Electrogenesis of sustained potentials

6 ELECTROGENESIS OF SUSTAINED POTENTIALS GEORGE G . SOMJEN

Department of Physiology and Pharmacology, Duke University, Durham, North Carolina 27710, U.S.A.

Contents 1. 2. 3. 4.

5. 6. 7. 8. 9.

Introduction Historic notes Conditions and sites of occurrence of SP shifts Sustained evoked potentials 4.1. The case for and against neural generators of evoked SP shifts 4.2. The case for and against glial generators of SP shifts 4.3. Rebuttal, summation and verdict in the case of glia versus neurones 4.4. If it be glia, what kind is it? 4.5. Spinal SP shifts and dorsal root potentials 4.6. Evoked SP shifts and oxidative metabolism The SP shifts of spreading depression Electrogenesis of other types of SP shifts General discussion Summary Appendix: a model simulating tbe hypothetical contribution of glia cells to extracellular potentiais. By R. JOYNERand G. SOMJEN 9.1. Assumptions 9.2. Method 9.3. Results

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Notes added in proof

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Acknowledgements

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References

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ELECTROGENESIS OF SUSTAINED POTENTIALS GEORGE G. SO~UEN Department of Physiology and Pharmacology, Duke University, Durham, North Carolina 27710, U.S.A.

1. Introduction

If one connects two points of the brain, or one point within the brain and another without, to the input of a differential amplifier, then between the two contacts there will lie uncounted "batteries" both in series and in parallel. Inprinciple the voltage of each battery could change independently from the others. The resistances connecting these sources of electricity could also vary and so impress voltage fluctuations on the amplifiers which would not easily be distinguished from those generated by the batteries. Contemplating such a multivariate system some might wonder whether attempts to detect the prime generators of voltages of the mammalian central nervous system (CNS)* are not inevitably doomed to fail. Yet though it no doubt is the most complex of all known natural systems, the brain is not built at random but to some very definite, if hard to decipher, blueprint. If there is regularity of structure, the expectation is justified that whenever voltage variations are seen to occur in a predictable manner, their mechanism may be discovered too. Yet even the most sanguine of the analysts of extraceliular potentials do not expect to find a unique explanation of, or a single source for, the many different kinds of sustained voltage shifts which have been discovered in the mammalian CNS. In this review the emphasis will be placed on those forms of sustained potential (SP) shifts for which a plausible, if not completely proven, theory can today be offered. These include in the first place those which can be evoked in the cerebral cortex and in the spinal cord by repetitive electrical stimulation of the cortical surface or of afferent pathways; and in the second place the potential shift associated with spreading depression. The many other types of SP phenomena which may or may not have features in common with the two which have just been mentioned will be briefly referred to but not discussed in detail. Our topic is the actual electrical mechanism of SP shifts, not the broader question whether extracellular potentials, phasic or sustained, are of the very essence of the working of the brain, or not. While abstaining in this article from the controversy of "signs versus signals" (see Uttal, 1967; also Somjen, 1972; and discussion in Katchalsky and Rowland, in preparation), the question will be raised whether the electrical currents associated with SP * Only three abbreviations will be used in this review, a n d I feel like apologizing oven for these few. SP stands for sustained potential; CNS for central nervous system; and D R P for dorsal root potential.

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shifts of the extracellular medium do or do not have an effect on the neurones lying in their path. The circumstances under which SP shifts are generated have been reviewed on a number of occasions (O'Leary and Goldring, 1959, 1964; Aladjalova, 1964; Rowland, 1967, 1968; Rowland and Anderson, 1971 ; Adey, 1969) and therefore I shall dwell on this aspect of our topic only briefly. The history of the subject will also receive perfunctory treatment only, because it has been covered well by Brazier (1963) and by Rowland (1968). Final prefatory remarks must concern the use of terms, and the definition of limits. Taking a practical approach, O'Leary and Goldring (1964) defined as "DC potentials" those voltage differences which can be recorded only by direct coupled measuring instruments. Besides, "DC", these potentials have also been called "steady", even though they are neither of the two. Since they are rarely if ever truly constant, and not always unidirectional either; since "steady shift" seems to be a contradiction in terms; since, furthermore, "infraslow aperiodic wave" although precise enough has a somewhat clumsy ring; the adjective, "sustained", seems the most appropriate. It is one of several which already have been used (e.g. by O'Leary and Goldring, 1964). Since the abbreviation "SP" is identical for both expressions, "sustained" and "steady" potential, it (and the adjectival form: SP shift) could be acceptable for all concerned. Apart from the somewhat trivial circumstance that special instrumentation is required for their detection, is there in fact something special about the apparent shifting of the electric "baseline"? Some think not. Aladjalova (1964) for example, appears to be thinking in terms of a continuum of brain waves in which "infraslow potential waves", oscillatory as well as "aperiodic", merge imperceptibly with the slow waves better known from conventional electroencephalography. If this were the case, there would be little justification to review the electrogenesis of SP shifts divorced from other types of evoked potential waves. As we shall see in the pages which will follow, there are reasons for assuming that SP shifts, or some of them, are in some respect different from other extracellular potentials 2. Historic Notes

As the phenomenon itself, interest in SP potentials has been waxing and waning slowly with time. SP shifts rose in the esteem of neuroscientists when it was expected that they might reveal aspects of brain functioning which are not reflected in the more customary recordings of electrical events. Krhler, for example (KShler and Held, 1949; Kfhler et al., 1955; K6hler and O'Connell, 1957), hoped to discover in the distribution of cortical "electrotonus" the embodiment of Gestalt or the cerebral counterpart of mental images. If that expectation was disappointed by the findings of Lashley et al. (1951) and of Sperry and Miner (1955; Sperry et al., 1955), other theories and other observations, before and after that time, gave periodic impetus to the study of SP shifts. It is generally believed (Brazier, 1963; Rowland, 1968) that SP shifts were amongst the earliest electrical events ever recorded from the mammalian brain. We do not know this for certain, because the earliest reports (e.g. Caton, 1875, 1877a, b; Beck, 1890; Gotch and Horsley, 1891) contained no illustrations and we can infer only from the verbal descriptions the nature of the phenomena that have been observed. Certainly the instruments which these authors had at their disposal were inert though sensitive, and therefore better suited to register slow voltage variations than the more rapid oscillations with which a later epoch was preoccupied. That Caton (1877a, b) found a potential difference between intact cortex

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and a cut surface of the brain is hardly remarkable. But he, as also Beck (1890) and Gotch and Horsley (1891), described changes of potential which seemingly were evoked by "natural" stimulation of sense organs, or by electrical stimulation of others parts of the nervous system. These may well have been voltage shifts of the kind we are now discussing. The first recordings of brain potentials to appear in print (Neminski, 1913) also had features which, if not artefacts, could have been SP shifts (some of Neminski's illustrations have been reprinted by Brazier, 1961). That significant electrical events of slow time course may be overlooked as a consequence of the use of condenser-coupled valve-amplifiers, was clear to some investigators at a time when few others used direct-coupled instruments. Among them were Fischer (1932), Hasama (1934), Adrian, (1936) and Dusser de Barenne and McCulloeh (1939). Fischer (1932) and Hasama (1934) persisted in the use of the string-galvanometer of Einthoven, with no other than optical amplification. With this instrument they detected cortical SP deflections evoked by sensory stimulation. Adrian (1936) used a Matthews oscillograph with a battery-coupled preamplifier; Dusser de Barenne and McCulloch (1939) used a vacuum-tube voltmeter. From the point of view of our narrative the next major event was the publication by Gerard (1936) and by Gerard and Libet (1940; also Libet and Gerard, 1941, 1962) of the suggestion that parallel arrays of neurones might act as electrically polarized sheets. In the resting state such sheets of cells would generate current "in parallel", causing a constant voltage difference between their dendritic and axonal sides. Modulations of this standing potential (see also Rischmaui and Vatter, 1969) would cause the SP shifts which can be recorded, for example, from the cortical surface. Gerard and Libet also speculated that the variations of the polarization current of such neuronal sheets could have an influence on the excitability of the fibers and of the other cells lying among the generator neurones. When Rusinov (1953; see also Morell, 1961, 1963) reported that artificial electrical polarization of the cerebral cortex can have long enduring effects on the behavior of neurones under the polarizing electrodes, interest in Gerard and Libet's (1940) suggestion was stimulated from a new point of view. The key question then became whether naturally occurring extracellular currents were ever intensive enough to influence the neural elements which they traverse. Terzuolo and Bullock (1956) supplied what seemed to be an affirmative answer, albeit based on experiments on an invertebrate nervous system. These authors found that a voltage gradient of ! mV/mm is sufficient to influence the activity of a neurone, provided that the latter has already been aroused by some input other than the current itself. Gerard and Libet's (1940) theory implied that the phasic waves of primary evoked potentials were generated by the same polarized neurones as were the SP shifts. The phasic potentials were believed to be modulations of the standing potential; in other words a phasic positive wave was thought to be caused by a momentary reduction of the standing negativity. Therefore a positive-going shift of the voltage of the cortical surface (whether caused by artificially applied current, or by natural processes in the neurone sheet itself) was expected to be associated with enhancement of the negative component of the phasic evoked potential and with a depression of the primary positive wave. Negative polarization was expected to have the obverse effects. According to Bishop (1949) and to Caspers (1959) this is exactly what happens, but Goldring and O'Leary (1951) found the situation rather more complex. Libet and Gerard (1962) themselves reported that the reciprocal correlation between SP and phasic evoked potentials is less than completely reliable, but they presented possible explanations of the deviations.

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Arduini et al. (1957) and Brookhart et al. (1958) also supported the idea that shifting of the potential of the cortical surface is generated by the same elements as the phasic driven potentials evoked by thalamic stimulation. Their theory held, however, that the SP shift arose by a process of summation of phasic potentials, and did not incorporate the idea of a standing "resting" polarization of the cortical neurone sheet, as postulated by Gerard and Libet (1940). As we have seen, there are two aspects to the neuronal polarization theories. First, that extracellular SP shifts are generated by neurones and second that, in their turn, neurones are influenced by extracellular current flow. Both propositions lead to the corollary that there must be a correlation between the activity of individual nerve cells and the SP shift in their neighborhood. The existence of such a correlation was both affirmed and denied by various investigators. This matter will be the subject of more detailed scrutiny later. Following the contributions of Gerard and Libet (1940) and of Krhler (Krhler and Held, 1949; Krhler et al., 1955), the discoveries made independently by Gumnit (1960; Gumnit and Grossman, 1961), by Rowland (1963); Rowland and Goldstone, 1963; Rowland et aL, 1967) and by Walter and associates (1964; Walter, 1968), that SP shifts occur in the cerebral cortex under circumstances which men and animals may experience outside the laboratory, once more directed the attention of researchers, especially of psychologists, to their study (e.g. Kornhuber and Deecke, 1965; Rebert et al., 1967; Rebert and Irwin, 1969; Marczynski et al., 1971). 3. Conditions and Sites of Oecurreaee of SP Skiffs

There is a standing potential between cerebrospinal fluid and the blood (Tschirgi and Taylor, 1958; Held et al., 1964; Pappenheimer, 1967) and it can be altered by changes of the blood gases and of the pH on either side of the blood-brain barrier (Goldensohn et aL, 1951; Held et aL, 1964; Wurtz and O'Flaherty, 1967; Woody et aL, 1970). These effects interest us only because they contaminate recordings whenever brain potential is referred to an "indifferent" extracerebral point. Ordinarily these blood-CSF potentials are smaller and change more slowly than the phenomena we are about to discuss; but it is well to be mindful of their existence. Besides alterations of blood (or CSF) composition, merely a change of the rate of flow of blood through nervous tissue will have some effect on the local potential (Besson et aL, 1970) and its distribution, partly because the bulk resistivity of the tissue will change and partly because of the standing potential between the two fluid components (intra- and extravascular) which was mentioned already. There are other sources of contamination which truly are trivial, yet of practical importance in designing experiments. Movements of the eyes and electrical activity of other muscles can produce voltages which are not negligible. Those working with awake animals have learned to avoid these pitfalls (see Rowland, 1968; Rowland and Dines, in press). Among the potentials which do arise within the substance of the central nervous system and were studied repeatedly for several years, are the ones caused by drugs (Goldring and O'Leary, 1951, 1954a, b; Tschirgi and Taylor, 1958; Eidelberg and Meyerson, 1964). Drugs are of interest not only because they cause SP shifts, but also because they influence the potentials evoked by other means (Goldring et aL, 1958, 1959a; Gerber, 1961; Strittmatter and Somjen, 1972 and in preparation; and Fig. 5). Very slow variations of brain voltage related to the sleep-wake cycle have also been observed (Caspers and Schulze, 1959; Kawamura and Sawyer, 1964; Kawamura and

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Pompeiano, 1969; Wurtz, 1966). To what degree these are caused by vascular and chemical factors, is as yet an unresolved problem (Kawamura and Sawyer, 1964; Wurtz, 1967; Wurtz and O'Flaherty, 1967; Besson et al., 1970). Since there is always a potential difference between CNS and non-neural tissue, modulations of this voltage, or changes of the tissue resistance between CNS and "earth", could create SP shifts which in "monopolar" recording arrangements could not be distinguished from bonafide brain potentials. The SP shifts which are evoked by sensory stimulation, or by electrical stimulation of afferent paths, certainly are generated within the nervous system itself. Figure 1 demonstrates this. The large and stable potentials illustrated in Fig. 1 were recorded by two micro-electrodes both of which were thrust into the substance of the spinal cord. One was lodged in a segment, the afferent nerves of which were stimulated by prolonged repetitive trains of pulses; the other in a segment some 5 cm distant from the activated one. Without deliberate stimulation there was no significant standing potential between these two electrodes, whereas there always is one between the CNS and an extraneural reference point. Since both electrodes were within the cord, neither the composition nor the flow of blood, nor the cells of the pia or ependyma, could have contributed materially to these SP shifts. Since there was no pre-existent standing potential, the SP shift was not a modulation of such a potential by a variation of resistance. The shape, magnitude, and timecourse of the SP shifts so recorded was indistinguishable from potential shifts recorded between an activated segment of the cord and a reference electrode on back muscle in the same experiment and during similar stimulation. In other words, even when referred to somatic tissue, this type of SP shift was demonstrably generated within the cord itself. We must next consider whether SP shifts could reflect electrochemical potentials consequent to changes of the electrolyte composition of extracelluiar fluid. Excited neurones are believed to add K + ions to, and to remove Na + ions from their extracellular environment. Inhibited neurones may, in addition, remove CI- from the extracellular phase (Eccles, 1963, 1964), but the latter effect is probably minor compared to the cation fluxes. If such ion gradients within the extracellular medium were the main source of SP shifts of the CNS, their significance would be quite different from that of electrical activity generated in cell membranes. Differences of ion concentrations in a watery medium could, in principle, give rise to voltage gradients by three mechanisms. First, a pair of electrodes immersed in two solutions of different composition could form a "concentration cell". But in order to register the voltage of a "concentration cell", the electrodes must be reversible with respect to the ion whose concentration contributes the electromotive force (Lakshminarayanaiah, 1969; Harris, 1972). In the case of neural tissue, one might expect transient but significant differences of the extracellular cation concentrations. Yet the Ag/AgCI junctions commonly used in electrophysiology are reversible only for CI- ions, not for Na + or K +. Besides, the Ag/AgC1 wires used in such experiments are not usually in direct contact with neural tissue, but are immersed in a "salt bridge" of constant composition. A second possible electrochemical mechanism which could simulate SP shifts would be a change of the tip potential of the recording micropipette electrode (Adrian, 1956; LaVallte et al., 1969). However, SP shifts of identical magnitude and timecourse can be recorded by two electrodes whose tips lie 30 to 60 pm apart in the extracelluiar phase of the spinal cord, even when one is filled with potassium citrate and the other with NaC1 (Somjen, 1970, and unpublished observations). Tip potentials of two micropipettes filled with unlike electrolytes could not be identical.

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The third possible spurious source of SP shifts could be diffusion potentials at liquid junctions. There is little doubt that such liquid junction potentials do occur in the extracellular phase of CNS tissue, the question only concerns their magnitude. Reliable estimates of the ion concentration in the extracellular fluid of mammals are hard to come by, but Orkand et al. (1966; also Kuffler and Nicholls, 1966) have suggested that the membrane potential ofglia cells is a precise function ofextracellular K + concentration. In the Appendix (section 9) we will argue that glia can act as a near-perfect potassium electrode only when isolated in an organ bath, and that in situ, in the CNS, estimates of increments of extracellular K + concentration based on measurements of glial depolarization probably fall short of true value. Nevertheless, as a first approximation, we can take into consideration that during intensive activation of the neural elements, glia cells undergo depolarization of 10 to 15 mV in the cortex and spinal cord of mammals (Karahashi and Goldring, 1966; Grossman and Hampton, 1968; Somjen, 1970). From the Nernst equation at 37°C such depolarizing shifts would correspond maximally to a 1.5- or 1.6-fold change of the ratio of intracellular over extracellular K ÷ concentration (Somjen, 1970). To avoid underestimating the K + effect, we will base our calculation on the assumption that extracellular K ÷ may double or even triple during intense neural activity. To simulate such a condition, we calculate the liquid junction potential for the following conditions. Assuming that in "resting" tissue N a + = 145 mEq/1 ; K ÷ ----- 5 mEq/1 ; and CI- = 150 mEq/1 ; and in "active" tissue: either Na + = 140 mEq/1 ; K + = 10 mEq/1 ; and CI- = 150 mEq/1 ; or: Na + -- 135 mEq/1; K + = 15 mEq/1; and CI- ----- 150 mEq/1. Then using Goldman's constant-field assumption, and the standard mobilities of the ions in water at 37 ° as the "permeability" terms, (Na ÷ = 64; K ÷ = 91 ; C1- = 95), we obtain a calculated voltage of about 0.16 mV for the former and 0.29 mV for the latter condition. Actual voltages of SP shifts in the spinal cord were frequently 20 times higher than these calculated potentials (Somjen, 1969, 1970). Even though the calculations contained a number of simplifying assumptions, the difference between calculated and observed values is so great that the contribution of diffusion potentials to SP shifts of the spinal cord can be dismissed as minor compared to other sources. In the cortex, where SP shifts are of smaller amplitude, the situation is less clear, and firm conclusions will be possible only when extracellular K ÷ concentrations have been measured accurately. At the peril of stressing the obvious, it should be said once more that the argument of the preceding three paragraphs concerns electrochemical potentials generated within the extracellular phase of the CNS. Effects of ion changes on cell membranes are a different case, which will be discussed, with special reference to glia cells, in section 4.2. SP shifts have been studied in the cerebral cortex more than in any other part of the neuraxis. They can be evoked here by stimulation of nonspecific as well as specific afferent pathways (Arduini et al., 1957; Goldring and O'Leary, 1957; Brookhart et al., 1958; Vanasupa et al., 1959), and also by direct stimulation of the cortical surface (Caspers, 1959; Goldring et aL, 1961). Natural stimuli, such as sounds or lights, are effective (Fischer, 1932; Hasama, 1934; Gumnit, 1960), even though not as powerfully and not as reliably as electrical stimulation. Spindle waves as well as recruiting waves are known to "ride" on negative shifts of the electrical baseline (Goldring and O'Leary, 1957; Brookhart et aL, 1958). Also, alerting the animal causes a change of the surface potential of the cortex (Caspers and Schulze, 1959; Kawamura and Sawyer, 1964; Kawamura and Pompeiano, 1969) as does electrical stimulation of the midbrain reticular formation (Vanasupa et al., t 959; Kawamura and Sawyer, 1964; Hayward et aL, 1966). In this context it is worth recalling that the

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association of a negative variation of the surface potential with a reduction of spontaneous voltage oscillations was already noted by Beck in 1890. What Beck saw could have been alerting with desynchronlzation, although it could have been spreading depression too. Electrical stimulation of the appropriate fiber tracts evokes SP shifts in the cerebellar cortex which are similar to those of the cerebral cortex (Van Gilder et aL, 1967) and also in diencephalon and brainstem (Garcia-Ramos and Rosenthal, 1967; Hayward et al., 1966). It is a curious circumstance that in spite of the intensive study of the electrophysiology of the spinal cord during the forties and the fifties, the extracellnlar SP shifts of this tissue went unnoticed or were ignored. Yet of all parts of the central nervous system it is here that they are of the largest amplitude, the most stable, and the most reliably evoked (Fig. 1, and Somjen, 1969, 1970; Strittmatter and Somjen, 1972). As was mentioned already (p. 204), besides stimulation of nerves or of sense organs, SP shifts of the forebrain are also associated with certain psychological states. Known examples are the expectation of a signal, or readiness to act (the contingent negative variation or CNV of Walter, 1968); the reinforcement of conditional stimuli (Morrell, 1961; Rowland and Goldstone, 1963); and the fulfilment of a biological need (the consummatory potential; Rowland et al., 1967). Whether SP shifts evoked by artefactual stimulation, and those associated with "natural" causes are generated by similar cellular mechanisms is, as it was mentioned earlier, another still-to-be solved problem. In the pages which follow we shall mainly deal with the SP responses to electrical stimulation. Cerebral pathology has SP shifts of its own. The uncommonly large shifts of potential associated with spreading depression were described first by the man who discovered this phenomenon (Le~o, 1944, 1951). Spreading depression will be discussed in some detail later (pp. 220-223). Electrical seizure discharges are usually associated with typical SP shifts. Noticed for the first time by Jasper and Erickson (1941), these SP shifts have been studied repeatedly (Gl6tzner and Gr~isser, 1968; Caspers and Speckmann, 1969; Goldensohn, 1969; Grossman and Rosrnan, 1971 ; Prince, 1971). Amongst the pathologic SP shifts can be counted those associated with anoxia and asphyxia (Van Harreveld, 1946; Collewijn and Van Harreveld, 1966).

4. Sustained Evoked Potentials

4.1. THE CASE FOR AND AGAINST NEURAL GENERATORS OF EVOKED SP SHIFTS It was at first taken for granted that neurones generate SP shifts. Nerve membranes were after all known to be electrically polarized and electrically responsive. They also were known to generate signals varying in duration between one millisecond (the spikes) to several, or several tens of milliseconds (the synaptie potentials). It was a natural step to assume that nerve cells could also be capable of maintaining electrical signals which last for seconds, and which could outlast the period of stimulation. There have also been observations of a close relationship between the SP shifts and the other more familiar types of neuraUy generated voltage fluctuations. We have mentioned already the association of desynchronization and surface-negative potential shifts (first noticed by Beck, 1890; see also Vanasupta et aL, 1959) and the inverse correlation between standing potential and the amplitude of evoked potentials and EEG potentials (el'. Bishop, 1949; Caspers, 1959; Libet and Gerard, 1962).

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With the advent of the era of single unit recording, reports at first favored the view of a close correlation between SP and the firing of single neurones. Fox and O'Brien (1965) found such a correlation between phasic evoked potentials and single cell discharge, and Robertson (1965) and Fromm and Bond (1964, 1967) found a similar correlation between slow spontaneous potential changes and unit firing. Li and Salmoiraghi's (1963) observations were, however, at variance with the others. They found the responses of individual neurones to identical stimuli to vary widely. While the firing of some ceils indeed showed a covariation with the SP of the surface of the cortex, or with that of its depth, with other cells this was not the case. With G. Marsh we have repeated these experiments (unpublished) and confirmed Li and Salmoiraghi's observations. Even though the firing rate of practically every neurone did change at a time when the SP in its vicinity was shifted by stimulation of one or other subcortical nucleus, there was no consistent relationship between SP shift and cell activity. While some ceils accelerated, others decelerated their discharge, and others again underwent biphasic or multiphasic changes. In sum, it seemed that the activity of the ceils and the level of SP of their neighborhood were both influenced by the subcortical stimulation, but in a disparate manner. It was therefore dillicult to see how the SP shift could have been the electrical result of the summed activity of masses of individual neurones, particularly of the neurones from which the recordings were made. There may be a simple explanation of the discrepancy of the two sets of experimental resuits. The "slow" potentials with which Robertson (I 965) and Fromm and Bond (1966, 1967) were comparing the unit spike activity occurred "spontaneously", and the ones with which Li and Salmoiraghi (1963) and Marsh and Somjen (unpublished) were dealing were evoked by electrical stimulation. The two kinds of SPs may have different electrical mechanisms. The most dramatic demonstration of the non-correlation of cell firing and SP shift can be given in the spinal cord. Here it is an easy matter to prepare several peripheral nerves for stimulation. One can then find neurones in the intermediate gray matter which are con= tinuously active without deliberate provocation. Such cells can either be inhibited or excited if the appropriate peripheral nerve is selected for stimulation. If now the baseline potential is recorded by a DC-coupled amplifier together with the spike potentials of the ceil, one can observe that the SP shift is always in the negative direction, regardless of whether the neurone is excited or inhibited. Furthermore, when both nerves of the antagonist pair are stimulated at the same time, the spike discharge takes an intermediate (halfway= excited) level, but the negative baseline shift is greater than with either stimulus (excitatory or inhibitory) alone (Somjen, 1969). Instead of sampling different units in succession, as was done by Li and Salmoiraghi (1963) and by Marsh and Somjen (unpublished) in the cortex, in the cord one thus can see opposite effects on one and the same ceil. Since excitatory and inhibitory postsynaptic currents have opposite electrical signs (Eccles, 1964), such results refute the idea that the neurones under observation could have generated the negative SP shifts. There are other reasons for doubting the role of neurones in generating this type of SP shifts. The classical rules of analyzing currents flowing in volume state that when potentials are caused by a current between dendrites and soma of neurones, the potentials should reverse sign when the el~trode is advanced from dendritic tree toward cell soma and beyond (Adrian, 1936; Renshaw et al., 1940; Bishop, 1949). This in fact is observed, as is well known, in the case of primary evoked potentials of the sensory receiving areas of the cerebral cortex. But as far as SP shifts arc concerned, it w a s noted by Goldring et al. (1959b),

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Li and Salmoiraghi (1963), Van Gilder et al. (1967), Rowland (1968; also Rowland and Anderson, 1971) and by Castellucci and Goldring (1970) that while some types do, others do not reverse sign when recording at increasing depth from the cortical surface. Even when source and sink areas of the SP shift could be detected within the gray matter, as was the case for repetitive stimulation of the cortical surface, its distribution was very different from the source-sink layering of the primary dendritic ("negative spike") response (Goldring et al., 1959b). As do those in the cortex, in the spinal cord too the orthodromically evoked phasic focal potentials which arc known to be related to the flow of synaptic currents appear to reverse polarity at a certain depth, but evoked SP shifts do not (Somjen, 1970). The observations mentioned in the preceding paragraphs concern the polarity of the SP shifts recorded against a distant "indifferent" reference electrode. More subtle and more complex are the considerations of potential gradients within the substance of gray matter. SP shifts of certain types are consistently negative throughout the cortical or spinal gray, when referred to such a distant point, but their magnitude varies considerably with depth in the tissue. Such potential gradients, it could be argued, may reflect the aggregate effect of stacked "dipoles" in the tissue, and such dipoles could be represented by polarized nerve cells. To complicate matters, the local gradients of potential may reverse sign with time. This is illustrated in Fig. 2, which shows tracings of potential recorded simultaneously at two depths in a spinal cord, and the difference between the two. Because the SP shift developed more rapidly in the superficial location but attained a higher final value at the deeper site, there first appears a negative-positive gradient, which then reverses to a positivenegative gradient. Interestingly, the phasic focal potentials which are visible with lowfrequency stimulation (Fig. 2A) in the superficial tracing are barely detectable in the deep record. These fast focal potential waves are the ones associated with synaptic potentials of neurones. These and similar observations suggest that the sustained potentials are composed of a faster but smaller and less constant component generated by ncurones, and a slower but larger component of non-neural origin which, it will be argued, is caused by glia (see next section and Fig. 7). The least compatible with the idea that ncurones are the main generators of extracellular SP shifts were the observations made with intracellular electrodes. With this method the potentials inside and outside of nerve cells can be compared, recorded either simultaneously using double electrodes, or in succession. Prolonged repetitive stimulation of afferent nerves, or of cerebral pathways, does evoke sustained synaptic potentials in neurones. These may be depolarizing or hyperpolarizing in direction, depending on the stimulus. There is, however, no correlation between the timecourse, the magnitude, or even the sign of such sustained membrane potential changes of ncurones, and the shifts of potential measured with an electrode located in their cxtracellular environment. This, in short, was the finding of Castellucci and Goldring (1970) in the cerebral cortex and of Somjen (1969, 1970; see also Fig. 4F) in the spinal cord. None of the arguments which we have discussed is entirely compelling by itself. We shall cross-examine each one later (pp. 215-216). Taken together they do seriously challenge the theory that neuroncs are the principal source of sustained potential shifts evoked by repetitive electrical stimulation. In the next section (4.2) the arguments in favor of a glial theory of SP shifts will be examined. But we must emphasize once more, and it is implied at all times, that the arguments apply only for the specific circumstances under which the investigations quoted have been conducted. There is reason to assume that in some instances

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the neural component is more important in generating an SP shift than the glial component. For example, the SP shifts evoked in the spinal cord by electrical tetanization of sensory nerves is depressed in deep anesthesia. In this state the residual SP shift assumes a "'waveform" which resembles neuronal sustained synaptic potentials much more closely than it does glial depolarizations. It is possible that in deep anesthesia the glial component is relatively more depressed than the neuronal component (Strittmatter and Somjen, in preparation). 4.2. THE CASE FOR AND AGAINST GLIAL GENERATORS OF SP SHIFTS Glia ceils were first distinguished from neurones in 1846 by Virchow (see Kuffler, 1967). Since that time the opinions varied from the one extreme, that they are just the "glue" holding nerve cells and fibers together, to the other extreme, that they could be the key to the very essence of the mind-brain enigma. These and other views have been reviewed by Galambos (1961, 1964), Kutiier and Nicholls (1966), Kuttler (1967) and Lasansky (1971). That glia might produce electrical waves was suggested by Tasaki and associates (see Hild et al., 1958; Hild and Tasaki, 1962), who observed glia cells cultured in vitro. The electrical responses they described were subsequently attributed to dielectric breakdown of the cell membrane (Wardell, 1966), caused by excessively strong stimulating currents. However, electrical responses of another kind were later observed in intact tissue instead of a culture by Kuffler and associates (Orkand et al., 1966). These authors saw gradual depolarizing potentials of glial elements of the optic nerve when they stimulated it repeatedly. The depolarizing potentials were slow to rise and even slower to decay. Hence when one followed another closely, the successive waves merged and summed. There was no refractory period and no all-or-none rule. Only when nerve fibers were excited, did the surrounding glia ceils respond. According to Kuffler and Potter (1964) and Kuffler et al. (1966) glia is insensitive to direct electrical stimulation. Unlike Hild et al. (I 958; Hild and Tasaki, 1962), Kuttter and associates have avoided the use of excessively intense currents in their attempts to stimulate glia cells. Orkand et aL (1966) attributed the depolarizing potentials of glia cells to an effect of potassium ions spilt from nerve fibers in the course of generating impulses. Indeed they (Nicholls and Kuffler, 1964; Kuffler et al., 1966; see also Hild et al., 1958; Wardell, 1966) could demonstrate that the membrane potential of gila in invertebrate and in amphibian nervous systems is dependent on the extracellular K + concentration, even more so than is the membrane potential of neurones. More recently a depolarizing effect of K + was demonstrated for mammalian glia cells as well (Krnjevi6 and Schwartz, 1967; Dennis and Gerschenfeld, 1969; Pape and Katzman, 1972). In a review of the physiology of glia, Kuffler and Nicholls (1966; also Orkand et al., 1966) suggested that such glial depolarization could well contribute to sustained extracellular potential shifts observed in the central nervous system of mammals. (Already suggested earlier by Galambos in 1961 and by Roitbak in 1963; but without much experimental evidence; for English language reference of the latter author see Roitbak, 1970.) In order to produce this effect, glia ceils would have to be electrically connected in such a way that a circuit be formed of which depolarized glial elements in an activated region and resting glia cells in inactive parts of the tissue would all be part. (See also Appendix, and Figs. 9, 10, 11.) Electrotonic continuity between glia ceils was demonstrated by Kuttter and

ELECTROGENESlS OF SUSTAINEDPOTENTIALS

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Potter (1964) in the leech and by Kuffler et al. (1966) in amphibia. Cohen (1970) estimated that in the optic nerve of amphibia 40 ~o of a depolarizing voltage generated by gila to one side of a "sucrose gap" could be recorded as an extracellular potential gradient along the nerve. In the absence of a sucrose gap some of this extracellular voltage would, of course, bc dissipated (see Fig. 11D). In the mammalian central nervous system glia cells are said to make up about half of the volume of the tissue (Kufiter and Nicholls, 1966). It is practically unavoidable that the tip of an exploring microelectrode should from time to time enter glia cells. Indeed since the dawn of the intracellular era (now entering into its third decade) it was a common observation that a penetrating microelectrode registered standing negative potentials from loci where no action potentials or synaptic potentials could be recorded. These negative standing potentials could have been caused by junction artefacts of one kind or another, or by capillaries and their endothelial linings. But it was a fair guess that in many instances these "silent" or "unresponsive" or "idle" resting potentials were the signs of the entry of the electrode tip into glia cells. Goldring and associates (Sugaya et al., 1964; Karahashi and Goldring, 1966; Castellucci and Goldring, 1970) were probably the first to describe depolarizing potentials of "idle cells" in the cerebral cortex of cats, which were similar to the glia] responses reported by Orkand et al. (1966) in the optic nerve of Necturus. These reports were followed by Ol6tzner and GriJsser (1968), by Grossman and Hampton (1968) and by Grossman et al. (1969). But while Karahashi and Goldring (1966) and Castellucci and Goldring (1970) suggested that the depolarizing potentials of "idle" cells could indeed supply the current of extracellular SP shifts, Grossman et al. (1969) thought this not to be the case. I learned about these findings after having been frustrated in attempts to relate SP shifts to cortical neurone function (Marsh and Somjen, unpublished). Turning to the spinal cord for an answer, the resemblance of the SP shifts of the cord and the records of glia] depolarization published by Orkand et al. (1966) seemed too striking to be dismissed as mere coincidence. Thus began a systematic exploration of the relationship of depolarizing potentials of "unresponsive" cells with the SP shifts recorded in their immediate vicinity (Somjen, 1969, 1970; Strittmatter and Somjen, 1972 and in preparation). It was found first, that the time course of the intra- and extracellular shifts of potential of these unresponsive cells was, if not identical, quite similar (see Fig. 3A). Second, that as afferent stimulation was varied, there was a precise inverse linear correlation between the amplitude of the potential shifts recorded simultaneously in and outside any one of these cells (Fig. 4). Third, there also was a correlation between the depolarizing potentials of different unresponsive cells and the extracellular SP shifts of their neighborhood evoked by repeating a constant standard stimulus. The latter relationship was demonstrated by recording at varying depths from the surface of the spinal cord (Fig. 3B). Fourth, that whenever the extracellular SP shifts evoked by a standard afferent stimulus were depressed by the administration of an anesthetic or anticonvulsant drug, the average depolarizing potentials of unresponsive cells were depressed to a similar degree. (See Fig. 5; and Somjen, 1970; Strittmatter and Somjen, 1972, and in preparation.) These observations, taken together, would seem to make the hypothesis of a glial generator of SP shifts quite believable. However, we owe the reader a fair presentation of the arguments which weaken the case. First of all there is some uncertainty of the identity of unresponsive cellular elements. In the cerebral cortex Krnjevi6 and associates (Kelly et al., 1967) and Grossman and Hampton

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214

GEORGEG. SOMJEN

(1968) have marked such cells with a stain released from microcapillary electrodes after completion of a recording. By this means they proved that the cells they called "unresponsive" or "silent" were indeed glia. In the spinal cord this demonstration is as yet lacking, and the identification hinges on the similarity of the properties of spinal unresponsive cells to those of the cortex. Also lacking is a demonstration of electrotonic continuity between glia cells of the mammalian CNS. The existence of "tight junctions" seen on electron micrographs (Brightman and Reese, 1969) is insufficient evidence, for it must be shown that such sites are indeed electrically conductive. In invertebrates and in cold-blooded vertebrates this proof has been rendered (see above; and Kuffler and Nicholls, 1966) but in mammals it has not yet. We might mention that there is an alternative to the hypothesis of an electrotonic syncitium, which also is compatible with the idea of glial electrogenesis of SP shifts. If processes of individual glia cells extended far enough to provide electrical continuity between active and inactive regions of the gray matter, these processes could provide the return path for the current which in the extracellular phase generates the SP shift. Grossman (personal communication) called attention to illustrations by Cajal (1952, Vol. 1, pp. 236237) showing glial fibers which answer this description; and Lenhossrk (1891) pictured glial fibers as radiating through practically the entire thickness of the spinal cord. A more serious challenge of the glial hypothesis of the generation of SP shifts came from Pollen (1969; see also discussion in Katchalsky and Rowland, in preparation). His argument is based on the low specific membrane resistance which glia cells were estimated to have (Trachtenberg and Pollen, 1970; Trachtenberg et al., 1972). Having such leaky membranes makes these cells, according to Pollen (1969), unlikely generators of surface potentials. Reservations could be raised concerning the estimates of specific membrane resistance of glia cells in situ. Two values are needed to calculate the specific resistance: one is the actual measurement of the "input" resistance "seen" by the intracellular microelectrode, the other is an educated guess of the surface area of the cell. The measurement of input resistance is less than completely reliable, due to bias in sampling and to variations according to method. Krnjevi6 and Schwartz (1967), for example, reported higher values of the input resistance for cortical glia cells than did Trachtenberg and Pollen (1970). But more importantly, the surface area of glia cells in situ has only been estimated indirectly. Accurate measurements would require three-dimensional reconstructions from serial electron micrographs of many such cells; a formidable task even if aided by a computer program. Finally, even if the size of the average glial cell were accurately known it would not help the problem at hand, for if there are low-resistance electrotonic junctions between cells, the "input resistance" measured by intracellular current injection pertains not to one cell but to the entire electrotonic network. In a tissue explant the surface area of cells can be estimated (Trachtenberg et al., 1972), but the properties of the membrane of cultured cells may deviate from the normal condition, as indicated by their low membrane potential. But granted that the membrane of glia may be ten to twenty times more leaky than the membrane of neurones, this would not refute the glial theory of SP generation. It will be shown in the Appendix (and Figs. 9, 10, and 11) that variations of the membrane resistance do influence the distance over which both intra- and extracellular potentials spread (i.e. their virtual "space constant") but not the ratio of the magnitudes of intra- over extracellular potential shifts. In fact, when the membrane is highly conductive, as glia is, according to Trachtenberg and Pollen (1970), both the intra- and extraceUular potentials are larger than when the membrane is more resistive. Thus the condition for glia cells to be able

ELECTROGENESlS OF SUSTAINEDPOTENTIALS

215

to supply extracellular current for the SP shifts is not that they have high membrane resistance, but (a) that there should be electrotonic continuity from active to inactive region, (b) that a large enough mass of glial cells should undergo depolarizing shifts simultaneously. If the membrane of these cells is highly conductive, as claimed by Trachtenberg and Pollen (1970), then the distribution of SP shifts which they generated would accurately coincide with the areas of intense neural activity. If, on the other hand, their membrane were highly resistive, then SP shifts would spread outside the activated areas. Under the conditions which enable sustained potentials to be evoked, we have seen neurones to be intensely activated (and/or inhibited) over wide areas of the gray matter (Li and Salmoiraghi, 1963; Marsh and Somjen, unpublished; Somjen, 1969, 1970). The neuropil of the first (superficial) cortical layer could hardly be exempt from such generalized activation, and this could easily explain the shifts of potential seen at the cortical surface. The cause of the depolarization of glial cells could be an elevation of the extracellular concentration o f K + ions, as was suggested by Orkand et al. (1966). K + is probably released from neurones whenever they generate action potentials, and also when they generate excitatory as well as inhibitory synaptic potentials (see, for example, Eccles, 1964). This fact could explain why the SP is shifting in the negative direction in the vicinity of excited as well as of inhibited neurones. Besides K + there could be other chemical agents released from neurones which may have depolarizing effects on the glial membrane. For example, Krnjevi6 and Schwartz (1967) found that gamma-amino butyric acid and acetylcholine, but not glutamate or noradrenaline, cause depolarization of"unresponsive cells" of the cortex. Nicholls and Kuffler (1964), and Kuffler et al. (1966) have found that in the leech and in amphibia the membrane potential of glia cells behaved almost as a perfect K + electrode. Their experiments were performed in vitro, with the entire preparation bathed in solutions of varying K + concentration. Similar results could not be expected in experiments in the intact nervous system, if glia indeed generated currents which are conducted from activated into inactivated regions. The reason will become clear from the computer simulation of an electrotonic network presented in the Appendix. The results of the simulation demonstrate that if cells are supplying current to the "extracellular" compartment, the voltage registered in the "intracellular" compartment must fall short of the voltage of the battery representing the "membrane electromotive force". In other words, the extracellular return path of the current acts as a shunt for the intracellular voltage (Fig. 9, lower left diagram and Fig. 11B). Glia could therefore act either as a near-perfect K + electrode, or as generator of extracellular SP, but it could not play both roles at the same time. In the preceding paragraphs we presented much evidence in favor and little against the notion that glia is a major supplier of extraceUular sustained currents. But since neurones have for long been regarded the sole generator of all voltages of the CNS, we should not dismiss them without re-examination of their case.

4.3. REBUTTAL, SUMMATION AND VERDICT IN THE CASE OF GLIA VERSUS NEURONES The arguments in favor of a glial origin of the SP shifts were based on the close correlation between extracellular SP shifts and glial depolarization, which was found under a wide variety of circumstances. The arguments against a neuronal origin were based on the lack of similar correlation between SP shifts and the electrical signals which are known to be of

216

GEOROEG. SO~EN

neuronal origin. Is then the neuronal hypothesis refuted beyond reasonable doubt? The answer is" not quite. The first doubts about neuronal SP generators arose, as we mentioned earlier, when it was found that the spatial distribution of "sources" and "sinks" of phasic evoked potentials did not coincide with those of evoked SP shifts (see p. 208; and Goldring et aL, 1959b; Castellucci and Goldring, 1970; and Somjen, 1969, 1970). With this finding it became dit~cult to assume that one and the same generator was responsible for both phenomena. It would, of course, be possible to draw a theoretical electrical circuit which could produce potential gradients of opposite polarity at different frequencies of alternation of current. The condition would be that a short time-constant filter should be incorporated as a bypass or shunt in such a way that it should reverse polarity for short-period potential waves but not for long ones. For the case in question, phase should be selectively reversed at about 100 Hz (since the approximate period of primary evoked potentials of the cortex and of the phasic focal potentials of the cord is around 10 msec) and not for 1.0 Hz or lower frequencies (since SP shifts rise and fall within a second or more). If the pyramidal cells of the cortex are considered, the synaptic potentials responsible for primary evoked potentials reverse polarity at about the level where the somata of large pyramidal cells lie (see Fig. 8). If the same cells were to generate SP shifts as well, the source of these very slow electrical shifts would have to be millimeters farther down the axon, at least for those SP shifts whose "source" cannot be detected within the cortical gray matter. In extracellular electrical recordings phasic and sustained potentials often appear together, the former seemingly riding upon the latter. The two can, however, be dissociated, not only in space, but also by the application of drugs (Goldring et al., 1959a). In the light of the known electrical characteristics of neurones, it seems unlikely that one cell could simultaneously generate two kinds of potentials of such different properties. As a subsidiary hypothesis one could, however, postulate that both phasic and sustained potentials were generated by nerve cells, but by nerve cells of different classes. To fit the experimental data one would then have to stipulate that the activity of the type of nerve cell which generated SP shifts was not detected by any currently available means other than the SP shifts. SP shifts would in that case not be expected to be correlated with other signs of neural activity, because they would not be generated by the same neurones. This hypothesis would, for example, put into a new light the fact that spike discharges recorded from extraceUular positions are not dependent on SP shifts. The SP shifts could perhaps be the product of neurones which do not generate spikes at all, only slow potentials, and whose presence would therefore not be recognized in experiments designed to detect spike discharges. Nonspiking neurones have indeed been discovered in the retinae of reptiles. The horizontal and bipolar cells have so been described (Werblin and Dowling, 1969). These neurones have no need of propagated action potentials since their axons are short enough for electrotonic conduction of synaptic potentials to suffice for signal transmission. Similar cells could well exist elsewhere in central gray matter. There is, however, a flaw in this argument. Nonspiking cells, if they exist in the spinal cord and cortex of mammals, are probably of small size since they are not detected by intracellular electrodes. Unless many were stacked endto-end and all polarized in the same direction, they could not generate the currents required for the potentials observed. There is no histologic evidence for such "Volta-piles" in the gray matter. If non-spiking neurones are unlikely candidates, could there be parts of ordinary nerve ceils which do generate spike discharges, but which are not detected by customary methods

ELECTROOENF~Sor SUSTAINEDPOT~NT~tS

217

for some other reason ? The answer again is, theoretically yes. It is true that the probes we use for recording do not readily find the smallest of neural elements, for example unmyelinated nerve fibers, unless special efforts are made. Sheets of parallel C-fibers, or o f dendritic fibers of fine caliber, could indeed be the source of SP potentials, but only if many were longitudinally polarized in the same direction. Unless and until structures conforming to these specifications are discovered in the central nervous system of mammals, the neural theory of SP potentials remains less plausible than the glial theory. 4.4. IF IT BE GLIA, WHAT KIND IS IT ? Classical neurohistology has distinguished at least three major types of neuroglia, and after some wavering of confidence, students of ultrastructure again recognize all three (Peters et al., 1970; Vaughn and Skoff, 1972). There may be additional variant subgroups. Certainly oligodendroglia cells behave very differently from astrocytes, and both have little in common with microglia. The question is, which of these are likely to generate SP shifts ? The answer, in short: no one really knows. Kelly et al. (1967) have left methyl blue marks inside unresponsive cells from which they had recorded. Histologie examination of the tissue showed the staining of nuclei resembling those of oligodendroglia. These authors did not study depolarizing potentials under conditions in which SP shifts could have been generated, but they did report that certain pharmacological agents caused depolarization of the unresponsive cells observed. Later, we (Kelly et al., 1969) found such cells' membrane resistance to be increased when exposed to excess Ca 2 + or Mg 2+. Oligondendroglia is closely associated with nerve fibers (Peters et al., 1970), and for that reason could be the first to be exposed to elevated K + in the wake of spike activity. On the other hand, astrocytes occupy a larger volume, and are therefore perhaps more accessible by microelectrodes. Grossman and Hampton (1968) were unable to determine whether their stain marks were left in astrocytes or in oligodendroglia, but when working in glial scar tissue of experimental lesions, G-rossman and Rosman (1971) believed to have been recording from astroeytes. The latter had properties similar to the silent cells of the normal cortex. There is of course no a priori reason why glia of all kinds could not generate SP shifts. 4.5. SPINAL SP SHIFTS AND DORSAL ROOT POTENTIALS Besides cell bodies and dendrites, neurones also have axons and we should not neglect an examination of these as possible generators of SP shifts. Especially so, since the terminal presynaptic portions of axons are believed to be the site of slow potential waves. Replying to a question raised by Jung in the discussion of a lecture, Eccles (1963) suggested that these may be the source of SP shifts of the cortical surface. In the spinal cord the slow axonal potentials are known as the primary afferent depolarization, and are believed to be the generator of the negative dorsal root potentials (DRP) of Barton and Matthews (1938) or D R V of Lloyd and Mclntyre (1949). The suggestion had to be rejected on the basis of three grounds. First, the waveform of the SP and of the D R P evoked simultaneously by the same afferent stimulation, were quite different. Second, as afferent stimulation was increased, the growth of the one did not correlate with the growth of the other. Third, after injection of picrotoxin the DRP was

218

GEORGEG. SOMJEN

depressed, sometimes almost to the vanishing point, but the SP shifts were at the same time somewhat enhanced (Somjen, 1970). The first two named reasons could, in themselves, not have decided the issue, but the third observation is difficult to reconcile with this suggestion. It is hard to conceive of a circumstance in which one variable could be the cause of another, when the decline of the first is accompanied by the growth of the second. Matthews and associates (see Borland et al., 1970) have recently suggested a link between glial activity and dorsal root potentials which would be the reverse of the one just considered: in their view DRPs are caused by glial cells. Even though at variance with the other currently better known theories of the DRP (Wall, 1962; Eccles, 1964), the opinion of the discoverer of slow root potentials (cf. Barton and Matthews, 1938) merits closer examination. The terminal arborizations of afferent fibers lie in the tissue which is permeated by the currents we think are generated by depolarizing gila cells. Given the right kind of electrical properties of the afferent fibers, the SP gradient could perhaps impress on them the electrical change which can be recorded as the dorsal root potential. If so, a transformation of waveform (i.e. of time course) must take place at the afferent fiber membrane. Apart from the need of postulating such a temporal transformation, none of the three arguments which refuted the idea of the DRP causing the SP shift need exclude the reverse hypothesis, that of the SP shift causing the DRP. Picrotoxin, for example, could block the DRP by acting on the membrane properties of the afferent fibers in such a way as to prevent the SP effect on the afferent terminals; in other words by "disconnecting" the one from the other. In that case the DRP would be depressed, but the SP shift would not. Borland et al.'s (1970) hypothesis is less easily reconciled with the effect of pentobarbital on dorsal root potentials and on sustained evoked potentials. In many, though not all preparations, a small amount (20 mg/kg body weight) of this barbiturate causes an enhancement of the DRP (see also: Eccles et al., 1963) while depressing the SP shifts (Somjen, 1970; Strittmatter and Somjen, 1972). Such enhancement of DRP and a simultaneous depression of SP by the action of pentobarbital has recently been recorded in the same cat (Somjen, unpublished). Similar non-correlation was also seen in unanesthetized spinal cords, as either the strength or the frequency of afferent stimulation was varied. Although these results are preliminary, they are mentioned here because of their bearing not only on the search for the generator of DRP, but also on the larger problem, whether extracellular current fields can ever have an influence on the neural elements which they might traverse. While a categorical answer cannot yet be given to the general problem, the conclusion seems justified that spinal DRP are not caused by SP shifts, and hence, by inference, not by glial activity. 4.6. EVOKED SP SHIFTS AND OXIDATIVE METABOLISM If it is true that SP shifts are generated by glia cells which are exposed to elevated extracellular K + levels, it could be expected that under the influence of such a deviation from the homeostatic condition the active transport of ions would be stimulated (cf. Hertz, 1966). If so, then the energy turnover of the tissue would also be increased. It is possible to test this proposition since J6bsis and associates (1971; Rosenthal and J6bsis, 1971) have invented a method of monitoring the oxidative activity of the cerebral cortex in situ, under relatively normal conditions. The method depends on the fluorometric detection of the ratio of NADHfNAD in the tissue, and has been described in detail in the papers quoted above.

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We have measured simultaneously the fluorescence of N A D H and the potential of the cortex during stimulation of either the thalamic relay to the particular cortical area which was under observation, or of the cortical surface itself (Rosenthal and Somjen, 1971; and in preparation). It was indeed found that with increasing intensity of stimulation both the reduction of fluorescence attributable to NADH, and the negative potential shift of the cortex increased pari passu, and that the two were closely and linearly correlated (Fig. 6).

220

GEORGEG. SOMJEN

While not "proving" anything, these observations are compatible with the potassium~ glia-SP hypothesis. In these experiments there was, of course, no way of distinguishing between oxidative metabolism of neurones and that of glia. It is equally plausible that the extracellular overflow of K + is cleared by the "pumping" of ions through the membrane of neurones, of glia, or of both. Several authors have spoken of glia acting as a "potassium-buffer" (Kuffler, 1967; Pollen and Trachtenberg, 1970), meaning that these cells could have the function of removing the excess K + from the vicinity of neurones and thus counteracting the depolarization of the latter. Kuflter (1967) thought the removal of K + to be a passive process, ions flowing along their electrochemical gradient. Active pumping of K + could, however, aid the process, in which case the depolarization of glia cells may be the servo signal regulating the activity of the potassium pump. It has also been suggested that failure of the "buffer'" mechanism may be the trigger for such pathological phenomena as seizures (Pollen and Trachenberg, 1970) and spreading depression (Grafstein, 1956; Bourke, 1969). There is only one point unexplained by the scheme just outlined. This is the matter of how the potassium lost by neurones is restored to them. In some way the K + extracted by gila from the extracellular medium would again have to be returned to the neurones, Electron microscopists find increasing numbers and types of non-synaptic junctions between cells, and it may not be long before glia-neurone junctions will be found where such unidirectional exchange of material could conceivably take place.

5. The SP Shifts of Spreading Depression A few years after discovering the phenomenon of spreading depression, Leilo (1944) described the surface-negative SP shift associated with it (Le~lo, 1951). The SP shifts of spreading depression are remarkable for their intensity. Frequently the maximal amplitude exceeds 30 mV (for a review of the literature on spreading depression see Marshall, 1959). Under certain circumstances the large surface-negative shift is preceded and followed by smaller positive-going shifts. Grafstein (1956) offered a theory to explain the phenomenon. She suggested that K + ions released from neurones during excessive activity may cause depolarization of other neurones which in their turn would add to the excess K + until depolarization proceeded to the point where cells became "inactivated" and hence temporarily inexcitable. Her theory was in good agreement with the following features of spreading depression: first, coincident with the accelerating negative shift of potential, many neurones are first intensely active before they fall silent. Second, once it is initiated, the process inexorably proceeds to completion; it has an eruptive, almost all-or-none type of course. Third, a variety ofchemical, mechanical and electrical stimuli are effective in triggering spreading depression. Fourth, its slow spread, gradually engulfing much or all the cortex of one hemisphere. This theory has recently been put to a direct test by Bureg and associates (Vysko~i! et al., 1972). They used potassium-sensitive glass electrodes to record the local K + concentration together with the potential of the cortex. They found that K + level rose to very high levels during spreading depression, but the onset of the rise of K + did not occur early enough to be the cause of the shift of potential. If confirmed, this observation argues against the idea that K + is the primary causal agent in the development of spreading depression. An alternative theory was offered by Van Harreveld (1959; also Van Harreveld and Fifkov~i, 1970, 1971). In common with Crrafstein (19563, Van Harreveld and Fifkovlt (1970,

ELECTROOENESIS OF SUSTAINEDPOTENTIALS

221

1971) think in terms of a vicious circle in which a stimulant chemical having been spilt from activated cells then causes more stimulation of these cells and of their neighbors with consequent further release of the stimulating compound. In Van Harreveld's (1959) view the exciting chemical is glutamate (Van Harreveld and Fifkov~i, 1970, 1971), a known stimulant of neurones. Cells exposed to a high concentration of glutamate gain Na + and water and thereby swell (Van Harreveld, 1966). Van Harreveld's theory, thus, also incorporates the idea of an imbalance of ion distributions, albeit in a sense different from Grafstein's. It is, of course, quite possible, and in fact probable, that loss of K + and gain of Na + go, under conditions of overstimulation and inactivation, hand in hand. Yet another variant to the several ionic theories of spreading depression has been offered by Bourke (1969). In his view the swelling is mainly one of glial cells, not of the dendrites as Van Harreveld proposed. Furthermore, the fluid entering the swollen cellular elements contains, according to Bourke (1969), much K ÷ as well as CI-, instead of Na + and CI- as Van Harreveld postulated. It must be mentioned, however, that Bourke's experiments were performed on sliced brain in vitro, and his findings may not be applicable to the intact brain. All three theories having merit, it seems worthwhile to try to reconcile them, since all have certain observations to support them. Could it be that spreading depression is a condition in which too much K + is discharged from neurones, which then take up Na + instead, in amounts which are too large to be handled by the ion pump. The K ÷ spilt from the nerve cells is removed by glia, which accumulates it together with CI-. The combined uptake of these two ions inevitably leads to swelling of the cells. And the initial trigger for the entire cycle may yet turn out to be an excess of glutamate. Proof of the swelling of cortical tissue during spreading depression came from direct observation of the movement of the cortical surface, from measurements of tissue impedance which increased as the depression developed, and from electron microscopy (Van Harreveld, 1966). In agreement with Van Harreveld, we (Rosenthal and Somjen, 1971 and in preparation) found an increase of the reflectance of the cortical surface at the time of onset of spreading depression (Fig. 6). We attribute the increased reflectance to paling of the surface and to the movement caused by the swelling of the cells. Later there follows a decrease of reflectance due to the reddening of the surface as the small vessels dilate. If the spreading depression is caused by an overload of the ion transport mechanism of cell membranes, could we detect this by monitoring the oxidative metabolism of the cortex ? To answer this question we (Rosenthal and Somjen, 1971 and in preparation) recorded the fluorescence and the potential of the cerebral cortex, and provoked spreading depression either by electrical stimulation or by the application of Ringer solution with abnormally high K ÷ concentration or, occasionally, observed "spontaneously" occurring spreading depressions in deteriorating cortical tissue. Under each of these conditions the onset of spreading depression was heralded by a decrease of the NADH-related fluorescence, which continued to a level greatly in excess of the fluorescence response observed under the usual "physiological" conditions of stimulation of the cortex. However, at variance with the expectation of a shortage of the supply of oxidative energy caused by a failure of the electron transport chain, the NADH-fluorescence continued to fall after the time when EEG activity ceased. In other words, oxidative metabolism was still increasing when the neuronal system had already failed (Fig. 6). Unfortunately this experiment decides only one half of the question we want to explore. For while the results seem to suggest that the depression is not caused by a failure of the electron transport chain, they neither exclude nor support a failure of the ion pump for

222

GEORGEG. SOMJEN

some other reason, for example, by the jamming of a "bottleneck" at the hypothetical "pores" or "channels" in the membrane through which ions are supposedly transported. The next question concerning the SP shift of spreading depression is the same as the one raised concerning the evoked SP shifts. Is it generated by neurones, by glia, or both? Galambos (1961) first suggested that glia might be involved, and Karahashi and Goldring (1966), Goldensohn (1969) and Higashida et al. (1971)have reported that glia cells are indeed depolarized during spreading depression. Grafstein's (1956) extracellular observations suggested that neurones are subject to a similar change, and this was confirmed by intracellular recording too (Collewijn and Van Harreveld, 1966; Goldensohn, 1969). The objections against the theory of neuronal generators of evoked SP shifts are not applicable to the SP shifts of spreading depression. In spreading depression most or all neurones seem to behave in identical fashion, proceeding in register through a cycle of excessive activity which is followed by inactivation, and which cycle is governed by progressive depolarization and repolarization. Furthermore, in spreading depression the negative potential shift of the cortical surface is complemented by a feeble but reproducible positive shift of the cortical depth (Le~o, 1951; Van Harreveld, 1966; Rosenthal and Somjen, in preparation). It seems that, in the balance, both glia and neurones are equally possible sources of the currents in this case. The combined depolarization of virtually all cells in the tissue, neurones as well as glia, seems to supply the intensive current causing the unusually large fluctuations of voltage seen in this condition. 6. Electrogenesis of Other Types of SP Shifts Have we, by analyzing SP shifts evoked by electrical stimulation of afferent pathways, also solved the mechanism of the SP shifts known as contingent negative variation (Walter, 1968) consummatory potential (Rowland, 1968) and the like (Kornhuber and Deecke, 1965; Rebert et al., 1967, Marczynski et al., 1971) ? Unfortunately, we have not. It is difficult to make the necessary electrical recordings in these cases because these potentials can only be observed in alert and uninjured animals, or in healthy human subjects. Data are nevertheless slowly accumulating, and sooner or later it will be possible to analyze the "natural" SP shifts as well. Rowland and Dines (in press) found that spike discharges seen in multi-unit recordings sometimes were and sometimes were not correlated with SP shifts. It was precisely this kind of inconsistency which has prompted the search for generators other than neurones in the case of the electrically evoked SP shifts. There is, however, another observation which seems to be more compatible with a neuronal generator of at least some of these more physiological SP phenomena. It was the observation by Rowland (1968) that the consummatory potential occasionally has opposite sign in the cortical depth compared to the surface (see also Marczynski et al., 1971). As we shall see in the Appendix, positive-going extracellular potentials could, evidently, also be generated by ghal syncitia. However the finding of an electrical "source" within the substance of the cortical gray is at least compatible with the idea that the SP shifts in this ease may be due to current generated by neuronal activity. 7. General Diaemdon Figure 7 presents a synthetic view of what probably takes place when evoked SP shifts

ELECTROGENESlSOF SUSTAINEDPOTENTIALS

223

f~

i . . . . .i. . . . . . ................

d I: b °- c

I

J

.......... ....................................................... U

.... " 7

f- ..................................

o, FIG. 7. Fact and theory: Schematic representation of recordings from glia cells (a), extracellular space (b) and neurones (c and d). a': "smoothed" version of a typical recording from an "unresponsive" cell in the neck of the dorsal horn of the spinal cord. Dotted line represents theoretical "pure" glial response, continuous line the actual depolarizing voltage shift; the difference is attributed to interference by synaptie current generated by neurones (c") traversing glial membrane, a ' : hypothetical contribution of glia to extracellular potentials, b': an idealized extracellular recording of actual voltage, which is the composite of a" and of c'. c': sustained excitatory postsynaptic potential of a neurone, which is postulated to contribute c ~ to the extracellular potential, d': transmembrane potential of neurone; whereas c' is recording referred to ground. (From Somjen, 1970.)

are recorded in the gray matter of the spinal cord. The situation may be similar in the cerebral cortex. In Fig. 7, b' is a smoothed version of SP shifts often recorded in the center of the dorsal horn from an extracellular position. In the same figure, c' shows the typical timecourse of a sustained excitatory synaptic potential often recorded from neurones when a sensory nerve was stimulated tetanically; and a' shows what an electrode lodged in a glia cell usually records under similar circumstances. These curves (a', b'andc')represent slightly idealized versions of actual recordings. The curves of a" and c" are hypothetical: they show the postulated contribution of glia and neurones to extracellular potential, the sum of which yields the actual extracellular SP (shown as b'). It will be noticed that a' has two tracings. The broken line represents the hypothetical behavior of glia cells in isolation; the solid lines are similar to an actual recording, and contain the sui generis glial potential algebraically summed with the extracellular component of the neural extracellular contribution (shown as c'). The reason for this representation of glia is that phasic focal potentials were frequently registered by electrodes lodged inside unresponsive cells. The phasic potentials seemed to have been "seen" through the passive membrane, for they had the same polarity and general configuration in intracellular records as those recorded in an extracellular position (Somjen, 1970). Figure 8 is also a hypothetical construct. It shows in simplified form the extracellular currents associated with three types of electrical activity of cells in central gray matter, of

224

GEORGEG. SOMIEN A.

B.

C. < - :: -, c

FIo. 8. Schematic representation of different types of extracellular potential in CNS. A: When a ncxve ~ is born at the axon hillock, it ~ extracellular current from the cell soma, dendrites, and from d o ~ ~ of the axon. There is thus an intense localized sink, and weak d ~ sourc~ of current. B: If many neurones are aligmedsimilarly, and are ~ in ~ Synaptic currents, they can create "ope~" exLracdtular current fields, w ~ can be ~ over wide areas by microelcctrodes. ~ which are not in "register" (indicated in diagram by single neurone in bllckgrolmd) can distort such fl~Ids; and so could cells lying in parallel, but with ~ t e d excitatory and/or inhibitory s ~ distributed according to another pattvrn ovex their surface. C: The c o n t r i ~ o n of depolarizing gila cells to extracellular currents. It is assumed that gila ~ in ~ part of diagram lie near active neuronm, are exposed to a high ¢oncmtr~ion of K+, and therefore draw current from glia lying below in an inactive region. Not shown in diagram, but essential to theory, is the existence of an intracellular retm'n current path which must trave~,semany gila cells through i n ~ ~ junctions; or else it would have to go through very long, ~ oriented, filamentous glial processes attached to each cell. which the cerebral cortex is a good example. In Fig. 8A is shown the extracellular current field surrounding a neurone as it fires a spike potential at the axon hillock region. There is a strong "sink" into which current flows near the point of activity, and rather less dense sources above and below it. In this case an exploring microelectrode registers a potential deflection when placed close to the cell, but not at a distance, because of the steep attenuation of the electrical field as currents are diluted in the conducting volume. Spikes are like point-sources in infinite space. For that reason the intracellular-to-extracellular ratio of voltage amplitudes of spike potentials is close to 50:1. Figure 8n is an impressionistic rendering of t h e synaptic currents generated by a row o f similarly aligned neurones. The intracellularly recorded amplitude of synaptic potentials is smaller than that o f spikes, but the extracellular potential can b e a s large or larger, and m a y be detected over a wide area, provided that a s u l f ~ e n t number of cells is acting in synchrony. Synaptic potentials o f m a n y cells can sum readily both in time and in space, ~ a u s e they last longer and because they arise over a larger portion of the neuronal surface area than do spikes.

ELECTROOENF.SmOF SUSTAINEDPOTENTIALS

225

Figure 8c illustrates how glia cells could be effective sources of widespread extracellular potential fields of relatively large amplitude. If, as many believe, glia forms a latticework of like elements in which all other cells are embedded, and ff glia cells all undergo depolarizing potentials together, then they could well be the source of the uniform and sustained potential fields which we were discussing. This geometry is also compatible with the 2:1 intra/extracellular voltage ratio observed in spinal gila cells (Somjen, 1970; Strittmatter and Somjen, 1972 and in preparation; and Fig. 5B). A formal and precise statement of the conditions of such a distribution of currents and voltages will be found in the Appendix. To match theory with actual findings, the reader should compare Fig. 3B with Figs. 9 and ll. A three-dimensional electrotonic net is not the only geometrical arrangement by which glia could act in the manner suggested here. We mentioned already (Grossman's reference to Cajal's illustration: see p. 214) that the processes of glia cells may extend over sufficiently large distances to link electrically active with electrically inactive regions, thus creating gradients of voltage and current. Nor is excess K + an essential ingredient in the hypothesis of glial potential generators. The theory is believable because we know that K + is released by neurones not only during excitation, but probably also during post-synaptic inhibition (cf. Eccles, 1964). It also is true that glia cells are depolarized by elevated extracellular K +. Not known is whether the amount of K + normally spilt by nerve cells and fibers is sufficient to raise the extracellular concentration to the level which would cause a sensible change of membrane potential. If it was not K +, it could be some other chemical agent released by neurones. Krnjevi6 and Schwartz (1967) found that acetylcholine and gamma-amino butyric acid caused depolarization of some unresponsive cells of the cortex. Besides these compounds, there could be others as yet not discovered. The question has repeatedly been asked since the dawn of electrophysiology, whether extracellular currents flowing in central nervous tissue could have an influence on the activity of neurones in their path. The reason that we still don't know the answer, is the lack of quantitative information. Terzuolo and Bullock (1956) have derived a practical figure. They found that a voltage gradient of 1 mV/mm is sufficient to influence, either to facilitate or to inhibit, the rate of firing of an already excited neurone. Much more intensive gradients are needed to initiate firing of a quiescent cell. This gradient, 1 mV/mm, was certainly exceeded in the potentials recorded from the spinal cord (see Figs. 2 and 3B and Somjen, 1970). There are two difficulties which prevent us from giving an affirmative answer to the question posed above. First, Terzuolo and Bullock's (1956) observations were made on crustacean neurones in tissues prepared in vitro. Whether they are applicable to the mammalian CNS is uncertain, to say the least. Strumwasser and Rosenthal (1960) experimented with extracellular currents in frogs' brains but they measured currents, and it is difficult to translate their values into voltage gradients. Besides, the large potentials recorded in the spinal cord were evoked by massive tetanic stimulation of most of the branches of the sciatic nerve. Milder stimuli, approaching physiological levels, caused very much smaller shifts of potential. In the cortex, unlike in the cord, potential shifts greater than 1.0 or 1.5 mV do not seem to occur, except in spreading depression. It in fact is our impression (Rosenthal and Somjen, in preparation) that any SP shift in excess of about 1.5 mV inevitably is followed--sometimes after a delay of seconds--by spreading depression. This then raises the question contemplated ever since the phenomenon was discovered, whether the electrical currents

226

GEORGEG. SOMJEN

seen in spreading depression have, in addition to or instead of chemical factors, a role either in provoking or in propagating the process. The fact that cuts in the cortex are not crossed by the wave of depression (cf. Marshall, 1959) does not exclude such a mechanism, for currents do not flow through a gap in the tissue in the same way as they do among closely packed cells. In spite of the large shifts of potential (sometimes as high as 8 mV) I never observed spreading depression in the spinal cord, and to my knowledge no one else has either. The cord can undergo seizures, but spinal paroxysmal activity is not followed by protracted postconvulsive electrographic silence. Newborn animals (Bureg, 1957; Schadr, 1959) are similarly immune to spreading depression. If we could explain what protects the cord and the immature brain, we could be on our way to understanding the pathogenesis, and perhaps be in the position to prevent, clinical episodes such as postconcussion coma and perhaps migraine. Several authors, including Hydrn (1967a, b), Galambos (1961), Hertz (1966), and Roitbak (1970) have speculated about possible neurone-glia interactions. We shall not discuss these theories, except by pointing out that the exchange of ions and other chemical, and/or electrical interactions, are key features in some of them. So far as postulates of an effect of SP shifts on the excitability of neurones is concerned, the considerations and cautions raised above are valid, whether the postulated generators of the SP shifts are glia cells, or neurones

8. Sammm'y The weight of evidence, as of this date, is in favor of the hypothesis of a predominantly glial generation of the SP shifts which are evoked by repetitive stimulation of afferent nerves or of fiber tracts. Unlike electrically evoked SP shifts, the SP shifts associated with spreading depression are probably generated by massive depolarization of all cells, glia as well as neurones. For SP shifts related to more physiological process, such as the contingent negative variation, the SP shifts of positive reinforcement and the related "consummatory potential", the evidence is insufficient even for a tentative conclusion. SP shifts of varying intensity are associated with a proportional increase of oxidative energy turnover. During spreading depression the electron transport of the oxidative enzyme chain is accelerated well beyond the level observed in healthy cortex. The cause of spreading depression is not a shortage of the supply of oxidative energy. Dorsal root potentials do not significantly contribute to SP shifts of the spinal cord, nor do SP shifts appear to cause dorsal root potentials. There is no convincing reason at this time to suggest that under normal conditions neurones are influenced by sustained currents flowing in the extracellular medium. The more intense extraeellular currents occurring during pathological conditions may possibly contribute to spreading depression and/or other types of paroxysmal activity. More quantitative data are badly needed to decide these fundamental questions. The curious immunity of the spinal cord and of the immature brain to spreading depression challenges the ingenuity of the pathophysiologist.

227

ELECTROGENF~IS OF SUSTAINEDPO~h'TI~.S

9. Appendix. A Model Simulating the Hypothetical Contribution of Glia Cells to Extracellular Potentials R. JOYNER and G. SOMIEN 9.1 A S S U M P T I O N S This model was designed to enable the exploration o f the electrical properties o f a netw o r k o f interconnected cells, such as glia is assumed to be. It is intended to reproduce the essential features o f the t o p o l o g y o f glia tissue in a sufficiently realistic fashion to d e m o n strate the m a n n e r in which variations o f the c o m p o n e n t s affect the behavior o f the circuit as a whole. W e did not attempt, however, to approximate the absolute magnitudes o f resistances and voltages in neural tissue but aimed instead at exploring the effect o f variations o f the relative magnitudes o f the various parts o f the network. Simplification was essential in the design o f the model. As a first approximation we represented the postulated three-dimensional network o f electronically coupled glia cells by a single row o f simulated "cells". The cytoplasm o f each cell was assumed to be isoI

"possive cells"

VI,

i v

I

I .

"active ce[{s"-

Vlz

Vh...

VI{

~{+,...

REj VEZ

VE]"'

VEi

VEh,i...

J,

IJ'pcssi~ecells'~l

Vl..,

VT.

_J....:. 1

VE,.IRE.q VE.

VI - Inlernol polenliols; VE - Externol potenliols; AE =- Shiflsofmemi~ronepolenliol; Ri-=Membraneresislonce; RE---Exlracellulorresislonce; RX ---Coupling resislQncebetweencells.

VE,=0 /

b ...... ~"Introcellu]or" e ~

f

g

g

o

b

RM=RE=Rx=I

RM=RE=I

Rx=IO

Cells"oclive":5, Cells "possive ":16

RM=IO; RE=I ; RX=;' . . . . d Cells ochve : I

e 5

_f 9

~

_h

±

RM=I

RM=IO

RM=IO0

RE=I ; RX=2 ; Cells"aclive":5

FIo. 9. Model representing a row of glia cells connected by electrotonic junctions; and the distribution of voltages inside and outside each row of cells which would occur ff the membrane of some but not all of the "cells" were the source of depolarizing EMFs. The voltage profiles were generated by computer simulation On the electrical network (upper half of the figure) each point Vtj represents the interior of one gila ceil: the cytoplasm is assumed to remain isoelectric. Each point F~j represents the extracellular locus in the neighborhood of such gila cells. "Active" cells are undergoing depolarizing potential shifts, "passive" cells serve as return paths for the electrotonic spread of current. The curves of the lower half of this figure are simulations of the spatial distribution of potentials occurring at the height of ghal depolarization, such as have been observed in actual experiment, and are illustrated in Fig. 3B. Each point on the simulated curves corresponds to one of the points V of the electrical network. For further explanation and definitions, see text of the Appendix.

228

GEORGEG. SOM.IEN

potential, and was represented by a single "point" in the network. An electrical steady state (dV/dt = 0) was taken to exist, and capacitive elements were therefore ignored. This was considered permissible because SP shifts are very slow compared to the time constants of biological membranes. The "resting" membrane potential was also neglected and only departures from the resting level were represented, by "batteries" inserted between "intracellular" and "extracelIular" compartments of some of the "ceils". These batteries thus stand for the sustained depolarizations of glia ceils, which were described in the main part of this article. In the illustrations (Figs. 9, I0 and 1I), these depolarizations were taken to be identical in all activated ceils, but the model could easily be made to fit a situation where the depolarization of different ceils would be a function of time and/or location. Figure 9 depicts the simplest possible representation of our problem. The internal potentials of glia ceils (Vz) are thought to be coupled one to another by electrotonic junctions shown as resistances (Rx). Other resistances represent the cell membranes (RM) and the extracellular medium (Re). Some cells are, and others are not, assumed to undergo depolarization and hence some do and others do not have a battery (AE) in series with the membrane resistance. Current spreads in such a network from "activated" into "passive" ceils in a manner similar to that found in a cable of infinite length. The network thus has a "length constant" which is given by: Ru ;~ = R~ + R--------~"

(1)

9.2 METHOD We have not actually buiR physical models like the ones shown in Figs. 9 and I0, bu. solved the currents and voltages in such networks by simulation on a PDP-15 computert (Readers interested in the program should contact R. Joyner at the address of this article.) The solution is based on the assumption of current loops flowing in each completed component circuit of four resistors (RMj, REj_ t, RMj_ l, R~_i) of the network. These current loops can be expressed as a set of simultaneous equations as follows:

11 (2Ru -[- Rx + RE) + 12 (--Ru) ---- 0

(2)

I,_2 (--Ru) + Ij_l (2RM + Rx + RE) + I~ (--Ru) = E

(3)

Is-1 (--RM) -I- I s (2RM + Rx + RE) + Is+x (--RM) -~ 0

(4)

Ij (--RM) +- Ij+x (2RM + Rx + R,O + Ij+2 (--RM) = --E

(5)

In-2 (--RM) + I,,_~ (2RM + Rx + RE) --- 0

(6)

where I s = current loop in thejth component circuit (flowing in Rxj, Ruj+~, REt and Ruj), VE~ ---- the external potential at thejth cell, V~j ---- the internal potential at the jth cell, Ru = m e m b r a n e resistances, RE Rx

---- extracellular resistances o f extracellular fluid, ---- electrotonic coupling resistances between cells,

E

= depolarizing potential shifts of glia cells.

ELECTROGENESmoF SUSTAINEDP ~

229

After solving these equations [represented by eqns. (2)-(6)], one can compute the voltages at the various points along the "extracellular" and "intracellular" compartments by the recursive relationships: V~, = 0

(7)

v~, =

(8)

-/,R~

) o o

Vz, = --Ij_zRE + VE,_, and

Ij_,Rx + V&_,

V,, =

(9)

Figure 9 shows the network and some typical solutions. In this version of the model the "extracellular compartment" was "grounded" at one end, and at this point the potential was defined as zero. One of the consequences of this simplification was that the network was asymmetrical. In real nervous tissue extracellular fluid is not at ground potential, but could be thought of as being separated from ground by finite resistance. The model of Fig. 10A incorporates this added feature. Rr represents here the resistance of tissue to ground.

A. "passivecells'

I

VI~

I

~

~Uz... 'WWVREz...

RTt~ _ ~

I

I~passivecells~l

M

Rxf... '

RM~

"active cells"

l

Rx, i V

1

+Zm

~Tz .,

I

. . . .

--RE

....

= i

"Zntracellular" - Prone

~

~

~---1L'/"

i:

.:

_

• :~.~'~

.:

"Exll~l%CnelUl°r"

~'~~_: ~ Rx ~

~Grou.d"

Rx Rx

Rx

FIG. 10. A. Model network similar to the one in Fig. 4, except that the extracellular compartments are separated from ground by "tissue resistance" (RT). B: Network as in A, except that a two-dimensional sheet of simulated "cells" takes the place of the one-dimensional row. In this network two sets of current loops can flow, and two sets of simultaneous equations must be solved to find the currents and the voltages in it. One set is similar to the ones shown previously [see eqns. (2)-(6)], and may be written for the general case as follows:

230

GEORGEG. SOMJEN

/~-i (R~) + /j (2RM + Re + Rx) + /#+t (--RM) --- --It, (RE)

(I0)

The second set of equations takes the general form:

Irj_, (--Rr) +

ITj (2gr + RE) +

Irj+~ (--Rr) = --I# (RE)

(I1)

where lrj is the current flowing in the "external" component circuits (through Re and Rr). In principle the model could be modified to represent, instead of a row of cells, a twodimensional sheet of cells, or even a three-dimensional latticework. Figure 10a gives an impression of the representation of a two-dimensional array. The components of this circuit are identical with those of Fig. 10A. Clearly, computations needed to solve the voltages in such a network (Fig. 10B) would be much more complex than the previous two versions of the model. It seemed to us that not much more information could be expected from solving the network of Fig. 10B than was already available from Fig. 10A, and the illustration is shown here only to give an impression of the nature of such a multidimensional model.

9.3 RESULTS In Figs. 9 and 11 are shown some of the voltage profiles derived from the solutions of the equations presented above. For the sake of simplicity and clarity, in each example identical

&VARYING MEMBRANE RESISTANCE

B:VARYINGCOUPLING RESISTANCE b

" InltaceI lutar . , , , b

"Exlrocellulor"

O: RM:IO ,

,/ " * ' "

_

",,~

_o:

Rx: I

b

C:VARYING EXTRACELLULAR RESISTANCE

b "-'--"

D :VARYING TISSUE-TO-GROUND RESISTANCE

a_.:RE=4 :

:

~ f

a R.:O '\

G

FIo. 11. "Intracellulax" and "extraceUulax" voltage profiles INnexated by computer simulation o f the network shown in Fig. 10A. In ~ cases 21 "cells" are represented, of which the central 5 are considered to be "activated" and the other 16 to be "passive". The values o f those n ~ , t a a c e s which are not shown on the labelins o f the i l l . r e above ate as follows: i n A : R~ --- 8, Rx = 1, R~ = 1; in B: R u = 10, Rr ~- 8, RB = 1; in C: R u = 10, Rr ---- 8, Rx = 1; and i n D : R u = 10, R x = 2, Re ----- 1. The horizontal lines of the ~ ~ t zero potential ( = ground potential). See ~ lelNnd o f Fig. 9.

ELECTROGE~ OF SUSTAINEDPOTENTIALS

231

components were used to simulate each individual "cell", except that some did and some did not have a battery. That varying the number of "activated" cells (i.e. the number of batteries) will alter the distribution of the voltage in the network, is hardly surprising (Fig. 9, below center). It is interesting to compare the voltage profiles of Figs. 9 and 11. The presence of tissueto-ground resistances (R~, in Fig. 10) raises part of the extracellular voltages above ground (i.e. to a positive level, see Fig. 11), which feature is missing from Fig. 9. This voltage drop is, of course, caused by current flow 'in RT. In this way the familiar "source" to "sink" flow of current becomes simulated by the model. Clearly, if extracellular fluid were grounded throughout its extension (Rr = 0), then its voltage would be forced to zero (Fig. 1IDa). With increasing tissue resistance (R~.) the extracellular potentials increase and, to a lesser degree, the intracellular voltages fall in the activated but rise in the inactive regions (Fig. 11I)). We mentioned already that varying the membrane resistances of the component cells (RM) has the effect of varying the "length constant" of the network (eqn. 1). Raising RM causes the voltages to spread farther, but at the expense of reducing both the intraceUular and extracellular voltages of the "active" region (Fig. 9, lower right-hand diagram, and Fig. 11A). If varying RM influences the voltages of intra- and extracellular compartments in the same sense, varying the coupling resistances (Rx) has effects on V1 which are opposite to those exerted on Ve. The lower the coupling resistances, the greater the voltages in the extracellular compartment, and also the farther spread the currents; but reducing Rx has also the effect of lowering intracellular voltages. Conversely, increasing the value of Rx depresses the levels of Ve (Fig. 9, left-hand diagram and Fig. 11B). If cells were insulated one from the other (Rx = oo), no current could flow in the extraceUular medium and there would be no extracellular potential gradient. It follows that, in order to generate extracellular current, the intracellular potential shifts must be reduced, for under these circumstances there exists a "shunt" from "activated" to "inactive" cells. Varying the resistances of the extracellular medium (Re) has effects which are the opposite of the ones caused by variation of the coupling resistances (Fig. 1lc). Variations of RE are obviously felt more strongly in the extracellular compartment than in the intracellular compartment. But as a working simplification one may state that the ratio of intracellular over extracellular potential shifts is influenced most strongly by the ratio of coupling resistances over extracellular resistances.

Notes Added in Proof 1. After completing the manuscript, my attention was called to recent work in four laboratories, demonstrating a rise in extracellular K + concentration in CNS tissue evoked by the kind of stimulation which also evokes SP shifts. All four teams cf workers used, each independently from the others, ion-selective glass electrodes to measure the K + levels. The references are: L. Vyklick37, E. Sykov,'i, N. K ~ and E. Ujec: Post-stimulation changes of extracellular potassium concentration in the spinal cord of the rat, Brain Res. 45: 608-611, 1972; D. A. Prince and H. D. Lux: Measurement of extracellular potassium activity in cat cortex, Brain Res., in press; K. Krnjevi6 and M. E. Morris, ExtraceUular K + activity and slow potential changes in spinal cord and medulla, Can. J. Physiol. Pharmacol., in press; and R. G. Grossman and Hubschmann (personal communication).

232

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Krnjevi~ and Morris actually demonstrated a correlation between SP shift and increment of the logarithm of the K + concentration. 2. Discussion of an article by R. M. Lebovitz: A theoretical examination of ionic interactions between neural and non-neural membranes, Biophys. J. 10, 423-444, 1970, was inadvertently and regrettably omitted from my review. 3. With reference to Bourke's theory of spreading depression (see p. 221) the reader should consult, in addition to the article quoted, R. S. Bourke, K. M. Nelson, R. A. Nauman and O. M. Young: Studies of the production and subsequent reduction of swelling in primate cerebral cortex under isosmotic conditions in vivo. Exper. Brain Res. 10, 427-446, 1970. 4. An article by W. Strittmatter and G. Somjen: Depression of sustained evoked potentials and of glial depolarization in the spinal cord by barbiturates and by diphenylhydantoin, quoted as "in preparation" in this review, is now "in press" in Brain Res,

Acknowledgements Thanks are due to Mrs. R. Hougom for typing the manuscript. Work reported here for the first time was sponsored by P.H.S. grant number NS 05330, with additional support by the United Health Services of North Carolina; and by P.H.S. grant NS 06729 to Dr. F. J6bsis, and GM 16718 to Dr. J. Moore.

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