/ . Embryol. exp. Morph. Vol. 33, 1, pp. 29-41, 1975
Printed in Great Britain
29
The effects of minute direct electrical currents on
cultured chick embryo trigeminal ganglia
By BETTY F. SISKEN 1 ' 2 AND STEPHEN D. SMITH 1
From the Department of Anatomy, University of
Kentucky College of Medicine, U.S.A.
SUMMARY
The effects of low levels of electric current were determined on organ cultures of 6- to
19-day-old chick embryo trigeminal ganglia. Current levels ranged from 0-00115 to 11-5 nA/
mm3; the time of electrical treatment varied from 2 h to 96 h.
Low levels of electricity were found to have at least three major effects on this system
of mixed cellular types:
(1) Outgrowth of nerve fibers from the explant was enriched. This resulted in a greater
number of fibers which were longer and more highly branched than those in control
cultures.
(2) Survival of neurons within the original explanted ganglion was enhanced by treatment
with electricity. The presence of healthy neurons was found in 931 % of the treated cultures
and in 53-5 % of untreated cultures.
(3) Neurons, fibers and non-neural cells were stimulated to grow in the direction of the
cathode. The rate of cathodal migration was calculated to be 01 mm/h (2-4 mm/day) in an
explant from 12-day-old embryo.
No differences were observed between surviving treated and control cultures in histochemical tests for acetycholinesterase.
A similarity between the action of low levels of electrical current and nerve growth factor
(NGF) is suggested.
INTRODUCTION
In recent years a number of attempts have been made (using a variety of
procedures) to induce limb regeneration in non-regenerating animals. Some
of the experiments which have achieved partial success have entailed the use
of low levels of electricity (Smith, 1967; Becker, 1972). In this study we have
attempted to ascertain how low levels of direct electrical current (0-0011511-5 nA/mm2) affect the nervous system of a non-regenerating animal, specifically
the sensory ganglia, which provide nerve fibers essential for the regenerative
process in vertebrates (Singer, 1952). The in vitro system chosen allows precise
control of the electrical level issued per chamber and permits visual and
chemical analyses within a relatively short time.
1
Authors'1 address: Department of Anatomy, University of Kentucky College of Medicine,
Lexington, Kentucky 40506, U.S.A.
2
Submitted in partial fulfillment of the requirements for the degree of Doctor of
Philosophy.
30
B. F. SISKEN AND S. D. SMITH
MATERIALS AND METHODS
Controls
Twenty-eight trigeminal ganglia of chick embryos ranging in age from 6 to
19 days were explanted onto 12 mm round glass coverslips (Corning) in 15 mm
Falcon plastic organ culture dishes containing 1-5 ml of tissue culture medium
and incubated in air at 37 °C. For microcinematography, the cultures were
established in Rose chambers, which required the use of 2 ml of medium.
Control Falcon dishes and Rose chambers were wired as in experimental cultures
(viz. Fig. 1), but no current was applied. The medium consisted of TC 199
(85%) and Fetal Calf Serum (10%) (Difco Co.), Streptomycin (0-012 g/ml),
Penicillin (1250 units/ml), and glucose (5 % to give 600 mg % final concentration).
All cultures used for scoring outgrowth and nuclear state were fixed in
3-5 % glutaraldehyde in 0-1 M cacodylate buffer pH 7-4, rinsed in 0-1 M cacodylate + 2 % sucrose, postfixed in 2 % OsO 4 -01 M phosphate buffer for 1 h
and subsequently stained en bloc with toluidine blue (0-1 %), Harris' haematoxylin, or Brachet's methyl green-pyronin Y (Pearse, 1968). Cultures prepared
for the Bodian technique for nerve fibers were fixed in 10 % neutral formaldehyde;
10 % formaldehyde, 2 % ammonium bromide; or 3-5 % glutaraldehyde (w/vol.
concentrations). Paraffin sections were made from explanted ganglia which
did not attach to the coverslip or from well-established ganglia by removing
their centers with a fine scalpel. Scoring of all sections was carried out by a
double-blind procedure; cultures were scored as healthy if over 50 % of the
total area of ganglion sectioned contained round sensory cell bodies surrounded
by satellite cells, and if the neurons were morphologically typical of those
found in the normal embryo at the corresponding stage of development.
Enzymic analyses were carried out without prior fixation on cryostat
sections of cultures and freshly removed ganglia of various ages. The procedure
of El-Badawi & Schenk (1967) was used to demonstrate the histochemical
localization of acetylcholinesterase, using tetraisopropyl pyrophosphoramide
(iso-UMPA) as an inhibitor of non-specific activity.
Phase contrast microcinematography of cells in Rose chambers was carried
out at 1 frame/minute. The temperature of the chambers was maintained at
37 °C by means of a thermistor controller oiled to the coverslip of the chamber
and connected to an infrared heating lamp. Temperature was stable to about
±0-5 °C.
Experimental
Cultures were also established in 15 mm Falcon organ culture dishes and
Rose chambers modified to deliver an electrical stimulus to the contents. The
lids of the Falcon dishes were pierced by two 28 gauge platinum wire electrodes
spaced 14 mm apart. These wires extended down into the incubation medium.
When the electrodes were coupled with a 14 V Eveready mercury battery
Electrical currents on cultured chick ganglia
31
Treatment
chamber
Picoammeter
Falcondish
©
©
©
©
Rose chamber
Fig. 1. Electrical treatment apparatus.
(+E-42) and an appropriate resistor, a non-uniform d.c. electrical field was
generated in the chamber by the electrodes. Precise current level was obtained
by inserting an appropriate resistor (Victoreen glass-encapsuled metal film)
between the battery and the electrodes. These resistors (108, 109, 1010, 1011,
1012 Ohm) were housed in a Silica gel-filled Teflon box with a common input
and separate output leads (Fig. 1A). The total current flow between the
electrodes was measured after allowing 3 h for electrode polarization. This
period of time was chosen empirically by observing the rate of change in
current flow with time in a test culture, then doubling the length of time
{ca. l^h) required for the drop in current flow to level off. Measurements
were made under shielded conditions with an RCA WV 511 A picoammeter
in series with the dish, and current levels of 0-00115 to 11-5 nA/mm2 in decade
steps were calculated to exist for a 1 mm2 spot directly between the electrodes
and at half the distance between them. It should be emphasized that the
current levels given existed only at that point, and that the field generated was
quite non-uniform. A non-uniform field was chosen, as it had previously
been shown (Becker & Murray, 1967) that uniform fields are not usually
effective when d.c. currents are involved. This fact has recently been confirmed
by Pilla (1974). At the highest current level (11-5 nA/mm2) the potential drop
across the chamber was 0-25 volts.
Rose chambers were wired by threading 2 platinum electrodes through the
gasket of the assembly (Fig. 1B). Observations of Falcon dish cultures had
32
B. F. SISKEN AND S. D. SMITH
Fig. 2. 12-day-old embryo trigeminal ganglion culture treated for 12 h at 11-5 nA,
4 days in vitro. Glutaraldehyde-OsO4 fixation. Toluidine blue stained, x 242.
indicated that current densities of 1-15 nA/mm2 and 11-5 nA/mm2 for time
periods of 12 h or longer produced the most notable effects associated with
electrical stimulation. All Rose chambers prepared for filming, therefore, were
wired at the 108 Ohm position, and received 11-5 nA/mm2 at the selected point.
Current levels were measured and calculated as for the Falcon dishes.
RESULTS
Control cultures
Within the first 10 h after explantation, nerve fibers, fibroblasts, and glial
elements emerged from the ganglion and attached the explant to the substrate.
By 24 h, the original explant was surrounded by a mat of nerve fibers, fibroblasts, and glial cells. This mat of cells formed a substrate for the typically
rounded sensory cells which, upon migration from the explant, became bipolar
or multipolar in form. As the neurons migrated over this mat, their processes
became attenuated. As a result the periphery of the explant appeared as a
network of nerve cells and long thin processes. A typical migrated neuron
remained connected by a centripetal process to the original ganglionic mass;
peripheral processes of such a neuron terminated on another neuron, a group
of neurons, flat supporting cells or the substrate.
33
Electrical currents on cultured chick ganglia
Fig. 3. 12-day-old embryo trigeminal ganglion culture untreated 4 days in vitro.
Glutaraldehyde-OsO4 fixation. Toluidine blue stained, x 242.
Mitosis in migrated 8-day-old embryo neurons was first observed after
30 h in vitro. Prior to division all processes withdrew into the cell body, which
then became spherical. The nucleus then underwent a division. The multipolar
shape was subsequently re-established by the outgrowth of new processes.
Cinematographic records of cultures prepared in Rose chambers showed that
all types of cells were engaged in active movements, often pulsatile, and that
mitotic activity in many cells remained high for at least 4 days in vitro.
The usual life expectancy of untreated sensory neurons after migration from
an explant was low (ca. 3 days).
Effect of treatment
Ar
Neurons
Fig. 2 illustrates a typical culture derived from a 12-day-old embryo subjected
to electrical treatment for 12 h at 11-5 nA/mm2. Fig. 3 is a control for comparison. Cultures treated with electricity exhibited a far greater preponderance
of fibers surrounding the original explant than non-treated sister cultures. This
effect was noticed especially in films of these cultures and will be discussed
later.
Table 1 illustrates the overall effect of electrical treatment on neuronal
3
EHB
33
34
B. F. SISKEN AND S. D. SMITH
Fig. 4. 13-day-old embryo trigeminal ganglion culture treated for 12 h at 0-115 nA,
5 days in vitro 6/*m sections, Haematoxylin and eosin. x 161.
Table 1. Summary of experiments
Age at
which
ganglia
Experimentals
Controls.
• 11,111
removed
to
r*i 11 ti i VP*
t U 1IU1 C (
(days)
6
7
8
9
10
12
13
14
15
16
17
19
Totals
Scoring after 4
<
days in culture
Current level (nA/mm2)
A
11-5
| 24*
I24
224
I24
I48
J12J74
I12
I24
I24
J50
12
115
0115
0-0115
1612
—
0
I24
0
I24
I24
I24
—
I24
0
I24
I12
224
—
2
24
I24
0
0
8
J24
] 24
J 24
I48
000115
I24
—
I24
I24
0
I12
]12]74
J 24
J12J74
0
0
0
I24
0
0
I12
0
0
J50
150
J 50
7
8
9
t
A
after 4 days
in culture
A
Healthy Dead Healthy Dead
1
2
7
0
3
1
0
0
0
1
4
0
3
1
5
0
0
2
0
2
0
2
5
0
1
0
7
1
2
1
5
0
3
0
2
0
2
0
1
0
0
0
5
0
0
3
41
3
15
13
* Superscript number indicates length of treatment in hours.
Rose chamber cultures not included in table.
35
Electrical currents on cultured chick ganglia
Fig. 5. 8-day-old embryo, trigeminal ganglion culture untreated.
Glutaraldehyde fixed, Bodian stain, x 930.
survival within the original explant. Histological sections of explants were
scored for the presence of healthy neurons as previously described and illustrated
in Fig. 4. Of 44 cultures that were treated electrically (including all current
levels, all ages, all treatment times), sections of 41 were scored as healthy.
In 28 control cultures, only 15 were scored healthy. This increased survival of
neurons within the original explant appears to be the result of electrical
stimulation. The state of differentiation of neurons from the experimental
explants was occasionally quite remarkable and paralleled that seen in those
from ganglia in vivo.
Under the culture conditions used in these experiments, neuronal differentiation (accumulation of Nissl substance, cell enlargement) in the cells that
have migrated away from the explant was rarely seen. Identification of such
cells as neurons was possible by a number of techniques, foremost of which
was the simple observation of osmium tetroxide-positive nerve fibers. Bodian
staining of cultures explanted at different ages also indicated that these cells
had an affinity for silver as opposed to the negative reaction of the non-neuronal
elements (Fig. 5).
No differences between surviving neurons in cultures treated for 24 h at
11-5 nA/mm2 and those in 24 h control cultures were observed in the
3-2
36
B. F. SISKEN AND S. D. SMITH
Fig. 6. 6-day-old embryo trigeminal ganglion culture, 3 days in vitro untreated,
unfixed. Acetylcholinesterase activity seen as precipitate in cells labelled SNE
(sensory neurons within explant), and SN (sensory neurons that have migrated
away from explant). x 447.
histochemical studies qualitatively localizing acetylcholinesterase. Acetylcholinesterase activity has been demonstrated histochemically in the developing
sensory ganglion cells of the chick embryo with the light microscope (Strumia &
Baima-Bollone, 1964) and the electron microscope (Pannese, Luciano, lurato
& Reale, 1971). It is localized in neuroblasts and neurons exclusively and has
been detected recently in the spinal ganglia of the chick embryo at three days
of incubation (Pannese et ah 1971). Therefore, a positive stain for this enzyme
was used to confirm the morphological evidence of the presence of neurons
in the emigrating population of cells in these cultures, and was obtained at
all ages used. The histochemical reaction for this enzyme was more intense
within the neurons of the original explant mass than in those neurons which
had migrated outward (Fig. 6). There was no reaction to this histochemical
test in non-neuronal cells.
Electrical currents on cultured chick ganglia
Fig. 7. Rose chamber culture of a 12-day-old embryo trigeminal ganglion 4 days
after explantation and constant treatment with 11-5 nA. Single frames obtained
from 16 mm film. A = x270. Sequence B-D occurs within 3 h, and depicts
neurons, fibers and underlying supporting cells migrating to the bottom of the
field (cathode). x261.
37
38
B. F. SISKEN AND S. D. SMITH
Rose chamber observations
Time-lapse cinematographic films of cultures from eight Rose chambers
were prepared for analysis of cell migration and mitotic behaviour. It was
found that the culture conditions in chambers stimulated a more luxuriant
outgrowth of cells and fibers in treated cultures than in the Falcon dishes
(compare Figs. 2 and 7). Electrical stimulation induced neuronal migration,
the production of extensive fiber networks (some in the form of long cables),
and the generation of a heavy ground mat of supporting cells. Further, the
cathodal side of the treated explants was always the area of most intense
migration and growth of neurons, fibers, and non-neuronal cells. In contrast
to control cultures which generally appeared round, treated cultures were
always eccentrically elongated toward the cathode.
Fig. 7 represents single frames from a sequence taken over a 3 h period.
These frames were obtained from a time-lapse movie of a 12 day explant
treated with ll-5nA/mm 2 for 96 h prior to filming. Frame A shows a field
of nerve fibers wherein a group of neurons supported by a mat of other cells
is approaching from the top of the field (part of the explant mass). Frames
B, C, and D show the front of the migrating mass with its advance fibers
passing through the middle of the field and beyond toward the cathode. The
rate of migration of these cells and fibers toward the cathode was approximately
0-1 mm/h (2-4 mm/day).
DISCUSSION
These experiments illustrated the effects of electricity on nervous tissue of
a non-regenerating animal. The results indicated that cultured trigeminal
ganglia respond to electricity in three distinct ways: by more profuse outgrowth of fibers, by prolongation of the survival of neurons within the original
explant, and by migration of neurons, their processes, and non-neuronal cells
to the cathode. Each of these results will be considered below.
Fiber outgrowth
Increased outgrowth of neuronal processes was the first indication that
the low levels of current employed were affecting the culture system. This
outgrowth was characterized by a greater number of fiber bundles as welt as
a greater number of branches per bundle. Grosse, Lindner & Schneider (1969)
subjected cultures of 10-day-old chick embryo trigeminal ganglia to an electrostatic field and counted and measured the nerve endings of the ganglion cells.
Their results showed that there was no effect on the number of differently
shaped endings. However, the size of the ring-shaped nerve endings was
significantly greater in electrically-treated cultures as opposed to controls. The
effect of electricity on nerve fibers thus appears to be similar to the welldocumented effect of nerve growth factor (NGF) on nerve fibers of sensory
Electrical currents on cultured chick ganglia
39
and sympathetic ganglia. Roisen, Murphy & Braden (1972) have published
quantitative data on the effects of NGF (as well as cyclic AMP and dibutyrl
cyclic AMP) on sensory ganglia in culture. They found that all three chemicals
increased the area of outgrowth, the number of neurites per culture, and the
diameter, length and degree of neurite arborization in their cultures. It thus
appears that profuse fiber outgrowth can be stimulated by a variety of agents;
chemical and electrical.
Neuronal survival
The second important effect of low levels of current was a protective effect
on neurons within the original explant. Table 1 illustrates that cultures treated
with electricity show almost a twofold increase in the number of cultures
termed 'healthy' over untreated cultures. Neuronal maturation and survival
have heretofore not been associated with liquid culture systems. Even in semisolid or solid (plasma clot) culture systems there is usually considerable degeneration within the original explant (Murray, 1965) which may be minimized
by supplementing the medium with NGF. Recent studies by Blood (1972)
suggest that NGF exerts its action on explanted embryonic spinal ganglia by
preserving neuroblasts that would have died, and by increasing neuronal cell
number and rate of maturation, the latter resulting in an overall increase in
neurite growth. We suggest that in our cultures, d.c. current protects neuroblasts and neurons from degeneration, thereby allowing a more luxuriant
outgrowth than in untreated cultures. These findings represent the first evidence
for a protective influence exerted on nerve tissue explants by non-chemical
means.
Cathodal migration
A third effect of electrical treatment was the migration of neuronal cells,
neurites, and non-neuronal cells to the cathode in Rose chamber cultures.
No information on the motive force behind this movement was obtained.
Further studies may determine whether the cathodal attraction influenced the
growing neuronal fibers or the non-neuronal cells to cause this movement.
In vitro studies of the effects of electricity on polarity of nervous tissue growth
date back to 1920 with Ingvar's report on the orientation of cell processes
along the direction of force in a galvanic field (Ingvar, 1920). This observation
was confirmed by Peterfi & Williams (1934) and Karssen & Sager (1934) but
disputed by Weiss (1934) who claimed that the galvanic field oriented the
fibrin of the plasma clot, i.e. he claimed that the outgrowing nerve fibers
oriented themselves parallel to the tension strains within the clot. However,
Marsh & Beams (1946) not only confirmed that the current itself provided
a stimulus for growth, but that the orientation of fiber outgrowth was always
more extensive in the cathodal direction. Further, if they rotated the cathode
40
B. F. SISKEN AND S. D. SMITH
90°, the fibers reorientated themselves to grow toward the new cathodal
position.
The outgrowth of neuronal fibers and descriptions of growth cones have
been extensively reported by Harrison (1907), Ramon y Cajal (1928), Hughes
(1953) and Nakai (1956). The rate of outgrowth found in our treated cultures
was 2-4 mm/day in a 12-day-old chick embryo culture. Some growth rates
reported in the literature are presented in the following table.
Table 2
Investigator
Harrison (1907)
Speidel (1933)
Cajal (1928)
Levi (1934)
Hughes (1953)
Nakai (1956)
Gutmann et al. (1942)
Gillilan (pers. comm.)
System
Culture, larval amphibian brain
In vivo, larval tail fin
In vivo, 3-day-old chick embryo
Culture, chick embryo
Culture, chick embryo, lumbar ganglia,
midbrain
Culture, chick embryo
In vivo, adult rabbit sciatic nerve
In vivo, human, nerve regeneration
Growth rate
(mm/day)
0-374-1-344
0199
0-240
0-564
1-324
0-648-1008
0-158-1-224
3-5-4-3
2-0-3-0
By comparison with this table, it is apparent that the growth rate of chick
embryo neurites treated with current exceeds that reported by other investigators
on chick embryos by a factor of from x 2 to x 10, and approximates that found
in adult (mammalian) forms. It is apparent that the effects of current are
comparable to those of NGF, for though no measurement has been reported
on cultures treated with NGF, the growth rate must be accelerated to result
in an increased length of neurite arborization within the same time period as
a control culture. The basis for the similarity is obscure. The effect may be
caused by a direct electrical stimulation of the cell, though recent research
(Pilla, 1974) indicates that a more probable explanation lies in the alteration
of cation concentration (especially Ca ++ ) which spreads outward from the
cathode rather like a moving boundary or wave front. Such an alteration in
cation concentration (or ratios, since cations will migrate at different rates in
the d.c. field) might be expected to cause profound changes in the cell membrane
boundary conditions, and hence in permeability, excitability, adhesiveness, and
ultimately in state of differentiation. That shifts in cation concentration can
cause shifts in state of differentiation has been clearly demonstrated by Pilla
(1974).
This research was supported in part by grant No. GM 16844 from the National Institute
of General Medical Sciences - National Institutes of Health.
Electrical currents on cultured chick ganglia
41
REFERENCES
R. O. (1972). Stimulation of partial limb regeneration in rats. Nature, Lond. 235,
109-111.
BECKER, R. O. & MURRAY, D. G. (1967). A method for producing cellular dedifferentiation
by means of very small electrical currents. Trans. N.Y. Acad. Sci. 29, 606-615.
BLOOD, L. A. (1972). Some quantitative effects of nerve growth factor on dorsal root ganglia
of chick embryos in culture. / . Anat. 112, 315-328.
BECKER,
CAJAL, RAMON Y. (1928). Degeneration and Regeneration of the Nervous System. Vol. 1.
Reprint .1959. New York, N.Y.: Hafner Pub. Co.
A. & SCHENK, E. (1967). Histochemical methods for separate, consecutive
and simultaneous demonstration of acetylcholinesterase and norepinephrine in cryostat
sections. / . Histochem. Cytochem. 15, 580-588.
GROSSE, G., LINDNER, G. & SCHNEIDER, P. (1969). Der Einfluss des elektrischem Feldes auf
in vitro kultiviertes Nervengewebe. Z. mikrosk.-anat. Forsch. 80, 260-267.
GUTMANN, E.,GUTMANN, L., MEDAWAR, P. B. &YOUNG, J. Z. (1942). The rate of regeneration
of nerve. / . exp. Biol. 19, 14-44.
HARRISON, R. (1907). Observations on the living developing nerve fiber. Anat. Rec. 1,
116-118.
HUGHES, A. (1953). The growth of embryonic neurites. A study on cultures of chick neural
tissues. / . Anat. 87, 150-162.
INGVAR, S. (1920). Reaction of cells to the galvanic current in tissue cultures. Proc. Soc. exp.
Biol. Med. 17, 198-199.
KARSSEN, A. & SAGER, B. (1934). Sur l'influence de courant electrique sur la croissance des
neuroblasts in vitro. Arch. exp. Zellforsch. 16, 255-259.
LEVI, G. (1934). Explantation, besonders die Struktur und die biologischen Eigenschaften
der in vitro gezuchteten Zellen und Gewebe. Ergeb. Anat. EntwGesch. 31, 125-707.
MARSH, G. & BEAMS, H. W. (1946). In vitro control of growing chick nerve fibers by applied
electric currents. / . cell. comp. Physiol. 27, 139-157.
MURRAY, M. (1965). Nervous tissues in vitro. In Cells and Tissues in Culture, vol. 2,
pp. 372-455. New York: Academic Press.
NAKAI, J. (1956). Dissociated dorsal root ganglia in tissue culture. Am. J. Anat. 99, 81-131.
PANNESE, E., LUCIANO, L., IURATO, S. & REALE, E. (1971). Cholinesterase activity in spinal
ganglia neuroblasts: a histochemical study at the electron microscope. / . Ultrastruct. Res.
36, 46-67.
PEARSE, A. G. E. (1968). Histochemistry, vol. 1, pp. 651-652. Boston, Mass.: Little, Brown
and Co.
PETERFI,T. & WILLIAMS, S. C. (1934). Elektrische Reizversuche an gezuchteten Gewebezellen.
II. Versuche an verschiedene Gewebekulturen. Arch. exp. Zellforsch. 16, 230-240.
PILLA, A. A. (1974). Electrochemical information transfer at living cell membranes. Ann.
N.Y. Acad. Sci. (in the Press).
ROISEN, F., MURPHY, R. & BRADEN, W. (1972). Neurite development in vitro. I. The effects
of adenosine 3'5'-cyclic monophosphate (cyclic AMP). / . Neurobiol. 3, 347-369.
SINGER, M. (1952). The influence of the nerve in regeneration of the amphibian extremity.
Q. Rev. Biol. 27, 169-200.
SMITH, S. D. (1967). Induction of partial limb regeneration in Rana pipiens by galvanic
stimulation. Anat. Rec. 158, 89-98.
SPEIDEL, C. C. (1933). Studies of living nerves. Am. J. Anat. 52, 1-74.
STRUMIA, E. & BAIMA-BOLLONE, P. L. (1964). AChe activity in the spinal ganglia of the
chick embryo during development. Acta anat. 57, 281-393.
WEISS, P. (1934). In vitro experiments on the factors determining the course of the outgrowing nerve fiber. / . exp. Zool. 68, 393-448.
EL-BADAWI,
(Received 17 April 1974, revised 3 July 1974)