Alteration of birefringence signals from squid giant axons by intracellular perfusion with protease solution

The optical signal, arising from a transient birefringence change associated with excitation, was recorded from a squid giant axon together with the membrane potential change, and the effect of removal of the axoplasm on the optical signal was examined. In an unperfused axon, repetitive stimulation at a frequency of about 100 Hz produced two kinds of optical response. The initial response had a brief, spike-like time course and was elicited by each stimulating pulse. The delayed response had a slow time course and the sign of decreased light intensity, and summated with repetitive stimulation. Most of the axoplasm was removed from interior of the axon by intracellular perfusion with solutions containing pronase at a concentration of 0.1 mg/ml. The delayed response could selectively be eliminated by perfusion with a pronase-containing solution for 2–8 min. The result was interpreted as showing that the delayed birefringence signal originates from axoplasm when its gel structure was transiently disturbed by an increased Ca²⁺ influx associated with excitation. When perfusion was further continued the duration of the action potential started increasing and often a prominent after-depolarization appeared. At this stage the initial optical response was again followed by a large show signal with the sign of increased light intensity. This reversed delayed response was tentatively assumed to originate from the membrane with some remaining axoplasm, but its cause is still not understood.     

Subaxolemmal filamentous network in the giant nerve fiber of the squid (Loligo pealei L.) and its possible role in excitability

A new technique utilizing the squid giant nerve fiber has been developed which permits direct examination of the inner face of the axolemma by scanning electron microscopy. The axoplasm was removed sequentially in a 15-mm long segment of the fiber by intracellular perfusion with a solution of KF, KCl, Ca++-containing seawater, or with pronase. The action potential of the fibers was monitored during these treatments. After brief prefixation in 1% paraformaldehyde and 1% glutaraldehyde, the perfused segment was opened by a lne could be related to information on the detailed morphology of the cytoplasmic face of the axolemma and the ectoplasm. The results obtained by scanning electron microscopy were further substantiated by transmission electron microscopy of thin sections. In addition, living axons were studied with polarized light during axoplasm removal, and the identification of actin by heavy meromyosin labeling and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis was accomplished. These observations demonstrate that a three-dimensional network of interwoven filaments, consisting partly of an actinlike protein, is firmly attached to the axolemma. The axoplasmic face of fibers in which the filaments have been removed partially after perfusion with pronase displays smooth membranous blebs and large profiles which sppose the axolemma. In fibers where the excitability has been suppressed by pronase perfusion, approximately one-third of the inner face of the axolemma in the perfusion zone is free of filaments. It is hypothesized that the attachment of axoplasm filaments to the axolemma may have a role in the maintenance of the normal morphology of the axolemma, and, thus, in some aspect of excitability.     

Effects of Redox and Sulfhydryl Reagents on the Bioelectric Properties of the Giant Axon of the Squid

The effects of internally and externally applied sulfhydryl reagents on the bioelectric properties of the giant axon of the squid Loligo pealeii and Dosidicus gigas were studied. Cysteine-HCl (400 mM, pH 7.3) was used to remove axoplasm from the perfusion channel. Oxidizing agents (1 to 60 mM) tended to increase the duration of the action potential and had a slow, irreversible blocking effect when perfused internally; the membrane potential was little affected. Reducing agents applied internally caused a decrease in the spike duration without affecting its height or the membrane potential, although at high concentrations there was reversible deterioration of the action potential. Both external and internal perfusion of mercaptide-forming reagents caused deterioration in the action and membrane potentials with conduction block occurring in 5 to 45 min. 2-mercaptoethanol reversed the effects. Thiol alkylating reagents, iodoacetate and iodoacetamide, were without effect. N-ethylmaleimide did, however, block. Tests with chelating agents for nonheme iron in the membrane brought about no change in the electrical parameters. The implications of the present findings with regard to the macromolecular mechanism of excitation are discussed.     

Internally perfused Myxicola giant axons showing long-term survival.

An improved method for internally perfusing the Myxicola giant axon based on removing the axoplasm by dispersing it in KCl-KF salt solutions is described. Proteolytic enzymes are not introduced. With this improved method perfused preparations show long-term stability of their electrical properties and the ability to generate action potentials for many hours. Mean initial values for resting membrane potential, action potential amplitude, and peak inward current were -68 mV, 118 mV, and 3.62 mA/cm2, respectively. Mean resting membrane resistance was 75% of that in intact axons. In one series of voltage clamp experiments, perfused preparations remained excitable for a mean period of 5 1/2 h, but this period could exceed 10 h. 4 min are needed for exchange of internal solutions. At least 50 mM KF is required both in the axoplasm liquefying solution and in the standard perfusate to obtain stable preparations.     

Fluorometric measurement of pyridine nucleotide reduction in the giant axon of the squid

By monitoring the fluorescence of the isolated giant axon of the squid Loligo pealei, it was possible to follow changes in its oxidation-reduction state as caused by the action of anoxia, cyanide, Amytal, and azide. The response to oxygen depletion was very rapid, the NAD within the axon being 90% reduced within 1–2 min. Cyanide and Amytal gave essentially similar results, although somewhat longer periods of time elapsed during their onset and washout periods. The extent of NAD reduction was essentially the same under conditions of anoxia and treatment with cyanide and Amytal. Azide was less effective in this respect, and at comparatively high levels of concentration (25–50 mM) gave values of 40% or less of the reduction observed with the other inhibitors. The application of ouabain and strophanthidin gave no observable NAD reduction. Variations in the time required to consume given quantities of dissolved oxygen before and after stimulation indicated an increase of 10–20% in oxygen uptake rate associated with activity, although this figure appeared to be a function of the surface-to-volume ratio of the axon. A biochemical analysis of axoplasm for oxidized and reduced pyridine nucleotide was made. Fluorometric examination of centrifuged axoplasm indicated that the NAD-NADH was largely confined to the mitochondria of the axon.     

The effects of nitroprusside and putative agonists on guanylate cyclase activity in squid giant axons

cGMP content of axoplasm from the giant axon of Loligo forbesi was investigated after subjecting the axon to various treatments. Repetitive electrical stimulation or depolarisation by high K+ caused no change in cGMP content. Glutamate and serotonin were also without effect. The nicotinic agonist carbachol (100 microM) increased cGMP levels by 90% (n = 5). A large transient elevation of cGMP content was evoked by external nitroprusside (10 nM-20 microM in intact axons. Nitroprusside injected into both extruded axoplasm and intact axons also increased cGMP content, the stimulation being considerably higher in intact axons where the axolemma was also present. Nitroprusside was also active in axons where the soluble cytoplasmic components were washed out by internal perfusion.     

Magnesium influx in dialyzed squid axons

Summary The influx of magnesium from seawater into squid giant axons has been measured under conditions where internal solute control in the axon was maintained by dialysis. Mg influx is smallest (1 pmol/cm² sec) when both Na and ATP have been removed from the axoplasm by dialysis. The addition of 3mm ATP to the dialysis fluid gives a Mg influx of 2.5 pmol/cm² sec while the addition of [Na] _i and [ATP] _i gives 3 pmol/cm² sec as a value for Mg influx; this corresponds well with fluxes measured in intact squid giant axons.The Mg content of squid axons is 6 mmol/kg axoplasm; this is unaffected by soaking axons in Li or Na seawater for periods of up to 100 min.     

Distribution of Acid Protease Activity in the Squid Nervous System

Abstract: Acid protease activity was measured in homogenized stellate ganglion, axoplasm extruded from the squid giant axon, homogenized fin nerves, and in lysed synaptosomes prepared from the optic lobe of the squid. At least two different acid protease classes were distinguished on the bases of their inhibitor profiles. Acid protease activity was present in each of the above tissues except extruded axoplasm. This result suggests that the acid protease activity found in our homogenized finnerves might be located not within the axons but rather in glial cells or extracellular tissue. The absence of acid protease activity in extruded axoplasm indicates that acid proteases are unlikely to play a significant role in the catabolism of intracellular proteins along the length of the axon.     

Alteration of Acetylcholine Penetration into, and Effects on, Venom-Treated Squid Axons by Physostigmine and Related Compounds

Choline and neostigmine markedly antagonize the effect of acetylcholine (ACh) on the action potential of the venom-treated squid axon, although they themselves have no effect on conduction. Physostigmine also antagonizes the blocking action of ACh at a concentration well below that which has any effect on conduction. In contrast, d-tubocurarine (curare) increases the effect of ACh on the action potential. Choline, neostigmine, and physostigmine markedly decrease the penetration of C¹⁴-labeled ACh into the axoplasm of the squid axon. Curare, in contrast, increases the penetration of ACh, whereas dimethylcurare gives variable results. The results provide an explanation why physostigmine and neostigmine do not influence the action of ACh on axonal conduction in a way similar to that observed at the junction. The additive effect of curare and ACh on the action potential may be due either to the greater rate of penetration of ACh or to an additive effect of the two compounds on the receptor, or to a combination of both factors.     

Effect of specific antibodies on the excitability of internally perfused squid axons

Giant axons from the squid Dosidicus gigas were internally perfused with rabbit antiaxoplasm antibodies and their effect upon the action potential and the membrane potential was studied. Necessary requirements for the antibodies to affect these parameters in a consistent manner were: (a) removal of the bulk of axoplasm from the perfused zone, accomplished by initially perfusing with a cysteine-rich (400 mM) solution, and (b) addition of small amounts of cysteine (30 mM) to the antibody-containing solution. When these experimental conditions were met, conduction block ensued generally within 3 hr of the first contact of the axon inner surface with the antibody Antineurofilament antibodies and nonspecific antibodies had no effect. External application of antiaxoplasm antibodies had no effect.     

Temperature Characteristics of Excitation in Space-Clamped Squid Axons

Temperature characteristics of excitability in the squid giant axon were measured for the space-clamped axon with the double sucrose gap technique. Threshold strength-duration curves were obtained for square wave current pulses from 10 µsec to 10 msec and at temperatures from 5°C to 35°C. The threshold change of potential, at which an action potential separated from a subthreshold response, averaged 17 mv at 20°C with a Q¹⁰ of 1.15. The average threshold current density at rheobase was 12 µa/cm² at 20°C with a Q¹⁰ of 2.35 compared to 2.3 obtained previously. At short times the threshold charge was 1.5·10^-8 coul/cm². This was relatively independent of temperature and occasionally showed a minimum in the temperature range. At intermediate times and all temperatures the threshold currents were less than for both the single time constant model and the two factor excitation process as developed by Hill. FitzHugh has made computer investigations of the effect of temperature on the excitation of the squid axon membrane as represented by the Hodgkin-Huxley equations. These are in general in good agreement with our experimental results.     

A study of the effects of externally applied sodium-ions and detection of spatial non-uniformity of the squid axon membrane under internal perfusion

Electrical properties of the axon membrane were examined under internal perfusion of squid giant axons with a dilute solution of NaF or CsF. The rate of propagation of the action potential was markedly enhanced when NaCl was added to the external CaCl₂ solution. The membrane conductance both at rest and during the action potential was increased with increasing Na-concentration in the external medium. In the perfusion zone of these axons, the action potentials in different parts of the membrane were found to terminate in a more-or-less spatially random and temporally irregular fashion. When the electric field outside the axon membrane was examined with hyperfine glass-pipette electrodes, small rectangular potential changes of uniform amplitude were observed. The small potential changes, which resemble those obtained by Bean in EIM-treated lipid bilayer, were interpreted as indicating spatial non-uniformity of the axon membrane during excitation. The importance of long-range electric interaction between different parts of the axon membrane is emphasized.     

Axoplasmic free magnesium levels and magnesium extrusion from squid giant axons

The free magnesium concentration in the axoplasm of the giant axon of the squid, Loligo pealei, was estimated by exploting the known sensitivity of the sodium pump to intracellular Mg2+ levels. The Mg- citrate buffer which, when injected into the axon, resulted in no change in sodium efflux was in equilibrium with a Mg2+ level of about 3- -4 mM. Optimal [Mg2+] for the sodium pump is somewhat higher. Total magnesium content of axoplasm was 6.7 mmol/kg, and that of hemolymph was 44 mM. The rate coefficient for 28Mg efflux was about 2 X 10(-3) min-u for a 500-mum axon at 22-25degreesC, with a very high temperature coefficient (Q10=4-5). This efflux is inhibited 95% by injection of apyrase and 75% by removal of external sodium, and seems unaffected by membrane potential or potassium ions. Increased intracellular ADP levels do not affect Mg efflux nor its requirement for Na+/o, but extracellularl magnesium ions do. Activation of 28Mg efflux by Na+/o follows hyperbolic kinetics, with Mg2+/o reducing the affinity of the system for Na+/o. Lanthanum and D600 reversibly inhibit Mg efflux. In the absence of both Na+ and Mg2+, but not in their presence, removal of Ca2+ from the seawater vastly increased 28Mg efflux; this efflux was also strongly inhibited by lanthanum. A small (10(-14) mol cm-2) extra Mg efflux accompanies the conduction of an action potential.     

Are axoplasmic microtubules necessary for membrane excitation?

Summary The excitability of the squid giant axon was studied as a function of transmembrane hydrostatic pressure differences, the latter being altered by the technique of intracellular perfusion. When a KF solution was used as the internal medium, a pressure difference of about 15 cm water had very little effect on either the membrane potential or excitability. However, within a few minutes after introducing either a KCl-containing, a KBr-containing, or a colchicine-containing solution as the internal medium, with the same pressure difference across the membrane, the axon excitability was suppressed. In these cases, removal of the pressure difference restored the excitability, indicating that the structure of membrane was not irreversibly damaged. Electron-microscopic observations of these axons revealed that the perfusion with a KF solution or colchicine-containing solution preserves the submembranous cytoskeletal layer, whereas perfusion with a KCl or KBr solution dissolves it. These results suggest that the submembranous cytoskeletons including microtubules provide an important mechanical support to the excitable membrane but are not essential elements in channel activities.     

Effects of narcotics on the giant axon of the squid

—Levorphanol (10^-3 M) reversibly blocked conduction in the giant axon of the squid and axons from the walking legs of spider crab and lobster. Similar concentrations of levallorphan and dextrorphan blocked conduction in the squid giant axon. Under the same experimental condition morphine caused an approximately 40 per cent decrease in spike height. Levorphanol did not affect the resting potential or resistance of the squid axon. Spermidine, spermine and dinitrophenol had little or no direct effect on the action potential nor did they alter the potency of levorphanol. Concentrations of levorphanol as low as 5 × 10^-5 M blocked repetitive or spontaneous activity in the squid axon induced by decreasing the divalent cations in the medium. After exposure to tritiated levorphanol, the axoplasm and envelope of the squid axon accumulated up to 500 per cent of the concentration of tritium found in the external medium, dependent on time of exposure, and other variables. At pH 6 the levels of penetration were 33-50% of those found at pH 8, which correlates with our observation that levorphanol is about 33 % as potent in blocking the action potential at pH 6. The penetrability of levorphanol was not affected by spermidine, dinitrophenol or cottonmouth moccasin venom. Levorphanol did not alter the penetration of [C¹⁴]acetylcholine nor did it render the squid axon sensitive to it. The block of axonal conduction by compounds of the morphine series is discussed both as to possible mechanisms and significance.     

Calcium measurement in the periphery of an axon

Aequorin was microinjected into squid giant axons, the axons were stimulated, and the change in light emission was followed. This response was compared with that found when the axon, in addition to being microinjected with aequorin, is also injected with the dye phenol red. Large concentrations of phenol red injected into axons result in a high probability that photons emitted by aequorin, when it reacts with Ca in the core of the axoplasm, will be absorbed before they escape from the axon; photons produced by the aequorin reaction at the periphery of the axoplasm are much less likely to be absorbed. This technique thus favors observing changes in Cai taking place in the periphery of the axon. Stimulation in 50 mM Ca seawater of an aequorin-phenol red-injected axon at 180 s-1 for 1 min produces a scarcely detectable change in Cai; the addition of 2 mM cyanide (CN) to the seawater produces an easily measureable increase in Cai, suggesting that mitochondrial buffering in the periphery is substantial. Making the pH of the axoplasm of a normal axon alkaline with 30 mM NH4+ -50 mM Ca seawater, reduces the resting glow of the axon but results in an even more rapid increase in Cai with stimulation. In a phenol red-injected axon, this treatment results in a measureable response to stimulation in the absence of CN.     

Restoration of membrane excitability of squid giant axons by reagents activating tyrosine-tubulin ligase

Summary Using squid giant axon, an experimental survey was performed on restoration of the membrane excitability which had been partially suppressed. Among reagents examined, a combination of 400mm KF, 50 m tyrosine, 1mm ATP, 1mm Mg ions and 5 m cAMP was found to induce the restoration of the excitability to a large extent. Further addition of a small amount of either porcine brain microtubule proteins or the squid axoplasm was found to support complete restoration. The experiments suggest that tubulin-tyrosine ligase contained in the porcine brain microtubule protein fraction or the squid axoplasm maintains the coupling between cytoskeletal structures and the plasma membrane.     

Transfer of molecules from glia to axon in the squid may be mediated by glial vesicles

Although the transfer of glial proteins into the squid giant axon is well documented, the mechanism of the transfer remains unknown. We examined the possibility that the transfer involved membrane-bound vesicles, by taking advantage of the fact that the fluorescent compound, 3,6-acridinediamine, N,N,N,',N'-tetramethylmonohydride [acridine orange (AO)], rapidly and selectively stains vesicular structures in glial cells surrounding the giant axon. We labeled cleaned axons (1–3 cm long) by incubation for 1 min in filtered seawater (FSW) containing AO. Because the AO was concentrated in glial vesicular organelles, these fluoresced bright orange when the axon was examined by epifluorescence microscopy. To look for vesicle transfer, axoplasm was extruded from such AO-treated axons at various times after labeling. During the initial 15 min, an increasing number of fluorescent vesicles were observed. No further increases were observed between 15 and 60 min post AO. The transfer of the fluorescent vesicles into the axoplasm seemed to be energy dependent, as it was inhibited in axons treated with 2 mM KCN. These results suggest that a special mode of exchange exists between the adaxonal glia and the axon, perhaps involving phagocytosis by the axon of small portions of the glial cells.     

Release of proteins from the inner surface of squid axon membrane labeled with tritiated N-ethylmaleimide

Proteins in the inner surface of the squid axon membrane were labeled by intracellular perfusion of [3H]N-ethylmaleimide (NEM), which forms covalent bonds with free sulfhydryl groups. The excitability of the axon was unaffected by the [3H]NEM perfusion. After washout of the unbound label, the perfusate was monitored for the release of labeled proteins. Labeled proteins were released from the inner membrane surface by potassium depolarization of the axon only in the presence of external calcium ions. Replacement of the fluoride ion in the perfusion medium by various anions also caused labeled protein release. The order of effectiveness was SCN- greater than Br- greater than Cl- greater than F-. The extent of labeled protein release by the various anions was correlated with their effects on axonal excitability. The significance of these results is discussed.