Protein Transport in Intact and Severed (Anucleate) Crayfish Giant Axons

Abstract: Using video-enhanced microscopy and a pulse-radiolabeling paradigm, we show that proteins synthesized in the medial giant axon cell body of the crayfish ( Procambarus clarkii ) are delivered to the axon via fast (∼62 mm/day) and slow (∼0.8 mm/day) transport components. These data confirm that the medial giant axon cell body provides protein to the axon in a manner similar to that reported for mammalian axons. Unlike mammalian axons, the distal (anucleate) portion of a medial giant axon remains intact and functional for >7 months after severance. This axonal viability persists long after fast transport has ceased and after the slow wave front of radiolabeled protein has reached the terminals. These data are consistent with the hypothesis that another source (i.e., local glial cells) provides a significant amount of protein to supplement that delivered to the medial giant axon by its cell body.     

Ionic Conductance Changes in Voltage Clamped Crayfish Axons at Low pH

Giant axons from the crayfish have been voltage clamped with an axial wire system. General characterististics of observed ionic currents under normal conditions are similar to those measured in other giant axons and in nodes of Ranvier. As the pH of the external bath is lowered below 7, a marked, reversible slowing of potassium currents is seen with little effect on sodium currents. The steady-state potassium conductance-voltage curve is shifted along the voltage axis in a manner consistent with the development of a hyperpolarizing surface charge. Results suggest that this potential shift accounts for part, though not all, of the observed increase in τ_n. From the behavior of the kinetics of the delayed current with external pH these alterations in potassium conductance are attributed to the titration of a histidine imidazole residue of a membrane protein. Chemical modification of histidine by carbethoxylation at pH 6 slows and strongly depresses potassium currents. The results suggest that in addition to the introduction of electrostatic forces, possibly resulting from a hyperpolarizing surface charge, protonation of a histidine group at low pH also alters the nonelectrostatic chemical interactions determining the ease with which potassium gates open and close. The evidence indicates that the modified histidine residue is closely associated with the membrane components involved in the control of potassium conductance.     

Integration of Shallow Gradients of Shh and Netrin-1 Guides Commissural Axons

During nervous system development, gradients of Sonic Hedgehog (Shh) and Netrin-1 attract growth cones of commissural axons toward the floor plate of the embryonic spinal cord. Mice defective for either Shh or Netrin-1 signaling have commissural axon guidance defects, suggesting that both Shh and Netrin-1 are required for correct axon guidance. However, how Shh and Netrin-1 collaborate to guide axons is not known. We first quantified the steepness of the Shh gradient in the spinal cord and found that it is mostly very shallow. We then developed an in vitro microfluidic guidance assay to simulate these shallow gradients. We found that axons of dissociated commissural neurons respond to steep but not shallow gradients of Shh or Netrin-1. However, when we presented axons with combined Shh and Netrin-1 gradients, they had heightened sensitivity to the guidance cues, turning in response to shallower gradients that were unable to guide axons when only one cue was present. Furthermore, these shallow gradients polarized growth cone Src-family kinase (SFK) activity only when Shh and Netrin-1 were combined, indicating that SFKs can integrate the two guidance cues. Together, our results indicate that Shh and Netrin-1 synergize to enable growth cones to sense shallow gradients in regions of the spinal cord where the steepness of a single guidance cue is insufficient to guide axons, and we identify a novel type of synergy that occurs when the steepness (and not the concentration) of a guidance cue is limiting.     

Assaying the Ability of Diffusible Signaling Molecules to Reorient Embryonic Spinal Commissural Axons

Dorsal commissural axons in the vertebrate spinal cord¹ have been an invaluable model system in which to identify axon guidance signals. Here, we describe an in vitro assay, "the reorientation assay", that has been used extensively to study the effect of extrinsic and intrinsic signals on the orientation of commissural axons². This assay was developed by numerous people in the laboratories of Jane Dodd, Thomas Jessell and Andrew Lumsden (see acknowledgements for more details) and versions of this assay were used to demonstrate the reorientation activities of key axon guidance molecules, including the BMP chemorepellent in the roof plate^3,4 and the chemoattractive activities of Netrin1⁵ and Sonic Hedgehog (Shh)⁶ in the floor plate in the spinal cord.Explants comprising 2-3 segments of the dorsal two-thirds of spinal cord are dissected from embryonic day (E) 11 rats and cultured in three dimensional collagen gels⁷. E11 dorsal spinal explants contain newly born commissural neurons, which can be identified by their axonal expression of the glycoprotein, Tag1⁸. Over the course of 30-40 hours in culture, the commissural axon trajectory is recapitulated in these dorsal explants with a time course similar to that seen in vivo. This axonal trajectory can be challenged by placing either test tissues or a COS cell aggregate expressing a candidate signaling molecule in contact with one of the lateral edges of the dorsal explant. Commissural axons extending in the vicinity of the appended tissue will grow under the influence of both the endogenous roof plate and signals from the ectopic lateral tissue. The degree to which commissural axons are reoriented under these circumstances can be quantified. Using this assay, it is possible both to examine the sufficiency of a particular signal to reorient commissural axons^3,4 as well the necessity for this signal to direct the commissural trajectory⁹.Download video file.^{(108M, mp4)}     

EXCITABLE MEMBRANE ULTRASTRUCTURE : I. Freeze Fracture of Crayfish Axons

Cross-sectioned and cross-fractured crayfish axons display regions in which axon and Schwann cell surface membranes are regularly curved and project into the axoplasm. At these regions (projections) the two membranes run precisely parallel, separated by a gap of 130–140 Å. Longitudinal fractures through the axons expose the inner fractured surface of either the internal (face A) or the external (face B) leaflet of axon and adjacent Schwann cell surface membranes. On both membranes the projections appear as elongated structures oriented with the long axis parallel to the long axis of the nerve fiber. On face A of the axon surface membrane they are seen as elongated indentations 0.5–1.2-µm long, 0.12–0.15-µm wide. The indentations contain parallel chains of globules. The chains repeat every 120–125 Å and are oriented obliquely in such a way that if one looks at the axon surface from the extracellular space, the axis of the chains is skewed counterclockwise to the long axis of the indentations by an acute angle (most often 55–60°). The globules repeat along the chain every 80–85 Å. Globules of adjacent chains are in register in such a way that the axis on which globules of neighboring chains are aligned forms an angle of 75–85° with the axis of the chains. The complex structure can be defined as a globular array with a rhomboidal unit cell of 80–85 x 120–125 Å. On face B of the axon surface membrane the complementary image of these structures is seen. The projections of the Schwann cell surface membrane also contain groupings of globules; however, these differ from those in the axonal projections in size, pattern of aggregation, and fracture properties. Several possible interpretations of the meaning of these membrane specializations could be proposed. They could be: (a) structures involved in the mechanism of excitation, (b) regions of presumed metabolic couplings, and (c) areas of cell-to-cell adhesion.     

Surface Charge of Giant Axons of Squid and Lobster

A method is described for the determination of the electrophoretic mobility of single, isolated, intact, giant axons of squid and lobster. In normal physiological solutions, the surface of hydrodynamic shear of these axons is negatively charged. The lower limit of the estimated surface charge density is -1.9 × 10^-8 coul cm^-2 for squid axons, -4.2 × 10^-8 coul cm^-2 for lobster axons. The electrophoretic mobility of squid axons decreases greatly when the applied transaxial electric field is made sufficiently intense; action potential propagation is blocked irreversibly by transaxial electric fields of the same intensity. The squid axon recovers its mobility hours later and is then less affected by transaxial fields. Eventually, a state is reached in which the transaxial field irreversibly reverses the sign of the surface charge. In contrast, there is no obvious effect of electric field on the mobility of lobster axons. The mobility of lobster axons becomes undetectable in the presence of Th⁴⁺ at a concentration which blocks the action potential, and in the presence of La³⁺ at a concentration which does not affect propagation. Quinine does not alter lobster axon mobility at a concentration which blocks action potential conduction. Replacement of extracellular Na⁺ by K⁺ is without effect upon lobster axon mobility. The electrophysiological implications of the results are discussed.     

Effect of Extracellular Potassium on Ouabain-Sensitive Consumption of High-Energy Phosphate by Crayfish Giant Axons: A Study of the Energy Requirement for Transport in the Steady State

Crayfish axons exposed to a high or low extracellular K+ concentration ([K+]o) maintain intracellular Na+ and K+ concentrations constant, for up to 3 h, by adjusting both the Na+/K+ transport "coupling ratio" and turnover rate in compensation for changes in ion fluxes due to altered electrochemical gradients. These findings give rise to the prediction that the steady-state consumption of high-energy phosphate (approximately P) [ATP and phospho-L-arginine (Arg-P)] is inversely proportional to the [K+]o, i.e., directly proportional to the product of membrane conductance and magnitude of the transmembrane electrochemical gradients for Na+ and K+. This investigation was designed to test this hypothesis. The [K+]o did not influence total approximately P consumption (Q approximately P) of the axon. For a [K+]o between 0.5 and 21.6 mM, Q approximately P averaged 52.8 +/- 4.7%/h (n = 44) of the initial [ATP] + [Arg-P]. Unlike total Q approximately P, the ouabain-sensitive portion of Q approximately P was markedly influenced by [K+]o. In 0.5 mM K+o, ouabain poisoning reduced Q approximately P to 8%/h, a result indicating that 85% of the total Q approximately P was ouabain sensitive. For 1.35 mM K+o, the ouabain-sensitive portion was 66%; at 5.4 mM K+o, 45%; and at 13.5 mM K+o, 41%. There was a small but significant increase in the ouabain-sensitive Q approximately P at 21.6 mM K+o, compared with Q approximately P at 5.4 mM K+o. The pattern of effect of [K+]o on Q approximately P was similar to its effect on the electrical power content of the Na+ and K+ electrochemical gradients. In contrast to the generally accepted Na+ flux (JNa)/approximately P stoichiometry of 3, an actual ratio of JNa/approximately P stoichiometry of approximately 33:1 was calculated for the experiments reported here, a result suggesting that cells in a zero-membrane current steady state utilize efficient energy conservation mechanisms that may not operate under non-steady-state conditions.     

Electrical Changes in Pre- and Postsynaptic Axons of the Giant Synapse of Loligo

Potential changes both in pre- and postsynaptic axons were recorded from the giant synapse of squid with intracellular electrodes. Synaptic current was also recorded by a voltage clamp method. Facilitation of postsynaptic potential caused by applying two stimuli several milliseconds apart was accompanied by an increase in the amplitude of the presynaptic action potential. Depression of the postsynaptic potential occurred without changes in the presynaptic action potential. Increase in the concentration of Ca in sea water caused an increase in amplitude of the synaptic current. On the other hand increase in Mg concentration decreased the amplitude of the synaptic current. In these cases no appreciable change in the presynaptic action potential was observed. Extracellularly recorded potential changes of the presynaptic axon showed mainly a positive deflexion at the synaptic region and a negative deflexion in the more proximal part of the presynaptic axon. Mechanism of synaptic transmission is discussed.     

Tetrodotoxin Blockage of Sodium Conductance Increase in Lobster Giant Axons

Previous studies suggested that tetrodotoxin, a poison from the puffer fish, blocks conduction of nerve and muscle through its rather selective inhibition of the sodium-carrying mechanism. In order to verify this hypothesis, observations have been made of sodium and potassium currents in the lobster giant axons treated with tetrodotoxin by means of the sucrose-gap voltage-clamp technique. Tetrodotoxin at concentrations of 1 x 10^-7 to 5 x 10^-9 gm/ml blocked the action potential but had no effect on the resting potential. Partial or complete recovery might have occurred on washing with normal medium. The increase in sodium conductance normally occurring upon depolarization was very effectively suppressed when the action potential was blocked after tetrodotoxin, while the delayed increase in potassium conductance underwent no change. It is concluded that tetrodotoxin, at very low concentrations, blocks the action potential production through its selective inhibition of the sodium-carrying mechanism while keeping the potassium-carrying mechanism intact.     

Removal of Potassium Negative Resistance in Perfused Squid Giant Axons

Squid giant axons, internally and externally perfused with solutions having potassium as the only cation, exhibit an approximately linear steady-state current-voltage relation. When small amounts of calcium and magnesium are present in the external potassium solution, the current-voltage curve is markedly nonlinear, exhibiting the rectification and negative resistance which have been observed for intact axons in isosmotic potassium solutions. The effects of perfusion and removal of external divalent cations are interpreted in terms of two components of current, a linear component and a nonlinear time-varying component. The former is increased and the latter diminished by the removal of the external divalent cations.     

Gap Junction Channels and Cardiac Impulse Propagation

The role of gap junction channels on cardiac impulse propagation is complex. This review focuses on the differential expression of connexins in the heart and the biophysical properties of gap junction channels under normal and disease conditions. Structural determinants of impulse propagation have been gained from biochemical and immunocytochemical studies performed on tissue extracts and intact cardiac tissue. These have defined the distinctive connexin coexpression patterns and relative levels in different cardiac tissues. Functional determinants of impulse propagation have emerged from electrophysiological experiments carried out on cell pairs. The static properties (channel number and conductance) limit the current flow between adjacent cardiomyocytes and thus set the basic conduction velocity. The dynamic properties (voltage-sensitive gating and kinetics of channels) are responsible for a modulation of the conduction velocity during propagated action potentials. The effect is moderate and depends on the type of Cx and channel. For homomeric-homotypic channels, the influence is small to medium; for homomeric-heterotypic channels, it is medium to strong. Since no data are currently available on heteromeric channels, their influence on impulse propagation is speculative. The modulation by gap junction channels is most prominent in tissues at the boundaries between cardiac tissues such as sinoatrial node-atrial muscle, atrioventricular node-His bundle, His bundle-bundle branch and Purkinje fibers-ventricular muscle. The data predict facilitation of orthodromic propagation.     

Interaction of Tetraethylammonium Ion Derivatives with the Potassium Channels of Giant Axons

A number of compounds related to TEA⁺ (tetraethylammoniumion) were injected into squid axons and their effects on g_K (the potassium conductance) were determined. In most of these ions a quaternary nitrogen is surrounded by three ethyl groups and a fourth group that is very hydrophobic. Several of the ions cause inactivation of g_K, a type of ionic gating that is not normally seen in squid axon; i.e., after depolarization g_K increases and then spontaneously decreases to a small fraction of its peak value even though the depolarization is maintained. Observations on the mechanism of this gating show that (a) QA (quaternary ammonium) ions only enter K⁺ channels that have open activation gates (the normal permeability gates). (b) The activation gates of QA-occluded channels do not close readily. (c) Hyperpolarization helps to clear QA ions from the channels. (d) Raising the external K⁺ concentration also helps to clear QA ions from the channels. Observations (c) and (d) strongly suggest that K⁺ ions traverse the membrane by way of pores, and they cannot be explained by the usual type of carrier model. The data suggest that a K⁺ pore has two distinct parts: a wide inner mouth that can accept a hydrated K⁺ ion or a TEA⁺-like ion, and a narrower portion that can accept a dehydrated or partially dehydrated K⁺ ion, but not TEA⁺.     

Bilaterally Symmetric Populations of Chicken dI1 (Commissural) Axons Cross the Floor Plate Independently of Each Other

Axons use temporal and directional guidance cues at intermediate targets to set the rate and direction of growth towards their synaptic targets. Our recent studies have shown that disrupting the temporal guidance process, by unilaterally accelerating the rate at which spinal dI1 (commissural) axons grow, resulted in turning errors both in the ventral spinal cord and after crossing the floor plate. Here we investigate a mechanistic explanation for these defects: the accelerated dI1 axons arrive in the ventral spinal cord before necessary fasciculation cues from incoming dI1 axons from the opposite side of the spinal cord. The identification of such an interaction would support a model of selective fasciculation whereby the pioneering dI1 axons serve as guides for the processes of the bilaterally symmetrical population of dI1 neurons. To test this model, we first developed the ability to “double” in ovo electroporate the embryonic chicken spinal cord to independently manipulate the rate of growth of the two bilateral populations of dI1 axons. Second, we examined the requirement for a putative bilateral interaction by unilaterally ablating the dI1 population in cultured explants of chicken embryonic spinal cord. Surprisingly, we find no evidence for a bilateral dI1 axon interaction, rather dI1 axons appear to project independently of each other.     

Ndfip Proteins Target Robo Receptors for Degradation and Allow Commissural Axons to Cross the Midline in the Developing Spinal Cord

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Current- and Voltage-Clamped Studies on Myxicola Giant Axons : Effect of tetrodotoxin

A new dissection procedure for preparing Myxicola giant axons for observation under voltage clamp is described. Preparation time is generally 40–45 min. 65–70% of the preparations attempted may be brought through the entire procedure, including insertion of the long internal electrode, and support an initial action potential amplitude of 100 mv or greater. Mean values for axon diameter, resting membrane potential, action potential amplitude, maximum peak inward transient current, and resting membrane resistance are 560 µ, —66.5 mv, 112 mv, 0.87 ma/cm² and 1.22 KΩ cm ² respectively. Cut branches do not seem to be a problem in this preparation. Behavior under voltage clamp is reasonably stable over several hours. Reductions in maximum inward transient current of 10% and in steady-state current of 5–10% are expected in the absence of any particular treatment. Tetrodotoxin blocks the action potential and both the inward and outward transient current, but has no effect on either the resting membrane potential or the steady-state current. This selective action of tetrodotoxin on the transient current is taken as an indication that this current component is probably carried by Na.     

Effect of ATP on the Calcium Efflux in Dialyzed Squid Giant Axons

Dialysis perfusion technique makes it possible to control the internal composition of squid giant axons. Calcium efflux has been studied in the presence and in the virtual absence (<5 µM) of ATP. The mean calcium efflux from axons dialyzed with 0.3 µM ionized calcium, [ATP]_i > 1,000 µM, and bathed in artificial seawater (ASW) was 0.24 ± 0.02 pmol·cm^-2·s^-1 (P/CS) (n = 8) at 22°C. With [ATP]_i < 5 µM the mean efflux was 0.11 ± 0.01 P/CS (n = 15). The curve relating calcium efflux to [ATP]_i shows a constant residual calcium efflux in the range of 1–100 µM [ATP]_i. An increase of the calcium efflux is observed when [ATP]_i is >100 µM and saturates at [ATP]_i > 1,000 µM. The magnitude of the ATP-dependent fraction of the calcium efflux varies with external concentrations of Na⁺, Ca⁺⁺, and Mg⁺⁺. These results suggest that internal ATP changes the affinity of the calcium transport system for external cations.     

The Action of Tetrodotoxin on Electrogenic Components of Squid Giant Axons

Voltage clamp measurements on squid giant axons show that externally applied puffer fish poison, tetrodotoxin, eliminates only the initial inward current component of spike electrogenesis and does not affect the subsequent outward current. The selective effect on Na activation, which is reversible, confirms the view that the movements of Na and K during spike electrogenesis occur at structurally different sites on the membrane. Spike electrogenesis is also blocked when tetrodotoxin is injected into the axon, but the interior of the membrane appears to be somewhat less sensitive to the poison. Differences in reactivity of various electrogenic membrane components to tetrodotoxin are discussed as signifying differences in chemical structures.     

The pH Dependency of Relative Ion Permeabilities in the Crayfish Giant Axon

The dependence of the membrane potential on potassium, chloride, and sodium ions, was determined at the pH's of 6.0, 7.5, and 9.0 for the resting and depolarized crayfish ventral nerve cord giant axon. In normal saline (external potassium = 5.4 mM), the dependence of the membrane potential on the external potassium ions decreased with lowered pH while that for chloride increased. In contrast, in the potassium depolarized axon (external potassium = 25 mM), the dependence of the membrane potential on external potassium was minimum around pH 7.5 and increased in either more acidic or basic pH. In addition, the dependence of the membrane potential on external chloride in the depolarized axon was maximum at pH 7.5 and decreased in either more acidic or basic pH. The sodium dependency of the membrane potential was small and relatively unaffected by pH or depolarization. The data are interpreted as indicating a reversible surface membrane protein-phospholipid conformation change which occurs in the transition from the resting to the depolarized axon.