The Role of the Caudal Photoreceptor in Crayfish © 2009 Auburn University Anna-Marie Kelemen Scripps College December 02, 2009

The Caudal Photoreceptor Two in the 6th abdominal ganglion Photosensitive[6] Increase firing when exposed to light Linked to crayfish mobility[3] The Caudal photoreceptors, pair of neurons located bilaterally in the 6th abdominal ganglion have been studied in the crayfish since the early 30s. They have been found to respond to both light and mechanical stimuli through the increase in locomotor activity. The picture on the bottom depicts the nerve cord and abdominal ganglion in the crayfish. As you can see the 6th abdominal ganglion is the last ganglion branching off into multiple root ends. The picture to the right is a photo depicting the relative location of the CPR in the 6th ganglion. © 1990 Simon & Edwards

Previous research Whelsh (1934) –response to light Theory: that CPR serves to warn of exposure of the tail to light and therefore to attack. Kennedy (1958) – Theory: photoreceptors are slightly modified neurons with photosensitive elements Uchizono (1962) – lamellar, honeycombed structures assumed to be photoreceptive elements in 6th ganglion neurons Simon (1989) – CPRs are trigger neurons for locomotor behavior Can choose the motion of response (Ex. forward vs. backward walking) Kendall Snyder (2009) Used LED to imitate light on 6th ganglion Light increased locomotion showing linkage to CPR Previous Research has led to some important conclusions in regards to the relationship between the CPR and crayfish mobility. Some of the bigger names in research have concluded the following: In 1934 John Whelsh published an article in which he first observed the CPR in response to varying degrees of light and assumed it to be used for safety. Donnald Kennedy followed in 1958 with the theory that the CPRs have photosensitive elements. In 1962 Koji Uchizono expanded on this and came to the assumption that the lamellar, honeycombed structures in in the 6th ganglion are photoreceptive elements and their properties are restricted exclusively to the 6th ganglion Ted Simon in 1989 theorized that CPRs are trigger neurons for locomotor behavior mediate a backward walking response. Last year Kendall Snyder used an LED to imitate light on the 6th ganglion. From this she noticed that light did increase locomotion and that selective illumination did not support Simon’s previous of backward walking. In addition, over the summer, Karin Westin managed to graph the response of the CPR to light. The picture to the right depicts Uchizono’s honeycomb structure labeled as H. © 1962 Uchizono

Locomotor Activity Light activation of CPR results in locomotor behavior of crayfish These are two graphs presenting Kendall’s research from last year in regard to locomotor activity. Both graphs have an initial pre-recording time of 30 seconds. In graph (a) we see that with no light input there is no locomotor response. However in graph (b) we see that locomotor behavior occurs in response to illumination after a latency of 20 seconds. Basically, these graphs show evidence of locomotor activity increasing in response to light. Initial latency is thought to be due to temperature. Though further research needs to be done to be sure. Figure 1. Single movement sample, crayfish 3. (a) No light onset. (b) Light onset after 30 seconds of recording. Dotted lines indicate time during which no movement may occur for trial to be counted.. © 2009 Kendall Snyder

Response to Light These graphs were produced by Karin Westin this summer. They show the electrophysiological activity of the CPR in response to light. The blip in the second row represents the onset and offset of light. The 1st row represents individual time responses to the light. Each bar is the measured group of responses at a specific time. Row 4 is a zoomed in portion of row 3. The time of illumination is the same as row 1. What we see is that after the short latency period after initial illumination, activity increases as a response to the light input on the CPR. It is also noteworthy to mention that activity continues to occur after illumination ceases as seen in the top graph. This activity gradually tapers off to normal. It is believed that this is due to after-discharge and proprioceptors. Overall, we can see that when illumination occurs an increase in CPR firing is the result. After discharge = resistance of response of muscle or neural element after ceasing of stimulation. Proprioceptors = sensory receptors in the nucleus, tendons, joints, etc. which detect the motion or positioning of the body or limbs through responding to stimuli. © 2009 Karin Westin

Hypothesis To show that the increase in locomotor activity in the crayfish is due to the illumination of the CPR. Show the relationship between muscle activity in relation to the 6th ganglion and illumination. It can be seen through the previous research that there appears to be a linkage between crayfish mobility and the CPR. The purpose of this experiment is to show that the increase in locomotor activity in the crayfish is in fact due to the illumination of the CPR. This can be done by showing the relationship between illumination and muscle activity.

Dissection Crayfish is sedated in ice bath for 10 minutes Dissection scissors used to cut off tail Tail is pinned to a dissection dish Swimmerets removed Remove all connective tissue Separate out nerve cord Nerve cord pinned to dissecting dish and soaked in saline Fine forceps were used to desheath the cord, removing connective tissues Ganglia exposed © 2008 Smith College Dissection of the crayfish is necessary throughout most of the experimental procedures. The specific species used in the this experiment was Procambarus clarkii otherwise known as Red Swamp Crayfish. The dissection is carried out by sedating the crayfish in an ice bath for 10 minutes inducing a hypothermic shock. Research has been shown that through this induced hypothermia crayfish show no response to painful procedures and are therefore assumed to not be in any pain. The 6th abdominal ganligon is found in the tail portion of the crayfish. This part is cut off and the swimmerets and all connective tissue are removed to expose the nerve cord as depicted in the top picture The nerve cord as shown in the bottom picture is then cut out from the tail and tightly pinned down in a dissecting dish filled with a saline solution. This saline solution is designed to mimic crayfish blood plasma. Fine forceps are then used to desheath the cord, removing connective tissues from around the ganglia to expose them. © 2008 Smith College

Methods and Materials Goal #1 To develop a new technique in which I can precisely read the responses of the CPR to light Use a suction electrode to locate the CPR in the 6th ganglion Accurately measure activity with and without light There are three proposed goals in this experiment. The first goal is to develop a technique in which I can precisely read the responses of the CPR. The current problem as evidenced in the graphs by Karin is that too many cells are being measured as seen by the varying sizes in action potentials. I hope to find the right electrode size and area in which I can measure the CPR more exactly with less background noise. This would involve the use of a suction electrode which is used to record or stimulate the submerged CPR. I would then measure the activity of the CPR in response to both light and no light to determine the electrophysiological behaviors of the CPR.

Methods and Materials Goal #2 To carry out a semi-intact preparation in which the animal is still alive Relationship between activation and movement through direct observation Crayfish displayed on back with ventral side facing upwards Constantly sedated with cold Fine silver wires twisted together and inserted into the muscles at the base of a walking leg Multiple muscles to see correlation between muscle activity and CPR firing Upon finding a successful technique, my next goal would be to carry out a semi-intact preparation in which the crayfish is still alive. This would allow me to observe the relationship between activation or illumination and physical movement of the crayfish. This procedure involves displaying the crayfish ventral side up and keeping it constantly sedated with cold. Twisted fine silver wires would then be inserted in the muscles at the base of a walking leg (as this is where increased locomotion is seen) to measure electrical activity as the CPR is illuminated. This would be repeated in multiple muscles to see if muscle activation and CPR firing are related.

Methods and Materials Goal #3 Assuming first two goals successful Knockout neurons and observe electrical behavior Sedate crayfish and open abdomen Sever nerve chord between the 5th and 6th ganglia Use suction electrode to measure activity Tells us whether any source of increase in muscle activity was actually caused by activity in the 6th ganglion Assuming that a correlation between muscle activity and CPR illumination is seen in step 2. The final goal for this experiment would be to try and knockout the neurons and observe behavior from that point. Knocking out the neurons would be difficult with a glass needle as the cell body is small and precision is key in this experiment. However, it could be feasible to sever the nerve cord between the 5th and 6th ganglia and then use the suction electrode procedure perfected at the beginning of the experiment to measure firing activity. Overall, what this experiment would essentially tell us is whether any source of increase in muscle activity was actually caused by activity in the 6th ganglion.

Acknowledgements I would like to thank Professor Copp for reviewing this presentation and for the encouragement and advice throughout this experiment. I would also like to thank Karin Westin for teaching me the dissection technique necessary for this experiment. I would also like to thank her for providing me with her past research so as to further my understanding and knowledge of the material.

Literature Cited Kennedy, D. & Preston, J.B. “Activity pattern of interneurons in the caudal ganglion of the crayfish.” 1960. The Journal of General Physiology. 43 (3): 655-670 Prosser, Ladd. “Action potentials in the nervous system of the crayfish.” 1935. The Journal of General Physiology. 19 (1): 65-73. Simon, Ted W. “Light-evoked walking in crayfish: Behavioral and neuronal responses triggered by the caudal photoreceptor.” 1989. The Journal of Comparative Physiology. 166: 745-755. Snyder, Kendall. “Light activation of the caudal photoreceptors: effects on locomotor behavior in Procambarus clarkii.” 2009. Senior Thesis in Neuroscience. 1-30. Uchizono, Koji. “The structure of possible photoreceptive elements in the sixth abdominal ganglion of the crayfish.” 1962. Brief Notes. 15 (1): 151-154. Whelsh, John H. “The caudal photoreceptor and responses of the crayfish to light.” 1934. Journal of Cellular and Comparative Physiology. 04 (3): 379-388. Yano, Takeshi; Ibusuki, Shoichiro; and Takasaki, Mayumi. “A comparison of intracellular lidocaine and bupivacaine concentrations producing nerve conduction block in the giant axon of crayfish In Vitro.” 2006. Anesthesia & Analgesia. 102 (06):1734-1738. (2005, February 08). Worms and fish feel no pain. Cape Times, 2005: 001.