Principle and task To use a nerve function model to study the following aspects of a nerve cell: INTRACELLULAR POTENTIAL AND ACTION POTENTIAL Comparison between low and high threshold levels Comparison between low and high stimulus levels MEMBRANE TIME CONSTANT AND LOW-PASS FILTERING Membrane time constant Low-pass filtering EXCITATORY SYNAPSE Depolarisation Temporal summation Spatial summation Synaptic amplification by terminal branches Effect of decreasing stimulus HEBBIAN SYNAPSE Synaptic learning and forgetting INHIBITORY SYNAPSE Hyperpolarization Spacial inhibitory-excitatory summation VETO SYNAPSE Figure: Typical experimental set-up. To set up the experiments, use the corresponding set-up drawings. 1
Equipment 1 Neurobiology Lab PC, Windows 95 or higher 65963-11 Set-up and procedure INTRACELLULAR POTENTIAL AND ACTION POTENTIAL (FIG. 1) Internet search keywords: Intracellular potential, resting potential, action potential, stimulus, nerve cell, neuronal stimulation. Action potential arises by influx of sodium ions through the sodium channels of the nerve cells. Stimulus movement along the axon occurs due to the consecutive influx of sodium ions along its cell membrane. The measurement method of this experiment differs from that of the other experiments so that action potential can be displayed (together with intracellular potential). In the other experiments (with the exception of the experiments dealing with the excitatory synapse) presynaptic signal strength is shown instead, along with intracellular potential. Experiment set-up according to Fig. 1 and Fig. 2: Software: select the fast measurement mode (trigger +25%, rising, data transfer to "Analog in 2", frequency 10 khz, 1024 values, show channels "Analog in 1" and "Analog in 2", X data: time, range ±10V for "Analog in 1" and ±0.1V for "Analog in 2"). The two experiments described here show the effect of the threshold level of the Neurosimulator and the stimulus level emitted by the operating unit. 2
Fig. 1: Experimental set-up 3
Fig 2: Window for settings a) Comparison between low and high threshold levels: Graph: maximum stimulus intensity (turn knob of operating unit to the right) and low threshold (here: 0) creates fast frequency of action potential (see Fig. 3). Graph: same stimulus intensity, but this time, increase threshold level, therefore lower frequency of action potential (see Fig. 4). b) Comparison between low and high stimulus levels: Perform the experiment comparing low and high stimulus intensities with one another, while keeping threshold at 0 (turn threshold knob to left). 4
Fig. 3 Fig 4 MEMBRANE TIME CONSTANT AND LOW-PASS FILTERING (FIG. 5) Internet search keywords: Resting potential, membrane time constant, low-pass filtering. Experiment setup (see Fig. 5 and Fig. 6): Here, as in all the other experiments in which the intracellular potential is measured together with a stimulus level, choose the normal measurement mode with the following settings: get value every 2 ms, start and end measurement on key press, show channels "Analog in 1" and "Analog in 2", X data: time, range ±10 V for both "Analog in 1" and for "Analog in 2", select the following displays: digital displays 1 and 2, diagram 1. 5
After clicking on button "Continue", an intracellular resting potential of -7 V is shown, which is 100 times the resting potential in a real nerve cell. Fig. 5: Experimental set-up 6
Fig. 6: Window for settings a) Membrane time constant (see Fig. 7) Graph: membrane time constant. Since nerve membranes have capacitative properties and electrical resistance, intracellular potential behaves like the charging and discharging of an electrical capacitor. In nerve cells, the time constant is 10 to 50 ms, i.e. this amount of time is needed for the intracellular potential to reach 63% of its highest level. b) Low-pass filtering (see Fig. 8) This phenomenon is used to allow that fast and short term signals are decreased and that intense (slow and long) signals can be transmitted = filtering of low-pass signals. Graph: low-pass filtering. Low stimulation frequency: intracellular potential changes can be fully reproduced. High stimulation frequency: individual stimulations are not reproduced any more and the intracellular potential remains unchanged. 7
Fig. 7 Fig 8 EXCITATORY SYNAPSE (FIG. 9) Internet search keywords: Depolarization, summation, temporal summation, spatial summation, EPSP. Set-up is as in "Intracellular potential and action potential. Software parameters are as in "membrane time constant and low-pass filtering. Threshold = 0. a) Depolarization (see Fig. 10) 8
Stimulations via excitatory synapses depolarize the cell membrane of the intracellular potential, i.e. the voltage gradient between inside and outside the nerve cell membrane becomes less negative. Graph: medium stimulus level. One stimulus. The intracellular potential builds up briefly and degrades again, as shown in "membrane time constant and low-pass filtering. Fig. 9: Experimental set-up 9
Fig 10 b) Temporal summation (see Fig. 11) Temporal summation makes use of the integrated loudspeaker of the operating unit (acoustic monitor). The signal helps find the level of stimulus at which no acoustic signal is emitted when pressing the stimulus button very briefly calibrate carefully by turning the stimulus knob counterclockwise and testing by pressing the button. Now, without changing the level of stimulus again, press the button for a longer time. The acoustic signal will again sound. Pressing the knob for a longer time is identical with multiple stimuli. Graph: temporal summation: the stimulus is so low that no action action potential is created. Only multiple stimuli create action potential. No significant increase of intracellular potential. c) Spatial summation Spatial summation again makes use of the integrated loudspeaker of the operating unit again. As in the previous experiment, the position of the stimulus knob is determined at which no signal is emitted, i.e. no action potential is created. This is done for a second stimulus channel which is connected to a second synapse (i.e. two white cables are now required instead of one). Then both stimulus buttons are pressed at the same time, creating action potentials. d) Synaptic amplification by terminal branches Again here, acoustic determination of action potentials. As in the two previous experiments, the position of the stimulus knob is determined at which no signal is emitted, i.e. no action potential is created. Then an additional cable (white) is attached to connect the excitatory synapse, which is connected to the stimulus, with the second excitatory synapse. This step is repeated again with the third excitatory synapse. Each time the signal gets more pronounced. 10
Fig 11 e) Effect of slowly decreasing stimulus (see Fig. 12) Decreasing stimulus leads to decrease of action potential frequency and reduction of depolarisation. Left measurement: maximum stimulus S frequency of action potential high and hyperpolarized intracellular potential. Right measurement: stimulus is reduced S frequency of action potential and intracellular potential back to normal. Fig 12 11
HEBBIAN SYNAPSE (FIG. 13) Internet search keywords: Hebbian synapse, synaptic learning, synaptic plasticity. Fig 13: Experimental set-up Synaptic learning and forgetting (see Fig. 14) In the anatomical sample the Hebbian synapse is located at the end of dendritic spines. It is an excitatory synapse with variable transmission behaviour. To perform the experiment, turn threshold button to left (0) and turn stimulus buttons of channels 1 and 2 to 75%. The first 6 spikes in the graph show: consecutive activation of the Hebbian and excitatory synapses. The next 12 spikes are created by simultaneous activation of the Hebbian and excitatory synapses and increase of depolarization. 12
The next 3 spikes show activation of the Hebbian synapse which is now above the level of initial activation. The last 2 spikes show activation of the Hebbian synapse after pressing the reset button to initiate synaptic forgetting: the Hebbian synapse has unlearnt the properties which it learnt when coupled with an excitatory synapse. Longterm potentation: activation of Hebbian synapse by complementary excitatory synapse can last several minutes up to several hours. In the Neurosimulator activation lasts about 10 minutes, unless the reset button is pressed. Fig. 14 INHIBITORY SYNAPSE (FIG. 15) Internet search keywords: IPSP, hyperpolarization. 13
Fig. 15: Experimental set-up In the anatomical sample the location of the inhibitory synapse is in the shaft of the dendrite of a nerve cell. Effect similar to excitatory synapses, however, its effect is inverse: the negative polarisation of the intracellular potential increases further (> 70 mv) = hyperpolarization = reduced excitation. For the two experiments, wire stimulation channels 1 and 3 with the two inhibitory synapses and stimulation channel 2 with one excitatory synapse. a) Hyperpolarization (see Fig. 16) Graph: activation of stimulation channel 1 (which is connected to an inhibitory synapse). Hyperpolarization. Thereafter return to resting potential. 14
b) Spacial inhibitory-excitatory summation (see Fig. 17) Sequential activation of all three stimulation channels to demonstrate spacial inhibition. At first activation of the excitatory synapse (depolarization), thereafter inhibition in two steps, by activating at first one, then the second inhibitory synapse. Keep buttons pressed. Setting up the experiment: medium setting for stimulation channels 1 and 3, maximum setting for stimulation channel 2 (which is connected to the excitatory synapse). Yellow cable which connects to computer interface must be plugged into stimulation channel 2. Fig. 16 Fig 17 15
VETO SYNAPSE (SEE FIG. 18) Internet search keywords: Veto synapse. The veto synapse does not bring about a change of the intracellular potential (so-called silent inhibition). Its effect is only on the excitatory synapse to which it is attached (socalled presynaptic inhibition or shunting inhibition). No spatial summation. Experimental setup: Connect stimulation channel 1 to excitatory synapse and stimulation channel 2 to veto synapse. The stimulation channel for the veto synapse is connected to the interface to show in the graph when the veto synapse is stimulated. Graph: threefold activation of the veto synapse while the excitatory synapse is continuously stimulated (Fig. 19). Fig 18: Experimental set-up 16
Fig. 19 17