J.Z. AND THE DISCOVERY OF SQUID GIANT NERVE FIBRES
Cambridge University rdk12{at}cam.ac.uk
|
J.Z., to give him the title by which he was universally known, initially
acquired an interest in cephalopods when working in Naples with Enrico Sereni
in 1932 on the axons in the mantle connectives and stellar nerves of octopus.
This led him to further studies at the Plymouth Marine Laboratory of some
structures in the mantles of squid that he tentatively identified as giant
nerve fibres (Young, 1936). In
the summer of 1936 he visited Woods Hole in Massachusetts, determined to prove
that these `curious structures' were in fact motor axons. With F. O. Schmitt
and R. Bear, he successfully examined the axoplasm of axons from the mantle of
the squid Loligo pealii with polarized light, but failed in attempts with
Ralph Gerard, Detlev Bronk and Keffer Hartline to make any oscilloscope
recordings of action potentials from single fibres. However, he and Hartline
did better one day when they found that application of a solution of sodium
citrate to one end of the supposed axons generated a rhythmic discharge at the
other, showing that they were indeed nerve fibres. He then made a careful
study of the anatomy of the mantles, and in his classical paper on `The
functioning of the giant nerve fibres of the squid'
(Young, 1938
), he showed that
the third order giant axons served to bring about the precisely coordinated
contraction of the mantle causing expulsion of a powerful jet of water
propelling the animals rapidly backwards or forwards according to the position
of the funnel, sometimes accompanied by a slug of `ink' to assist the animal's
escape.
Having confirmed that the squid giant axons did conduct action potentials,
and having with R. J. Pumphrey in 1938
(Young and Pumphrey, 1938)
looked at the effect of their diameter on the rate of conduction, the only
respect in which J.Z. subsequently involved himself in research on the ionic
basis of conduction was to measure their electrolyte content
(Young and Webb, 1945
). He
did, nevertheless, devote many years to an important series of observations at
the Zoological Station in Naples on the mechanism of memory in octopus. And
always interested in the animal as a whole he was working vigorously in the
laboratory till the very end of his life on a wide range of problems. He will
also be remembered as a teacher of great distinction, and as the author of two
outstandingly wise and well-written textbooks on vertebrates and
invertebrates.
It was, however, the introduction of giant nerve fibres by J.Z. that
enabled the biophysics and biochemistry of excitable membranes to be properly
studied in depth, which was said by Alan Hodgkin in 1973 to have done more for
axonology than any other single advance in technique during the previous 40
years. J.Z. neatly summed up the impact that the discovery of giant motor
axons would have on the field when he wrote `on account of their enormous
size [the squid's giant nerve fibres] provide unique opportunities for study
of the functioning of single neuromuscular units'
(Young, 1938).
The first step in the exploitation of squid axons was taken in 1938 at
Woods Hole by Kacy Cole and H. J. Curtis
(Cole and Curtis, 1939) when
they showed using external electrodes that during the passage of an impulse
there was a rise and fall of the membrane conductance whose time course was
very similar to that of the action potential. Then in the summer of 1939, both
Curtis and Cole (1940
,
1942
) at Woods Hole, and Alan
Hodgkin and Andrew Huxley (Hodgkin and
Huxley, 1939
) at the Laboratory of the Marine Biological
Association in Plymouth, succeeded in slightly different ways in pushing long
glass tubes, 0.1 mm in diameter and filled with K+ solutions, for
some distance into the axons and thus recording the potential internally from
an undamaged part of the membrane. To their great surprise they found that at
the peak of the conducted impulse the membrane potential did not, as was
expected, fall close to zero, but was in fact substantially reversed.
After the end of six years of war that had interrupted biological research,
the problem of accounting for the reversal of potential at the peak of the
spike still remained unsolved. In writing up their 1939 experiments at greater
length, Hodgkin and Huxley
(1945) presented four elegantly
argued alternative explanations, in none of which it was obvious that they had
any faith. But then Alan Hodgkin dared to suggest that the permeability of the
membrane to Na+ ions might undergo a transient increase. Working
with squid giant axons at Plymouth in 1947, he and Bernard Katz were able to
establish that the sodium theory was sound
(Hodgkin and Katz, 1949
). As
has been described vividly by Hodgkin in his autobiography Chance &
Design (Hodgkin, 1992
),
the great experimental triumph that came next was his and Huxley's development
at Plymouth of the voltage-clamp technique for the quantitative analysis of
the relationship between current and voltage in an excitable membrane
(Hodgkin and Huxley,
1952
).
There followed a series of research projects on related questions, for
example the measurement of the net movements during the nerve impulse of
sodium and potassium by Keynes and Lewis
(1951); the establishment by
Hodgkin and Keynes (1955a
) of
the existence of the sodium pump; studies by Caldwell, Hodgkin, Keynes and
Shaw (1960
) on the dependence
of the sodium pump on a supply of phosphate-bond energy from ATP and arginine
phosphate; the discovery of Hodgkin and Keynes
(1955b
) in Cambridge, using
cuttlefish giant axons, of the manner in which K+ ions diffused in
single file through the voltage-gated potassium channels in nerve membranes;
and to crown Hodgkin's direct participation in experiments on squid axons, the
development of a method for perfusing them with a variety of solutions after
squeezing out the axoplasm as described by Baker, Hodgkin and Shaw
(1962
), in order to carry out
further rigorous tests of the ionic theory.
During the 1960s and 1970s, experiments on squid giant fibres continued to
occupy many axonologists, an advance of particular interest being the records
made for the first time independently at Woods Hole by Armstrong and Bezanilla
(1974) and at Plymouth by
Keynes and Rojas (1974
), of
the sodium gating current. The existence of such currents generated by the
transmembrane movements of the charged gating particles had been predicted by
Hodgkin, but they had not previously been recorded because of their small size
relative to the ion currents. Then in 1984 Numa and his colleagues in Kyoto
had succeeded, as described by Noda et al.
(1984
), in cloning the sodium
channel gene of the electric eel, and soon the primary amino acid sequences of
the voltage-gated sodium, potassium and calcium channels in a great many
animals were known. What is more, it turned out that the channel proteins
could readily be expressed in Xenopus oocytes, where their properties
could conveniently be examined by the patch-clamping techniques first
developed by Neher and Sakmann
(1976
). Research on these
lines is now being vigorously pursued in many laboratories all over the world
on the properties of ion channels gated not only by membrane potential, but
also by other agents.
Such work could be regarded as the ultimate offspring of J.Z.'s
introduction of giant axons to biologists, though few of its practitioners
have ever seen a squid. But as a postscript it may be added that for technical
reasons the time resolution obtained when voltage-clamping a squid giant axon
is appreciably better than when voltage-clamping a patch of oocyte membrane,
and for the best records yet made of the time course of the rise and fall of
the sodium gating current in a squid axon the reader should refer to those
obtained in the old-fashioned way by Keynes and Elinder
(1998) and their
colleagues.
References
Armstrong, C. M. and Bezanilla, F. M. (1974).
Charge movement associated with the opening and closing of the activation
gates of the Na channels. J. Gen. Physiol.
63,675
-689.
Baker, P. F., Hodgkin, A. L. and Shaw, T. I. (1962). The effects of changes in internal ionic concentrations on the electrical properties of perfused giant nerve fibres. J. Physiol. Lond. 164,355 -374.[Medline]
Caldwell, P. C., Hodgkin, A. L., Keynes, R. D. and Shaw, T. I. (1960). The effects of injecting `energy-rich' phosphate compounds on the active transport of ions in the giant axons of Loligo.J. Physiol. Lond. 152,561 -590.[Medline]
Cole, K. S. and Curtis, H. J. (1939).
Electrical impedance of the squid giant axon during activity. J.
Gen. Physiol. 22,649
-670.
Curtis, H. J. and Cole, K. S. (1940). Membrane action potentials from the squid giant axon. J. Cell. Comp. Physiol. 15,145 -157.
Curtis, H. J. and Cole, K. S. (1942). Membrane resting and action potentials from the squid giant axon. J. Cell. Comp. Physiol. 19,135 -144.
Hodgkin, A. L. (1992). Chance & Design. Reminiscences of Science in Peace and War. Cambridge: Cambridge University Press.
Hodgkin, A. L. and Huxley, A. F. (1939). Action potentials recorded from inside a nerve fibre. Nature 144,710 -711.
Hodgkin, A. L. and Huxley, A. F. (1945). Resting and action potentials in single nerve fibres. J. Physiol. Lond. 104,176 -195.
Hodgkin, A. L. and Huxley, A. F. (1952). A quantitative description of membrane current and its application to conduction and excitation in nerve. J. Physiol. Lond. 117,500 -544.[Medline]
Hodgkin, A. L. and Katz, B. (1949). The effect of sodium ions on the electrical activity of the giant axon of the squid. J. Physiol. Lond. 108,37 -77.
Hodgkin, A. L. and Keynes, R. D. (1955a). Active transport of cations in giant axons from Sepia and Loligo.J. Physiol. Lond. 128,28 -60.[Medline]
Hodgkin, A. L. and Keynes, R. D. (1955b). The potassium permeability of a giant nerve fibre. J. Physiol. Lond. 128,61 -88.
Keynes, R. D. and Elinder, F. (1998). On the slowly rising phase of the sodium gating current in the squid giant axon. Proc. R. Soc. Lond. B 265,255 -262.[CrossRef][Medline]
Keynes, R. D. and Lewis, P. R. (1951). The sodium and potassium content of cephalopod nerve fibres. J. Physiol. Lond. 114,151 -182.[Medline]
Keynes, R. D. and Rojas, E. (1974). Kinetics and steady-state properties of the charged system controlling sodium conductance in the squid giant axon. J. Physiol. Lond. 239,393 -434.[Medline]
Neher, E. and Sakmann, B. (1976). Single-channel currents recorded from membrane of denervated frog muscle cells. Nature 260,799 -802.[Medline]
Noda, M. et al. (1984). Primary structure of Electrophorus electricus sodium channel deduced from cDNA sequence. Nature 312,121 -127.[Medline]
Young, J. Z. (1936). The structure of nerve fibres in cephalopods and Crustacea. Proc. R. Soc. Lond. B 121,319 -337.
Young, J. Z. (1938). The functioning of the giant nerve fibres of the squid. J. Exp. Biol. 15,170 -185.
Young, J. Z. and Pumphrey, R. J. (1938). The rates of conduction of nerve fibres of various diameters in cephalopods. J. Exp. Biol. 15,453 -466.
Young, J. Z. and Webb, D. A. (1945). Electrolyte content and action potential of the giant nerve fibres of Loligo. J. Physiol. Lond. 98,299 -313.