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.

Evidence for the glia-neuron protein transfer hypothesis from intracellular perfusion studies of squid giant axons.

Incubation of intracellulary perfused squid giant axons in [3H]leucine demonstrated that newly synthesized proteins appeared in the perfusate after a 45-min lag period. The transfer of labeled proteins was shown to occur steadily over 8 h of incubation, in the presence of an intact axonal plasma membrane as evidenced by the ability of the perfused axon to conduct propagated action potentials over this time-period. Intracellularly perfused RNase did not affect this transfer, whereas extracellularly applied puromycin, which blocked de novo protein synthesis in the glial sheath, prevented the appearance of labeled proteins in the perfusate. The uptake of exogenous 14C-labeled bovine serum albumin (BSA) into the axon had entirely different kinetics than the endogenous glial labeled protein transfer process. The data provide support for the glia-neuron protein transfer hypothesis.

Rapid mechanical responses of the dark-adapted squid retina to light pulses.

Dark-adapted squid retinas respond to brief light pulses with early and late mechanical responses. These responses are mechanical counterparts of the early and late receptor potentials.

Heat generated by the dark-adapted squid retina in response to light pulses.

A rapid increase in the temperature of the dark-adapted squid retina evoked by a brief light pulse was detected with a pyroelectric detector. The amount of heat generated by the retina in response to a pulse of blue light of moderate intensity was far greater than that produced by direct conversion of the stimulating light by the retinal pigments into thermal energy. D-Glucose in the medium was required to maintain the ability of the retina to produce light-evoked thermal responses.

Changes in extrinsic fluorescence in squid axons during voltage-clamp.

Fluorescence changes in squid axons were examined after staining with rhodamine B, pyronin B, or 8-anilinonaphthalene-1-sulfonate by intracellular application. Gradual changes in fluorescence were detected during both hyperpolarizing and depolarizing voltage-clamp pulses. Abrupt changes were often observed at the onset and at the end of voltage-clamp. Possible sources of artifact in optical measurements of this type and some implications of the findings are discussed.

Effects of tetrodotoxin on excitability of squid giant axons in sodium-free media.

The effect of tetrodotoxin on excitability of internally perfused squid giant axons immersed in various sodium-free media was examined. Action potentials were found to be suppressed by this substance, with or without sodium ion in the external medium. Tetrodotoxin showed a strong suppressive effect upon action potentials produced in media containing salts of only divalent cations (CaCl(2), CaBr(2), SrCl(2), or BaCl(2)). Our findings concerning the action of tetrodotoxin do not support the separate-channel hypothesis for excitable membranes.

Swelling of nerve fibers associated with action potentials.

Swelling of nerve fibers during the action potential was demonstrated by three different methods. Generation of a propagated nerve impulse in a crab nerve produced an outward movement of 50 to 100 angstroms of the nerve surfce and a rise in swelling pressure on the order of 5 dynes per square centimeter. In squid giant axons, the amplitude of the observed outward movement of the surface was small.

Fluorescence changes during conduction in nerves stained with Acridine Orange.

Nerves from spider crabs anid squid fluoresce when staiined with Acridine Orange. The intenisity of fluorescence increases during nerse conduction. Prolongation of the electric response in the squid axon is associated with a fluorescence change of similar duration. These findings suggest that the physicochemical properties of the macromolecules around the dye molecules in the nerve membrane drastically change during the process of nerve conduction.

Fast axonal transport in squid giant axon.

Video-enhanced contrast-differential interference contrast microscopy has revealed new features of axonal transport in the giant axon of the squid, where no movement had been detected previously by conventional microscopy. The newly discovered dominant feature is vast numbers of "submicroscopic" particles, probably 30- to 50-nanometer vesicles and other tubulovesicular elements, moving parallel to linear elements, primarily in the orthograde direction but also in a retrograde direction, at a range of steady velocities up to +/- 5 micrometers per second. Medium (0.2 to 0.6 micrometer) and large (0.8 micrometer) particles move more slowly and more intermittently with a tendency at times to exhibit elastic recoil. The behavior of the smallest particles and the larger particles during actual translocation suggests that the fundamental processes in the mechanisms of organelle movement in axonal transport are not saltatory but continuous.

Evidence for the utilization of extracellular [gamma-32P]ATP for the phosphorylation of intracellular proteins in the squid giant axon.

Proteins in the squid giant axon were labeled with 32P by in vitro incubation of isolated axoplasm with radioactive [gamma-32P]adenosine triphosphate (ATP) and separated by polyacrylamide sodium dodecyl sulfate gel electrophoresis. The two major phosphorylated regions on the gel had molecular weights of 400,000 and 200,000. These two peaks appear to be neurofilament proteins of squid axoplasm. The same set of proteins was phosphorylated in the axoplasm regardless of whether the [gamma-32P]ATP was applied in situ intracellularly or extracellarly. These results suggest that ATP in the extracellular space is, by some ATP-translocation mechanism, utilized in the process of intracellular phosphorylation. Measurements of the apparent influx of ATP across the squid axon membrane yielded results consistent with the view that ATP in the extracellular fluid could be transported into the axoplasm.

Protein release from the internal surface of the squid giant axon membrane during excitation and potassium depolarization.

The proteins in the perfusate collected from intracellularly perfused squid giant axons were analyzed after being labeled with radioactive 125-I-labeled Bolton-Hunter reagent. The rate of protein release into the perfusate was found to be increased by the following electrophysiological manipulations of the axons: (1) repetitive electrical stimulation at 60 Hz in axons perfused with normal potassium fluoride-containing solution or at 0.125 Hz in axons perfused with tetraethylammonium containing solution, (2) perfusion with 4-aminopyridine solution which induces spontaneous electrical activity in the axon, and (3) depolarization of the axon induced by raising the external potassium concentration. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the proteins released under these conditions yielded molecular weight profiles different from those of the extruded axoplasmic proteins. These observations indicate that there exists, in close association with the axonal membrane, aparticular group of proteins, the solubility of which is readily affected by changes in the state of the membrane.

An approach to the study of intracellular proteins related to the excitability of the squid giant axon.

The technique for covalently labeling proteins with 125I-labelled Bolton-Hunter reagent was used to determine the quantities of proteins released from the axoplasmic side of the squid axon membrane. The reagent could be introduced into the interior of the axon by the technique of intracellular perfusion, the radioiodination reaction being carried out in situ. Alternatively, the reaction could be carried out in vitro, i.e., by mixing the reagent with samples of proteins dissolved in the intracellular perfusion fluid collected from the axon. This technique was found to be sensitive enough to permit analysis of a large number of protein samples collected from a single axon. By the method of sodium dodecyl sulfate polyacrylamide gel electrophoresis, it was found that proteins of approx. 56 000 daltons were released into the perfusate when a solution of potassium chloride or potassium bromide was introduced into the interior of an axon. Suppression of axonal excitability was associated with this release of proteins. The significance of these findings in relation to the structure and function of the axon is discussed.

Cation interdiffusion in squid giant axons.

Radiotracer techniques were used to study the influxes and effluxes of various univalent cations in internally perfused squid giant axons. Membrane currents and conductances were determined by the voltage-clamp technique under analogous internal and external conditions. Both sodium-containing and sodium-free internal and external media were studied. Membrane impedance was measured with an ac impedance bridge to determine the general magnitude and time course of the impedance loss which accompanied the excitation process in both varieties of external media. Maximum transmembrane currents were found to be of comparable magnitude to the charge transfer associated with the peak interdiffusion flux measured under the same conditions. The product of the membrane resistance and the interdiffusion flux remained constant over a wide range of resistance and flux values, both at rest and during activity, both in sodium-containing and sodium-free media. The implications of these findings for excitation theory are discussed.

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.

Synthesis and study of conformationally defined enantiomers of local anesthetics and conformationally defined enantiomers of fluorescent dyes designed to label electrically excitable membranes.

Conformationally defined enantiomeric local anesthetics and fluorescent dyes were synthesized. Neither the local anesthetics nor the fluorescent probes showed stereospecificity in interacting with nerve membranes. The fluorescence signals generated by the dyes showed excellent correlation with the time course and shape of the nerve action potential.

Large mechanical changes in the bullfrog olfactory bulb evoked by afferent fiber stimulation.

The isolated olfactory bulb of the bullfrog responds to olfactory nerve stimulation with large mechanical changes in the bulb. The sequence of these changes represents: (1) swelling of the bulb associated with action potential production in the secondary neurons; (2) shrinkage during powerful inhibition of the secondary neurons; and (3) swelling accompanied by repetitive firing of action potentials in the bulb. The effects of bicuculline and Cl-free Ringer on these mechanical changes were examined. The shrinkage of the bulb is closely related with the inhibitory postsynaptic potentials (IPSPs) in the mitral cells.

Swelling of frog dorsal root ganglion and spinal cord produced by afferent volley of impulses.

Electrical stimulation of the frog sciatic nerve was found to produce rapid, transient swelling of the 8th and 9th dorsal root ganglia followed by prolonged swelling of the spinal cord. Swelling of the ganglion is analogous to the rapid mechanical change observed in invertebrate axons during excitation. The mechanical change observed in the spinal cord is probably related to prolonged depolarization of the primary afferent fibers near their terminals.

Heat production associated with synaptic transmission in the bullfrog spinal cord.

By using heat detectors made with pyroelectric film, rapid heat production by the bullfrog spinal cord in response to dorsal root stimulation has been demonstrated. The heat production rises to its peak in about 100 ms after the arrival of afferent impulses and falls slowly with a time course comparable to that of the dorsal root potential. Stimulation of the ventral roots produces no detectable heat. The heat production was reversibly suppressed by immersion of the cord in a low Ca2+, high Mg2+ salt solution, indicating that the underlying exothermic process is associated with intraspinal synaptic transmission. The source of this 'synaptic heat' is located near the boundary between the dorsal column and the substantia gelatinosa in the vicinity of the stimulated dorsal roots.

Mechanical changes in the amphibian spinal cord produced by afferent volleys of nerve impulses.

A volley of impulses entering the toad spinal cord via large myelinated fibers in the lumbar dorsal roots was shown to evoke a contraction of the cord, which lasted for about 100 ms or more. A volley entering the cord antidromically via the ventral roots produced only a small, brief contraction. When two electric shocks were delivered to the same dorsal roots at a short interval, the contraction associated with the second shock was small; a period of about 1 s was required for a full recovery. When two shocks were applied separately to two neighboring dorsal roots, the contraction associated with the second shock was partially or totally occluded. Electric polarization of the dorsal root fibers produced mechanical changes in the cord. The effects of magnesium salt, GABA, glutamate and several other neuropharmacological agents on the contractile process were investigated. The experimental findings suggest that the contractile process is related to the phenomenon of primary afferent depolarization.