GIU Physiology Lab Manual PDF, PHTH 105, Winter 2024

Faculty of Physical Therapy PHTH 105 Winter 2024 PHYSIOLOGY LAB MANUAL Lab 2 Nerve Experiments (1) Name: _______________________________ ID: _____...

Faculty of Physical Therapy PHTH 105 Winter 2024 PHYSIOLOGY LAB MANUAL Lab 2 Nerve Experiments (1) Name: _______________________________ ID: ________ Tutorial number: _______  Faculty of Physical Therapy PHTH 105 Winter 2024 Objectives: 1- Describe the four phases of action potential and identify the state of the Na+ and K+ voltage-gated channels in each phase. 2- Discuss the refractory period in terms of its cause and its importance. Introduction The basic unit of the nervous system is the individual nerve cell or the neuron. Neurons operate by generating electrical signals or impulses, which are transmitted from one part of the body to another. The impulse is normally conducted along the axon at a constant amplitude and velocity, until it reaches axon terminals. Action Potential An action potential (AP) is a rapid, large change in membrane potential following stimulation with an adequate (threshold) stimulus. It consists of four different phases: (1) Latent Period: the time between application of stimulus and beginning of depolarization. Membrane is at RMP (-70 mV), where both Na+ and K+ voltage-gated channels are closed, but K+ ions continue to flow outside through the non-gated channels of the cell membrane. (2) Depolarization: loss of polarity and reversal of polarity. During depolarization, Na+ voltage-gated channels open and Na+ influx occurs. At the beginning, depolarization is slow until it reaches firing level (= threshold) (-55 mV), then it suddenly becomes rapid, where more and more Na+ channels open. Membrane potential reaches +35 mV. (3) Repolarization: membrane potential moves back towards RMP. At the peak of the action potential, Na+ channels slowly become inactivated, and repolarization starts. K+ channels slowly start to open, and K+ efflux increases to even higher levels than at rest. (4) Hyperpolarization: a prolonged period of mild hyperpolarization (-80 mV). When the membrane has repolarized almost completely, the gates of the K+ channels close slowly. Thus, some K+ ions still flow out after RMP has been reached. Concentrations of Na+ and K+ are then restored by the Na+-K+ pump, and gradual return to RMP is achieved. 2  Faculty of Physical Therapy PHTH 105 Winter 2024 Refractory Period During the ascending limb of action potential and 1/3 of descending limb, the Na+ channels initially open and then go into an inactivated state. Any stimulus, no matter how strong it is, will not produce a second action potential and the membrane is said to be in its absolute refractory period (the nerve cell is then unexcitable). Following the absolute refractory period, there is an interval during which a second action potential can be produced but only if the stimulus is of stronger intensity, this is the relative refractory period: it corresponds to the period from the end of absolute refractory period to the end of action potential. It occupies the last 2/3 of repolarization until the end of hyperpolarization. NB: The action potential propagates only in one direction towards the axon terminal due to the refractory period. 3  Faculty of Physical Therapy PHTH 105 Winter 2024 Practical application Aim The aim of this experiment is to record the action potential and the refractory period in a frog’s sciatic nerve. Principle of the experiment Experiments are conducted on simulated frog’s sciatic nerve using a simulation program (SimNerv®). The nerve can be electrically stimulated by a stimulator. We can adjust the number of stimuli, durations and intensities. Results are then displayed by an oscilloscope. Experimental setting 7 1 2 6 3 4 5 Search for “SimNerv®”, then open the simulation program. The virtual lab appears on the screen, and shows the experimental chamber (A), stimulator (B), and oscilloscope (C). 4  Faculty of Physical Therapy PHTH 105 Winter 2024 The experimental chamber (A): - Open the chamber (1) by clicking once on the lid. - Place one of the nerves, which are present in a nutrient solution inside a petri dish (5), inside the chamber and then close the lid. The lid should always be closed, otherwise you will catch interference voltages. - The chamber contains two sets of electrodes: stimulating electrodes (2), on the left end receiving electric stimuli from the stimulator, and recording electrodes (4) on the right end sending the electrical changes occurring in the nerve to the oscilloscope (C). - A ruler (3) is also present in the chamber. The electrodes can be moved along the ruler by shifting their colored handles with the pressed mouse button. The stimulator (B): It is used to deliver electrical stimuli to the nerve. - The mode of the electrical stimuli can be varied between single or twin (in case of double stimuli). - The amplitude and duration of the stimulus pulses can be varied. - Time delay can be set for delivery of a double pulse (used with “mode” switch to "twin"); this sets the time between the start of the first and the start of the second pulse (stimulus). - The current direction can be set using the "polarity" switch, with “invert” allow immediate switching of the polarity of the stimulating electrodes. The oscilloscope (C): It is a digital storage instrument with two channels and time-base. - Channel 1 (6) shows the stimulus. - Channel 2 (7) is connected with the electrodes of the experimental chamber and thus shows the nerve response to a stimulus. - With the knobs, the channel sensitivity of each channel can be adjusted. - Time-base is adjusted in millisecond from the button (8). - The base line of the channels can be regulated by CH1 and CH2 (9) that can be moved up and down. 5  Faculty of Physical Therapy PHTH 105 Winter 2024 Basic settings Switch on the stimulator and the oscilloscope, by clicking on the power button in each one. Open the chamber by clicking once on the lid. Place one nerve (5) inside the chamber and then close the lid. Ensure that the stimulating and recording electrodes are at a distance ranging from 7 to 9 cm apart from each other. Ensure that the “store” button on the oscilloscope is not switched on. Drag CH1 (9) (on the oscilloscope) to the bottom of the screen and set its sensitivity to 500 mV/Div. Drag CH2 (9) (on the oscilloscope) to the lower part of the screen but above CH1 and set its sensitivity according to the experiment. For each experiment, make the required adjustments before starting your work. Experiment 1: Action potential Adjustments: Oscilloscope Time-base: 1 ms/div Channel 1: 500 mV/div Channel 2: 1 mV/div Stimulator Mode: single Polarity: normal Trigger: normal Amplitude: start with 200 mV (multiplier is adjusted at X10) Duration: 1 ms (multiplier is adjusted at X1) Delay: off 6  Faculty of Physical Therapy PHTH 105 Winter 2024 - Stimulate the nerve by pressing the red button in the stimulator “Start”. - Apply stimuli of slowly rising amplitudes (by 10mV) until action potential appears on the screen. - Once AP is reached click on “Store”. - Continue to increase the intensity of the stimulus until no further increase in the amplitude of the action potential is observed. Experiment 2: Refractory period Adjustments: Oscilloscope Time-base: 1 ms/div Channel 1: 500 mV/div Channel 2: 1 mV/div Stimulator Mode: twin Polarity: normal Trigger: normal Amplitude: 400 mV (multiplier is adjusted at X10) Duration: 1 ms (multiplier is adjusted at X1) Delay: start with 7 ms and decrease until refractory period is reached (multiplier is adjusted at X1) - Stimulate the nerve. - Gradually reduce the delay (by 2 ms every time) and stimulate the nerve. The amplitude of the second action potential will start to decrease. - Determine the stimulus delay at which the relative refractory period appears. - Determine the stimulus delay at which the absolute refractory period appears. 7  Faculty of Physical Therapy PHTH 105 Winter 2024 Experiment 1: Action potential (a) Fill the following table with your results: (2 marks) Results Stimulus strength in (mV) Threshold stimulus needed to obtain AP Maximum stimulus (b) Draw and label the action potential obtained with a maximum stimulus. (2 marks) 8  Faculty of Physical Therapy PHTH 105 Winter 2024 (c) Mention the ionic conductance in each phase of the action potential and its cause. (3 marks) _____________________________________________________________ _____________________________________________________________ _____________________________________________________________ _____________________________________________________________ _____________________________________________________________ _____________________________________________________________ _____________________________________________________________ _____________________________________________________________ _____________________________________________________________ _____________________________________________________________ Experiment 2: Refractory period (a) The stimulus delay at which the relative refractory period appears equals: ms (1 mark) (b) The stimulus delay at which the absolute refractory period appears equals: ms (1 mark) (c) Give an explanation for the absolute refractory period. (1 mark) 9