Flicker Fusion with Light Stimulus in Crayfish, Procambarus clarkii.

Abstract

Methods

Results

A 150 mL beaker was placed on the 250 mL beaker and secured with wax. The tungsten electrode wires were attached to the sides of the 150 mL beaker with wax to support the weight of the wires and hold them in place. One tungsten electrode was placed into the right eye of the crayfish. This recording electrode was inserted about 1.5 mm into the bottom surface of the eye. A second tungsten electrode was inserted into the cavity of the left antennae. A silver chloride pellet ground electrode was inserted into the posterior end of the cephlothorax to reduce noise from the electrical interference (Figure 4).

Table 1. Specifications of tungsten electrodes. Electrodes are sharpened by etching.

1999-2000 A-M system, Inc. Catalog No.	Diameter inches	Diameter (uM)
716500	.008	200

The light-emitting diode (LED) was placed on a 100 mL beaker and secured with tape. The LED was placed 1.5 cm from the eye at 90 degrees to the recording electrode (Figure 5 ). Each LED color was placed in the same position. 

Recording
The electrodes were attached to ADInstruments Powerlab bioamp and Scope V. 4.0 was used for recording. The PowerLab was also utilized as the stimulator by modulating the pulse interval of the LED. The settings for recording in the scope were identical for each LED color, the pulse interval was the only variable that varied. The settings in were as follows:

Scope:
     10 repetitions averaged in each recording to reduce noise. 
     Range: 500uV
     Time Base:
     -400 HZ
     -1024 Samples
     -2 Seconds
Stimulator:

     Double mode
     -500ms Delay- time period before first stimulus
     -25ms Duration A
     -25ms Duration B
     -2.5V Amplitude A
     -2.5V Amplitude B
 

ÝThree wavelengths of LEDs (Red, Yellow, Green) were used to stimulate the crayfish eye (Table 1). 

ÝTable 2. Specifications of each LED. 

	Yellow	Green	Red
Power dissipation at 25C	60mW	75mW	60mW
Forward current at 25C	20mA	25mA	20mA
Forward voltage at 10mA	2.1V	2.1V	2.0V
Luminous intensity at 10mA	6.3mcd	6.3mcd	6.3mcd
RadioShack catalog no.	276-021A	276-022A	276-041A

Experiment

Spontaneous Activity
The ADInstruments Chart program was used to record spontaneous extracellular activity in the eye after it was covered was the dark box. This recording was performed by covering the set-up with no LED stimulation. 

Electrical Response
The experimental setup was covered by a cardboard box, wrapped with aluminum foil, to prevent external light from entering the crayfish eye. The crayfish eye was stimulated by light from the LED. The response to each wavelength LED was recorded separately. The pulse interval from each LED was varied by manually modifying it in the scope stimulator settings. The interval between light pulses was varied for each LED until flicker fusion was found. Flicker fusion was the point at which we only got one electrical response from the crayfish eye with double pulse stimulation. Double responses were observed up to the point of fusion with double pulse stimulation, this allowed us to distinguish fusion from flicker responses.

Human Flicker Fusion

The human subject sat approximately 30cm from the LED stimulus (Figure 6).

Figure 6. Experimental setup for human subject.

The point of flicker fusion of humans was tested for the three wavelengths of light on one human volunteer by pulsing the lights and changing the interval between pulses, then asking the volunteer when the twin pulses appeared as one.  The subject was shown a single pulses and double pulses haphazardly. The subject was not told which pulses were single or double. Fusion of LED stimulus was deemed the interval at which the human subject was unable to distinguish the double pulses as separate stimuli.