Introduction: The giant nerve fibres are relatively simple to study because of their large diameter.

The large diameter of the giant nerve fibres makes intracellular recording convenient and the large electrical current produced during an action potential makes the extracellular recording very easy (Fullard, 1978). Most annelids have giant nerve fibre (Stough,1926). The giant fibres in Earthworm, Lumbricus terrestri is made of three fibres, two lateral fibres and one median fibre. Giant fibre lies on the dorsal side and runs through the entire length of the ventral nerve cord (Bullock, 1945).Giants fibres is a chain of short compound axons and the two lateral fibres are connected anastomoses (Bullock, 1945). Giant nerve fibres are best known for their high speed, motor pathways and they are known for their escape response (Bullock, 1945). Stimulation at any end of the worm will cause a rapid contraction in the length of the entire animal and there will be simultaneous contraction in the entire segment of the longitudinal muscle. (Stough, 1930). The median fibre starts from the anterior part of the suboesophageal ganglions, which is just posterior to circumoesophageal . In addition, the lateral giant fibres lie on either side and at the same level as the median giant fibre(Stough, 1926). Median nerve fibre receives sensory information from the anterior part of the worm and sends the information to the posterior part of the worm. Whereas the lateral nerve fibre receives information from the posterior part only and sends the information to the anterior part. (Stough,1930). Temperature is one the major ecological factors, that increases the velocity of chemical reaction in poikilotherm(Lagerspetz, 1974). Acclimatization is a biochemical, physiological or anatomic change in the animal due to new environmental condition (Burggren et al., 2001). Threshold stimulus is influx of sodium that causes the deplorization of the membrane , an action potential is generated when the deporization reaches a certain threshold. (Burggren et al., 2001). Absolute refractory period is when the membrane is absolutely insensitive to any stimulus. The relative refractory period is when a membrane might be stimulate by a strong stimules(Burggren et al., 2001).

The purpose of the experiment was to analyze the action potential of Earthworms lateral and median giant fibres, determine the conduction velocity , the refractory period , the relation between the duration of the stimulus and the threshold voltage and the effect of the temperature. A study done in 1974 by Lagarspetz on frog nerve by shows that at higher temperature the conduction velocity increases relatively to the lower temperature (Lagerspetz, 1974). It can be hypothesized that the conduction velocity should decrease with the decrease in temperature in the earthworm giant fibre.

Material and Methods.

The experiment was performed with Tanvi Mehta. In the experiment, the equipment used were Grass P15 A.C. preamplifier, Grass SD stimulator, Dell computer and PC. The serial number was 04106J and the model is SD92A. Data not obtained in the lab were provided the teaching instructor, Matt Jackson. In the experiment, Earthworm, Lumbricus terrestris, was narcotized in 10% ethanol for 5-1 minutes and before dissection it was washed by worm ringer solution. Then, with a fine scissor make a cut on either side of the earthworm, to produce a thin strip of ventral skin 4-6 cm long, with the nerve cord attached only. Wash it with earthworm ringer solution. Add 1-2mm of the worm ringer solution to the nerve cord chamber below the electrode. Then place the preparation on the electrode and cover the nerve cord. Set up the recording apparatus as shown in figure 4.43. in the lab manual with the appropriate setting. Then, using the computer program, the time for the stimulus artifact of the median and lateral giant nerve was recorded. Then the duration and the voltage were measured for the median and the lateral nerve . In addition, the absolute refractory and the relative refractory period were measured for the median nerve. Then the strength- duration curve was generated using various duration and threshold voltage. The preparation was kept in the refrigerator for 12.5 minutes and then reading were taken for conduction velocity and refractory period.

Results: The threshold potential for median giant was 0.216mV and for lateral giant was 422mV and the duration were 1.991ms and 2.323ms respectively. The conduction velocity for the median giant was higher than lateral giant. The conduction velocity for the median giant was 10.55m/s and for lateral giant it was 9.04m/s. The duration of stimulus artifact for median and lateral giant was 1.25ms and 3.841ms respectively. The absolute refractory period for the median giant was 4.469ms and relative refractory period was 3.429ms-4557ms, with a voltage of 0.3745V and a duration of 1.0ms. Figure 1, shows a relationship between threshold voltage and duration in giant fibre of the earthworm,Lumbricus terrestri. The threshold woltage decreases as the duration increases. When the preparation was exposed to cold, the conduction velocity was 15.83m/s at a voltage of 0.335V, the voltage for the absolute refractory period was 0.335V, the duration was 0.2ms and the period was 5.5ms. When the preparation was recovered at room temperature, the conduction velocity was 16.53m/s, at a voltage of 0.335V. The absolute refractory period was 4.5ms, the duration was 0.2ms and the voltage was 0.335V. The conduction velocity increases from the initial room temperature, to the cold temperature and then to the recovery temperature. The sample size was 1.

4 3.5 Threshold voltage (mV) 3 2.5 2 1.5 1 0.5 0 0 10 20 30 40 50 Duration (ms)

Figure 1: The relationship of duration and threshold voltage in Earthworm, Lumbricus terrestris. The giant nerve fiber was placed on the electrode and the measurements were taken using the software Picoscope 6, and the preamplifier and the stimulator were set according to the instruction in the lab manual. The result of the stimulus was shown on a Dell computer PC. The room temperature was 20.1C. The sample size was 1. Discussion: The experiment shows that median giant has a lower threshold potential, lower duration and higher conduction velocity than lateral giant fibre. The size or the diameter has a significant effect on the conduction velocity whether it is myelinated or unmyelinated. Study from 1945 shows that diameter of the median giant fiber was 80.0m and the diameter of the lateral giant fiber was about 44m (Rushton, 1945). As a result median giant transmits faster impulse at a lower threshold than lateral giant fiber(Robert, 1962). In addition, the conduction velocity depends on the membrane ahead of it, and how fast it travels that brings it to threshold by the local current. The ionic current is much faster than channel mechanism, so a cell tends to increase conduction

velocity. (Burggren et al, 2001). Length constant is defined by the square root of membrane resistance divided by internal resistance. As the diameter increases, the internal resistance decreases, which increases the length constant and conduction velocity (Bullock, 1945). Conduction velocity can be increased by myelination, as it electrically insulates the axon except at the nodes of Ranvier , the insulated axons have higher membrane resistance, so again the length constant and conduction velocity increases. As a result higher conduction velocity have be achieved by larger diameter and myelination (Burggren et al ,2001). These results are consistent with a research done in 1978, which showed that mean rate of conduction of the median giant fiber in the anterior-posterior direction was 32.2m/s and mean rate for the lateral giant fiber in the posterior- anterior direction was 12.6m/s. (Drewes et al., 1978). It shows that attached nerve cord has a higher conduction velocity than an isolated preparation. Further, median giant fiber do have a higher conduction velocity than lateral giant fibres. The absolute refractory period of the median giant was longer at a colder temperature, when compared to room temperature. This might be because the longer time frame allowed the median giant fiber to depolarize. In addition, the median giant fiber is myelinated, so it deplorizes for a longer time period, as the current cannot leave. As a result there is an increases in the absolute refractory period(Burggren et al, 2001) The strength duration curve shows that as the duration increases the voltage decreases. It shows that when the membrane was exposed to high voltage for a longer time, that additional voltage was not necessary to produce an action potential. When the giant fiber was placed from a room temperature to a colder temperature and was then allowed to recover. The conduction velocity increased over the time, at room

temperature the conduction was 10.55m/s, at colder temperature was 15.82 m/s and at a recovery temperature was 16.55m/s. As a conduction velocity is proportional to the diameter, an increase in diameter should increase the conduction velocity. But a research from 1974, shows that a giant fiber accilimated at 5C and at 19C for a two week period, showed no significant statically difference in the diameter of the giant fiber.(LAgerspetz, 1974). As a result, it can be concluded that there was other factors that effect the conduction velocity.