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The glia-neuronal interaction: Some observations
J. psycbiot. Res., 1971, Vol. 8, pp. 219-224.
Pergamon Press. Printed in Great Britain.
THE GLIA-NEURONAL
INTERACTION:
SOME OBSERVATIONS ROBERTGALAMEOS Department of Neurosciences,University of California at San Diego, La Jolla, California
UNTIL some 10 years ago, the functions performed by glial cells in the brain and peripheral nerves lay almost entirely in the realm of speculation. As an example, CAJAL,in an early paper entitled “Anatomical Mechanisms of Thought, Association and Attention”l attributed two different roles to the cortical glia, each dependent upon the motility of glial processes which his extensive microscopic examinations of stained material seemed to him to support. He supposed, on the one hand, that glial processes might insinuate themselves between pre- and post-synaptic elements and by impeding the flow of ‘nervous currents’ there produce mental relaxation and natural or induced sleep. Retraction of the neuroglial pseudopods would allow reestablishment of the synapse and “the brain would pass from the relaxed to the active stage.” These glial contractions, which might occur “automatically or by an act of will” in his scheme, could direct associative processes in specific directions. “The unpredictable turns that associations sometimes take-the fading away of ideas and words, the momentary halting of speech, the obsessive persistence of memory, the repression of an idea or experience as well as all types of erroneous motor reactions and other psychological phenomena can be understood . . . by supposing the neuroglia of the gray matter serve as an insulating and switching mechanism for nervous currents, permitting connections when they are active, and acting as insulators during repose.” His second hypothesis holds that when the perivascular astrocytes contracted, their endfeet attached to the walls of brain capillaries would pull on the wall of the vessel, enlarge its lumen, and thus increase local blood flow. To explain attention, he hypothesized that such contractions were voluntarily initiated in the cortical zone appropriate for the perceptions or recollections in order to produce and sustain the increased metabolism there. Apparently these ideas soon lost their appeal for CAJALsince he does not mention them in the two volumes on neuroanatomy that insure for him a permanent place in the history of science.2 Though SCHLEICH~ restates the glial switchboard idea in a fanciful way, neither of Cajal’s ideas seems to have received much currency nor, to my knowledge has either of them been directly tested experimentally. This despite Cajal’s statement about them at the end of his paper: “Needless to point out, a hypothesis represents a new path, opened up by experiments and observations, and even if it does not immediately reveal the truth, it 219
220
ROBERTGALAMB~~
always leads to investigations and criticisms that bring us nearer to it. Our future investigations may not cotirm these hypotheses, but their outcomes will provide valuable information. Negative findings will limit the number of tenable hypotheses and reduce the possibility of making unproductive investigations in the future.” As of 10 years ago, the glial were generally supposed to function as nutritive and supportive cells in the normal brain, and to proliferate to repair it after damage. Recent solid data about glia morphology and glia physiology generally support these ideas and put them in a realistic framework (see, for example, F~~EDE,~KUFFLER.~)These data include biochemical, microscopic and electrophysiological information that clarifies both the specialized features of the cells themselves and the interactions that presumably go on among them, and between them and the neurons with which they are so intimately associated. The remainder of this paper will examine a few of these new facts. In a review as brief as this, the developing body of information on macromolecular synthetic activities of glia must be given short shrift. The pioneer, and still the principal student of this problem, is Holger Hyden. Using micro techniques developed for the assay of quantities of material contained in a few nerve or glia cells, he has developed a picture of the ebb and flow of RNA, enzyme and protein synthesis in these cells during functional activities of animals such as rats and rabbits. Evidence for altered synthesis in glia has, for instance, been correlated with disease in man in a study of the glia from patients with basal ganglia disorders (GOMIRATO and HYDEN~), with sleep in the rabbit (HAMEZRGERetuL7), and with learning in the rat (HYDEN and EGYHAZI~). From similar analyses made upon the neurons associated with these glia, some details of the active chemical interactions going on in what he calls the glia-neuron functional unit are beginning to emerge (HYDEN~*~O). In an otherwise excellent review (KUFFLER and NICHOLLS~~),Hyden’s
attempts to map out
the special biochemical properties of glia in the functioning brain receive, in this writer’s opinion, unnecessarily harsh treatment. At the light microscope level direct evidence for an active proliferation of glial cells during function has been provided by several studies. For example, both ALTMAN and DAP and DIAMOND et al.13 count more glial nuclei in the somewhat thicker cortex of rats raised
communally in enriched, challenging environments than in the cortex of littermate controls raised alone under conditions where there is little for them to do and experience. The implication of these studies, that a relationship exists between glial multiplication and the functional requirements of nerve cells, has been tested in other kinds of experiments. MIJRRAY,~~ for instance reports that dehydration in the rat stimulates hypothalamic glial proliferation only in the supraoptic nucleus (100 per cent in two weeks) and in the posterior pituitary (about 25 per cent), which is to say, in precisely the regions predicted from other evidence to be stimulated into activity by dehydration. From such studies one can assert that, in some cases at least, one result of the glial-neuronal interactions taking place during increased functional activity is the stimulation of glial mitoses. Why more neuronal activity should require the increased glial metabolism implied by an increase in cell number is an unanswered question (but see FRIEDE~). Thanks to the electron microscope the cytoplasmic characteristics of the different types of glia and the varied contacts their membranes make with one another and with the neurons they attend have been greatly clarified (e.g. GFCAY,~~MUGNAINI and WALBERG’~
THE GLIA-NEURONAL INTERACDON: SOMEOBSERVATIONS
221
and others). It is by now an old story pictured in the textbooks that peripheral Schwann cells and central oligodendroglia invest their axons and create the myelin sheath around them (see S&n&’ for a discussion of this biosynthetic event). Without this sheath the rapid saltatory conduction upon which central integration depends is impossible, as is so evident in a disease like multiple sclerosis where the sheath is abnormal or absent. The increased transverse resistance offered by the myelin sheath provides the basis for this saltatory conduction, namely, depolarization at the nodes only. Here at least is a clear case where the two cells must work together to produce a functionally important end product. Not all axons are myelinated, however, and the ones with and without myelin both display extensive glia-neuron membrane contacts where functional interactions might occur. What transpires across the membrane of an unmyelinated peripheral nerve fiber which is everywhere surrounded by the membrane of a Schwann cell (CAUSEY~*)? Myelinated axons possess this same intimate membrane-to-membrane contact, and therefore, if an interesting interaction takes place in the unmyelinated fiber it may do so in the myelinated form as well. Other curious details to which no functional significance has yet been attached include the complete absence of glial cytoplasm at central nodes and its presence in peripheral ones, and the investment of some cell bodies in the CNS by astrocytic processes and of others by oligodendroglial membrane (PALAY~~). Turning now to the contacts between glial cells (gliapses; GALAMBO$~), it is clear that specialized regions called gap junctions abound in the nervous systems of many animals (MUGNAINI and WALBERG,l6 BRIGHTMAN and REESE~~).Gap junctions differ from synaptic, desmosomal and tight junctions in being limited to glial membranes, in having five layers with a space of 20-3OA intervening between the middle pair, and in providing the most likely anatomical substratum for the low-resistance pathway needed to explain the easy passage of electrical currents between one glial cell and its neighbor. If these gap junctions should indeed be areas through which ions pass freely, their number and distribution in a particular volume of brain would determine how much, and in what direction, any currents generated by neuronal activity would flow. We shall return to this matter shortly. Another morphological specialization of glia is revealed after serial reconstructions of electron microscopic pictures of such regions as the lateral geniculate (COLONNIER and GUILLERY~~)and the granular layer of the cerebellum (ECCLES et ~1.~~). In these structures, limited collections of pre- and post-synaptic elements turn out to be completely invested by sheets of glial membrane. Such synaptic regions may lie tightly packed beside one another, each wrapped into a kind of package by the thin glial processes. It has been speculated that this glial covering, like the shell of an egg, protects what is inside from undesirable outside influences or, like the walls of a stove, prevents what goes on inside from spreading into the surroundings. The many important electrophysiological observations upon glia made by the group associated with Kuffler are summarized in the review already cited (KU~LER and NICHOLLS~~). Using microelectrode methods on the leech and mudpuppies-forms with especially large cells that simplify intracellular recording-they have shown the properties of glial membranes to differ from those of neurons in four ways important for understanding the reciprocal interactions between the two cell types. First, glial membranes show graded
222
ROBERT GALAMBOS
depolarizations, never spikes. Second, their resting potential (regularly 90 mV as opposed to 70 in the neurons) is remarkably sensitive to external K+ concentration, responding to increases by depolarizing in a manner almost exactly predicted for a K+ electrode by the Nernst equation. Third, the glial membrane depolarizes as impulses traverse adjacent axons in just the manner, and to the degree that would be predicted by the release of K+ from the neuron during the impulse. Fourth, experimentally injected current passes freely from one glial cell to the next, presumably by way of the gap junctions already discussed. These facts, derived from a series of elegant experiments that is still in progress (e.g. NICHOLLS and BAYLOR~~), have led to the following important new generalization about glia. During normal neuronal activity, the K’ released by neuronal activity into the extracellular space produces a drop in glial membrane potential (proportional to log K+ concentration) at that site. Current then flows to this region from undisturbed membranes via the extracellular space and the glial cytoplasm until the K+ concentration returns to its resting level. The time course, as well as the metabolic and electrical consequences of this dynamic interaction are questions for which answers are being sought in several laboratories. For instance, electrical currents generated by such glial depolarizations should produce recordable potentials at a distance from the site of their initiation. Evidence that the well-known negative afterpotential recorded from the surface of mixed nerves originates from this source has in fact been assembled (ORKANDet dz5). The question of whether some unknown fraction of the voltages recorded from the surface of the brain arises in a similar manner has been raised, (ORKAND,26 POLLEN~~)and recordings made from the so-called idle or silent cells in cat cortex contribute to the answer. These silent cells, identified by dye-marking as glia, resemble the glia or lower forms in several ways: their membrane resting potentials range up to 90 mV; they are electrically inexcitable; and they depolarize when neurons are active nearby. Reasonable values for the speed, magnitude and extent of K+ movement across their membranes in cat cortex have been established and interpreted as showing the glia to regulate K+ concentration at synaptic regions there (TRACHTENBERG and POLLEN*~) as they do in invertebrate ganglia (NICHOLLSand BAYLOR~~). If such regulation of K+ in the synaptic environment is indeed a major function of the glia, then the glia found in cortical scars might be unable to perform this function; POLLEN and TRACHTENBERG~~ have suggested the essential lesion of focal epilepsy in man to be just this failure to buffer extracellular K+ concentration normally. Depolarizations in the cortical silent cells accompany spontaneous brainwave spindles as well as the neuronal activity evoked by either thalamic or direct cortical stimulation. These responses seem in all important ways analogous to the glial responses of lower forms. Several experimental observations have provided information on the possible contribution of these glial depolarizations to the slow waves recordable with large electrodes from brain and elsewhere. The most recent of them (CASTELLUCCI and GOLDRING~~) calls attention to the striking resemblance between the silent-cell depolarizations and certain steady potential shifts recorded from the cortical surface during seizure discharge and brain stimulation. The authors suggest “ . . . that glia lying closest to a focus of neuronal activity in the cerebral cortex become depolarized by local release of K+ and draw current from neighbouring ‘source’ glia to which electrical linkage occurs in all directions.” It seems likely that the possibility of a glial contribution to all the various spontaneous and evoked
THE GLIA-NEURONAL INTERACTION : SOMEOBSERVATIONS
223
electrical responses recordable from nervous tissue will in time be directly tested. It has already been claimed that the b-wave of the electroretinogram, an event long considered to be a manifestation of neuronal acitivity, is due solely to depolarization of the Mtiller fiber, a glial cell in the retina (MILLER and DOWLING~~). The experiments of WALKER and HILD~~ on mammalian brain cells in culture show low-resistance junctions to exist via glia over distances up to 200 pm. Surprisingly, a similar electrical coupling seems to exist between these glia and certain neurons in the culture which are incapable of action potentials and in still other ways display glia-like properties. Such studies with cultured cells also provide opportunities for verifying and testing the variety of current hypotheses of glial function. It has been shown, for instance, that the impedance of glial clumps is altered by pharmacologically active molecules in the bath (WALKER and TAKENAKA~~), that enzyme activity increases in astrocytes after Na+ (but not K+) additions to the medium (FRIEDE~~), and that 0, consumption of glial clumps, but not of nerve cells, declines rapidly in Na+-free medium and rises two or three-fold when K+ is added (HERTz~~). Such experiments validate evidence available from the electrophysiologists on the sensitivity of glia to K+ ions, suggest the wide range of metabolic activities that could be initiated in glia by activity of adjacent neurons, and point to the possibility that glia may respond selectively to biologically important molecules. On this last point, ROITBAK,~~ working with cat cortex, has shown that morphine and other analgesics reduce or abolish potentials that seem to originate in glial depolarizations; hypothesizes on the basis of this evidence that the glia are related to the mechanism pain since drugs that relieve pain act upon them.
he of
The studies epitomized above illustrate some of the new hypotheses about glia and new experiments upon them which Cajal encouraged 75 years ago. These ideas and experiments have their origins in the inclusive biochemical generalizations, insights into fine structure provided by the electron microscope, and electrophysiological data that have developed during David Rioch’s lifetime of devotion to the nervous system. He has seen the view of nervous system function shift from that of all-or-nothing, purely neuronal interactions to the current conception where neurons and glia cooperate to store and deliver the variety of actions which nervous systems produce. If, like Cajal, he applauds what is heuristic in hypothesis, he may well accept the glia-neuron hypothesis, for it has already led to many new and surprising observations on the nervous system.
REFERENCES 1.
CAJAL,S. R. Algunas conjeturas sobre el mecanismo anatomic0 de la idecion asociacion y atencion. Revta
Med.
Cirug. prdct.
36, 497, 1895.
CAJAL,S. R. Histologic du Systeme Nerveux de I’Homme et des Vertebres, Maloine, Paris, 2 ~01s. 1909, 1911. Reprinted, Madrid: Consejo Superior de Investigaciones Cientificas, Vol. l-1952, Vol. 11-1955. 3. SCHLEICH,C. L. Vom Schaltwerk der Gedanken. S. Fischer Verlag, Berlin, 1918. 4. FRIEDE, R. L. Enzyme histochemistry of neuroglia. In: Progress In Brain Research, Biology of Neuroglia, DE ROBERTIS,E. D. and CARREA,R. (Eds.), Vol. 15, p. 35, 1965. 5. KUFFLER,S. W. Neuroglial cells: physiological properties and a potassium mediated effect of neuronal activity on the glial membrane potential. Proc. R. Sot. B. 168, 1, 1967. 6. GOMIRATO, G. and HYDEN,H. A biochemical glia error in the Parkinson disease. Brain 86,773, 1963. 2.
224
7. 8. 9. 10. 11. 12. 13. 14.
15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.
28. 29. 30. 31. 32. 33. 34. 35. 36.
ROBERTGALAMBOS HAMBERGER,A., HYDEN, H. and LANG, P. W. Enzyme changes in neurons and glia during barbiturate sleep. Science 151, 1394, 1966. HYDEN, H. and EGYHAZI, E. Glial RNA changes during a learning experiment in rats. Proc. nutn. Acad. Sci. U.S.A. 49, 618, 1963. HYDEN, H. Biochemical changes accompanying learning. In: The Neurosciences, QUARTON,G. C., MELNECHUK,T., SCHMITT,F. 0. (Eds.), p. 765. The Rockerfeller University Press, New York, 1967. HYDEN, H. RNA in brain cells. In: The Neurosciences, QUARTON,G. C., MELNECHUK,T., SCHMFIT, F. 0. (Eds.), p. 248. The Rockefeller University Press, New York, 1967. KUFFLER, S. W. and NICHOLLS,J. G. The Physiology of Neuroglial Cells. Ergebn. Physiol. 57,1,1966. ALTMAN, J. and DAS, G. D. Autoradiographic examination of the effects of enriched environment on the rate of glial multiplication in the adult rat brain. Nature 204, 1161, 1964. DIAMOND,M. C., LAW, F., RHODES, H., LINDNER,B., ROZENSWEIG,M. R., KRECH, D., and BENNETT E. L. Increases in cortical depth and glia numbers in rats subjected to enriched environment. J. camp. Neurol. 128, 117, 1966. MURRAY, M. Effects of dehydration on the rate of proliferation of hypothalamic neuroglia cells. Expl. Neurol. 20,460, 1968. GRAY, E. G. Ultra-Structure of synapses of the cerebral cortex and of certain specializations of neuroglial membranes. In: Electron Microscopy In Anatomy, BOYD,J. D., JOHNSON,F. R., LEVER, J. D. (Eds.), p. 54. Edward Arnold, London, 1961. MUGNAINI, E. and WALBERG,F. Ultrastructure of Neuroglia. Ergebn. Anut. EntwGesch. 37, 194, 1964. Shah, M. E. The metabolism of myelin lipids. Adv. Lipid Res. 5,241, 1967. CAUSEY,G. The Cell of Schwunn. E. & S. Livingston, Edinburgh and London, 1960. PALAY, S. L. Morphology of neuroglial cells. In: Basic Mechanisms of the Epilepsies, JASPER,H. H., WARD, A. A. and POPE, A. (Eds.), p. 747. Little, Brown, Boston, 1969. GALAMBOS,R. A glia-neural theory of brain function. Proc. natn. Acad. Sci. U.S.A. 47, 129, 1961. BRIGHTMAN,M. W. and REESE, T. S. Junctions between intimately apposed cell membranes in the vertebrate brain. J. Cell BioZ. 40, 648, 1969. COLONNIER, M. and GUILLERY, R. W. Synaptic Organization in the Lateral Geniculate Nucleus of the Monkey. Z. Zellforsch. mikrosk. Anat. 62, 333, 1964. ECCLES, J. C., ITO, M. and SZENTAGOTHAI,J. The CerebelIum as a Neuronal Machine. SpringerVerlag, New York, 1967. NICHOLLS, J. G. and BAYLOR, D. A. Long lasting hyperpolarization after activity of neurons in leech central nervous system. Science 162,279, 1968. ORKAND, R. K., NICHOLLS,J. G. and KUFFLER, S. W. Effect of nerve impulses on the membrane potential of glial cells in the central nervous system of amphibia. J. Neurophysiol. 29, 788, 1966. ORKAND, R. K. Neuroglial-neuronal interactions. In: Basic Mechanisms of the Epilepsies, JASPER, H. H., WARD, A. A. and POPE, A. (Eds.), p. 737. Little, Brown, Boston, 1969. POLLEN, D. A. Discussion on the generation of neocortical potentials. In: Basic Mechanisms of the Epilepsies, JASPER, H. H., WARD, A. A. and POPE, A. (Eds.), p. 411. Little, Brown, Boston, 1969. TRACHTENBERG,M. C. and POLLEN, D. A. Neuroglia: biophysical properties and physiologic function. Science 167, 1248, 1970. POLLEN, D. A. and TRACHTENBERG,M. C. Neuroglia: gliosis and focal epilepsy. Science 167, 1252, 1970. CASTELLUCCI,V. F. and GOLDRING,S. Contribution to steady potential shifts of slow depolarization in cells presumed to be glia. Electroenceph. clin. Neurophysiol. 28, 109, 1970. MILLER, R. F. and DOWLING, J. E. intracellular responses of the Mhller (glial) cells of mudpuppy retina: their relation to b-wave of the electroretinogram. J. Neurophysiof. 33, 323, 1970. WALKER, F. and HILD, W. J. Neuroglia electrically coupled to neurons. Science 165, 602, 1969. WALKER, F. D. and TAKENAKA, T. Electric impedance of neuroglia in vitro. Expl. Neurol. 11, 277, 1965. FRIEDE, R. L. The enzymatic response of astrocytes to various ions in vitro. J. Cell Biol. 20, 5, 1964. HERTZ, L. Neuroglial localization of potassium and sodium effects on respiration in brain. J. Neurochem. 13, 1373, 1966. ROITBAK,A. I. Further analysis of slow surface-negative potentials of the cortex: action of X-rays and analgesics. Acta Biol. exp., Vars. 29, 125, 1969.
Pergamon Press. Printed in Great Britain.
THE GLIA-NEURONAL
INTERACTION:
SOME OBSERVATIONS ROBERTGALAMEOS Department of Neurosciences,University of California at San Diego, La Jolla, California
UNTIL some 10 years ago, the functions performed by glial cells in the brain and peripheral nerves lay almost entirely in the realm of speculation. As an example, CAJAL,in an early paper entitled “Anatomical Mechanisms of Thought, Association and Attention”l attributed two different roles to the cortical glia, each dependent upon the motility of glial processes which his extensive microscopic examinations of stained material seemed to him to support. He supposed, on the one hand, that glial processes might insinuate themselves between pre- and post-synaptic elements and by impeding the flow of ‘nervous currents’ there produce mental relaxation and natural or induced sleep. Retraction of the neuroglial pseudopods would allow reestablishment of the synapse and “the brain would pass from the relaxed to the active stage.” These glial contractions, which might occur “automatically or by an act of will” in his scheme, could direct associative processes in specific directions. “The unpredictable turns that associations sometimes take-the fading away of ideas and words, the momentary halting of speech, the obsessive persistence of memory, the repression of an idea or experience as well as all types of erroneous motor reactions and other psychological phenomena can be understood . . . by supposing the neuroglia of the gray matter serve as an insulating and switching mechanism for nervous currents, permitting connections when they are active, and acting as insulators during repose.” His second hypothesis holds that when the perivascular astrocytes contracted, their endfeet attached to the walls of brain capillaries would pull on the wall of the vessel, enlarge its lumen, and thus increase local blood flow. To explain attention, he hypothesized that such contractions were voluntarily initiated in the cortical zone appropriate for the perceptions or recollections in order to produce and sustain the increased metabolism there. Apparently these ideas soon lost their appeal for CAJALsince he does not mention them in the two volumes on neuroanatomy that insure for him a permanent place in the history of science.2 Though SCHLEICH~ restates the glial switchboard idea in a fanciful way, neither of Cajal’s ideas seems to have received much currency nor, to my knowledge has either of them been directly tested experimentally. This despite Cajal’s statement about them at the end of his paper: “Needless to point out, a hypothesis represents a new path, opened up by experiments and observations, and even if it does not immediately reveal the truth, it 219
220
ROBERTGALAMB~~
always leads to investigations and criticisms that bring us nearer to it. Our future investigations may not cotirm these hypotheses, but their outcomes will provide valuable information. Negative findings will limit the number of tenable hypotheses and reduce the possibility of making unproductive investigations in the future.” As of 10 years ago, the glial were generally supposed to function as nutritive and supportive cells in the normal brain, and to proliferate to repair it after damage. Recent solid data about glia morphology and glia physiology generally support these ideas and put them in a realistic framework (see, for example, F~~EDE,~KUFFLER.~)These data include biochemical, microscopic and electrophysiological information that clarifies both the specialized features of the cells themselves and the interactions that presumably go on among them, and between them and the neurons with which they are so intimately associated. The remainder of this paper will examine a few of these new facts. In a review as brief as this, the developing body of information on macromolecular synthetic activities of glia must be given short shrift. The pioneer, and still the principal student of this problem, is Holger Hyden. Using micro techniques developed for the assay of quantities of material contained in a few nerve or glia cells, he has developed a picture of the ebb and flow of RNA, enzyme and protein synthesis in these cells during functional activities of animals such as rats and rabbits. Evidence for altered synthesis in glia has, for instance, been correlated with disease in man in a study of the glia from patients with basal ganglia disorders (GOMIRATO and HYDEN~), with sleep in the rabbit (HAMEZRGERetuL7), and with learning in the rat (HYDEN and EGYHAZI~). From similar analyses made upon the neurons associated with these glia, some details of the active chemical interactions going on in what he calls the glia-neuron functional unit are beginning to emerge (HYDEN~*~O). In an otherwise excellent review (KUFFLER and NICHOLLS~~),Hyden’s
attempts to map out
the special biochemical properties of glia in the functioning brain receive, in this writer’s opinion, unnecessarily harsh treatment. At the light microscope level direct evidence for an active proliferation of glial cells during function has been provided by several studies. For example, both ALTMAN and DAP and DIAMOND et al.13 count more glial nuclei in the somewhat thicker cortex of rats raised
communally in enriched, challenging environments than in the cortex of littermate controls raised alone under conditions where there is little for them to do and experience. The implication of these studies, that a relationship exists between glial multiplication and the functional requirements of nerve cells, has been tested in other kinds of experiments. MIJRRAY,~~ for instance reports that dehydration in the rat stimulates hypothalamic glial proliferation only in the supraoptic nucleus (100 per cent in two weeks) and in the posterior pituitary (about 25 per cent), which is to say, in precisely the regions predicted from other evidence to be stimulated into activity by dehydration. From such studies one can assert that, in some cases at least, one result of the glial-neuronal interactions taking place during increased functional activity is the stimulation of glial mitoses. Why more neuronal activity should require the increased glial metabolism implied by an increase in cell number is an unanswered question (but see FRIEDE~). Thanks to the electron microscope the cytoplasmic characteristics of the different types of glia and the varied contacts their membranes make with one another and with the neurons they attend have been greatly clarified (e.g. GFCAY,~~MUGNAINI and WALBERG’~
THE GLIA-NEURONAL INTERACDON: SOMEOBSERVATIONS
221
and others). It is by now an old story pictured in the textbooks that peripheral Schwann cells and central oligodendroglia invest their axons and create the myelin sheath around them (see S&n&’ for a discussion of this biosynthetic event). Without this sheath the rapid saltatory conduction upon which central integration depends is impossible, as is so evident in a disease like multiple sclerosis where the sheath is abnormal or absent. The increased transverse resistance offered by the myelin sheath provides the basis for this saltatory conduction, namely, depolarization at the nodes only. Here at least is a clear case where the two cells must work together to produce a functionally important end product. Not all axons are myelinated, however, and the ones with and without myelin both display extensive glia-neuron membrane contacts where functional interactions might occur. What transpires across the membrane of an unmyelinated peripheral nerve fiber which is everywhere surrounded by the membrane of a Schwann cell (CAUSEY~*)? Myelinated axons possess this same intimate membrane-to-membrane contact, and therefore, if an interesting interaction takes place in the unmyelinated fiber it may do so in the myelinated form as well. Other curious details to which no functional significance has yet been attached include the complete absence of glial cytoplasm at central nodes and its presence in peripheral ones, and the investment of some cell bodies in the CNS by astrocytic processes and of others by oligodendroglial membrane (PALAY~~). Turning now to the contacts between glial cells (gliapses; GALAMBO$~), it is clear that specialized regions called gap junctions abound in the nervous systems of many animals (MUGNAINI and WALBERG,l6 BRIGHTMAN and REESE~~).Gap junctions differ from synaptic, desmosomal and tight junctions in being limited to glial membranes, in having five layers with a space of 20-3OA intervening between the middle pair, and in providing the most likely anatomical substratum for the low-resistance pathway needed to explain the easy passage of electrical currents between one glial cell and its neighbor. If these gap junctions should indeed be areas through which ions pass freely, their number and distribution in a particular volume of brain would determine how much, and in what direction, any currents generated by neuronal activity would flow. We shall return to this matter shortly. Another morphological specialization of glia is revealed after serial reconstructions of electron microscopic pictures of such regions as the lateral geniculate (COLONNIER and GUILLERY~~)and the granular layer of the cerebellum (ECCLES et ~1.~~). In these structures, limited collections of pre- and post-synaptic elements turn out to be completely invested by sheets of glial membrane. Such synaptic regions may lie tightly packed beside one another, each wrapped into a kind of package by the thin glial processes. It has been speculated that this glial covering, like the shell of an egg, protects what is inside from undesirable outside influences or, like the walls of a stove, prevents what goes on inside from spreading into the surroundings. The many important electrophysiological observations upon glia made by the group associated with Kuffler are summarized in the review already cited (KU~LER and NICHOLLS~~). Using microelectrode methods on the leech and mudpuppies-forms with especially large cells that simplify intracellular recording-they have shown the properties of glial membranes to differ from those of neurons in four ways important for understanding the reciprocal interactions between the two cell types. First, glial membranes show graded
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ROBERT GALAMBOS
depolarizations, never spikes. Second, their resting potential (regularly 90 mV as opposed to 70 in the neurons) is remarkably sensitive to external K+ concentration, responding to increases by depolarizing in a manner almost exactly predicted for a K+ electrode by the Nernst equation. Third, the glial membrane depolarizes as impulses traverse adjacent axons in just the manner, and to the degree that would be predicted by the release of K+ from the neuron during the impulse. Fourth, experimentally injected current passes freely from one glial cell to the next, presumably by way of the gap junctions already discussed. These facts, derived from a series of elegant experiments that is still in progress (e.g. NICHOLLS and BAYLOR~~), have led to the following important new generalization about glia. During normal neuronal activity, the K’ released by neuronal activity into the extracellular space produces a drop in glial membrane potential (proportional to log K+ concentration) at that site. Current then flows to this region from undisturbed membranes via the extracellular space and the glial cytoplasm until the K+ concentration returns to its resting level. The time course, as well as the metabolic and electrical consequences of this dynamic interaction are questions for which answers are being sought in several laboratories. For instance, electrical currents generated by such glial depolarizations should produce recordable potentials at a distance from the site of their initiation. Evidence that the well-known negative afterpotential recorded from the surface of mixed nerves originates from this source has in fact been assembled (ORKANDet dz5). The question of whether some unknown fraction of the voltages recorded from the surface of the brain arises in a similar manner has been raised, (ORKAND,26 POLLEN~~)and recordings made from the so-called idle or silent cells in cat cortex contribute to the answer. These silent cells, identified by dye-marking as glia, resemble the glia or lower forms in several ways: their membrane resting potentials range up to 90 mV; they are electrically inexcitable; and they depolarize when neurons are active nearby. Reasonable values for the speed, magnitude and extent of K+ movement across their membranes in cat cortex have been established and interpreted as showing the glia to regulate K+ concentration at synaptic regions there (TRACHTENBERG and POLLEN*~) as they do in invertebrate ganglia (NICHOLLSand BAYLOR~~). If such regulation of K+ in the synaptic environment is indeed a major function of the glia, then the glia found in cortical scars might be unable to perform this function; POLLEN and TRACHTENBERG~~ have suggested the essential lesion of focal epilepsy in man to be just this failure to buffer extracellular K+ concentration normally. Depolarizations in the cortical silent cells accompany spontaneous brainwave spindles as well as the neuronal activity evoked by either thalamic or direct cortical stimulation. These responses seem in all important ways analogous to the glial responses of lower forms. Several experimental observations have provided information on the possible contribution of these glial depolarizations to the slow waves recordable with large electrodes from brain and elsewhere. The most recent of them (CASTELLUCCI and GOLDRING~~) calls attention to the striking resemblance between the silent-cell depolarizations and certain steady potential shifts recorded from the cortical surface during seizure discharge and brain stimulation. The authors suggest “ . . . that glia lying closest to a focus of neuronal activity in the cerebral cortex become depolarized by local release of K+ and draw current from neighbouring ‘source’ glia to which electrical linkage occurs in all directions.” It seems likely that the possibility of a glial contribution to all the various spontaneous and evoked
THE GLIA-NEURONAL INTERACTION : SOMEOBSERVATIONS
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electrical responses recordable from nervous tissue will in time be directly tested. It has already been claimed that the b-wave of the electroretinogram, an event long considered to be a manifestation of neuronal acitivity, is due solely to depolarization of the Mtiller fiber, a glial cell in the retina (MILLER and DOWLING~~). The experiments of WALKER and HILD~~ on mammalian brain cells in culture show low-resistance junctions to exist via glia over distances up to 200 pm. Surprisingly, a similar electrical coupling seems to exist between these glia and certain neurons in the culture which are incapable of action potentials and in still other ways display glia-like properties. Such studies with cultured cells also provide opportunities for verifying and testing the variety of current hypotheses of glial function. It has been shown, for instance, that the impedance of glial clumps is altered by pharmacologically active molecules in the bath (WALKER and TAKENAKA~~), that enzyme activity increases in astrocytes after Na+ (but not K+) additions to the medium (FRIEDE~~), and that 0, consumption of glial clumps, but not of nerve cells, declines rapidly in Na+-free medium and rises two or three-fold when K+ is added (HERTz~~). Such experiments validate evidence available from the electrophysiologists on the sensitivity of glia to K+ ions, suggest the wide range of metabolic activities that could be initiated in glia by activity of adjacent neurons, and point to the possibility that glia may respond selectively to biologically important molecules. On this last point, ROITBAK,~~ working with cat cortex, has shown that morphine and other analgesics reduce or abolish potentials that seem to originate in glial depolarizations; hypothesizes on the basis of this evidence that the glia are related to the mechanism pain since drugs that relieve pain act upon them.
he of
The studies epitomized above illustrate some of the new hypotheses about glia and new experiments upon them which Cajal encouraged 75 years ago. These ideas and experiments have their origins in the inclusive biochemical generalizations, insights into fine structure provided by the electron microscope, and electrophysiological data that have developed during David Rioch’s lifetime of devotion to the nervous system. He has seen the view of nervous system function shift from that of all-or-nothing, purely neuronal interactions to the current conception where neurons and glia cooperate to store and deliver the variety of actions which nervous systems produce. If, like Cajal, he applauds what is heuristic in hypothesis, he may well accept the glia-neuron hypothesis, for it has already led to many new and surprising observations on the nervous system.
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