Visual Coding in the Blowfly H1 Neuron Tuning Properties and Detection of Velocity Steps in a new Arena Jeff Moore and Adam Calhoun TA Erik Flister UCSD Imaging and Electrophysiology Course Prof David Kleinfeld 1 Introduction The blowfly visual system is frequently used in vision experiments and is attractive because of its simple preparation and small scale H1 is well suited for these experiments because of its large size and its ease to record from Because it is four synapses away from the retina it may be seen as similar to cells in visual cortex It responds primarily to horizontal full field motion and is directionally tuned to regressive motion from the back to the front of the eye 1 This important system allows the fly to remain stable and avoid objects while in flight We sought to characterize various properties of the cell both to test the capabilities of our arena and the capabilities of the fly Stimuli were presented by changing three parameters movement speed spatial frequency and refresh rate These experiments should expose some of the limits of the fly s ability to perceive these events 2 Methods 2 1 Fly preparation The blowfly Calliphora was ordered from Ward s Natural Science Rochester NY The pupae were housed in clear plastic water containers They were placed in a small dish to let hatch A dish of water and a dish of sugar were also placed in the containers One end of the container was cut off and replaced with pantyhose Cutting off part of the pantyhose allowed easy access to the flies without letting them escape while still letting them breathe The last several batches of flies failed to hatch for unknown reasons despite changing many of the housing elements When a fly was needed it was trapped in a test tube with a small air hole It was then anesthetized by placing it in the freezer for eight minutes Upon removing the fly its legs wings and antennae were quickly removed These wounds were covered in wax to prevent movement from vestigial remnants of the limbs The fly was then waxed to the platform with its head positioned off of it The head was pressed forward so that the back of the head was pointing vertically and then waxed down with the proboscis The back of the head behind the eye was then exposed After initially taking out the mucouslike substance we found we were better able to record from H1 when we left it in A small incision was made on the back for the reference wire While in the rig a saline solution was intermittently placed in the opening The solution is described in 2 2 2 Stimulus delivery The stimulus was delivered through a system of LEDs Ninety six columns of LEDs were arranged in an arc encompassing 270 degrees around the center of the fly platform This was controlled by three NI DAQ cards two NI DAQ 6224 cards and one NI DAQ 6229 card National Instruments Austin TX The output of the three cards was fed through a custom designed box to switch the signal from six cables to two These cards were controlled from a MATLAB program Natick MA through the NIDAQmx C library All digital writes to the cards were synchronized We broke every frame of our stimulus down into one hundred subframes to digitally control perceived brightness If we wanted an LED to display a brightness at say half maximum the LED would be turned on for approximately fifty randomly chosen subframes Since the NI DAQ cards were capable of 1MHz output this allowed us to show frames of stimuli at 10kHz Subframes were calculated using an algorithm that converted from arbitrary spatial and temporal waveforms to desired LED intensities The brightness was gamma corrected with a gamma of 2 2 Our software allows for individual trial lengths ranging from 1ms to 15s memory buffer limit 2 3 Recording We used the MATLAB Data Acquisition Toolbox to acquire data using two analog inputs One input was wired into the rightmost LED This LED was non functional but a signal was sent to it at the start of every trial In this way we could ensure synchronization of the input from the electrode with the start of each trial The other input came from an amplifier filter which amplified the voltage between the active and reference electrodes filters were set all the way open Trials with movement artifacts were removed manually from the dataset To find H1 a sine grating stimulus was used The grating moved with a constant speed of 100 deg s switching direction every second The fly was placed on the rig tilted upward and in the direction of the side of the head being recorded from allowing us access to the cell The electrode 3 MOhm Tungsten MicroProbe Inc was then inserted until a characteristic spiking sound was heard from motion in the regressive direction to the contralateral eye 2 4 Stimuli Several stimuli were used All had a characteristic sine wave in space which was moved at constant speed To test flicker we presented a stimulus moving at 50 deg sec with 10 cycles at artificial frame rates of 5Hz 10Hz 30Hz 70Hz 100Hz 300Hz 700Hz 1000Hz and 10000Hz We tested spatial frequencies moving at 100 deg sec with a refresh rate of 10 kHz The number of cycles appearing on the screen was 3 5 8 20 40 and 60 To test preferred speed we displayed a sine wave with a spatial frequency of 10 cycles and a refresh rate of 10 kHz Speeds used were 10 deg sec 20 deg sec 40 deg sec 60 deg sec 75 deg sec 100 deg sec 200 deg sec 300 deg sec 500 deg sec and 700 deg sec 3 Results 3 1 Raw Data and Spike Sorting Stimuli presented to the fly consisted of 20 consecutive trials of progressive motion followed by regressive motion top right A sample recording is shown in Fig 3 1 top left The same recording is separated into trials middle left and sample spikes are shown middle right To get spike times we applied a threshold voltage and counted upward threshold crossings In several recordings for each electrode placement in each fly we checked plots of peak vs trough spike amplitude with different thresholds Fig 3 2 top left and right and chose thresholds so that only one cluster was seen in each plot right We also checked the spike autocorrelation to make sure there was a dip near 0 lag lasting several milliseconds corresponding to the refractory period bottom Fig 3 1 Sample recording 75 deg s set top raw recording and triggers middle left separated by trials middle right single spikes bottom left raster plot of spike times and bottom right spike histogram 3 2 Flicker vs Motion Because the flies were presented with apparent motion stimuli