Depolarization of cortical glial cells during electrocortical activity

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DEPOLARIZATION OF CORTICAL GLIAL CELLS DURING ELECTROCORTICAL ACTIVITY

ROBERT G. G R O S S M A N AND TOM H A M P T O N

Division of Neurological Surgery and Department of Anatomy, University of Texas Southwestern Medical School at Dallas, Texas (U.S.A.) (Accepted May 21st, 1968)

INTRODUCTION

It has long been thought that the glial cells of the cerebral cortex might exhibit electrical activity that is related to the normal functioning of the cortex. If cortical glial cell activity does occur as a normal physiological event it would be important to know its relationship to neural activity, and to know if glial activity contributes to the electrocorticogram. Recently the so called idle 6,11 or unresponsive7,8 cells which can be penetrated by fine micropipettes inserted into the cortex have been marked by the intracellular release of dye and appear to be glial cells7. These cells do not exhibit the postsynaptic and spike responses which can be evoked by adequate stimuli in neurons6,8,11. However, these cells have been reported to exhibit very slow depolarizations in response to strong direct cortical and thalamo-cortical stimulationL It has also been reported that these cells undergo depolarization during cortical seizure activity1~ and during spreading depression of cortical activity6. To further investigate the relationships between glial cell activity and electrocortical potentials we have recorded the intracellular potentials of synaptically and intracellularly inexcitable cortical cells during spontaneous and thalamically evoked electrocortical activity and have marked inexcitable cells with intracellularly deposited dye. METHODS

Intracellular recordings were made in the sigmoid gyri of 20 cats anesthetized with pentobarbital. A complete description of the methods used for animal preparation and recording has been published previously4. Micropipettes with DC resistances of 20-40 Mf~, filled with 1.5 M potassium citrate were used for intracellular recording and micropipettes filled with a saturated solution of fast green dye in 1 M potassium acetate were used for both recording from and intracellular marking of cellslL Penetrated cells were identified by their responses to intracellularly passed currents and to antidromic and orthodromic stimulation. A bridge circuit was used for recording intracellular potentials while simultaneously passing depolarizing current through Brain Research, 11 (1968)316-324

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Fig. 1. Photomicrographs of inexcitable cells marked by the intracellular release of fast green dye through the recording micropipette. A, A cell with an intensely marked nucleus and weakly marked cytoplasm. B, A marked nucleus without arty marked cytoplasm. Calibration lines: 10/~.

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the micropipette a, and the cerebral peduncle and the nucleus ventralis lateralis of the thalamus were stimulated with bipolar electrodes to evoke antidromic and postsynaptic cortical responses. Recordings were obtained from 8 to 15 inexcitable cells in each experiment. Inexcitable cells were marked in 4 experiments by passing a current of 0.5 #A for 4-6 min through the micropipette with the pipette connected to the negative terminal of the generator 5,7,15. The brains of these animals were perfused with saline and then with 10 % formalin. After fixation for 12 h serial frozen sections 80 # in thickness were cut through the cortex. The sections were mounted on slides, dried, cleared in dioxane, and covered with permount. The slides were examined with light and phase contrast microscopy. RESULTS

Identification of &excitable cells Inexcitable cells were penetrated at all depths in the cortex, including the outer 100/a. The penetration of inexcitable cells was signalled by the recording of intracellular potentials without the spike discharges and synaptic noise generally observed following the penetration of neurons. In one quarter of the cells studied the membrane potential increased in the 1st rain after penetration and then became stable. Cells with the highest potential on penetration were the most stable. The longest period of recording obtained in an inexcitable cell without deterioration of the membrane potential was 10 min, in a cell with a resting membrane potential o f - - 8 5 mV. Postsynaptic potentials and spike potentials of the type evoked in sigmoid gyrus neurons by pyramidal tract and thalamic stimulation were not found in inexcitable cells. Depolarizing potentials and spike discharges could not be evoked in inexcitable cells by intracellularly passed depolarizing currents of up to 5 • 10-9 A. In contrast, Betz cells regularly responded with spike discharges to the passage of depolarizing currents of 2 . 1 0 -9 A. Intracellular recordings were obtained from 257 electrically and synaptically inexcitable cells with membrane potentials larger than --50 mV and 74 of these cells had membrane potentials of --80 to --95 mV. Six dye marked inexcitable cells were found on microscopic examination of the cortex, all in areas of the cortex where the cells penetrated had exhibited slow depolarizations of membrane potential, as described below. The marked cells were in the grey matter of the cortex and all but one were adjacent to capillaries. The general appearance of these cells when examined with the oil immersion objective at a magnification of 1250 × is shown in Fig. 1A. The marked cells had intensely stained oval nuclei which had average measurements of 4 # × 7/~. A well-developed nuclear membrane was present, and the nucleoplasm was granular. The cytoplasm was lightly stained and was vacuolated. The cell diameters were about twice the diameter of the nuclei. Ten additional marked cells were found with severely disrupted cytoplasm. In some of these cells only the nucleus of the cell was well marked (Fig. 1B). The marked inexcitable cells resembled unstained glial cells seen with the phase contrast microscope in frozen sections of the cortex.

Brain Research, 11 (1968) 316-324

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Fig. 2. Slow depolarization of an inexcitable cell during electrocortical spindle burst activity. Depth of cell, 0.8 m m in the posterior sigmoid gyrus. A, Penetration. B, At the first arrow the micropipette was advanced 2 #. The membrane potential first fell abruptly and then declined more slowly. At the second arrow the micropipette was again advanced 2 # and the penetration of a neuron was recorded. Note the bursts of spike discharges recorded in synchrony with the spindle bursts of the electrocorticogram.

Slow depolarization of inexcitable cells Spontaneous slow depolarizations of the resting membrane potential were found in 22 ~o of the 257 inexcitable cells studied (Fig. 2). These depolarizations were associated with spindle burst activity in the electrocorticogram. Cells that exhibited slow depolarizations had higher membrane potentials in general than those that did not exhibit definite depolarization. 45 ~o of the cells that exhibited slow depolarizations had membrane potentials greater than --80 mV while only 29 ~o of the cells that did not exhibit slow depolarization had membrane potentials greater than --80 mV. The amplitudes of slow depolarizations ranged from 1 to 5 mV, and their durations ranged from 3 to 5 sec. Their amplitudes and durations were directly related to the amplitudes of the spindle bursts during which they occurred. During the first 1-3 sec of the spindle burst there was a gradual increase in the amplitude of the slow deBrain Research, 11 (1968) 316-324

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polarization, which generally reached a peak about 1 sec later than the peak amplitude of the spindle burst. The depolarizations then decreased and the membrane potential returned to the resting level in 1-2 sec after the end of the spindle burst. Hyperpolarization of inexcitable cells associated with electrocortical activity was not observed. There was no relationship between slow blood pressure oscillations or respiratory movements and slow depolarizations. When the micropipette tip was moved just outside of an inexcitable cell, slow depolarizations were not recorded (Figs. 2B, 3C, D). Slow depolarizations were also evoked during cortical augmenting responses produced by low frequency (8/sec) thalamic stimulation, and during desynchronization of cortical activity produced by high frequency (100/sec) thalamic stimulation. With high frequency thalamic stimulation a smooth depolarization of up to 10 mV was produced (Fig. 3B). When thalamic stimuli were delivered with the micropipette B.P. mm.Hg

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Fig. 3. Slow depolarization of inexcitable cells during thalamo-cortical stimulation. A, Penetration of a cell 0.6 mm deep in the posterior sigmoid gyrus. Note change of chart speed at the arrow. A and B, Responses to stimulation of the nucleus ventralis lateralis and the pyramidal tract (PT) at the frequencies given. C, Exit from the cell and control stimulation of the nucleus ventralis lateralis. D, Another cell. The upper set of traces shows the intracellular responses during stimulation of the nucleus ventralis lateralis at 8/sec. The lower set of traces shows the responses recorded immediately outside of the cell. Brain Research, 11 (1968) 316-324

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tip immediately outside of an inexcitable cell only the intracortical field potentials evoked by thalamic stimulation were recorded, without any subsequent slow depolarization (Figs. 3C, D). Two types of experiments were carried out to obtain information about the relationships between inexcitable cells and adjacent neurons. In the first type of experiment, when large depolarizations were recorded in an inexcitable cell, an attempt was made to record the thalamically evoked action potentials of adjacent neurons within the inexcitable cell (Fig. 4). The evoked spike discharge of an adjacent neuron was recorded in 3 inexcitable cells which exhibited slow depolarizations of large amplitude. The amplitudes of the spikes were 1-3 mV when recorded within these inexcitable cells. However, in the majority of inexcitable cells only intracortical field potentials were recorded during the surface positive and negative waves of the primary response evoked by a single thalamo-cortical volley. The amplitude and form of the field potentials were the same when recorded within and outside of inexcitable cells. In the second experimental situation, when slow depolarizations of an inexcitable cell were recorded, the micropipette was advanced 2-4 #. In a few cases, it was possible to penetrate an adjacent neuron with this movement of the micropipette and to record spike discharges and postsynaptic potentials in synchrony with individual waves of the spindle bursts of the electrocorticogram (Fig. 2B). A ECo,

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Fig. 4. Cortical neuronal activity recorded within an inexcitable cell. Depth of cell, 0.4 mm in the anterior sigmoid gyrus. A, Oscillograph recording of a slow depolarization during stimulation of the nucleus ventralis lateralis at 10/sec, followed by two slow depolarizations occurring during spindle bursts. B, Spike responses of a neuron evoked by a single shock stimulation of the nucleus ventralis lateralis which were recorded in this inexcitable cell. Line a, ECoG. Line b, IntraceUular potentials, RC coupled. Line c, Zero reference line for intracellular potentials DC coupled recorded in Line d. DISCUSSION As previously reported by Kelly et al. 7, dye marked nuclei which were the size of glial nuclei were found in sections of the cortex after releasing dye in inexcitable cells. However, in other cells both intact and partially disrupted cytoplasm was seen. A puzzling feature of the appearance of the cells found was the lack of staining of any definite cytoplasmic processes. It is possible that the current used for marking passed out of the cell largely through the somatic membrane and failed to carry dye into fine cellular processes branching from the soma. In addition, the cytoplasm did not stain as well as the nucleus, a characteristic reaction of glial cytoplasm with most cell Brain Research, 11 (1968) 316-324

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stains. The marked cells had the dimensions and appearance of glial cells. It is known that the morphology of glial cells varies with the methods of perfusion and fixation used to study them 1, and that even when they are studied with silver stains there are many glial cells which appear to be transitional forms which cannot be classifiedlL For these reasons, as well as the distortion of the cell produced by penetration and passing of current, it was not possible to identify the marked cells as either astrocytes or oligodendrocytes. The inexcitable cells clearly differed from neurons in their electrical behavior as well as in their morphology. The membrane potentials of one-third of the inexcitable cells studied ranged from --80 to --95 mV, while the membrane potentials of motor cortex neurons in general do not exceed --75 mV (refs. 9, 14). The slow depolarizations of inexcitable cells which followed a spindle burst had the form of an envelope of the burst, while in contrast in cortical neurons fast postsynaptic potentials occur with each individual wave of the burstL Evidence was obtained that some of the cells undergoing slow depolarization were adjacent to at least one neuron firing in synchrony with electrocortical waves. Depolarization of glial cells in amphibian optic nerves has been reported to occur following optic nerve activity, and evidence has been presented that this effect is mediated by potassium ions released by the discharging optic nerve fibers 10. The temporal relationship between slow depolarization of inexcitable cells and the activity of neurons suggests that the diffusion of ions from active neurons may also produce the slow depolarization of cortical inexcitable cells adjacent to them. It has been suggested that if glial cells were connected by low resistance pathways the depolarization of a localized group of glial cells could draw current from a distal part of the glial pathway and set up a potential that could be recorded by external electrodes. Evidence has been presented that the negative afterpotential following the compound action potential of the optic nerve of Necturus is contributed to by glial depolarization 10. Depolarization of cortical inexcitable cells, which appear to be glia, does occur during normal electrocortical activity. However, the depolarizations are slower than the 0.5-30 c/sec activity recorded in conventional electroencephalographic tracings. Glial depolarization might contribute though to slow potential changes recorded from localized areas of the cortical surface during prolonged afferent or direct cortical stimulation s . SUMMARY

Intracellular recordings were obtained from 257 inexcitable cells in the sigmoid gyri of the cat. Inexcitable cells were characterized electrophysiologically by an absence of postsynaptic and depolarizing potentials following thalamo-cortical and intracellular stimulation with current strengths adequate to evoke responses in neurons. Iontophoretic deposit of dye successfully marked 6 inexcitable cells, and revealed them to be small cells which had strongly marked oval nuclei surrounded by a small amount of lightly marked cytoplasm. These cells had the dimensions and appearance of cortical glial cells. One third of the inexcitable cells penetrated had membrane Brain Research, 11 (1968) 316--324

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potentials ranging from - - 8 0 mV to - - 9 5 mV a n d 2 2 ~ of the cells penetrated exhibited slow depolarizations of m e m b r a n e potential which followed electrocortical spindle bursts. Some cells u n d e r g o i n g slow depolarization were f o u n d to be adjacent to active neurons. It is suggested that depolarization of some glial cells occurs as a n o r m a l physiological event during electrocortical activity. ACKNOWLEDGEMENTS This investigation was supported in part by U.S. Public Health Service G r a n t NB-05429-04 from the N a t i o n a l Institute of Neurological Diseases and Blindness. The valuable technical assistance of Mr. Buren A. Whitten, Jr. is gratefully acknowledged.

REFERENCES 1 CAMMERMEYER,J., Differences in shape and size of neuroglial nuclei in the spinal cord due to individual, regional and technical variations, Acta anat. (Basel), 40 (1960) 149-177. 2 CREUTZFELDT,O. D., WATANABE,S., AND LUX, H. D., Relations between LEG phenomena and potentials of single cortical cells. II. Spontaneous and convulsoid activity, Electroenceph. din. Neurophysiol., 20 (1966) 19-37. 3 FRANK, K., AND FUORTES, M. G. F., Stimulation of spinal motoneurones with intracellular electrodes, J. Physiol. (Lond.), 134 (1956) 451-470. 4 GROSSMAN,R. G., CLARK, K., AND WHITESIDE,L., The influence of thalamic stimulus parameters on primary and augmenting cortical intracellular potentials, Brain Research, 5 (1967) 273-288. 5 HOLUBAR, J., HANKE, B., AND MALIK, V., Intracellular recording from cortical pyramids and small interneurons as identified by subsequent staining with the recording microelectrode, Exp. Neurol., 19 (1967) 257-264. 6 KARAHASHI,Y., AND GOLDRING,S., Intracellular potentials from 'idle' cells in cerebral cortex of cat, Electroenceph. clin. Neurophysiol., 20 (1966) 600-607. 7 KELLY, J. S., KRNJEVB~, K., AND YIM, G. K. W., Unresponsive cells in cerebral cortex, Brain Research, 6 (1967) 767-769. 8 KRNJEVI6,K., AND SCHWARTZ,S., Some properties of unresponsive cells in the cerebral cortex, Exp. Brain Res., 3 (1967) 306-319. 9 Lux, H. D., AND POLLEN, D. A., Electrical constants of neurons in the motor cortex of the cat, J. Neurophysiol., 29 (1966) 207-220. 10 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 (1966) 788-806. ll PHILLIPS,C. G., lntracellular records from Betz cells in the cat, Quart. J. exp. Physiol., 41 (1956) 58-69. 12 RAMON-MOLINER,E., A study on neuroglia. The problem of transitional forms, J. comp. Neurol., 110(1958) 157-171. 13 SUGAYA,E., GOLDRING, S., AND O'LEARY, J. L., lntracellular potentials associated with direct cortical response and seizure discharge in cat, Electroenceph. clin. NeurophysioL, 17 (1964) 661-669. 14 TAKAHASHI,K., Slow and fast groups of pyramidal tract cells and their respective membrane properties, J. NeurophysioL, 28 (1965) 908-924. 15 THOMAS,R. C., AND WILSON, V. J., Marking single neurons by staining with intracellular recording microelectrodes, Science, 151 (1966) 1538-1539.

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