Crayfish Electrophysiology Lab Report

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Five different external K+ concentrations ranging from 5.4 mM to 60 mM were used to bathe the crayfish preparation. Membrane potential was recorded from the fibres in two muscle groups in the crayfish abdomen the medial deep extensors and the lateral deep extensors. The solution containing 5.4 mM K+ was standard crayfish saline (van Harreveld's solution). Resting membrane potentials of the two muscle groups in van Harreveld's solution were 64.50 ± 6.64 mV in the lateral deep extensors and 67.80 ± .71 mV in the medial deep extensors. The resting potentials of these two muscle groups were not significantly different (P = 0.78 using an unpaired Student's t-test). No significant difference was observed between the membrane potentials of the lateral and medial deep extensors at any of the external K+ concentrations used in this study.

Depolarization with increase in external K+ concentration was observed in both muscle groups. Membrane potential in the lateral deep extensor muscle fibres increased at a rate of 4.17 ± 6.645 mV/10-fold increase in external K+ concentration, and in medial deep extensor muscle fibres it increased at a rate of 40.5 ± 5.4 mV/10-fold increase in external K+ concentration (fig 1). The rates of increase of membrane potential for these two groups of muscles were not significantly different (P = 0.551), but their elevations (ie, y-intercepts) were significantly different at a 5% level of confidence (P = 0.04165).

Discussion

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Membrane potential is defined as the difference in potential between the outside and inside of the cell, with the potential of the exterior defined arbitrarily as 0 mV (Randall et al., 00). The Nernst equation is used to calculate the membrane equilibrium potential for single ions. It states that the equilibrium potential depends upon the absolute temperature, the charge on the permeant ion and the ratio of the concentration of the ion on either side of the membrane (Randall et al., 00). For K+ ions at 0°C the Nernst equation has the approximate form

Resting potential = 58 log(Kout/ Kin) mV

where Kout is the K+ ion concentration outside the membrane and Kin is the K+ ion concentration inside the membrane ( Randall et al., 00; ZOO 485 Lab Manual, 00).

Potassium equilibrium resting potential has a negative sign because even a minute leakage of K+ from the inside of the cell due to a K+ concentration gradient will cause the interior of the cell to become more negative (Randall et al., 00). One would therefore expect that because increase in external K+ concentration would result in decreasing K+ gradient, potassium equilibrium resting potential would become less negative with increasing external K+ concentration. In the present study the same crayfish preparation was used with the variety of external K+ concentrations and therefore the internal K+ concentration was assumed to be constant. Therefore it can be hypothesized that when resting membrane potential (in mV) is plotted as a function of log external [K+], the relation will have a slope of 58 mV/10-fold increase in external [K+] (Randall et al., 00; ZOO 485 Lab manual, 00). The relation between resting membrane potential and external K+ concentration in this experiment was found to be 4.17 ± 6.645 mV/10-fold increase in external K+ concentration in the lateral deep extensor muscle and 40.5 ± 5.4 mV/10-fold increase in external K+ concentration in the medial deep extensor muscles (fig 1). These two slopes are not significantly different, and combine to provide a pooled slope of 41.5 mV/10-fold increase in external K+ concentration.

This pooled slope is has a percentage difference of % from the hypothesized slope of 58 mV/10-fold increase in external K+ concentration. The most important factor which could explain this difference is the influence on the membrane potential of other ions with equilibrium potentials different from that of K+. Most membranes are permeable to several ionic species. These species may be asymmetrically distributed across the membrane causing concentration gradients and thus affecting the membrane potential (Randall et al., 00). It is often better, therefore, to calculate membrane potential using the Goldman equation, a generalization of the Nernst equation extended to include the relative permeabilities of K+, Na+ and Cl-, the major ionic species in the intracellular and extracellular compartments (Randall et al., 00). Other, unidentified factors also affect measurement of membrane potential (Hinkle et al., 171). Hinkle et al. (171) performed a similar study of crayfish membrane potential. They used the Goldman equation to calculate membrane potential, but found marked discrepancies between observed and expected membrane potential. They found that the membrane repolarized more predicted.

Fig shows the experimentally determined relations of log external [K+] vs. membrane potential in lateral and medial deep extensor muscles, along with the hypothesized relation between membrane potential and log external [K+] with a slope of 58 mV/10-fold increase in external K+ concentration. If a reliable relationship between membrane potential and log external [K+] can be obtained, this type of plot can be used to determine internal concentration of K+. Values for external [K+] and resting potential can be obtained from this plot. When these values are put into the Nernst equation, Kin is the only remaining variable. Kin can therefore be calculated using the Nernst equation.

Due to technical problems the experiment studying synaptic potentials at the crayfish neuromuscular junction was not performed. This experiment was meant to provide a map of excitatory and inhibitory junction potentials in a muscle, which would then be used to hypothesize the function of the muscle as related to its structure. However detailed studies of this sort were undertaken by Kennedy and Takeda (165a; 165b). They studied reflex control of the crayfish abdominal flexor muscles. These muscles are divided into two systemssuperficial tonic muscles and a set of larger, deeper muscles which do not display tonic contractions (Kennedy and Takeda, 165a). almost all muscle fibres are triply innervated. Each receives endings from the 'motor giant' axon, a specific non-giant axon and a common inhibitor (Kennedy and Takeda, 165a). The deeper set of muscles is responsible for the 'tail-flip' reflex reaction, in which is the first few powerful flips of the tail provide a fast escape from danger (Kennedy and Takeda, 165a). This reaction is caused by the motor giant axons, which seem to function exclusively as escape mechanisms. These axons play almost no role in continuous swimming, but provide the powerful thrust in the first few strokes that serves to accelerate the crayfish away from its position of rest (Kennedy and Takeda, 165a). They provide this extra thrust by causing greater depolarization of the muscle fibre than that which would result from the non-giant axons alone, and they also cause this depolarization event to remain for a longer period of time (Kennedy and Takeda, 165a). The superficial abdominal flexor muscles are responsible for all graded contractions in the crayfish (Kennedy and Takeda, 165b). They receive complex, polyneural innervation and show a great deal of spontaneous activity (Kennedy and Takeda, 165b). If the experiment measuring and identifying synaptic potentials in a crayfish abdominal muscle had been performed, it would have been possible to infer the function of the muscle.

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