. I MINUTE

Fig. 4. Electrical activity induced by the injection of 10 MI of 1 M KCI (10 #mol) in physiological saline into the lateral ventricle. The cortex was stained with 0.5 mM oxonol V in physiological saline for 30 min. Traces (1) and (2) are, respectively, the oxonol V and pyridine nucleotide fluorescence signals. The fluorescence excitation and emission parameters were the same as those given in Fig. 1. The KCl-induced signals in this experiment persisted for 2.5 h at which time the supply of paper in the recorder was exhausted. Light guide diameter: 1 mm.

Figs. 3 and 4, respectively. The cyclic signals persisted for at least 30 min in each of these experiments. Again, appropriate control injections using ethanol were made to ensure that no signal was due to this solvent.

Seizure activity episodes to which the pyridine nucleotide and oxonol V optical responses were similar to those shown in Fig. 4 could also be generated by the topical application of 1 M KC1 at the remote skull opening. The signal to noise ratio in these experiments, however, was approximately 3 times lower than that obtained in the data shown in Figs. 1-4 that was obtained by the injection of seizure-inducing

agents into the lateral ventricle. The 580 nm wavelength of the extrinsic probe exci-

tation radiation is coincident with the visible absorp- tion bands of oxy and deoxy hemoglobin, and this wavelength is also near a maximum in the oxy-deoxy difference spectrum of this pigment 2. Accordingly, in a series of separate experiments, the oxonol fluorescence signal and the reflectance at 580 nm were monitored during seizure activity episodes that developed spontaneously after nitrogen administration (described in the next section) or were induced by intraventricular injection of bicuculline in order to ob- tain an estimate of possible blood volume change

72 SECONDS Z u_ 0 u. o (1)

;~ ;~ AF/F- 38.3 % (OX-V)

AF/F~5.6 % (REFLECTANCE) vM~

o (2)

_1 h

AF/F= 26.7% (OX-V)

~/ /~AF/F = 56.7 % (OX-V) (I) / ,. " /

'L. AF/F- 6.0% (REFLECTANCE) ] (2) / , ,

(2)

I MINUTE

Fig. 5. Trace (1) illustrates the increase in the oxonol V fluorescence intensity signal caused by nitrogen administration; the increase was reversible by allowing the animal to recover by breathing air and as indicated by the 'N 2 off' notation was followed by oscillations in the dye emission signal characteristic of spreading depression. Trace (2) is the reflectance signal that undergoes only small changes over this experiment. The cortex was stained with 0.5 mM oxonol V in physiological saline for approximately 10 min. The dye fluorescence excitation and emission parameters were the same as those given in Fig. 1; the reflectance was monitored at 580 nm. Light guide diameter: 1 mm.

contributions to the probe fluorescence intensity signal during seizure activity. Data from 16 different measurements yielded a mean value for the fluorescence and reflectance changes of 37 (+11) and 2.7 (+1.1) %, respectively. The reflectance change in particular is small compared to the massive fluorescence intensity changes shown in Figs. 1-4. In con- trast to the case of pyridine nucleotide fluorescence 12,18, the latter reflectance change suggests that the contribution from cortex blood volume alterations to the oxonol V signal is comparatively minor under the conditions employed in the indicated measurements. This conclusion is also supported by the data contained in Fig. 5 that is described in the next

paragraph. The effect of anoxia on the pyridine nucleotide and

oxonol V fluorescence signal from the gerbil cortex was investigated by administering nitrogen by means of a cannula implanted in the trachea of the animals as previously described. Nitrogen inhalation caused an increase in the fluorescence intensity from both oxonol V and pyridine nucleotide that reverted to the control level when the animal was allowed to recover by breathing air for several minutes; these data are not shown. The fluorescence intensity increase could be obtained in repeated administrations of nitrogen

followed by nominally 10-min recovery periods. The average increase in the pyridine nucleotide and oxo- nal V signals was 29 (+4) and 18 (+2) %, respectively. The percentage pyridine nucleotide fluorescence increase observed in these experiments compares fa- vorably with the 25% increase reported by Mayevs- ky, et al. TM in work with the mongolian gerbil and is in the lower end of the 40 (+10) % increase range reported by Mayevsky and Chance 19 in the conscious

rat model. In a separate series of experiments, the MB-2

spectrometer was configured such that the oxonol V fluorescence intensity and the reflectance signal at 580 nm could be monitored. Under conditions in which nitrogen was administered over a time period sufficient to induce convulsions in the gerbil, significantly larger dye fluorescence intensity increases, 52 (+11) %, than those previously described were observed. In a number of cases, after the cessation of nitrogen administration, the spontaneous development of oscillations in the oxonol V fluorescence signal, characteristic of spreading depression, the maximal

355

amplitudes, 36 (S.D. 8) %, of which were compar- able to the nitrogen-induced fluorescence intensity increases, but only small corresponding reflectance signal changes, 3.7 (S.D. 4.5) %, were observed at 580 nm over the time period in which apparent spreading depression was present. A typical record is shown in Fig. 5 in which the apparent beginning of anoxia-induced spreading depression is marked by the 'N2 off' notation. In experiments in which both the pyridine nucleotide and the oxonol V fluorescence intensity were monitored, the spontaneous formation of spreading depression after nitrogen administration could be observed in both fluorophore emission signals; similar behavior of the pyridine nucleotide signal after a brief period of ischemia has been reported by Mayevsky, et al. 2°.

A limited series of investigations in which potential-sensitive probes other than oxonol V were employed in the gerbil preparation have been performed. Oxonol VI, the propyl derivative of oxonol V, proved to have low sensitivity to drug-induced seizure activity in these experiments. This probe also exhibits a 4-fold smaller fluorescence quenching in substrate-supplemented submitochondrial particles than does oxonol V 26. The cyanine diS-C3-(5 ) exhi- bited a small ( - 5 %) fluorescence intensity change at the onset of seizure activity but has not been studied in as much detail as oxonol V which has proved to be the most sensitive of the probes thus far employed.

The prevalence of spikes in the two records shown in Fig. 1 caused by intraventricular injection of bicuculline suggests ictal discharge as the origin for these signals. Similar behavior of the fluorescence signal from the probe RH414 has been observed by Or- bach, et al. 23 in investigations of the rat visual cortex, but the small-amplitude transient signals observed in the latter case developed and decayed on a faster time scale than that observed in the work described in this communication. The time resolution of the records illustrated herein is likely limited by the 600 ms RC time constant employed to improve the signal to noise ratio of the fluorophore signals. The inter- vals with little or no change in the optical signals illustrated in Fig. 1 may be due to interictal periods characterized by minimal spiking; contributions from spreading depression in the latter regions, however, cannot be excluded nor can signal components origi-

356

nating from seizure activity in subcortical regions. The local, topical application of both bicuculline and picrotoxin to the cerebral cortex has been reported to prevent the invasion of spreading depression into the loci of the drug-induced epileptiform activity 6'14. The experimental protocols used in the referenced work, however, differ somewhat from those used in the measurements reported in this communication. Short-lived spiking activity is clearly less prevelant in the optical records obtained by the use of picrotoxin and KC1 shown in Figs. 3 and 4, respectively. The period of the cycles present in the records shown in these figures is significantly longer than that observed in the majority of the data in Fig. 1 and may be due to either extended periods of low interictal activity, to spreading depression of to a combination of these phenomena. The behavior of the data shown in Fig. 5 is similar to the observations reported by Mayevsky, et al. 2° and identified as spreading de-

pression. A more detailed analysis of the physiological origin of the optical signal changes associated with both the pyridine nucleotides and the extrinsic probes must await investigations in which EEG recordings will accompany optical measurements.

The fluorescence signal from the extrinsic probe oxonol V closely follows that from the intrinsic pyridine nucleotides in seizures generated using several different agents. The response of this probe to anoxia also followed that of the pyridine nucleotides. The observation, Fig. 1, that the massive signal associated with both fluorophores induced by bicuculline injection into the lateral ventricle can be eliminated by the uncoupler CCCP suggests that the source of this signal is related to a membrane potential gradient(s). The uncoupler, however, would be expected to dissipate all cellular potentials such as any plasma membrane charge gradients in addition to the mitochondrial potential. In related work, however, the response of both fluorophores to spreading depression could be abolished by the more specific mitochondrial electron transport inhibitor rotenone and could not be reinitiated by repeated intraventricular injections of bicuculline. The preceding results suggest that the origin of the oxonol V signal is at least in part mitochondrial. The 30-40% pyridine nucleotide fluorescence intensity decreases observed in Figs. 1 and 4, for example, likely correspond to an essential- ly complete state 4 to state 3 transition since signals of

similar magnitude are observed under conditions of near complete anoxia induced by nitrogen inhalation (Fig. 5). Since this apparent transition appears to be associated with the restoration of cellular ion gradients 17, the oxonol V intensity loss that is observed during the apparent mitochondrial state transition would in this model reflect the response of this probe at least in part to a mitochondrial membrane potential reduction that accompanies a state 4 to state 3 transition due to the increased demand for ATP pro- duction in the latter state; the latter increased utiliza- tion of ATP may at least in part be due to activation of the Na pump 6. The effect of bicuculline in particular and probably picrotoxin as well may be ex- plained by postulating that these agents stimulate the consumption of ATP in the cytoplasm of cortex neu- rons; the latter stimulation would be expected to lower the phosphocreatine/phosphate ratio value that is ultimately translated into an ADP stimulation of res- piration. The latter stimulation would occur at the expense of the NADH level, i.e. a pyridine nucleotide intensity loss would result that experimentally is observed to be closely followed by a similar reduction in the oxonol V fluorescence intensity. The fluorophore signals in this interpretation are thus indirectly sensitive to the accumulation of ADP.

In isolated pigeon heart mitochondria, oxonol V undergoes an ATP- and succinate-dependent intensity change that can be reversed by CCCP, and in submitochondrial particles, NADH causes alterations in the probe emission intensity. The nature of the substrate-induced probe fluorescence intensity changes (i.e., an increase or decrease in intensity) is a func- tion of the dye to membrane concentration ratio. At low dye to membrane ratios, a substrate-induced oxonol V fluorescence intensity increase can be observed that is reversible by uncouplers and electron transport inhibitors 27. Thus, under the latter conditions, a decrease in fluorescence intensity accompanies a reduction in mitochondrial A ~u, an observation that is consistent with the behavior of this probe in the gerbil cortex. The origin of the fluorescence intensity change in the isolated organelle and in submitochondrial particle preparations is a change in the amount of probe associated with the preparation membrane as indicated by alterations in the dye- membrane dissociation constant and the maximum number of membrane binding sites available to the

probe. The extent to which this mechanism is appli-

cable in the cortex appl icat ion is uncertain, but does

offer a working model for the behavior of the oxonol

probe during seizure activity. The oxonol V fluores-

cence response, however , may not be directly re la ted

to mi tochondr ia l A T P a s e activity in the cortex since

the drug-induced probe signal could not be elimi-

nated by the ATPase inhibi tor oligomycin. The la t ter

observat ion may merely reflect the inaccessibil i ty of

the mi tochondr ia l A T P a s e to ol igomycin injected in-

t raventricularly. The lack of an effect of this inhibi tor

on the f luorescence signals from several probes em-

ployed in in situ studies of mi tochondr ia in cultured

cells has been noted by Johnson, et al. 13. Al though

1 Bashford, C.L., The measurement of membrane potential using optical indicators, Biosci. Rep., 1 (1981) 183-196.

2 Bashford, L., Barlow, C., Chance, B., Smith, J., Silberstein, B. and Rehncrona, S., Some properties of the extrinsic probe oxonol V in tissues. In A. Scarpa, P.L, Dut- ton and J.S. Leigh (Eds.), Frontiers of Biological Energet- ics, Vol. 2, Academic, New York, 1979, pp. 1303-1311.

3 Bashford, C.L. and Smith, J.C., The use of optical probes to monitor membrane potential. In S. Fleischer and L. Packer (Eds.), Methods in Enzymology, Vol. LV, Aca- demic, New York, 1979, pp. 569-586.

4 Bashford, C.L. and Thayer, W.S., Thermodynamics of the electrochemical gradient in bovine heart submitochondrial particles, J. Biol. Chem., 252 (1977) 8459-8463.

5 Blasdel, G.G. and Salama, G., Voltage-sensitive dyes re- veal a modular organization in monkey striate cortex, Na- ture (London), 321 (1986) 579-585.

6 Bures, J., Von Schwarzenfeld, I. and Brozek, G., Blockage of cortical spreading depression by picrotoxin loci of parox- ysmal activity, Epilepsia, 16 (1975) 111-118.

7 Chance, B. and Baltscheffsky, H., Respiratory enzymes in oxidative phosphorylation, J. Biol. Chem., 233 (1958) 736-739.

8 Chance, B., Cohen, P., Jobsis, F. and Schoener, B., Intra- cellular oxidation-reduction states in vivo, Science, 137 (1962) 499- 508.

9 Chance, B. and LegaUis, V., A spectrofluorometer for recording of intracellular oxidation-reduction states, IEEE Trans. Bio.-Med. Electron., 10 (1963)40-57.

10 Cohen, L.B. and Salzberg, B.M., Optical measurements of membrane potential, Rev. Physiol. Biochem. Pharmacol., 83 (1978) 35-83.

11 Grant, F,C., Spitz, E.B., Shenkin, H.A., Schmidt, C.F. and Kety, S.S., The cerebral blood flow and metabolism in idiopathic epilepsy, Trans. Am. Neurol. Assoc., 72 (1947) 82-86.

12 Jobsis, F.F., O'Conner, M., Vitale, A. and Vreman, H., Intracellular redox changes in functioning cerebral cortex. I. Metabolic effects of epileptiform activity, J. Neurophys- iol., 34 (1971) 735-749.

13 Johnson, L.V., Walsh, M.L., Bockus, B.J. and Chen, L.B., Monitoring of relative mitochondrial membrane potential in living cells by fluorescence microscopy, J. Cell. Biol., 88 (1981) 526-535.

14 Korovela, V.I. and Bures, J., Blockage of cortical spreading depression in electrically and chemically stimulated

357

the el iminat ion of the cyclic f luorescence intensity

signals from both pyridine nucleot ides and oxonol V

by ro tenone suggest a mitochondria l origin for these

signals, the possibil i ty that the extrinsic p robe signal,

in part icular , contains contr ibut ions from other

charge gradients cannot be excluded. Oxonol V, nev-

ertheless, appears to be a promising extrinsic indica-

tor of electrical activity originating from epi lept i form

behavior in the gerbil prepara t ion .

These investigations were suppor ted by Heal th

and Human Services Awards GM30552 and RR07171.

The authors thank the reviewers for helpful com-

ments.

areas of cerebral cortex in rats, Electroencephalogr. Clin. Neurobiol., 48 (1980) 1-15.

15 Leao, A.A.P., Spreading depression of activity in the cerebral cortex, J. Neurophysiol., 7 (1944) 359-370.

16 Loskota, W.J., Lomax, P. and Verity, M.A., A Stereotaxic Atlas of the Mongolian Gerbil Brain, Ann Arbor Science Publishers, Ann Arbor, MI, 1974.

17 Lukyanova, L.D. and Bures, J., Changes in pO 2 due to spreading depression in the cortex and nuclear caudatus of the rat, Physiol. Bohemosiov., 16 (1967) 449-455.

18 Mayevsky, A. and Chance, B., Repetitive patterns of metabolic changes during cortical spreading depression of the awake rat, Brain Research, 65 (1974) 529-533.

19 Mayevsky, A. and Chance, B., Measurements of extracel- lular potassium, ECoG, and pyridine nucleotide levels during cortical spreading depression in rats, Brain Research, 76 (1974) 347-349.

20 Mayevsky, A., Friedli, C.M. and Reivich, M., Metabolic, ionic and electrical responses of gerbil brain to ischemia, Am. J. Physiol., 248 (1985) R99-R107.

21 Mayevsky, A., Zarchin, N., Kaplan, H., Haveri, J., Hasel- groove, J. and Chance, B., Brain metabolic responses to ischemia in the mongolian gerbil: in vivo and freeze trapped redox scanning, Brain Research, 276 (1983) 95-107.

22 Meyer, J.A. and Pornoy, H.D., Post-epileptic paralysis, Brain, 82 (1959) 163-185.

23 Orbach, H.S., Cohen, L.B. and Grinvald, A., Optical map- ping of electrical activity in rat somatosensory and visual cortex, J. Neurosci., 5 (1985) 1886-1895.

24 Penfield, W., Von Santhra, K. and Cipriani, A., Cerebral blood flow during induced epileptiform seizures in animals and man, J. Neurophysiol., 2 (1939) 257-267.

25 Schmidt, C.F., Kety, S.S. and Pennes, H.H., The gaseous • metabolism of the brain of the monkey, Am. J. Physiol., 143 (1945) 35-52.

26 Smith, J.C., Potential-sensitive molecular probes in energy transducing organelles. In L.M. Loew (Ed.), Spectroscopic Membrane Probes, CRC Press, Boca Raton, FL, in press.

27 Smith, J.C., Russ, P., Cooperman, B.S. and Chance, B., Synthesis, structure-determination, and energy-linked spectral responses of the extrinsic probe oxonol V in mem- branes, Biochemistry, 15 (1976) 5094-5105.

28 Waggoner, A.S., Optical probes of membrane potential, J. Membrane Biol., 27 (1976) 317-334.

29 Waggoner, A.S., Dye indicators of membrane potential, Annu. Rev. Biophys. Bioeng., 8 (1979) 47-68.

Documents

Seizure activity and cortical spreading depression monitored by an extrinsic potential-sensitive molecular probe