Replacement of the axoplasm of giant nerve fibres with artificial solutions

Abstract

In 1937 Bear, Schmitt & Young showed that substantial quantities of axoplasm could be squeezed out of the cut end of a giant nerve fibre of Loligo. This technique has been widely used for obtaining samples of axoplasm, but little attention has been paid to the electrical properties of the thin sheath which remains after the contents of the nerve fibre have been extruded. Since extrusion involves flattening the axon with a glass rod or roller it is natural to suppose that the membrane would be badly damaged by such a drastic method of removing axoplasm. However, in the autumn of 1960 impulses were recorded from extruded sheaths which had been refilled with isotonic solutions of potassium salts (Baker & Shaw, 1961) and on further investigation it turned out that such preparations gave action potentials of the usual magnitude for several hours (Baker, Hodgkin & Shaw, 1961). Tasaki and his colleagues at Woods Hole have also been successful in perfusing the giant axons of Loligo pealii and several methods, some evidently developed before ours, were described by Oikawa, Spyropoulos, Tasaki & Teorell (1961). We have made no serious attempt to compare different methods and all the experiments described here were carried out by perfusing sheaths from which the bulk of the axoplasm had been removed by extrusion. The first paper deals with technique and with some general properties of perfused fibres, including histology and electron microscopy. The second is concerned with the electrical effects of changing the internal solution and ends with a discussion of both sets of results. METHODS MaterialGiant axons from large specimens of Loligo forbe8i were used throughout the investigation. The mantle lengths of the squid were usually 30-50 cm, and the diameters of the axon 700-900 u. Living squid were used occasionally but as a rule we employed refrigerated mantles. In this technique live squid are decapitated as soon as the trawl is brought up and

A comparison of the phosphorus metabolism of intact squid nerve with that of the isolated axoplasm and sheath.

“…Immersion in isotonic KCI rapidly depleted the axoplasm of orthophosphosphate Pi and energy-rich phosphate compounds. This observation, together with the action of calcium, indicates that the axolemma was not extruded along with the axoplasm and this has been confirmed by examination in the electron microscope of the sheath left behind after extrusion (Baker, Hodgkin & Shaw, 1962).…”

“…Many small branches of the giant fibre have to be cut during the dissection and great care was exercised to ensure that these were severed well away from the axon. After cannulation at the distal end and extrusion of axoplasm through a cut at the ganglion end, the fibres were perfused by the method of Baker et al (1962) with a solution containing 175 mm potassium sulphate and 10 mr phosphate buffer, isotonicity being maintained by sucrose (solution A, Table 4). The action potential was used as a criterion of the condition of the perfused axon.…”

“…Because this peripheral reaction only constituted a small fraction of the total phosphate break-down, changes in its activity were difficult to measure accurately. This was overcome by using perfused squid nerves (Baker et al 1962), where the rate of Pi release from perfused phosphate compounds was entirely dependent on the activity of the residual peripherally located phosphatase. Although rather few successful experiments have been made, the results are included in the present paper because of their particular relevance to the subject under investigation.…”

“…The sites of cyanide-sensitive energy-rich phosphate synthesis were most probably the mitochondria which are scattered evenly throughout squid protoplasm (Geren & Schmitt, 1954;Villegas & Villegas, 1960;Baker et al 1962 Fig. 1 with Fig.…”

Excitation of Squid Giant Axons in Hypotonic and Hypertonic Solutions

“…They were also recorded with a pen recorder through a transient digital time convertor. Osmolalities of solutions were changed by raising or reducing the concentration of non-electrolytes, that is, glycerol, glucose, or sucrose, or by adding NaCl to the external solution (TASAKI and TAKENAKA, 1964;BAKER et al, 1962;CHANDLER and HODGKIN, 1965). Concentrations of electrolytes were kept constant during one series of experiments except for those with hypertonic NaCl solutions.…”