BIO325 Laboratory Guide #14 (2024)
SYNAPSES I:
ELECTRONIC SIMULATIONS OF ELECTRICAL SYNAPSES
falls under category
B
For this exercise we will be using the Single Compartment Resistor Capacitor Model (SCRCM) boards. Individual circuits on this board will model simplified spherical presynaptic and postsynaptic compartments or cells. You will need one of these boards, assorted cables including several alligator clip jumper cables, and 1 Kohm and 10 Kohm resistors serving as the electrical synapses themselves. Stimulation will be applied as square wave current (as opposed to voltage) pulses via the Grass stimulator, connected through a "constant-current" unit. Triggering and recording will be controlled by the PowerLab Scope application. We will be primarily interested in the effects of electrical synapse connections on presynaptic and postsynaptic current/voltage relationships.
I. ELECTRICAL SYNAPSES - ELECTRONIC SIMULATION
A. Initial Setup
1) Startup the PC (if necessary), turn on the PowerLab box, and launch the Scope application.
2) Set up the Scope software for triggering stimuli and recording responses as follows. Open the Input Amplifier dialog box for each channel, turn all filters off, and set the Range to 200mV. Hit the START button to trigger a single sweep. Pull down the arrow to the left of each trace and under Set Scale choose 120mV and -20mV. In the Time Base box set Time: to 50 msec and Samples: to 2560. Under the Setup menu choose Sampling . . ., then set Mode: to Single and Source: to User. Under the Setup menu select Stimulator . . ., then set the stimulator Mode: to Pulse, Delay to 0, Duration to 1 msec and Amplitude to about 4 Volts. Under the Display menu select Axis Labels . . ., then label CHA as "Cell 1" and CHB as "Cell 2". Finally, under the Display Menu select Display Settings . . ., then set the Graticule to a grid pattern and Channels A and B to attractive colors.
3) You will be using the PowerLab stimulator only to trigger the electronic stimulator. To do this, simply connect the Output + of the PowerLab box to the TRIGGER IN of the electronic stimulator with a BNC cable. All subsequent changes in stimulation settings will be made ONLY on the electronic stimulator.
4) Connect the electronic stimulator through the Analog Stimulus Isolator (constant-current unit - CCU) as follows. Connect the output cable to the stimulator, plug the double banana plug end into a double banana-to-BNC adapter (paying attention to correct plug polarity!!), and plug this into the SIGNAL IN on the CCU. Attach a long red alligator test clip lead into the positive (red) output jack of the CCU. Attach a long black alligator test clip lead into the negative (black) output jack of the CCU. MAKE VERY SURE THAT THE FREE ENDS OF THESE TWO OUTPUT LEADS DO NOT TOUCH, ESPECIALLY WHEN A STIMULUS PULSE IS BEING DELIVERED!!
5) To set up the recording input cables for the PowerLab box, first connect a BNC to black double banana cable to the CH1 input of the PowerLab box and a BNC to red double banana plug to the CH2 input. Attach a yellow alligator test clip lead to the live banana lead (the side without the tab) at the other end of each cable. Attach a green alligator test clip lead to the common banana lead (the side with the tab) at the other end of each cable.
6) To establish initial settings for the electronic stimulator, first make sure that the stimulator MODE is set to OFF, then turn on the stimulator. Set the stimulator DELAY to 10 ms, the DURATION to 20 ms, and the VOLTS to 1.0 Volts. Set the STIMULUS to REGULAR, and the POLARITY to NORMAL and the OUTPUT to MONO(POLAR).
7) Finally, set up the CCU as follows. Set the CCU POLARITY to + and the RANGE to 10 microamps/volt. Turn on the CCU. The ERROR light should flash briefly; if it remains on, immediately turn off the CCU and consult with the instructor.
B. Cell Size and I/V Relationships
TO AVOID DAMAGE TO THE STIMULATOR AND/OR CCU, IT WOULD BE A GOOD IDEA TO TEMPORARILY TURN OFF THE CCU WHENEVER YOU REWIRE YOUR SIMULATED CELLS.
A1 and A2 each model a small cell with a relatively high membrane resistance (Rm = 10Kohm) and a low membrane capacitance (Cm =.1microF).
1) Connect the red (positive) CCU banana test clip lead to the "INSIDE" of Cell A1 on the SCRCM board and the black (positive) CCU banana test clip lead to the "OUTSIDE" of Cell A1. Connect your two recording leads for PowerLab CH1 across Cell A1 - yellow (CH1+ live) goes to the INSIDE and green (CH1+ common) goes to the OUTSIDE.
2) Deliver a single square wave pulse to Cell A1 using the Scope Start button. The recorded trace should a response which has been rounded off at both ends; showing capacitive exponential rising and falling phases. Adjust the stimulator voltage to produce a response trace exactly 100 mV in amplitude., starting its rise at 10msec and starting its fall at 30msec.
Q1: What amplitude current do you have to inject into Cell A1 to get a membrane potential change which is exactly 100 mV in amplitude? To calculate the stimulus current amplitude, multiply the voltage output of the stimulator (~ 1.0 V) times the I/V conversion ratio of the CCU (10 microA/V).
B1 and B2 each model a cell with 3.16x the diameter of Cell A1. This means that Cm for Cell B1 is 10 times larger and Rm for cell B1 is 10 times smaller than the comparable values for Cell A1.
3) Attach both the CCU and CH1 recording leads to Cell B1 (paying close attention to polarity), and determine the I/V relationship for input as above. It will be better to turn the CCU up to .1milliA/V, rather that turning the stimulator amplitude up.
C1 and C2 each model a cell with 10x the diameter of Cell A1. This means that Cm for Cell C1 is 100 times larger and Rm for cell C1 is 100 times smaller than the comparable values for Cell A1.
4) Attach both CCU and recording leads to Cell C1 (paying close attention to polarity), and determine the I/V relationship for input as above. Again, it will be better to turn the CCU up to 1milliA/V, rather that turning the stimulator amplitude up.
Q2: What amplitude currents do you have to inject into Cell B1 and Cell C1 to get membrane potential changes which are exactly 100 mV in amplitude? Is this what you would expect based on the differences between Cell A1, Cell B1, and Cell C1 and the relationship expressed in Ohms Law (V = IR)?
Q3: Do Cells A1, B1, and C1 all show the same exponential rise and fall time constants? Is this what you would expect, given their relative Rm and Cm values? Can you justify your expectation mathematically (hint: what is the formula for computing the time constant of an RC circuit?
C. Non-Rectifying Electrical Synapse
You will now simulate two similar-sized cells connected via an electrical synapse. Cells A1 and A2 have identical resistances and capacitances. The "OUTSIDES" of the two cells are already connected together within the circuit board. This simulates the common extracellular space.
1) Connect the "INSIDES" of the two cells together with a 10 Kohm resistor (brown-black-orange bands – green alligator clip leads). This simulates an electrical synapse between the cells.
2) Connect Cell A1 to PowerLab CH1 and Cell A2 to PowerLab CH2 (remember to connect the inside of each cell to the + PowerLab input).
3) Connect the CCU output leads across Cell A1, paying attention to polarity as before. Cell A1 will serve as the directly stimulated presynaptic cell and Cell A2 will serve as the synaptically-driven postsynaptic cell.
4) To start, uncouple the two cells by unclipping one end of the 10 Kohm resistor. Turn the CCU back down to 10 microA/V and adjust the stimulator amplitude to produce a trace for the presynaptic cell (recording channel A) of exactly 100 mV. Clear your display, set the display to Show Overlay, and trigger a single sweep.
5) Synaptically reconnect the two cells via the 10 Kohm resistor and trigger a new sweep.
You should notice two things. First, there should be an attenuation (reduction) in the voltage response of the presynaptic cell (trace A). The reduction in amplitude of the presynaptic response with a synapse RPrS relative to the presynaptic response without a synapse present RPrN, expressed as a fraction of the latter, is the presynaptic attenuation [(RPrN - RPrS) / RPrN ]. Second there should be a voltage response in the postsynaptic cell (trace B). The ratio of the postsynaptic response RPoS to the presynaptic response RPrS is the synaptic gain (RPoS/RPrS).
Q4: Calculate the presynaptic attenuation and the synaptic gain for a 10 Kohm "synapse" in this simulation. Why is there a presynaptic attenuation when two cells are electrically connected together (think in terms of where the injected current goes)?
Q5: Both presynaptic attenuation and synaptic gain should have values between 0 and 1. Can you explain why this is so, based on a consideration of current flow between the cells?
6) Replace the synaptic connection with a 1 Kohm resistor (brown-black-red bands – yellow alligator clip leads) and trigger a new sweep.
Q6: Calculate the presynaptic attenuation and the synaptic gain for a 1 Kohm "synapse" in this simulation. Did increasing the conductance (decreasing the resistance) of the electrical synapse from 10 Kohm to 1 Kohm increase or decrease the presynaptic attenuation? How about the synaptic gain? Can you explain both of these effects in terms of relative current flow?
7) Save your overlaid sweeps at this point, perhaps to a file called “A1 to A2”.
A non-rectifying synapse is one in which both the presynaptic attenuation and the synaptic gain are relatively independent of the direction of current flow through the synapse (i.e. which cell is presynaptic and which is postsynaptic). A rectifying synapse is one in which presynaptic attenuation and/or synaptic gain is dependent upon the direction of current flow through the synapse.
Q7: Would you expect electrical synapses connecting similar-sized cells to be rectifying or non-rectifying?
8) Clear the display and repeat your three sweeps with Cell A2 (trace B) serving as the presynaptic (directly stimulated) cell and Cell A1 (trace A) serving as the postsynaptic cell. To do this move ONLY the two stimulating leads.
9) Save these overlaid sweeps (“A2 to A1”), measure response amplitudes, and calculate presynaptic attenuations and synaptic gains.
Q8: Did your simulation results agree with your predictions? Based on this simulation are electrical synapses between similar-sized cells rectifying or non-rectifying?
10) Repeat the entire experiment above with cells B1 and B2 via 10Kohm and 1Kohm resistors. Save these overlaid sweeps (“B1 to B2” and "B2 to B1"), measure response amplitudes, and calculate presynaptic attenuations and synaptic gains.
11) Now repeat the experiment for cells C1 and C2. Save these overlaid sweeps (“C1 to C2” and "C2 to C1"), measure response amplitudes, and calculate presynaptic attenuations and synaptic gains.
Q9: Does the size of the cell influence the attenuation and gain for non-rectifying synapses?
D. Rectifying Electrical Synapse
You will now simulate electrical synapses between two cells of dissimilar size, using Cells A1, B1, and C1.
Q10 Would you expect electrical synapses connecting dissimilar-sized cells to be rectifying or non-rectifying (see definition above)? Why or why not?
1) Turn off the CCU and disconnect all of your stimulation and recording leads from the SCRCM hardware board.
2) Connect Cell A1 to PowerLab CH1 and Cell B1 to PowerLab CH2 as above. Connect the CCU output leads across Cell A1, paying close attention to polarity.
3) Turn on the CCU. Adjust the stimulator (if necessary) to produce a 100 mV response in Cell A1. Clear the PowerLab display and trigger a single sweep.
4) Now connect the inside of Cell A1 to the inside of Cell B1 via the 10 Kohm "synaptic" resistor. Trigger a new sweep.
5) Replace the 10 Kohm resistor with the 1 Kohm resistor and trigger a third sweep. Save your superimposed sweeps.
6) Repeat this experiment with Cell A1 serving as the presynaptic cell and cell C1 serving as the postsynaptic cell. Save this set of sweeps.
Q11: What are the presynaptic attenuation and synaptic gain for this small presynaptic Cell A1 synaptically connected to the larger postsynaptic Cell B1? What about for the much larger cell C1? (You may have to decrease the range of display channel B to answer these questions accurately - but be sure to return it to 200 mV before proceeding.)
7) Uncouple the resistors between the cells and move both the CCU output leads and the CH2 input leads to Cell B1.
8) Adjust the CCU and electronic stimulator to produce a 100 mV response in Cell B1. Clear the PowerLab display and trigger a single sweep.
9) Now connect Cell B1 to Cell A1 via first the the 10 Kohm, then the 1 Kohm "synaptic" resistors, triggering a new sweep for each. Save your superimposed sweeps.
10) Repeat this experiment with Cell C1 serving as the presynaptic cell and Cell A1 serving as the postsynaptic cell and save this final set of sweeps.
Q12: What are the presynaptic attenuations and synaptic gains for the larger presynaptic Cell B1 synaptically connected to the smaller postsynaptic Cell A1? What about for the much larger Cell C1?
Q13: In which direction is presynaptic attenuation greater (small -> large or large -> small)? In which direction is the synaptic gain greater? Do these results agree with your predictions?
Q14: What does this tell you about the relative abilities of large and small cells to "drive" each other; i.e. the ability of membrane potential changes in a cell to profoundly influence the membrane potential on a synaptically connected cell of a different size?
Q15: Invertebrates often have "command neurons" which function to obligatorily drive multiple sets of "motor neurons" via electrical synapses, in order to produce rapid, coordinated muscular contractions for escape behaviors. All other things being equal, would a large or a small cell/axon function better as a "command neuron"? Why? Should the electrical synapses connecting command to motor neurons be low resistance (large) or high resistance (small)?
Q16: What stereotyped behaviors in the crayfish are mediated by command neurons?
Q17: So far we have considered myelination of small diameter axons to be a better solution than large axon diameter both anatomically and energetically to the problem of increasing AP propagation velocity. Do your observations in this simulation suggest unique advantages of large diameter axons with electrical synapses over myelination for invertebrate neurons mediating stereotyped "escape" responses? If so, what are the advantages, in terms of relative threshold and ability to drive target motor neurons? Why is each of these an advantage?
Q18: In summary, what are five outstanding properties of command neurons with giant axons ending in electrical synapses (one has to do with axon diameter, one has to do with stimulation threshold, one has to do with AP propagation speed, one ability to serve as current "source" for driving target neurons, and one has to do with the metabolic expense of maintaining the cell)?
Q19: In the crayfish abdomen one of the two giant axons on each side of the abdomen is continuous down the length of the abdomen. The other is actually a "daisy chain" of segmental giant axons, connected end-to-end with low resistance electrical synapses. What is the metabolic or cellular advantage of such a system?
Q20: Can you think of a structure in vertebrates which is highly dependent on multiple interconnections via electrical synapses?
Q21: Generally speaking, what are the relative advantages and disadvantages of electrical synapses vs. chemical synapses? In other words, what can you do with an electrical synapse that you can't do with a chemical synapse, and vice-versa?
E. Shutting Down
Turn off the CCU and the stimulator. Close Scope and turn off the PowerLab box. Disconnect all of the cables.
II. PREPARATION OF THE LAB DATA SHEET
Your data sheet should include all THREE of the items described in the boxes above. Make sure that the axes of all of the graphs and print-outs are labeled and calibrated. You should certainly discuss your results and the answers to the questions with your partners and others in the lab. However, please work independently when you prepare your data sheet.
The writeup
for this lab
falls under category
B