Bundles of fibres were set up in a bath (0.5ml) suitable for rapid changeover of solutions (2ml/s)(Figure 2.1). A small stainless steel hook was placed through the tendon at one end of the preparation and attached to a semiconductor force transducer (Akers, model AE875, SensoNor a.s. Horten, Norway). The other tendon was attached to forceps held perpendicular to the preparation by Narashige micromanipulators, allowing for easy length adjustment. Tension could be induced either by the application of high potassium solutions or by direct electrical
stimulation to produce twitches and tetani.
The small volume bath (0.5 ml) was mounted on a thermostatically controlled water jacket which maintained temperatures of bathing solutions at
22 ± 1 °C throughout the experiments. The changeover of solutions was achieved by an electronic switching solenoid designed and built at the ANU. Solutions containing normal or elevated potassium were flowed into the bath (max. rate=2
ml/s) via an inlet located close to the forceps. The equilibration time for solutions was less than 0.5 sec. A motor driven pump (Neuberger, miniport pump) removed solutions through an outlet located close to the force transducer. platinum electrodes were fitted to the long walls of the bath and extended over the entire length of the preparation.
Experiments were controlled by an interface between an Osborne 386 personal computer and an isolated stimulator (designed and built at the ANU). A software program ("MUSCON") written within the department enabled parameters and data for each experiment to be saved in digital form for later analysis.
At the beginning of each experiment the length of the preparation was adjusted to achieve maximal twitch height. During recovery phases of experiments, fibres were stimulated continuously at a frequency of 0.1 Hz. Pulse durations were usually less than 1.0 msec and pulse amplitudes around 70V. Fused tetani could be achieved by trains of pulses (50-70 Hz) for a period usually less than 1.5 s. This produced maximal contractions with a plateau phase.
CHART R EC O R D ER O S C IL L O S C O P E FO R C E TRANSDUCER P r e p a r a t i o n AMPLIFIER STIMULATION A -D CONVERTER ISOLATED ST1M UATOR PULSE TRIGGER, DATA ACQUISITION & ANALYSIS
Figure 2.1: Diagram illustrating the main features involved in the stimulation, recording and analysis of force in contraction studies.
Specific contractures, tetani and twitches were saved digitally and a continuous record of each experiment was displayed on a digitizing oscilloscope (Tektronix 5223) and plotted on a two channel chart recorder (7402A, Hewlett- Packard Co., Palo Alto, CA, USA). The chart recorder had a frequency response of 55 Hz for full-scale deflections and 125 Hz for 10mm (twitch height) deflections.
2. 7.1 Two-microelectrode voltage clamp
Intracellular glass microelectrodes of 4-8 Mft were filled with 2.5 M KC1 and used to point voltage clamp single muscle fibres. The design layout of circuit is shown in Figure (2.2). Operational amplifiers (TL074 op-amps) 1 and 2 are unity gain voltage followers (buffers), providing a low output resistance from a high resistance voltage source (ie. a lOkO potentiometer). The outputs of these buffers, together with the input signals of the voltage electrode (em) and reference (bath) electrode (e^) respectively were fed into low-pass filters 3 and 4, also constructed from the TL074 chip. The purpose of the low-pass filters was to eliminate high frequency noise.
The output of op-amp 3 was fed into a unity gain op-amp (5) constructed * *
from an OP-07C chip . This allowed the membrane potential, Vm, to be monitored.
In addition, the output of op-amp 3 was also fed, together with the output of op-amp 4, into a unity gain differential amplifier, 6. The membrane potential, Vm, was then displayed on an oscilloscope and also fed back into a summing operational amplifier, 7. A command potential, -Vc, and the membrane potential, + V m, were compared at the summing point. The command p o ten tials the required membrane potential; and“^ a combination of a holding potential (-V^) and a brief depolarising test pulse (-Vc).
The feedback system ensures/ that the actual membrane potential (Vm) aasthe same as the required membrane potential (Vfo or Vc). If the two signals &*/* unequal (ie: non-zero signal following subtraction) then the difference ws fed back through a variable gain voltage amplifier and applied to a current passing microelectrode inserted in the muscle fibre. The gain of the clamp toM be changed so that depolarisation of the muscle fibre membrane closely resembles the applied
rectangular pulses. The 10% to 90% response time of the voltage clamp was 100
p s .
The inputs to the differential amplifier had switches which could isolate either part of the circuit (em or e^). A lOkQ 10-tum potentiometer on the non-
I m + I O + H U. F ig u re 2 .2 T h e t w o m ic ro e le c tr o d e v o lt a g e c la m p s e t- u p . A f u ll d e sc ri p ti o n is g iv e n i n th e te xt .
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inverting inputs of the buffers was used to "zero" the potential on the tip of the microelectrodes before insertion of em into the fibre.
The bath ground electrode for the system was a chloride coated, tightly coiled, silver wire attached to a virtual earth current to voltage convertor. Voltage, current and command signals were continually displayed on an oscilloscope.
*
( ) The TL074 is a low noise operational amplifier with low input bias and offset currents and fast slew rate. The low harmonic distortion and low noise make this amplifier ideally suited for high fidelity and audio pre-amp applications. Each amplifier on the chip has JFET inputs for high input impedance.
( ) The OP07C is an operational amplifier with ultra-low input offset voltage. The differential inputs with wide input voltage range and good CMRR provide accurate results in high-noise environments.
2. 7.2 Capacitance Neutralization
Each voltage microelectrode (em or e^) input was connected to a capacitance neutralisation circuit, so that the response time of a biological amplifier system to microelectrode input signals was not limited by stray capacitance arising from various sources, eg; between the drain and gate of FET input transistors,long connecting wires, capacitance across the microelectrode wall to the bath, etc.
With capacitance neutralization the current through the stray capacity is compensated for with current generated by a variable amplified output (1 - 10X) applied through a fixed capacitor to the input. Problems occur in adjusting the compensation correctly so as not to distort the input signal by inducing oscillations due to overcompensation, and also from the injection of current noise with the compensating signal. Optimal performance of the capacitance neutralization gives a
maximum bandwidth response with minimal overshoot.
2.7.3 Contraction Threshold measurement
Thin sheets of intact muscle fibres (section 2.3) bathed in the low chloride control solution (Table 2.1) were pinned out on a Sylgard 184 (Dow Coming) lined bath (volume 3 ml) prior to microelectrode insertion.
A high resolution (800 lines/inch) video camera (National WV-1800/B) attached to a phototube on a compound microscope (Zeiss) allowed the preparation to be observed on a television monitor (National WV-5410) connected to the video
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camera. The microscope was fitted with a 16 X air objective (Zeiss) and a 16 X 12.5mm working distance eyepiece (Zeiss). Calibration of the optical system with a stage m ic ro m e te r^ /.^ overall magnification ^ 5 0 0 . The microscope, stage and objective were earthed to a central point and positioned on a vibration-free air table.
Micromanipulators (Narashige) placed on either side of the microscope objective were used to position the voltage recording and current passing
microelectrodes. These electrodes were inserted into opposite edges of a muscle fibre at a separation of 50-100 fim (Figure 2.2). This configuration was the most appropriate in order to avoid nonuniformities in membrane potential around the current microelectrode (Adrian, Costantin & Peachey, 1969; Eisenberg & Johnson,
1970; Dulhunty, 1982). A third microelectrode was placed in the bathing solution in close proximity to the voltage recording microelectrode.
When the clamp gain was increased, the muscle fibre membrane was point voltage clamped in the region between the voltage recording and current passing electrodes. Fibres were usually clamped at a holding potential of -80mV (unless otherwise stated). Rectangular depolarizing (test) pulses of varying duration and
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CHAPTER 3
ACTIVATION AND INACTIVATION OF CONTRACTION IN NORMAL AND DENERVATED RAT SOLEUS AND EDL.