234

Excitability of Neurons and Glial Cells

ICHIJI TASAKI

Laboratory of Neurobiology, National Institutes of Mental Health, National Institutes of Health,

Public Health Service, U.S. Department of Health, Education, and Welfare, Bethesda, Md. ( U.S.A.)

Electrophysiological properties of mammalian neurons and glial cells have been

investigated in this laboratory by using the tissue culture technique and by introducing
microelectrodes into thin brain slices. The cells are observed with a phase microscope
when the microelectrodes are inserted. The results from tissue culture materials,
obtained in collaboration with Dr. Hild, were published elsewhere (Hild and Tasaki,
1962). Our experiments on brain slice preparations from newborn guinea-pigs are
still in progress. In this article we wish to review the present state of our knowledge
concerning mammalian neurons and glial cells as examined by various microphysiologi-
cal techniques.

Electrophysiological properties of neurons

Since about 1952 a large number of papers have been published dealing with the
resting and action potentials of single nerve cells in the mammalian nervous system
(Eccles, 1957; Frank and Fuortes, 1961). Almost all of these investigations were
carried out by introducing hyperfine (glass or metal) recording electrodes into various
parts of the mammalian central nervous system without direct visual observation
of the cells under study. Although this method of 'blind' recording yielded a large
volume of important knowledge of the function of the nerve cells, it has become
increasingly clear that the information about the exact position of the microelectrode
relative to the cell under study is indispensable for proper interpretation of the
results obtained (Spyropoulos and Tasaki, 1960). Our study of tissue culture material
was designed to examine electric responses of the neuron soma and dendrites under
direct visual observation.
Cultivation of the neural elements used for the present investigation was done
partly in the Tissue Culture Laboratory of the University of Texas Medical Branch in
Galveston and partly in the Laboratory of Neuroanatomical Sciences of the National
Institute of Neurological Diseases and Blindness. A phase-contrast microscope was
used to view various parts of neurons and glial cells, as well as the tips of the stimu-
lating and recording electrodes.
When a hyperfine glass microelectrode is pushed into the cell body of a neuron,
 N E U R O N A N D GLIA EXCITABILITY 235

there is a sudden D.C. shift representing the resting potential of the cell. By delivering
brief pulses of either inward- or outward-directed currents through the stimulating
microelectrode, it is possible to evoke all-or-none action potentials in the cell. Action
and resting potentials of such impaled neurons deteriorate with time. It is possible
to record the electric response of the cell body and dendrites extracellularly with a
microelectrode that makes contact with the cell surface.
In recording action potentials from the surface of neurons, there is no progressive
deterioration of the responses. In some neurons in tissue culture, there are repetitive
responses in the absence of external stimuli. Fig. 1 shows three examples of such
spontaneously fired action potentials recorded from the neuron surface. Note that
the configurations of the action potentials of these three neurons are very different
from one another. The differences in configuration arise mainly from variations in
the pressure one has to apply to the neuronal surface with the recording microelectrode

A B

c
3
<

Fig. 1. Several types of spontaneously fired action potentials recorded with glass microelectrode
making contact with the surface of neuron somata. Upward deflections represent positivity of re-
cording electrode. Records A were taken from neuron in 13-day-old culture of rat cerebellum, records
B from 10-day-old culture of rat cerebellum, and record C from 15-day-old culture of rat cerebellum.
Sudden change in configuration of recorded action potentials in record C was induced by pushing
recording electrode harder against neuron surface. Oscilloscope sweep speed was same for all records;
deflection sensitivity for B was same as that for C. (From J. Neurophysiol., 25, (1962) 284).
References p . 2411242
236 I. T A S A K I

in order to record properly the action potentials. If the tip of the recording electrode
is not making direct contact with the neuronal surface, the recorded responses would
be too small in amplitude to be recognized. If, on the contrary, the tip of the recording
electrode presses too strongly against the neuronal surface, the chance of injuring the
neuron is great. It is extremely difficult to control the pressure applied to the neuronal
surface by the tip of the recording electrode. This situation illustrates how risky it is

5 msec
Fig. 2. Action potentials recorded at surface of dendrite on stimulation of neuron soma. Stimulating
shocks were 0.05 msec in duration, approximately 20 ,uA at threshold and repeated at rate of 7/sec.
Arrangement of electrodes (both extracellular) and major, clearly discernible, portion of dendrite
are shown. Ten-day-old culture of cat cerebellum. Ax = axon; S = stimulating electrodes; R =
recording electrode. (From J. Neurophysiol., 25 (1962) 290).
 NEURON A N D G L I A E X C I T A B I L I T Y 237

to interpret the configuration of electric responses recorded without direct visual

observation of the neurons under study.
The electric responses recorded from the surface of neurons show a strong negative
phase. This negativity indicates that there is an inwardly directed current in the portion
of the membrane under the recording microelectrode. There is little doubt that the
surface membrane of the cell body of a neuron in tissue culture is electrically excitable.
It is possible to elicit action potentials of the cell body by stimulating the neuron
directly or by delivering electric shocks to the dendrites.
A recording microelectrode pressing the surface of a dendrite often picks up spon-
taneously fired action potentials. If, under such circumstances, stimulating electric
shocks are applied to the neuron soma or to some portion of the dendrites of the
same neuron, it can be shown that ‘dendritic potentials’ obey the all-or-none law, At
least in large cerebellar neurons under tissue culture, the basal portions of the den-
drites-are capable of carrying nerve impulses without decrement.
Fig. 2 illustrates an example of the all-or-none responses induced by stimulation
of the cell body and recorded from the surface of a dendrite. It is interesting to note
that the conduction velocity along the basal portion of the dendrite is of the order
of 0.1 msec, that is, roughly 1/1000 of the velocity in the largest mammalian myelin-
ated nerve fiber.
There has been controversy among neurophysiologists concerning the excitability
of the neuron soma and dendrite. Some investigators believe that the membranes of
the cell body and dendrite do not participate in production of action potentials
(Freygang, 1958; Furshpan and Furukawa, 1962). Others believe that the surface
membrane of the neuron is excitable (Fatt, 1957; Araki and Terzuolo, 1962). As far as
neurons in tissue culture are concerned, the problem is now settled: the soma and
dendrite membranes are electrically excitable. Some physiologists, however, seem to
believe that the neuronal membrane is inexcitable in vivo because of the presence of
numerous synapses which are absent in tissue culture material.

Physiological properties of glial cells

It is well-known that glial cells in tissue culture show spontaneous pulsatory move-
ments (Canti et al., 1935; Pomerat, 1951; Hild, 1954; Chang and Hild, 1959). We
have seen that a mass of glial cells in tissue culture respond to strong electric shocks
with a slow contraction. The phase of active contraction is 1 to 3 min in duration and
the phase of relaxation is of the order of 10 min. Occasionally, we have seen syn-
chronous twitches in the mass of glial cells in the absence of external stimuli.
For demonstration of contractability of glial cells, it is necessary to use the time-
lapsewmovie technique because of the sluggishness of the process. A preliminary
report on glial contractability has been published by Chang and Hild (1959). Fig. 3
is reproduced from their paper.
Attempts were made to demonstrate contractions of glial cells in freshly excised
brain slices. Two methods were devised for this purpose: the first method is to detect
slow changes in optical properties of the brain slice in response to electric stimulation.
References p . 241/242
238 I. T A S A K I

Fig. 3. Three selected film frames of a motion picture sequence showing the effect of electrical stimu-
lation on an isolated astrocyte. Eight-day-old culture of kitten cerebellum. Phase contrast. Stimulating
electrode with a tip diameter of approximately 12 p ; distance from the cell body approximately 24 p.
(a) Immediately before stimulation. The cell body shows normal size and configuration; (B) 1.7 min
after stimulation. The cell body is markedly contracted and the processes slightly thickened due to
shifting of cytoplasm from the cell body into the processes; (C)7.5 min after stimulation. The cell
body has expanded again and regained its original configuration. The total duration of this con-
tractile response was 7.5 min which represents the shortest duration observed in the course of this
investigation. (From J. cell. comp. Physiol., 53 (1959) 142).
 NEURON AND GLIA EXCITABILITY 239

If a dense layer of glial cells undergo a slow contraction following electric stimulation,
one might expect a change in the light intensity scattered by the tissue; measurement
of the intensity of the scattered light with a photomultiplier tube will then give
information about the contractability of the glial cells. An alternative method is to
measure the electrical impedance of a brain slice at the time when a slow contraction
of the mass of glial cells is expected to take place.
The results obtained by these two different methods were consistent; there was a
clear indication thzt the light-scattering property undergoes a transient change
following a strong electric shock. The duration of the ‘contraction phase’ as detected
by the optical method was close to that seen in tissue culture material. During the
‘contraction phase’ there was a rise in the electric impedance of the brain slice measured
with alternating current of 60 to 120 cycles/sec. The time course of ‘relaxation’ was
obscured by a slow, continuous change in optical and electric properties of the slice.
The resting membrane potential of a glial cell can be determined by piercing the
cell with a glass microelectrode. Both with tissue culture material and brain slices of
the cat, values between 50 and 70 mV (negative inside) can be obtained in clean pene-
trations. The resting potential can be reversibly reduced by raising the potassium-ion
concentration in the surrounding medium.
The membrane resistance of the glial cells can be determined byrintroducing two
glass microelectrodes into a single glial cell in tissue culture. The surface membrane
of a glial cell of about 15 p diameter shows a resistance of 0.6 MQ to 1.7 MQ to
penetrating electric current. The membrane obeys Ohm’s law in a wide range of
current intensity under these experimental conditions. Multiplying the surface area
of a glial cell by the resistance mentioned above yields a membrane resistance for a
unit area of the order of 10 Qcm2. This value is far smaller than the generally accepted
value of the membrane resistance of various neurons. The estimation of the surface
area of a glial cell is difficult; therefore our estimation of the membrane resistance
for a unit area is subject to a large error.
The time constant of the glial membrane is of the order of 0.5 msec. This value,
combined with our estimation of the membrane resistance, leads to an unexpectedly
large membrane capacity. It is at present impossible for us to evaluate the effect of
the glial cell processes upon the electric measurements. It is possible, therefore, that
a large correction has to be introduced when the effects of these numerous processes
are taken into proper consideration.
A strong electric shock applied to a glial cell with a large extracellular electrode is
known to bring about a reversible, transient reduction in the resting potential of the
cell. The degree of reduction depends on the intensity of the applied shock. There is
evidence indicating that this reduction in the resting potential is associated with a
simultaneous reduction in the membrane impedance. The intensity of current needed
to induce such a potential variation seems too large to link the phenomenon with
physiological events in living brain tissue. However, the ‘electric responses’ of glial
cells appear to signal the beginning of a sequence of events leading to contractile
responses. The temporal relationship between the electric and contractile processes
in the glia1:cells is in some respect similar to that observed in smooth muscles.
References p . 2411242
240 I. T A S A K I

Some observations on amoeba and slime molds

With a view to elucidating electrophysiological properties of single cells that demon-
strate primitive contractile movements, various measurements were made on Amoeba
proteus, Chaos chaos and on Physarum polycephalum. There were a number of simila-
rities between mammalian glial cells and these primitive cells.
The resting potentials of the amoebae have been determined with glass micro-
clectrodes and were found to be between 40 and 70 mV (Bingley and Thompson, 1962).
The membrane resistance and capacity of the amoebae were determined with two
intracellular microelectrodes; the values of approximately 1 to 3 K O cm2 and 10
pF/cm2 were obtained (Tasaki and Kamiya, 1964). In response to strong electric
shocks, a kind of localized response could be induced. In the absence of external
stimuli, both amoebae and slime molds showed slow but vigorous electric activity.

I I I I I I
0 5 10 15 20 25
TIME (Min)
Fig. 4. Time course of potential difference across an amoeba (upper trace) and simultaneous hydro-
static pressure difference required to keep the amoeba stationary (lower trace). (From Tasaki and
Kamiya, 1964).

Fig. 4 is an example of records showing the time course of the potential difference
across an amoeba (upper trace) and the hydrostatic pressure difference required to
keep the cell in a small capillary (lower trace). The upper trace of the figure shows a
succession of rapid potential changes repeated at irregular time intervals. There are
simultaneous changes in the membrane impedance associated with the potential
changes. The potential changes, produced by depolarization localized in limited areas
of the membrane, are suppressed by application of anesthetics, KC1, etc. Attempts
are now being made to see if there are spontaneously induced potential changes in
mammalian glial cells.
The lower trace in Fig. 4 can be regarded as a measure of the motive force of
amoeboid movement. It is interesting that these movements in amoebae can not be
suppressed by application of anesthetics or of solutions of MgClz or CaC12. This
situation is analogous to that in the mammalian glial cell; previously, we have seen
 N E U R O N A N D GLIA EXCITABILITY 24 1

that glial cells in tissue culture continued to pulsate under the action of anesthetics
and divalent cations.

SUMMARY A N D CONCLUSION

Various technical difficultiesare encountered in the electrophysiological investigation

of neurons and glial cells in the mammalian central nervous system under direct visual
control. Using a tissue culture technique and a method of preparing thin brain slices,
progress has been made in research along this line.
The surface membrane of the soma and dendrites of neurons in tissue culture is
capable of responding to electric stimuli with all-or-none action potentials. Electric
responses have been recorded from neurons in brain slices of newborn guinea-pigs.
Glial cells in tissue culture sometimes respond to strong electric stimulation with
a slow contraction followed by a gradual relaxation. The duration of the contractile
phase is 1 to 3 min and that of relaxation is of the order of 10 min. Experimental
evidence is presented indicating the existence of contractile responses in the glial
cells in brain slices. In a dense mass of glial cells, there are occasionally synchronous
contractions in many cells in the absence of external stimuli, suggesting the presence
of some kind of physiological interaction among the cells. It is interesting that the
duration of this slow contractile process in glial cells is comparable to that of Leao’s
spreading depression in the cerebral cortex.
The resting potential of glial cells in tissue culture and in brain slices has been
determined with the use of glass microelectrodes. The membrane resistance and
capacity have been estimated by introducing two microelectrodes into single glial
cells. The membrane of the glial cell appears to be far more permeable to various
cations than the neuronal membrane. Strong electric shocks applied to glial cells
with an extracellular electrode produce a reversible reduction of the resting potential;
this potential change appears to be the first step in the sequence of events leading to
a contractile response.
Similarities are pointed out between mammalian glial cells and fresh water amoebae.

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