Indian Journal of Experimental Biology
Vol. 42, January 2004, pp. 43-47
Ca2+-free medium enhances the magnitude of slow peak in compound action
potential of frog sciatic nerve in vitro
Maloy B Mandai* & Shripad B Deshpande
Department of Ph ysio logy, Institute of Medical Sciences, Banaras Hindu University, Varanasi 221 005, India
Received 2 1 April 2003; revised 2 Seprember 2003
The present in vestigat ion was carried out to know the effect of Ca 2+ on different peaks of compound act io n potential
(CAP) representing the fibers having different conduction ve locity. CAP was recorded from a thin bundle of nerve fibe rs
obtained from desheathed frog sciatic nerve. Suction electrodes were used for stim ulating and recording purposes. In Ca2+ free amphi bi an Ringer, two distinct peaks (Peak-I and Peak-II) were observed. The threshold, conduc tion veloci ty (CV),
amplitude and duration of Peak-l were 0.32±0.02 V, 56±3.0 m/sec, 2. 1 ±0.2 mV and 0.75±0.1 ms, respectively. The
Peak-11 ex hibi ted ten times greater threshold, eigh t times slower CV, three times lower amplitude and four times greater
duration as compared to Peak-!. Addition of 2 mM Ca2+ in the bathing medium did not alter CAP parameters of Peak-I
excepting 25% reduction in CV. But, in Peak-ll there was 70-75 % reduction in area and amplitude. The concentrationattenuation relation of Peak-ll to various concentrations of Ca2+ was nonlinear and 50% depression occurred at 0.35 mM of
Ca2+. Washing with Ca2+-free solu tion with or without Mg 2+ (2 mM)/verapamil (1 0 J.tM} could not reverse the Ca 2+- induced
changes in Peak-II. Washing with Ca2+ -free solution containing EDTA restored 70% of the response. The results indicate
that Ca2+ differentially influence fast and slow conducting fibers as the activity of slow conducting fibe rs is greatly
suppressed by external calcium .
Keywords: Calcium, Calcium antagonists, Compound action potential , EDTA, Frog sciatic nerve
Calcium plays a key role in controlling multiple
physiological processes including the maintenance of
neuronal excitability. It is known that the fibers
having different conduction velocities exhibit
differential susceptibility to conduction block by
various external agents such as pressure, local
anaesthetics and hypoxia1.2. Local anaesthetics and
hypoxia are known to influence the neuronal activity
via Ca2+ mediated mech anisms 3.4 . Further, the
membrane properties including the conductance and
rate constants are different in slow conducting and
fast conducting fibers 5 . Earlier experiments using
single fiber recording technique revealed the
inhibitory effect of Ca2+ on Na+ permeability and
facilitatory effect on K+ permeability in fast
conducting frog myelinated fibers 6·7 • But, the effect of
Ca 2+ on different fiber types has not been resolved.
However, there has been Jot of rethinking to
understand the role of calcium on nerve action
potential generation 8 . This study has therefore been
undertaken to know the influence of Ca2+ on different
peaks of the compound action potential of frog sciatic
*For correspondence :
Tel: +91-542-2312 099
Fax : +91-542-2367 568 I 2368 174
E-mail: [email protected]
nerve using suction electrodes (for recording and
stimulating purposes). This method enables one to
record the potentials without altering the population
activity. Besides the above-mentioned advantage, the
temperature, pH and the composition of the solution
could be easily maintained.
Materials and Methods
Animals and dissection- Procedure followed for
recording the compound action potential from frog
sciatic nerve was described earlier9· 10 . Briefly, Rana
tigrina frogs were pithed, sciatic nerve was dissected
free from the adjoining tissues and placed in a
petridish contammg amphibian Ringer solution
devoid of Ca2+. The connective tissue and sheath
surrounding the nerve were cleared carefully. After
desheathing, the nerve fibers could be separated easily
to several bundles. A thin bundle containing a small
group of nerve fibers of 25-30 rnm length was taken
and placed in a Perspex chamber (vol =0.5 ml), which
was superfused continuously with Ringer solution at
2-3 ml/min. The temperature of the chamber was
maintained at 21 °C. All the experiments were
performed according to the guidelines of Institute of
Medical Sciences, Banaras Hindu University,
Varanasi.
44
INDIAN J EXP BIOL, JANUARY 2004
Stimulation and recording- Suction
electrodes
were prepared from the borosilicate glass capillary
tubes. The cut ends of the nerve bundle were gently
sucked into the glass capillary tube filled with Ringer
solution . The diameter of the capillary tube was made
appropriate to the diameter of the nerve bundle by fire
polishing. The suction electrode at one end was used
for stimulating purpose (rectangular pulses <0.2 ms
duration at the rate of 0.1 Hz were obtained from S-11
Grass stimulator) and the other end was for recording
the potential using a common Ag-AgCl bath
electrode. The thresholds for Peak-I and Peak-II were
determined and were around 0.3 and 3.0 volts,
respectively. Five times the Peak-II threshold was
used to elicit the response. The potentials from the
recording electrodes were amplified via an amplifier
(Grass Microelectrode Amplifier, P-16) and then
digitized by usi ng DMS-708A AD converter
(Dyanalog Microsystems, India) and were displayed
on a computer using Unkelscope software (MIT,
Massachusetts) for storage and analysis. The signals
were also monitored on an oscilloscope (BPL, India)
simu ltaneously .
Experimental protocol- Averaged signal of five
successive potentials was recorded at every step. The
Ca 2+-free Ringer was used unless mentioned
otherwise. The initial recordin gs were made after the
stabilization of the preparation for 1 to 1.5 hr. Then
2
the preparation was exposed to 2 rnM of Ca + for 30
min and CAP was recorded. Subsequently, the nerve
was washed with Ca 2+-free solution up to 120 min.
Since the effect of calcium was not reversible, the
ti ssue was used only once. In a separate series, after
exposing the nerve to 2 rnM of Ca2+, it was washed
with Ca 2 +-free solution containing ethylenediamin etetraacetic acid (EDT A) or Mg 2+ (both 2 mM) or
verapamil (I 0 !lM) for 30 min. In another series of
2
experiments, after the initial recording in Ca +-free
medium, the nerve was exposed to the increasing
concentrations of ci+ (0.1 to 2 mM) to observe the
2
effect of different concentrations of Ca + on Peak-1
and Peak-II.
Drugs and solutions- Verapamil and EDTA were
obtained from Sigma Chemicals (USA). Ringer
sol ution had the following composition (in mM):
NaCl, 116; KCl, 2.0; Na 2 HP04, 1.3; NaH2P04 0.7;
pH=7.3. CaCb, 2H 20 was added to achieve the
desired concentration of ci+.
Analysis and statistics- Stimulation of one end of
the nerve bundle evoked a compound action potential
(CAP) with various peaks. The threshold (Th),
latency, rise time (RT), total duration (Dur),
amplitude (Amp) and area were computed using
Unkelscope software. The conduction velocity (CV)
of different peaks of compound action potential was
calculated from latency and the length of the nerve
segment. Comparisons between the groups were
performed using Student' s t test (paired/unpaired). A
P < 0.05 was considered significant.
Results
Characterization of CAP parameters in Ca 2+ }ree
medium- Evoked responses of the nerve bathed in
ci+-free Ringer solution showed two distinct peaks
of potentials (Fig. 1). The first peak had a latency of
around 0.4±0.01 ms and termed as Peak-1. The
second peak appeared after 3 ± 0.13 ms and refened
as Peak-11. The threshold, conduction velocity, rise
time, amplitude and duration of peaks I and II were
distinctly different as shown in Fig 1. The Peak-1 was
characterized by low threshold, shorter latency (faster
conduction velocity), greater amplitude and shorter
duration as compared to Peak-H. The threshold
voltage required for Peak-11 response was nearly ten
times higher than that required for Peak-I.
External Ca 2+ attenuated Peak-ll- Superfusion
with the Ca2 + containing Ringer did not alter Peak-1
area, amplitude, rise time and duration significantly
(P>0.05), but the CV was reduced by 25% (P<0.05,
Fig. 2). On the other hand, Peak-Il showed a
remarkable and significant decrease (70-75%) in area
J\
100.-------------------------~
=
Peak-1
=Peak-11
~
(I)
......
(I)
E
I
~
10
~
ro
D...
D...
<{
u
Th
Amp
(V)
(mv)
RT
(ms)
Our
(ms)
CV
(m/s)
Fig. I - Compound act ion potential (CA P) parameters in Ca 2+free Ringer solution. Actual potential showing the Peak-1 (first
peak) and Pcak-11 (second peak) is inserted in the box . The
horizontal calibration= 5 ms and vertical calib ration= 1 mV.
Histograms showing mean± SE values of threshold (Th) in vo lts,
ampl itude (Amp) in mY, ri se time (RT) in ms, duration (Our) in
ms and conduction velocity (CV) in meters/sec of Peak-! and
Pcak-11 were obtained from 8 different preparations. The
threshold strengths for Peak-J and Peak-11 were determined and a
single strength of stim ulu s (5 times the peak-If threshold) was
used to record the CAP in a given experiment
MANDAL & DESHPANDE: CALCIUM SENSITIVE PEAK IN COMPOUND ACTION POTENTIAL
and amplitude. However, there was about 65 %
increase in the rise time (Fig. 2). There was no
alteration in duration of Peak-IT. Increase in the
stimulus strength failed to increase the magnitude of
Peak-II in preliminary experiments (n = 3).
Gradual increment in the Ca 2+ concentration of the
bathing solution attenuated the Peak-II area, in an
exponential manner (Fig. 3). The concentration of
Ca2+ to prod uce 50% depression (C-50) was around
0.35 mM (Fig. 3). Increase of Ca 2+ above 1 mM did
not alter the attenuation any further. Washing with
Ca2+-free Ringer for 120 min could not reverse the
effect of Ca2+ (Fig. 4). Therefore, no further
experimentation could be done with this preparation.
45
Discussion
This investigation reveals the existence of fibers
with differential sensitivity to external calci um. It is
evident that slow conducting fibers are extremely
sensitive to the calci um. Further, the present
observations demonstrate that the generation an d
propagation of CAP can still occur in the absence of
Ca2+. However, under the physiological condi tion
0
0.2
0.5
1.0
Extracellular Ca2+ (mM)
EDTA reversed the Ca 2+ -induced effect on peak llIn thi s set of experiments also, the Ca2+ (2 mM)
decreased Peak-II area and amplitude and increased
the RT as observed earlier (Fig. 2). Superfusion of the
nerve with Ca2+-free medium containing EDTA
(2 mM) reversed the attenuati on to nearly 75 %. Even
the increased RT was also restored to the initial level
(Fig. 5).
Q)
Q)
<!T
+
100
N
(IJ
u
0
~
___..
50
Calcium antagonists failed to reverse the effectIn this series, exposi ng the nerve to normal amphibian
Ringer (Ca 2+ 2 mM) for 30 mjn produced the effects
sirrnlar to those observed in Figs 2 and 5. The Ca 2+induced attenuatio n could not be reversed by washing
with Ca2+-free medium containing Mg 2+ (2 mM) or
verapamjl ( I 0 11-M; a L-type calci um chann el
antagonist) as shown in Fig. 6.
=Peak-!
Peak-11
=
I
.:.:::
(IJ
Q)
a..
0.0
0.5
1.0
1.5
2.0
Extracellular Ca2+ (mM)
Fig. 3 - Exponential attenuation of Peak-II magnitude by
extracellular Ca 2+. Actua l poten tials were obtained from single
experiment after superfusing the Ringer solution conta ining
increasing Ca2+ concentrations (0-1 mM). Nerve was allowed to
equilibrate at each concentration for 30 min . The horizontal
calibration=5 ms and vertical calibration= 1 mY . The mean±SE
(n = 6) values are shown in th e lower graph. The concentrati on to
produce 50% depression was 0.35 mM of Ca 2+.
~
Q)
200.--------------~
rn
rn c
....
0
Q)
Q)
a.
rn
=
=Wash
+Ca
150
2
•
Q)
E ....
ro Q)
.... Q)
ro .._ 100
Area Amp
RT
Our
cv
Fig. 2 - Effect of Ca 2+ (2 mM) on compound action potential
(CAP) parameters. Histograms showing mean ± SE va lues (n=8)
of area, amplitude (A mp), ri se time (RT), duration (Dur) and
conduction velocity (CV) of Peak-1 and Peak-Il were ob tained
from 8 different preparations. In an individual experiment, a
si ngle strength of stimulus (5 times the pcak-II threshold) was
used to record the CAP as menti oned in Fig. I legends. The va lues
are expressed as% of Ca 2+-frcc res ponse (contro l). An asteri&k (. )
indicates significan t difference from Ca2 +-free response (?<0.05;
Student's !lest for paired observations).
o..-.,
- +
~ Nco
ro U
Q).._
0..
so
0
Area
Amp
RT
Our
cv
Fig. 4-No reversal of Ca 2+-induced depression of the Pez.k-11 by
washing with Ca 2+-free Ringer. Histograms show mean ± SE
(n = 6) of area, ampli tude, rise time, duration and conduction
velocity ofPeak-11. Area and amplitude even after wash remained
similar to pre-wash leveL
INDIAN J EXP BIOL, JANUARY 2004
46
(Ca 2+ 2 rnM), the Peak-Il of CAP is not at its
maximum activity.
In the present study two distinct peaks could be
recorded in Ca2+-free medium. The first peak (Peak-!)
0
Q)
(j)
(j)
Qj
Q)
E
c
g_
150
2.0
0 + EDTA
2
Extracellular Ca + (mM)
=
=
+ Ca 2+
+EDTA
(j)
~
~ ,__
al
ro
0...'7
100
-+
~Nrn
)il u
O...b
50
~
Area
Amp
RT
Our
cv
Fig. 5 - EDT A-dependant reversal o f th e C a 2+-induced
depression of Peak-II. Actual potentials recorded in C a 2+- free, 2.0
mM Ca 2+ and Ca 2+- free solution containing EDTA (2 mM)
medium are shown in the to p row . The nerve was all owed to
equilibrate at each solution for 30 min. Please note the reversal of
depressio n in the presence of EDT A. The ho rizontal
calibration= 5 ms and vertic al calibrati on= l mY. Hi stograms
de pict mean ± SE (n=6) values of area, amplitude (Amp), ri se
time (RT), durati o n (Dur), co nducti o n velocity (CV) of Peak-II in
the presence of c}+ (2 mM) and in the presence of EDTA. An
asterisk (. ) indic ates sig nificant difference from th e response in
presence of Ca2 + (P < 0.05 ; Student's t test for paired
observati o ns ).
=
=
c::::=:J
+ Verapamil
+ Mg2+
+ Ca 2 +
Amp Area
Our
RT
CV
Fig. 6 - No reversal of the Ca2+-induced depress ion of Peak-II
by Mg2+ or verapami l. Histog ram s show mean ± SE from four
di fferent preparations. The values after wash with Mg 2+ o r
verapamil were not sig nificantly di fferent from the Ca 2+ response
(? > 0.05 , Student' s t test for paired observations).
has a conduction velocity of 50-60 rnlsec. In frog this
corresponds with the CV of Aa fibers having fiber
diameter between 15-20 J..lm recorded at simi lar
temperature and reported earlier 11. The second peak
(Peak-11) has conduction velocity of 7-10 m/sec. Thi s
corresponds to Ay fibers with 3-5 J..lm fiber diameter
as reported previously 11 • However, in the present
experiments the AP peak could not be recorded
perhaps due to merging of this peak with Aa on
account of shorter segment of nerve used as described
12
elsewhere . The threshold for Peak-1 was very low
(0.3V) and that for Peak-11 was 10 times higher.
In presence of Ca2+, the magnitude of Peak-11 was
decreased by 70% with no alteration in Peak-I
(Fig. 2). However, calcium increased (almost
doubled) the threshold and slowed the conduction
velocity of Peak-I. The attenuation of Peak-Il was not
due to the increased threshold, as hi gher strength of
stimulus failed to increase the magnitude any further.
Thus, the effect may be due to mech anisms mediated
by Ca 2+. Further, experiments demonstrated th e
exponential fall in the magnitude of Peak-II with the
increasing Ca2+ concentration. The concentration as
low as 0.35 mM of Ca 2+ (i.e. l /6 1h the physiological
concentration) attenuated the Peak-11 by 50%
(C-50 =0.35 mM) as seen in Fig. 3. Further, after
exposure to Ca2+ medium , the effect of Ca 2+ on PeakIl could not be reversed despite a long wash ( J 20 min)
with Ca 2+ -free Ringer. These observations suggest
that the Ca 2+-induced changes were irreversible and
not in a concentration dependent manner.
The rise time is a function of Na+ conductance5 .
Therefore, the sign ificant increase in rise time of peak
II (Fig. 2) indicates the changed membrane properties
of slow conducting fibers by Ca 2+. Simi larly, the
decrease in conduction velocity in Peak-1 may be due
to decrease in maximal Na+ conductance5 · 13 .
Investigations 7' 14 ' 15 on frog single myelinated nerve
fiber have depicted that external Ca2+ concentration
influences Na+ permeability. The increased excitability
exhibited in low calcium may be due to such
increased permeability of Na+. However, the effect
may also be due to Na+ dependent K+ conductance 16 .
The Ca2+ mediated effect has been explained on the
basis of neutralization of fixed surface charges on the
axonal membrane7 · 17 - 19 . It was also suggested that
Ca2+ may affect the gating conformation of Na+
channels by occupying specific site of the channel
protein in frog myelinated nerve fibers 20 . The fai lure
of reversal of Peak-II attenuation by washing with
2
Ca +-free Ringer or by calcium channel antagoni sts
MANDAL & DESH PANDE: CALCIUM SENSITIV E PEAK IN COMPOUND ACTION POTENTIAL
signifies the non-involvement of Ca2+ permeability
mediated mechanisms. Further, the ability of EDT A
to reverse the changes indicates that membrane bound
Ca2+ is chelated by EDT A. The low C-50 (0.35 rnM),
non-linear concentration-response
pattern
and
irreversibility of the Ca2 + effect suggest that Ca2+
attachment to the membrane can not be displaced
easily. These observations support the earlier
postulations of neutrali zatio n of fixed charges on the
surface membrane or the negative surface charges
near sodium channel 21 by extern al Ca2+. This may be
the reason for the protection offered by Ca2+ free
medium to the hypoxic-injur/·22 -24 . Further, these
reports revealed the ineversible nature of Ca2+ action.
Although from the present study it is difficult to
asce11ain the underlying mechanism of Ca2+-induced
attenuation of Peak-II, we were able to demon strate
the unique behavior of slow conducting fiber to
external Ca2+.
In conclusion, it may be suggested that external
Ca2+ concentration in phys iological ranges prevent the
slow conducting fibers from attaining the max imum
actJVJty and may pl ay an important role m
determining the vulnerability of slow conducting
fibers to various pathological conditions.
Des hpande S B , Kumar P, Sachan A S, Dube S N &
Dasgupta
S,
Diisopropylphosphorofluoridate-ind uced
depression of co mpo und action potential of frog Sciatic
nerve in vitro is mediated through the inhibition of
cholinesterase activity, 1 Appl Toxicol, 16 (1996) 497.
10 Deshpande S B, Indi an red scorpion (Mesobuthus tamulus
concanesis, Pocock) venom prolongs repolarization time and
refractoriness of the compound action potential of frog
sciatic nerve in vitro through calcium dependent mechani sm,
Indian 1 Exp Bioi, 36 ( 1998) II 08.
11 Hutchinson N A, Ka les ZJ & Smith R S, Conduction velocity
in myeli nated nerve fibers of Xenopus laevis , J Physiol, 208
9
(1970) 279.
12 Brinely Jr F J, Exc itat ion and conduction in nerve fibers, in
Medical physiology edited by V B Mountcas tle (C V Mosby
Company , St. Lo ui s, USA) 1974, 34.
13 Stein R B & Pearson K G. Predicted amplitude and form of
14
15
16
17
2
3
4
5
6
7
8
action potentials recorded from unmyelinated nerve fibe rs, J
The or Bioi, 32 (197 1) 539.
Hille B, Pharmacuiogicai modification s of the sodium
channels of frog nerve, J Gen Physiol, 51 (1968) 199.
Brismar T , Effects of ionic concentration on permeab ility
properties of nodal membrane in myelinated nerve fibers of
Xenopus laevis, Potential c lamp experiments, Acta Physiol
Scand, 87 (1973) 474.
Poulter M 0, Hashiguchi T & Padjen A L, Ev idence for a
sodi um-dependent potassium conduc tance in frog myelin ated
axon, Neuroscience, 68 ( 1995) 487.
D ' Arrigo J S, Possible screening of surface charges on
crayfish axons by polyvalent metal ions, 1 Physiol , 23 1
(1973) 117.
18 Mozhayeva G & Naumov A P. Effect of surface charge on
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