Blowfly Visual System in the H1 Neuron Differentiation of Random and Preferred Motion Stimulation By Christian Bick and Nicholai Yonan TA Jeff Moore UCSD Biophysics and Neurophysiology Course Prof David Kleinfeld Introduction The visual system of the Blowfly in our case calliphora and sarcophaga can be easily studied due to the Blowfly s abundance and small scale The visual system of the Blowfly is comprised of numerous neurons but we are interested in the H1 neuron the H1 neuron is a relatively large neuron making it easy to find and record from with our equipment Full field visual motion in the regressive direction triggers the H1 neuron and the fly uses this to maintain equilibrium during flight The H1 Neuron is located four synapses away from the retina making it analogous to the cells in the visual cortex another reason it is widely used to study visual systems 4 This similarity led us to conduct an experiment that is comparable to an experiment that tested the ability of macaques to distinguish between random and coherent motion 1 In the coherent motion experiment macaques were presented with random dot motion in two dimensions with varying levels of motion in a single direction The macaques were then trained to choose the direction in which the coherent motion was traveling Results varied from monkey to monkey but a common trend signified that the macaques could differentiate and accurately choose the direction of coherent motion at levels of coherence at approximately 13 percent We hoped to accomplish the same thing the main aim of our experiment was to find out how well the neuron could differentiate between random horizontal motion and horizontal motion in the preferred direction Varying ratios of random motion and motion in preferred direction were presented to the fly in order to monitor this Procedures I Fly Preparation The Blowflies were ordered from several vendors Ward s Natural Sciences would send us a vile of 100 fly pupae while our second vendor Forked Tree Ranch would send us hatching fly pupae All the flies regardless of development were then place in a rectangular container with small dishes of sucrose and water in order to provide them with sustenance upon hatching Two days after the flies had hatched they were ready to be recorded from A fly would be caught and placed into test tube the test tube containing the fly was then placed into the freezer for approximately six to seven minutes This procedure anesthetizes the fly while anesthetized the fly s wings and legs are removed Hot wax is then used to secure the fly on a metal platform this wax also serves to cover and prevent the movement of vestigial limbs that could remain from their previous amputation The fly s proboscis was used to secure the head in a position that would make the back of the head easier to access Wax was placed on the proboscis which was then adjoined to lower abdomen of the fly exposing the back of the fly s head A scalpel in conjunction with a microscope was used to remove the exoskeleton covering the back of the fly s head doing so exposed the inside of the fly s head on the contra lateral side The fly s brain is covered with air sacks which we removed exposing the fly s brain the location of the H1 Neuron Fly saline1 See Appendix was used to prevent the brain from drying up With the completion of the preparation the fly was placed into the LED Arena to be recorded from A small incision was made into its back as a reference for recording after the incision was made a 3 electrode is used to record extra cellular activity from H1 II Stimulus Presentation A stimulus was presented to the fly via an LED Arena this Arena was comprised of 96 LED columns controlled by 3 separate NI DAQ cards All together the 3 degree LED columns combined to create a 288 degree visual arc which would surround the fly The program MATLAB was used to control the LED columns via a NI DAQmx C library We would then vary the brightness of the cards by breaking the LED column into sub frames Our timing setup was based on a 1 Hz clock with a 1 kHz frame rate This was presented with a varying brightness intensity of 1000 The brightness was then gamma corrected to a gamma of 2 2 After the general layout of the stimulus was created it became possible to vary its arrangement and overall appearance In order to test how well H1 differentiates between random motion and motion in the Figure 1 Fly in LED Arena Figure not to scale preferred direction our stimulus would be presented with varying correlations of motion in the preferred and random orientations III Recording Recoding took place once the fly was placed in the arena and upon its exposure to fullfield visual stimulation A 3 electrode was inserted into the back of the fly s brain and ultimately its neuropile The high resistivity of the electrode allowed us to measure over very minute areas within the nueropile thereby eliminating extra cellar noise from other neurons This electrode was connected to an amplifier with a gain of 3000 which in turn was connected to a speaker A reference stimulus consisted of a number of wide vertical bars moving in positive direction for 2 seconds and then reversed to move in the negative direction for 2 seconds This was done to test whether or not we had been able to locate H1 which was signified by a repeated clicking sound on the speaker when the test signal moved in the preferred direction Upon locating the neuron we would begin to send our desired stimuli to the LEDs All of the fly s response to our stimuli was recorded using MATLAB Data Acquisition Toolbox Chronux Cold Spring Harbor Laboratory was used to process the data 2 3 The baseline of the raw data was first using the locdetrend function from Chronux All data was concatenated for spike sorting using Chronux Spikes were detected with a threshold of 3 standard deviations from the total data a spike interval of 1ms was captured Waveforms were sorted with a modified version of Chronux 2 0 the automatically generated clusters were used Spike counts were extracted and the ROC curves were calculated similar to the way they were done in 1 VI Stimulus The stimulus presented to the fly was a mix of fixed direction horizontal line motion and random walk line motion Coherence levels between the stimuli were varied by multiples of 10 80 20 70 30 60 40 50 50 40 60 30 70 and 20 80 for random preferred motion Coherent motion was presented to the flies at a constant 80 deg sec which is