Desensitization and Recovery of Crayfish Photoreceptors Upon Delivery of a Light Stimulus

Summary

A protocol for the study of desensitization and sensitivity recovery of crayfish photoreceptors as a function of circadian time is presented.

Abstract

A method to study desensitization and recovery of crayfish photoreceptors is presented. We performed intracellular electrical recordings of photoreceptor cells in isolated eyestalks using the discontinuous single electrode-switched voltage-clamp configuration. First, with a razor blade we made an opening in the dorsal cornea to get access to the retina. Thereafter, we inserted a glass electrode through the opening, and penetrated a cell as reported by the recording of a negative potential. Membrane potential was clamped at the photoreceptor's resting potential and a light-pulse was applied to activate currents. Finally, the two light-flash protocol was employed to measure current desensitization and recovery. The first light-flash triggers, after a lag period, the transduction ionic current, which after reaching a peak amplitude decays towards a desensitized state; the second flash, applied at varying time intervals, assesses the state of the light-activated conductance. To characterize the light-elicited current, three parameters were measured: 1) latency (the time elapsed between light flash delivery and the moment in which current achieves 10% of its maximum value); 2) peak current; and 3) desensitization time constant (exponential time constant of the current decay phase). All parameters are affected by the first pulse.

To quantify recovery from desensitization, the ratio p2/p1 was employed versus time between pulses. p1 is the peak current evoked by the first light-pulse, and p2 is the peak current evoked by the second pulse. These data were fitted to a sum of exponential functions. Finally, these measurements were carried out as function of circadian time.

Introduction

In order to be perceived as a visual stimulus, light reaching the eyes must be transduced into an electrical signal. Hence, in all visual organisms, light triggers a transduction ion-current, which in turn produces a change in the membrane potential of photoreceptor cells, the so-called receptor potential. Due to this, the light sensitivity of the eye primarily depends on the state of the light activated conductance, which can be either available to be activated or desensitized.

In crayfish photoreceptors, light triggers a slow, transient, ionic current¹. Upon illumination, the transduction current arises after a lag....

Protocol

NOTE: The experiments comply with the Laws of Animal Protection of Mexico.

1. Experimental Setup

1. General connections
   1. Connect the amplifier to a suitable computer through an analog-to-digital converter and use an oscilloscope to monitor the experiment (Figure 1).
   2. Connect the photostimulator to the A/D converted.
2. Recording chamber
   1. Place the recording chamber on top of an anti-vibration table and locate it inside a Faraday cage.
      NOTE: This prevents mechanical vibration and electrical noise that may affect the recording. Our Faraday cage w....

Results

First, a representative receptor potential of crayfish photoreceptor cells is obtained (Figure 4). Afterwards, a test light-flash was applied to trigger the light transduction current (Figure 5). The cationic transduction current¹ activates after a lag, reaching a maximal and thereafter slowly drops into an absorbing desensitized state from which it slowly recovers.

Discussion

The crayfish has proven to be an excellent model due to its ability to survive under non-natural conditions. There is easy access to in vivo and in vitro electrophysiological analyses. In addition, crustaceans are a favorable group for neurobiological research in the field of comparative chronobiology²¹.

In this paper, the study of desensitization and recovery of the light-activated transduction-current of crayfish photoreceptor cells is shown using th.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Axoclamp2A	Axon Instruments Inc		Amplifier
Digidata 1200 Interface	Axon Instruments Inc		Digitizer
Oscilloscope TDS430A	Tektronix		Analogic Oscilloscope
Photostimulator PS33 Plus	Grass		Lamp
Puller PC-100	Narishige		Micropipette Puller
Puller P-97	Sutter Instruments		Micropipette Puller
Glass Capillary Tube Kimax-51	Kimble Products	34502	0.8, 1.10, 100 mm
HS-2 Headstage	Axon Instruments Inc		Headstage
Micromanipulator MX-4	Narishige		Mechanical Micromanipulator
Stereoscopic Microscope	Zeiss		Microscope
pClamp	Axon Instruments Inc		Data acquisition software for digidata 1200 interface
Clampfit	Axon Instruments, Inc		Analysis software linked to pClamp
Origin	OriginLab Corp.		Data analysis and graphing software
Sodium Chloride	Sigma	S7653	>99.5%
Potassium Chloride	Sigma	P-9333	Minimum 99%
Magnesium Sulfate	Sigma	M7506	Minimum 99.5%
Calcium Chloride	Sigma	C5080	Minimum 99.0%
Hepes	Sigma	H7523	>99.5%
Sodium Hydroxide	Sigma	S8045	98.00%
Sodium hypochlorite solution	Sigma	425044	Available chlorine, 10-15%