BIO325 Laboratory Guide #18 (2024)
SENSORY PROCESSING I:
ELECTRORETINOGRAM (ERG) OF THE FLY EYE
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In previous labs you have recorded and modeled both action potentials and postsynaptic potentials resulting from current flow across neuronal membranes. In contrast, field potentials are summed potentials derived from current flow through the extracellular space surrounding multiple active cells and may be recorded with electrodes at some distance from the actual cells. Structured field potentials are produced only when neurons are oriented perpendicular to the surface, arranged in sheets or arrays of parallel cells, and activated simultaneously in such a way as to produce temporary electrical dipoles across each layer. In this lab you will record the electroretinogram (ERG) in the common flesh fly Sarcophaga bullata. The ERG is a complex field potential produced by the superficial retina and deeper visual processing layers. In our case, the ERG will represent the extracellular manifestation of the peripheral visual system's response to a simple square-wave flash of light delivered to the intact eye of the fly, as recorded between the surface and the depths of the eye.
In the insect compound eye photoreceptor cells are packaged across the retina into multiple discrete ommatidia stacked in parallel across the retina. Each ommatidium is like a small, self-contained camera, comprised of a superficial corneal lens surface, a funnel-like light-gathering chamber, and a retinal sheet containing a very few photoreceptor cells surrounding a central “eccentric cell”. The photoreceptor cells are electrically coupled to the eccentric cell. Individual photons striking photopigment molecules in the receptor cells generate tiny depolarizing potentials called “Yeadle bumps”, analogous to the miniature endplate potentials seen at the neuromuscular junction. A brief light flash directed into the ommatidium will produce a prolonged depolarizing receptor potential in each of the receptor cells and an electrotonically summed potential in the central eccentric cell. These project, in turn to a deeper layer of parallel cells in the lamina and more complexly organized cells of the medulla.
Both the retina and the lamina have the requisite sheet-like architecture required to contribute to the ERG, provided that a large area of the retina is stimulated simultaneously. The retinal contribution is a slow negative-going component that develops over the first several hundred milliseconds of a light flash and decays back to baseline over a similar time course after the light flash terminates. The onset and decay time-courses are subject to "centrifugal" control from the optical lobe of the supraesophageal ganglion (brain). The underlying laminar processing layer produces a positive-going transient at the onset of the light and a negative-going transient at the termination of the flash.
The amplitudes of the insect ERG components are dependent upon several factors. The most obvious is the intensity of the light flash stimulus; brighter flashes produce higher amplitude receptor potentials. However, this is not the end of the story. In the real world, background illumination may vary over several orders of magnitude, and the fly must be able to visually detect and discriminate moving objects and shadows across this entire range. The mechanism for this involves the visual photopigment molecules themselves. Prolonged light exposure “bleaches” photopigment molecules, making them insensitive to photic stimulation. Similarly, in the dark, photopigment molecules unbleach, making the population of molecules in each cell more sensitive to light. This is analogous to the sort of automatically adjustments to ambient light levels that modern cell phone cameras accomplish. As you might guess, in the dark-adapted retina, the response to a standard flash of light is maximal, and this response amplitude is diminished substantially by light-adapting the eye.
The insect visual system also must process information which changes rapidly in time. A standard measure of the maximum temporal resolution of the visual system is the flicker fusion frequency (FFF); basically the frequency at which a flickering light is perceived as a constant or steady one. Human FFFs are typically around 40-50 Hz. In contrast, flying or predatory insects may have FFFs of 100 Hz or higher.
In this lab you will record the ERG as the potential between two micropipette electrodes in an intact, restrained fly. The live microelectrode will be placed against the surface of the retina and the reference microelectrode will be imbedded in the thorax of the fly. The light flash will be produced by a square-wave electrical current pulse to a light-emitting diode and delivered to the vicinity of the eye by a fiber-optic light pipe. Background light levels will be controlled by enclosing the entire Faraday cage in a black cloth drape.
I. RECORDING PREPARATIONS
A. Fly Mounting
1) Choose an active, healthy fly. Anesthetize it by placing it on a CO2 bed.
2) When the fly stops moving, secure it ventral side down to a glass microscope slide, using modeling clay. Make sure that both the head and upper thorax are accessible but immobilized. Also make sure that you have not obstructed the spiracles along the abdomen (in order to allow the fly to continue to breathe).
3) Mount the slide with attached fly to the glass disk of the dissecting microscope stage using more modeling clay. Center the eye of the fly in the microscope field.
B. Recording Setup
1) Pull both 1.0mm and 1.5mm electrodes. Pre-fill the tips by standing them upside-down in a small beaker of fly saline for ~5 minutes. Back-fill one of each size electrode with fly saline and mount it in its respective half-cell holder. The 1.0mm microelectrode will serve as the live electrode and the 1.5mm microelectrode will serve as the reference electrode.
2) Carefully plug the live 1.0mm half-cell holder into the Neuroprobe amplifier head stage on one micromanipulator. Mount the rod of the 1.5mm reference half-cell holder to the other micromanipulator. Clip one end of the shielded silver reference cable to the headstage shield, plug the other end into the 1.5mm headstage jack, and ground the cable shield to the cage.
3) Position and advance the reference electrode to impale the dorsal thorax of the fly. Position and advance the live electrode so that it is just touching the corneal surface of the eye.
4) Carefully apply a tiny droplet of saline at the contact point between the live electrode and the retina.
5) Using the third micromanipulator, position the fiber-optic light pipe so that it is pointed directly at the eye and is as close as possible to the eye surface.
6) Turn on the PowerLab box. Launch Scope from the "Fly ERG" settings file. Verify the following initial connections and settings:
Time Base: 5 seconds at maximum samples/second
CH A: Input from Neuroprobe x1; 50mV; no filters - this monitors the ERG
CH B: Input from Powerlab + Output; 10 V; no filters - this monitors the stimulus
PowerLab Stimulator: single pulse; 1000 msec delay; 2000msec duration;
4V amplitude
PowerLab + Output: Connected to both Input CH B and light-pipe/diode cable
Note: It is particularly important that NEITHER channel be set to AC Filter the recorded signal. Make SURE that the AC box is NOT checked for either channel.
7) Set Scope Sampling to Repetitive sweeps and hit START. Verify that the Stimulator is, in fact producing a light flash during each sweep, with the appropriate timing and amplitude (CH B trace). DO NOT GO ABOVE 10 VOLTS STIMULUS AMPLITUDE!
8) Temporarily set the Stimulator Amplitude to 0V. Turn on the Neuroprobe amplifier. START repetitive sweeps on Scope. Zero the ERG trace (CH A) using the Neuroprobe DC Balance knob. The trace may show a bit of baseline "drift" between sweeps. If there is excessive noise or instability in the trace, reposition your electrodes and/or consult the instructor. You will need a fairly stable baseline before you can continue with active recording.
9) STOP Scope sweeps, reset Sampling to Single, and set Stimulus Amplitude back to 4 volts.
10) You should now be ready for light-flash stimulation and ERG recording.
II. RECORDING THE ERG
A. Basic ERG
1) Scope should be set up to record both Channels A and B, with CH B recording the light flash and CH A recording the ERG response.
2) All ERG trials should be conducted with the fly in the dark. To do this, simply drop the drapes in the front of the cage prior to initiating a stimulating/recording sweep.
3) Trigger a single light flash at ~4V - 5V intensity. DO NOT GO ABOVE 10 VOLTS STIMULUS AMPLITUDE! The ERG response should appear as a slow negative going wave, with positive and negative transients at he onset and offset, respectively. If you have a stable baseline, but one or both of these components is not present in the ERG, that usually means that the fly is dead or dying. Consult with the instructor, and/or replace the fly as necessary. The instructor can also show you how to digitally filter the trace to remove very high-frequency fluctuations
4) Set Scope Sampling to Multiple with 8-16 samples and a 30 second delay between samples. Hit START and collect the set of traces.
5) The time-locked average of these traces is an averaged evoked potential (AEP). Scope calculates this automatically and displays it as the (X bar = mean) trace.
Note: If you have a lot of baseline fluctuation, you may get some traces which are non-representative - or in technical terms "bad". These traces may not start from a flat zero line, and/or may have slow voltage swings clearly unrelated to the stimulus or response. It is OK to temporarily delete such traces prior to producing your final AEP for printing.
Q1: Explain why the ERG retinal slow potential on these traces. isdownwards (negative)
on these traces. Recall that you are recording extracellularly from near the corneal
surface of the eye relative to a point in the body deep to the eye. How does a
negative-going extracellular ERG correspond to a depolarization of the
photoreceptors?
B. Stimulus Intensity Coding
1) Make sure that you have saved the traces from the previous section. Clear the screen. Re-zero the baseline at 0 V stimulus intensity as necessary, then clear the display. Set the Display to SHOW OVERLAY to superimpose recorded traces
2) Make sure that the front drapes are down, placing the fly in the dark.
3) Start with the Stimulus Voltage set to 2.5 Volts. At ~1 minute intervals trigger and record single ERG responses. Increment the Stimulus Voltage up by 0.25 Volts between samples. Continue until the ERG response reaches a maximal amplitude which is not increased by further increase in stimulus intensity. DO NOT GO ABOVE 10 VOLTS STIMULUS AMPLITUDE!
Q2: Does the ERG amplitude increase as the stimulus intensity is increased? At what point does the ERG amplitude max out, so that further increases in stimulus intensity do not produce larger ERG responses?
Produce a clean set ERG traces in response to increasing light stimulus intensity. Make sure that both axes are correctly labeled (it is fine to leave the Y axis as simply Stimulus Volts). Label each trace with the corresponding stimulus intensity.
Data Sheet Item #2b:Measure the amplitude of the slow retinal component of each trace (measured right before the onset of the terminal negative transient). Plot the magnitude (absolute value) of this amplitude (in mV) as a function of stimulus light intensity (in V).
C. Dark-Adaptation
1) Make sure that you have saved the traces from the previous section. Clear the screen. Re-zero the baseline at 0 V stimulus intensity as necessary, then clear the display. Set the Display to SHOW OVERLAY to superimpose recorded traces. Make sure that the drapes are completely closed.
2) Set the stimulus intensity back to ~ 4V - 5V.
3) Turn on the external fiber-optic light to illuminate the fly eye. Leave the light on for at least 1 minute to fully light-adapt the eye.
4) Turn off the external illumination and IMMEDIATELY trigger a single ERG sweep (0 seconds sweep).
5) Repeat steps 3 & 4 with the following waiting times between external light offset and ERG stimulation/recording:
10 seconds
20 seconds
30 seconds
1 minute
2 minutes
5 minutes
10 minutes
Q3: Does the fly visual system dark adapt? I.e. is there an increase in ERG amplitude as the waiting time in the dark increases?
Q4: What is the cellular/chemical mechanism of dark adaptation and light adaptation? Of what advantage to the fly are light and dark adaptation by the retina?
Produce a clean set ERG traces in response to increasing dark adaptation time. Make sure that both axes are correctly labeled. Label each trace with the corresponding dark-adaptation interval.
Data Sheet Item #3b:
Measure the amplitude of the slow retinal component of each trace (measured right before the onset of the terminal negative transient). Plot the magnitude (absolute value) of this amplitude (in mV) as a function of dark adaptation time (in seconds).
D. Flicker Fusion Frequency (FFF)
1) Make sure that you have saved the traces from the previous section. Close Scope, then re-launch it using the "Fly ERG FF" settings file. Verify the following initial connections and settings:
Time Base: 2 seconds at maximum samples/second
CH A: Input from Neuroprobe x1; 50mV; no filters - this monitors ERG
CH B: Input from Powerlab + Output; 10 V; no filters - this monitors stimulus
PowerLab Stimulator: multiple pulses; 10 pulses; 500 msec delay;
50 msec duration; 50 msec interval; 4V amplitude;
20 pulses
PowerLab + Output: Connected to both Input CH B and light-pipe/diode cable
Note: This will produce a 20Hz stimulus pulse train for just under 1 second, comprised of 20 pulses at 25msec duration and 25msec intervals, with a duty cycle (ON:OFF durations) of 1:1 or 50%.
2) Temporarily set the stimulus voltage to zero and re-zero the trace, using the Neuroprobe DC Balance knob. Set the stimulus voltage back to ~4V - 5V (whatever amplitude you have been using as your standard for previous recordings).
3) Make sure that the drapes are fully closed and dark-adapt the eye for at least 1 minute.
4) Trigger a single ERG stimulation/recording sweep. Although the slow component of the ERG response will still extend across 1 second, the ERG trace itself will be broken up into 10 discrete responses (CH A) to 10 discrete flashes (CH B). Internally label this trace as "10 Hz stimulation".
5) Repeat steps 3 & 4 as you increase the stimulation frequency by ~ 10 HZ increments, labeling each trace as you go. Use the following table to keep the total stimulation period close 1 second and the duty cycle at 50%. You will have to increase the number of pulses as you decrease both the pulse and interval durations. The upper limit of 100 pulses will shorten the overall ERG length for high stimulation pulse rates.
Approx. Freq. Pulse width in msec Interval in msec # of pulses
10 50 50 10
20 25 25 20
31.25 16 16 30
38.5 13 13 40
50 10 10 50
62.5 8 8 60
71.4 7 7 70
83.3 6 6 80
100 5 5 100
125 4 4 100
166.7 3 3 100
250 2 2 100
Note: Flicker fusion would be evidenced by the the disappearance of gaps between individual responses, or and or the disappearance of the onset and offset transients. This would indicate that the visual systems is missing or "skipping" individual light pulses and perceptually fusing those flashes together into a single, continuous flash.
Q5: As the flicker frequency increases, what happens to the ERG? Is there evidence of flicker fusion at high flicker frequencies, as specified above?
Q6: What does this imply about the ability of the fly to accurately track rapidly-moving objects or scenery?
III. SHUTTING DOWN
Complete the following steps before leaving the lab:
1) Make sure that you have saved all of your data to the hard drive, then quit Scope. Turn off the amplifier and the PowerLab box.
2) Turn off the amplifier and the stimulator.
3) Make sure that both the microscope and fiber-optic lights are turned off.
4) Properly dispose of your fly. Perhaps your roommates need a pet?
5) Dispose of the electrodes and rinse out the half-cell holders.
IV. PREPARATION OF THE LAB DATA SHEET
Your data sheet should include all FOUR 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.
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