3.1
MATERIALS & METHODS (For Chara & Nite11a Experiments)
3.1.1
Procurement and Culture of Chara and Nite1la:
Nitella species was procured from local seasonal pond within
Jawaharlal Nehru University Campus. They were planted in big
(18" high x 9" diameter} jars containing 2" soil (mixed with
humus and manure}at the bdttom. Jars were always kept filled
with tap water which was intermittently flushed out to clear
off the debris and the increasing salt concentrations. The
contaminating
phytoplanktons
(mainly blue-green
and
other
alga}which menacingly keeps growing in such conditions, were
controll-ed biologically by co-culture of some zooplanktons
like Cyclops, Daphnia, Cypris and other species. Direct sunlight at the window for 5 to 6 hours daiiy is sufficient for
their luxurient growth. By this easy
wa~
the Nitella culture
can be maintained in the laboratory through out the year.
Even in .the laboratory the most suitable season for
\
its
flourishing growth is from the month of July to September.
Chara,
on the other hand could not be cultured
laboratory by
in the
above mentioned method and it was always
procured· from the basin of Hindon river.
It could survive
for a long time in the laboratory.
3.1.2
Preparation:
Green internodal cells of diameter 1 mm and 4-6 em long were
isolated from the plant and kept in artificial pond water
106
(APW) for a day or two before the experiments. To minimize
disturbances while mounting, all the leaflets in whorls and
other branches at the nodes, were cut out carefully. The internodal cells suffer some shock in the process, but recover
completely after 4 and 5 hrs. Also the cell takes some time
to get adapted to a change of medium i.e. from the jar w·ater
(where it was grown)
to APW.
The preparation was made at
least 4 to 5 hrs before starting the experiment after which
it became stabilized. Internodal cells of roughly the same
size - 3 em were used.
3.1.3
Chemicals and Solution:
In all the ·experiments the bathing medium was the same (APW)
with different pH adjusted by adding NaOH or HCl solution
for alkaline and acidic ranges.
APW consists of 0.1 mM of
each KCl, NaCl and cac1 2 (Shimmen and Tazawa, 1982).
Temperature of the chamber in all experiments was
in the
. range of 25° to 28°c.
Following sequences of pH and K+ concentration in the perfusate.were used:
1.
pH in APW : 3 • 5; 4 • 0 ; 4 • 5 ; 5 • 0 ; 5 • 5 ; 7 • 5 ; 8 • 0 ;
8.5; 9.0; 9.5; 10.0; 10.5 and 11.0.
2.
K+(mM) in APW: 0.01; 0.1; 0.5; 1; 2; 3; 4; 5 and
10 mM K+ concentration.
Instruments and Procedure for Electrophysiological
3.1.4
Recording in the Chara and Nitella cells:
i)
Recording Chamber
Electrophysiological experiments were conducted in an airconditioned room;
the ambient temperature was maintained at
25°c to 28°c and the humidity at 35 to 40%. Maximum care.was
taken
was
to avoid mechanical
on ..
The
vibrations while
the
experiment
recording chamber partially covered with a
metallic {Faraday} cage for reducing electromagnetic noise,
was housed on a vibration free concrete bench. The recording
set up was adequetely grounded.
The principle of electrophysiological
recordings have been
extensively described by Findlay and Hope
Ravindran
{1983}.
Fig.
(1976}
and by
3.1 illustrates the principle ·of
recording of membrane potential.
ii}
Microelectrode Puller:
A vertical pipette puller{Model 700C David Kopff Instruments,Tujunga,California, USA) with standard 2.5 turns Nichrome
wire coil was used to pull rnicroelectrodes of the desire;d
tip diameter,
tip resistance and
tip potential.
1
Kwikfil
1
{WPI, New Haven, USA}glass capillaries with outside diameter
1.0 and 1.2 mm were used for making microelectrodes.
iii) Microelectrode Holders:
WPI,
MEH-lS rnicroelectrode holders were
the rnicroelectrodes.
WPI
used·. for
rnicroelectrode holders
coupling
provide
a
108
Tf?= Tip Resistance
TP =Tip Potential
ZBL= Zero Base Li~
BLS =Base Line Shift
·J~omv
Electrode~
in first
cell
fig.
Electrode in $econd cell
Sr,nin
3.1.
Electrode
.removed
A diagrammatic representation of the
sequence of events during the intracellular
recording~
convenient coupling of fluid
filled
glass microelectrodes
and high input impedance amplifiers.
A reversible
(non -
polarizing)electrode (Ag-AgCl)provides-the interface between
the electrolyte in the micropipette and the amplifier input.
These holders can be conveniently connected to the probe of
WPI,
M-701 microprobe amplifier discussed below.
electrode (Model RCl, WPI)
cons~sted
Reference
AG-AgCl pellet encapsu-
lated in epoxy. This electrode is supplied with a 1.8 meter
length of flexible wire. The net de potential in the circuit
resulting from the Ag-AgCl half cells was generally less
than 1 mv.
iv)
Electrometer:
WPI, M-701 Microprobe system used in the present study is a
precision broad banrl electrometer with current passing circui try included.
This instrument is eminently sui table for
the measurement of living cell membrane potentials with
fluid filled micropipette electiodes. The miniature probe is
designed for mounting in a micromanipulator.
allows the probe to be placed
~s
This feature
close to the source of
potential as possible, thereby enhancing its sensitivity.
'
v)
Micromanipulator and Microscope Assembly:
Leitz
(Laborlux-2, Wetzlar, W.Germany) precision micro-
manipulator was used· for driving' the probe carrying the
microelectrode into the cell. A binocular microscope (Leitz)
horizontally mounted on the platform Of the micromanipulator
enabled visual monitoring of the microelectrode.
109
vi)
Oscilloscope:
The output of WPI M-701 amplifier was connected to the input
of a 5113 Tektronix Dual Beam Storage Oscilloscope and to a
strip chart Recorder, LKB. The line voltage was regulated by
a voltage stabilizer with automatic + 10 volts cut off. (see
also in section 3.2.3-iii).
The 5Bl2N Dual Time Base amplifier was used with the oscilloscope. It provides the wide range of sweep rate 0.2 micro
second/division to 5 second/division.
The vertical plug-in
used was 5A22N differential amplifier (Tektronix).
vii) Chart Recorder:
For a few recordings, a simple single channel LKB chart recorder. was used.
The set up for electrophysiological studies is shown in
Fig.3.2.
3.1.5
Chamber for Giant Algal Cells:
The prepared internodal cells of Ni tell a
immersed in the
bathing solution in a transparent glass chamber (3.5 x 2.5 x
0.5cm) was kept on the microscope stage (Leitz Laborlux-2).
The bathing solution was constantly perfused in the chamber
.to keep the ionic concentration maintained.
Since the ex-
periment was to be done in light condition the chamber was
illuminated by a 50 W halogen lamp.·
Insertion of microelectrode into the cell was done with the
help ·of the micromanipulator and was monitored through the
110
·Electrometer
:strip Chart recorder
or Oscillascaptl
o = Outer Solution
'Yc . . o
Plasma membrane
v=Vacuole
'\f'v . . c
Tonoplast
:i-t---
Fig ••• 3.2.:-
Cell wall=
Free space
• The apparatus for trcmsmembrane-
elec-(opotential measurements. The "indifferent electrode"·
is placed in the bathing medium while the "different
electrode" is inserted in to the cell, in variably up
to the vacuol·e. The latter is connected to the active
probe of the
ele~tometer.
The output of electometer
is cdnnected to the strip- chart recorder (see text for
further detail ).
binocular eye piece of the microscope. The PD was recorded
on oscilloscope/strip chart recorder when the PD had stabilized. The time required for full stabilization was about 15
minutes after insertion of the microelectrode.
The recording system was calibrated periodically with a
standard voltage source(Grass stimulator,S-44).A diagramatic
representation of the electrical circuit is given in Fig.3.2
3.1.6
Measurement in Chara Cells:
For the preparation of Chara cells, the cortical cells which
encapsulate the internodal cell, were first removed by careful peeling off operation. This was done under stereo microscope (Carl-Zeiss/Jena) with the help of a fine blade tip
and fine tweezers.
After this the internodes were kept in
APW for minimum 24 hrs to recover from the shock before
starting the experiment. The rest of the procedure followed
were the same as in the case of Nitella.
3.1.7
Procedure for the Membrane Potential.Measurement:
Electrodes drawn
in the vertical electrode puller were
filled with 3M KCl.Prior to the penetration of microelectrodes in the cells,the electrode tip resistance was measured
by interconnecting the microelectrode tip and reference
electrode through lOOrnM KC1.Passing a constant PC current of
. 1 nA through the circuit by the
speci~l
microelectrode resi-
stance testing device provided in the WPI microprobe system,
111
gave the tip resistance. Electrodes with tip resistances of
5-50 M
were used for membrane potential measurements.
Electrodes with tip potential of less than + 10 mv alone
were U$ed for measurements. Tip potential was cancelled by
appropriate compensating adjustment
(position knob)
in the
microprobe system to establish the zero base line. With this
procedure the true membrane potential was recorded upon
penetration of the microelectrode into the cell.
When the •different, microelectrode
connected
to
the probe
contacted the surface of the internodal cell the open circuit noise disappeared. Deeper penetration of the electrode
into the cell led to a sudden negative deflection in the recorder. This indicated the entry of the electrode tip into
the cell interior. After the initial dip,
the PD gradually
became more and more negative and then stabilized, approximately within 15 minutes. The final steady value of PD
was taken as cell's resting potential, EM. After the recording of the PD of algal cells, the microelectrode was removed
from the cell and the change in the tip potential difference
recorded. This gave the shift in the .base line. This value
was either substracted from or added to the above value of
EM. , The entire sequence of events are shown in Fig.
Corrections for
tip potentials were generally needed.
3 .1.
In
certain cases after the initial dip, the measured PD was of
small. magnitude and did not show any further
change,indicat~
ing a possible damage to the cell or microelectrode. To fur112
ther establish the validity of the observed PD measurements,
the microelectrode was mechanically disturbed, once the
had
stabilised.
This
invariably
depolarised
the
PD
membrane
potential. The fact that microelectrode tip was not damaged
was verified by remeasuring its tip resistance. In the earlier stage of developing the EM measurement technique a test
for the authenticity of the.technique was performed by the
addition of Sodium azide (1 mM) in the bathing medium. The
membrane potential depolarised upon this treatment within a
few minutes,confirming that the observed PD was not a mere
artifact.
3.1.8
Procedure for
the. Measurement
of Velocity of
Cytoplasmic Streaming:
Cytoplasmic particles of different sizes ranging from 0. 5
micron to 10 micron were visible in the internodal cell with
a magnification (lOx X lOxi while most of these move at
roughly the same velocity we chose to observe medium size
particle for
the determination of Velocity of cytoplasmic
streaming. For this purpose we used an ocular scale inserted
in the place of one eye piece of microscope. With the help
of a stage micrometer the scale of the ocular was calibrated
for the amplification used •. The time taken by selected
cytoplasmic particles for
traversing and scales of the
'
ocular were recorded with
th~
'
help of stopwatch.
113
3.2
MATERIALS AND METHODS (for Muscle Experiment):
3.2.1
Experimental Material:
The
frog
sartorius muscle
is
chosen
for
physiological
experiments in view of the many advantages it offers. First
of all frogs are easily available in good numbers, and are
easy to keep in the laboratory for longer times. Of all the.
muscle types, sartorius muscle is relatively easy to dissect
and the fibres within it run parallel to the long axis.It is
small enough to allow adequate oxygenation and contains a
(glycogen)reserve for many hundreds of contractions. Considering all these points we also chose sartorius muscle for
our study on muscle.
a.
Dissection and Mounting:
Being superficially located on the ventral side of the thigh
region, the sartorius muscle of frog is very easy to dissect
out. Tendons at both ends are kept intact for tying to the
thread properly. For weights above 100 gms we observed that
the simple knot was not _helpful. To overcome this problem we
used to weave the thread with tendon and the connective
tissue terminating there, by means of a sewing needle. Care
was taken that muscle fibres would not get injured in the
process. The extra connective and other tissue debris were
removed all around the muscle and to some extent between the
fibres so that- the perfusing
muscle fibres.
~edium
could reach a maximum of
Fine dissection work was done . under stereo
114
microscope
ringer
(Carl-Zeiss/Jena)
solution.
with
the
muscle
immersed
in
The muscle preparation having threads
stitched on both ends, was transferred to the muscle chamber
(Section-3.2.3 [i]). One end was tied with the fixed hook in
the chamber and the other was tied with the ring attached to
the sliding thread on which the weight was hung.
The nerve
entry point of the muscle, evident by its cut portion, was
always kept above so that the stimulating electrode (insect
pin) could be inserted into it.
b.
Storage:
An advantage with frog muscle is that it is durable. In the
Ringer's
solution
(pH-70)
with
antibiotic-s
and
phosphate
buffer at 20°C to 40°C temperature, it could be stored upto
2 days or more in good working condition.
3.2.2
Chemicals and Perfusing Solutions:
The chemicals used in various perfusing media were mainly:
NaCl, KCl, CaC1 2 , Na 2 HP0 4 , NaH 2 P0 4 and DNFB (2, 4dinitrofluorobenzene).
The control solution was Muscle Ringer's solution (Squire,
1981) which consisted of 125 mM of NaCl, 2.5 mM of KCl, 1.2
mM of cac1 2 and 1 mM of phosphate buffer(in case of buffered
solution).
For
pH
experiment,
the
pH
of
solution
was
adjusted by NaOH and HCl for all ranges of pH on either
(alkaline and acidic) side. For DNFB treatment, 3.8 x ln- 4 M
115
concentration of DNFB was dissolved in Ringer's solution.
The temperature at which all the experiments were done was
between 18 to
20°c.
3.2.3
Instrumental Set-up:
(i]
Muscle Chamber:
Muscle chamber (Fig. 3.3) was fabricated in our laboratory.
It consisted of a
mounting-cum-perfu~ion
chamber,with a hook
for the attachment of one ligament of muscle and a thread
going over the pulley with one side containing a loop to·
which the other movable free end of muscle could be fixed.
The pulley scavenged from a Japanese tape deck was fixed in
such a position that the hook, the side hole in the chamber
(see below)
and the upper edge of the pulley were in the
same horizontal line.
The thread attached to the movable end of the muscle passed
over the pulley through a hole in the bottom of the side
compartment of the muscle chamber. This compartment was partitioned by a plastic wall from the chamber containing the
muscle in the bathing medium apart from a hole through which
the thread could pass. The thread went over the pulley and
dropped vertically downwards from the hole.
It could carry
the desired weight hung to a loop at its end. A 5 mm broad
and 3 cm.long strip of blackened photographic film was stuck
on the thread at about 1 em above the weight carrying loop.
116
_.:;r'
·U·
·.
.·
v-
._o: 0 0
:.
0 0
o o·
.:
oo
v
DO
0
=a
0
STIMULATOR
.OSCILLOSCOPE
/
0
G
0
B
1·1USCLE SHM-lBER
PHOTO DIODE BOX
. STAND
Figure 3,3 : Diagram of the instrumental set up used for the muscle experiment. The
muscle chamber constitutes two chambers (not apparent in the diagram) one inside
the other. The outer c~amber has an inlet and outlet io regulate the ice cold water
to keep the temperature of the bathing medium constant.
0
0
c:J
It had a very fine 0.5 mm x 3 mm slit cut out at the centre
of the strip. The muscle chamber was mounted on a metallic
stage. Below the chamber and in between the upper and lower
part of the stage a box containing two photodiodes at one
face was positioned. The photodiodes faced the side carrying
the thread with the attached weight.· A black photographic
paper with two slits of 3 mm size and an interslit distance
of 5.0 mm covered the upper and the lower photodiodes. The
output voltage from the photodiodes were recorded using the
Tektronix Oscilloscope {Section-3.2.3[iii]}.
A 6 vol t/24 Watt.
bulb together with a. lens were used for
getting a parallel beam of light of constant intensity.
Though we used the Carl-Zeiss transformer {220 to 6 volt}its
output
~as
given to a AC-DC converter which provided a
ripple free DC current to the lamp.
A parellel beam of light fell homogeneously on the two slits
over the photodiodes.If the photographic strip was displaced
by a pull on the end of the thread in the muscle chamber,
the fine beam of light through the slit passed over the two
slits of the photodiode chamber sequentially so that
two
peaks of voltage became visible on the oscilloscope screen.
[ii] Stimulator:
Grass stimulator model SD9 was used for ·stimulating the
muscle.
with 1
It contained a frequency range from 1 Hz to 1000 Hz
Hz to 100 Hz variable setting and 1, 10 and 100 fold
control knobs.The voltage
rang~
available was 100 mv to 200V
117
with 1 to 20 fold variable range and 0.1,
1. 0 and 10 fold
control knobs.The range of the duration of square pulses was
2 to 20 ms with 0.01, 0.1, 1.0 and 10 fold variable control
knobs.
Pulses could be given either repetitively or singly.
The output of the .stimulator was connected both to the
external trigger socket of the oscilloscope (see below) and
to the muscle chamber for stimulating the muscle. The ground
of the stimulator was connected to a silver wire
running
along the length of the muscle chamber. It remained immersed
in bathing medium and contacted the lower side of the muscle
along its whole length.The stimulating output terminal was a
flexible wire attached to a fine insect pin.
[iii]
Oscilloscope:
Tektronix storage oscilloscope model 5111 was used in the
experiments.The amplifiers used were Time Base model SB12N,
Dual Time Base Vertical Amplifier No.1 model 51A8N,Dual
Trace Amplifier,
Vertical
Amplifier
No.2
Model
5A22N,
Differential Amplifier.
In
the
Time Base we
triggering
(Source A)
used
trigger
mode-A for
and positive slope.
I
external
The horizontal
deflection of the electronic beam was triggered with the
help of output of the stimulator connected to the external
trigger input of· the Time Base.
The scanning velocity was
chosen according to the requirement between 10 and 50 ms per
division.
The output of the photodiodes was connected to
the differential amplifier (5A22N). Channel I of the other
118
amplifier was used for registering pH (in terms of mV) and
the Channel II for looking at the train of pulses from the
stimulator for the purpose of standardization of experiment
at the beginning.
3.2.4
Experimental Procedure
[i ]
Mounting:
The frog muscle preparation with threads tightened to the
tendons
was
transferred
in
muscle
chamber
ringer solution, with the nerve entry 9oint
filled
fa~ing
with
upwards.
One side was connected to the hook fixed to the wall of the
chamber while the other end was tied to the ring of the
thread which carried a load of 5 gms. The length of thread
between the tendon and the ring was adjusted
in a way that
the muscle achieved its slack length.It may be noted that no
pull was exerted on the muscle due to the load when the
muscle was at its slack length because the metallic ring to
which the thread was attached could not pass through the
whole in the side wall of the muscle chamber. Therefore, the
muscle worked against the load only when it shortened.
A
minimum amount of pull (wt. 1 gm) was required to keep the
thread carrying the photographic
film strip stretched so
that both the optical alignment and the mechanical functioriitig of the pulley were appropriate during the course of
..
shortening of muscle.
119
The stimulating electrode(insect pin)was carefully implanted
at the site of the cut end of the major nerve and running
parallel to it for some length i.e. for about 2 to 3 mm.
Before starting the experiment a 1 volt stimulus of 10 ms
duration was applied to elicit a twitch response in order to
see that the electrode insertion was proper. Thereafter 5 gm
weight was replaced by weights corresponding to the desired
sequence of load.
[ii] Perfusion:
The
experimental
chamber
wit,h
the
solution.Mult~ple
solution was
help of
introduced
syringe
into
replacing
the muscle
the
Ringer
exchanges were made if and when considered
necessary.
[iii] Procedure for Recording:
All our experiments consisted in recording the velocity (v)
of
isotonic contract ion for
different perfusing solutions
and/or prior treatments as a function of the loads (P) on
the muscle.The types of stimulus(fequency/duration/ voltage)
were also changed in the case of some recordings. The forcevelocity relation v(P) was determined for a sequence of
weights
attached
to
the
thread
carrying the photographic
strip by measuring the time taken for the light beam through
the moving slit.to cross over successfully the lower and the
upper slits over the photodiodes, as the muscle pulled the
weight upon stimulation.
As the
.fin~
b~am
of the light
through the slit in the photographic strip cross the lower
120
and the upper photodiode, two current pulses were obtained
giving two deflections on the oscilloscope screen.
When the
muscle resumed its slack length after the stimulation period
was over,the photographic film strip move downwards (due to
the attached weight) giving rise to two more current pulses.
The peak to peak distance between the first two pulses on
the oscilloscope screen was measured by a plastic scale in
inches. Knowing the scanning speed of the Time Base and the
number
of
divisions
in
graticule
of
the
oscilloscope
(corresponding to inches), the time ( .dt) taken for the slit
to traverse the known inter-slit distance ( £1 X) between the
photodiode opening V= ..1 x/ t:. t, was determind. Since we kept
the slit in the photographic plate about 1 em below the
lower slit of the photodiode system and also" because the
muscle usually shortened by sufficient length to cause the
slit in the photographic strip go beyond the upper slit of
the photodiode system by more than 1 em,
the velocity
recorded by us was representing the middle stages of the
velocity of shortenning.
121