Garfish Olfactory Nerve Research Articles

Line Tensions Caused by Voltage-Induced Axon Swelling Propel Non-Myelinated Action Potentials

Voltage-induced swelling of action potential (AP) accompanies shrinkage along the tubular axon. Longitudinal tensions appear at both boundaries of AP of nerve axon, and the line tension at the front boundary has been shown to be greater than that at the rear boundary. The difference of tensions has been certified by previously observed diphasic heat behavior of AP. The propagation velocity of AP is determined by the balance between the difference of tensions and the hydrodynamic resistance of AP by ambient water. The bilayer lipid membrane is liberated from the strong electrostriction of resting potential to depolarized AP. An elastic model of both boundaries of AP has been proposed, which describes the relation between diphasic heat behavior of AP and generated line tensions. The propagation velocity of HEK-293 cells, squid giant axon (SGA), and garfish olfactory nerve (GON) has been analyzed and reliably explained by the proposed line tension model. The energy cost of propelling APs of SGA and GON has been shown to be greater than that of the electric energy.

Electrothermal Equivalent Three-Dimensional Finite-Element Model of a Single Neuron.

We propose a novel approach for modelling the interdependence of electrical and mechanical phenomena in nervous cells, by using electrothermal equivalences in finite element (FE) analysis so that existing thermomechanical tools can be applied. First, the equivalence between electrical and thermal properties of the nerve materials is established, and results of a pure heat conduction analysis performed in Abaqus CAE Software 6.13-3 are validated with analytical solutions for a range of steady and transient conditions. This validation includes the definition of equivalent active membrane properties that enable prediction of the action potential. Then, as a step toward fully coupled models, electromechanical coupling is implemented through the definition of equivalent piezoelectric properties of the nerve membrane using the thermal expansion coefficient, enabling prediction of the mechanical response of the nerve to the action potential. Results of the coupled electromechanical model are validated with previously published experimental results of deformation for squid giant axon, crab nerve fibre, and garfish olfactory nerve fibre. A simplified coupled electromechanical modelling approach is established through an electrothermal equivalent FE model of a nervous cell for biomedical applications. One of the key findings is the mechanical characterization of the neural activity in a coupled electromechanical domain, which provides insights into the electromechanical behaviour of nervous cells, such as thinning of the membrane. This is a first step toward modelling three-dimensional electromechanical alteration induced by trauma at nerve bundle, tissue, and organ levels.

Mechanical surface waves accompany action potential propagation.

Many diverse studies have shown that a mechanical displacement of the axonal membrane accompanies the electrical pulse defining the action potential (AP). We present a model for these mechanical displacements as arising from the driving of surface wave modes in which potential energy is stored in elastic properties of the neuronal membrane and cytoskeleton while kinetic energy is carried by the axoplasmic fluid. In our model, these surface waves are driven by the travelling wave of electrical depolarization characterizing the AP, altering compressive electrostatic forces across the membrane. This driving leads to co-propagating mechanical displacements, which we term Action Waves (AWs). Our model allows us to estimate the shape of the AW that accompanies any travelling wave of voltage, making predictions that are in agreement with results from several experimental systems. Our model can serve as a framework for understanding the physical origins and possible functional roles of these AWs.

Mechanical Surface Waves Accompany Action Potential Propagation

Many diverse studies have shown that a mechanical displacement of the axonal membrane accompanies the electrical pulse defining the action potential (AP). We present a model for these mechanical displacements as arising from the driving of surface wave modes in which potential energy is stored in elastic properties of the neuronal membrane and cytoskeleton while kinetic energy is carried by the axoplasmic fluid. In our model, these surface waves are driven by the travelling wave of electrical depolarization characterizing the AP, altering compressive electrostatic forces across the membrane. This driving leads to co-propagating mechanical displacements, which we term Action Waves (AWs). Our model allows us to estimate the shape of the AW that accompanies any travelling wave of voltage, making predictions that are in agreement with results from several experimental systems. Our model can serve as a framework for understanding the physical origins and possible functional roles of these AWs. Action potentials in neurons are accompanied by a mechanical displacement of the axonal membrane. Here, the authors present a model to describe these as surface waves which are driven by changes in charge separation, and compare their model to published experimental results from squid giant axons and garfish olfactory nerve bundles.

Characterization of phospholipase A 2 and acyltransferase activities in squid ( loligo pealei) axoplasm: comparison with enzyme activities in other neural tissues, axolemma and axoplasmic subfractions

Phospholipase A 2 and acyltransferase were assayed and characterized in pure axoplasm and neural tissues of squid. Intracellular phospholipase A 2 activity was highest in giant fiber lobe and axoplasm, followed by homogenates from retinal fibers, optic lobe and fin nerve. In most preparations, exogenous calcium (5 mM) caused a slight stimulation of activity. EGTA (2 mM) was somewhat inhibitory, indicating that low levels of endogenous calcium may be required for optimum activity. Phospholipase A 2 was inhibited by 0.1 mM p-bromophenacylbromide, and was completely inactivated following heating. The level of acylCoA: lysophosphatidylcholine acyltransferase activity was higher in axoplasm and giant fiber lobe than in other neural tissues of the squid. K m (apparent) and V max (apparent) for oleoyl-CoA and lysophosphatidylcholine were quite similar for axoplasm and giant fiber lobe enzyme preparations. Acyltransferase activity was inactivated by heat treatment, and greatly inhibited by 0.2 mM p-chloromercuribenzoate, and to a lesser extent by 20 mM N-ethylmaleimide. Phospholipase A 2 activity was present in fractions enriched in axolemmal membranes (separated from squid retinal fibers and garfish olfactory nerve) from both tissues, and it was also highly concentrated in vesicles derived from squid axoplasm. In all three preparations, phospholipase A 2 activity was stimulated by Ca ++ (5 mM) and inhibited by EGTA (2 mM). In addition, axoplasmic cytosol (114,000 g supernatant) retained a substantial portion of a Ca ++-independent phospholipase A 2, active in the presence of 2 mM EGTA. Acyltransferase activity was present at high content in both axolemma membrane rich fractions, and among subaxoplasmic fractions and axoplasmic vesicles.

Rapid structural chances in nerve fibers evoked by electric current pulses

Using the garfish olfactory nerve, the time-courses of lateral expansion (swelling) and birefringence changes in nerve fibers have been examined at the site of application of electric current pulses. The effects of various chemical agents on these non-electrical signs of rapid structural changes in the nerve fibers have been examined. These studies have shown that a pulse of outwardly-directed current through the superficial layer evokes a fast increase, followed by a slow, gradual increase, in the water-content of the nerve fibers. The full significance of these findings in studies of the process of nerve excitation is discussed.

Rapid mechanical and thermal changes in the garfish olfactory nerve associated with a propagated impulse

Mechanical and thermal changes associated with a propagated nerve impulse were determined using the garfish olfactory nerve. Production of an action potential was found to be accompanied by swelling of the nerve fibers. The swelling starts nearly at the onset of the action potential and reaches its peak at the peak of the action potential. There is a decrease in the length of the fibers while an impulse travels along the fibers. The time-course of the initial heat was determined at room temperature using heat-sensors with a response-time of 2-3 ms. Positive heat production was found to start and reach its peak nearly simultaneously with the action potential. The rise in temperature of the nerve was shown to be 23 (+/- 4) mu degrees C. In the range between 10 degrees and 20 degrees C, the temperature coefficient of heat production is negative, primarily due to prolongation of the period of positive heat production at low temperatures. The amount of heat absorbed during the negative phase varies widely between 45 and 85% of the heat evolved during the positive phase. It is suggested that both mechanical and thermal changes in the nerve fibers are associated with the release and re-binding of Ca-ions in the nerve associated with action potential production.

Survival and subsequent regeneration of olfactory neurons after a distal axonal lesion.

If an axonal lesion is made close enough to the cell body, injured olfactory neurons degenerate and are replaced by new nerve cells arising from undifferentiated mucosal basal cells. Therefore, under these conditions neural regeneration occurs through a process similar to neuronal development. The use of the long garfish olfactory nerve has revealed that neuronal death is not an inevitable consequence of an axonal injury and that the extent of cell death depends on the distance between the site of injury and the perikaryon. A lesion located up to 40 mm from the cell body induces the death of all mature neurons. Between 60 and 100 mm an increasing proportion of neurons survive the injury (from 60 to more than 90%) and are able to regenerate their distal segment. During regeneration, two populations of growing axons have been characterized. The fastest growing fibres (5.8 mm per day) correspond to the small population of neurons which were already growing at the time of the lesion and are able to survive an injury at any distance along the nerve. The majority of the regenerating fibres grow at 0.8 mm per day and corresponds to the damaged mature neurons. Elongation velocities were not affected by the distance from injury to cell body or by the mode of neural repair (development or regeneration). The initial delay between the injury and the beginning of elongation increases linearly with distance at a rate of 1 day cm-1 and is independent of the elongation velocity of the growing neurons. This indicates that the mechanism responsible for the beginning of axonal growth is initiated at or near the cell body and involves the entire axonal stump and not only the area surrounding the crush site. The ability of nerve cells to survive an injury may depend on the length of the axonal stump remaining attached to the cell body and on the level of protein synthesis at the time of the crush. From preliminary results it can be hypothesized that the length of the initial delay is determined by a reorganization of the cytoskeletal elements in the proximal axonal stump.

Raman spectroscopy of nerve fibers. A study of membrane lipids under steady state conditions

The molecular structures of different nerve fibers kept in good physiological conditions were studied by laser Raman spectroscopy. For myelinated nerves like the rat sciatic nerve, the Raman spectrum is dominated by bands due to the lipid component of the myelin sheath. The temperature dependence of these bands does not reveal any thermotropic phase transition between 0 and 40 degrees C. There is, however, with temperature, a linear increase in the intermolecular disorder that is accompanied by an increase in the number of gauche bonds of the phospholipid acyl chains. For unmyelinated nerves such as the lobster leg nerve, the C-H stretching region of the Raman spectrum is covered by bands arising from the protein component of the axoplasm. However, for the garfish olfactory nerve that has a high density of excitable membranes, phospholipid bands are observed and can be used as intrinsic structural probes of the excitable membranes. The relative intensity of these bands is also temperature dependent.

Influence of temperature on slow flow in populations of regenerating axons with different elongation velocities.

The slow intra-axonal transport mechanisms may be subdivided into 4 elementary processes: (a) protein synthesis; (b) release of the slow material from the cell body; (c) transport of the molecules along the axon; and (d) deposition of the material from the moving phase into the immobile axonal constituents. These 4 components of the slow flow mechanisms are differently affected by temperature and react differently to axonal regeneration. They have been studied at temperatures ranging from 10 to 35 degrees C, in 3 different populations of regenerating C-fibers of the garfish olfactory nerve. These 3 classes of axons have very different elongation properties as determined by elongation rates ranging from a minimum of 0.3 mm/day at 14 degrees C to a maximum of 12.4 mm/day at 35 degrees C. From this study several properties of the slow intra-axonal transport mechanism can be underlined: (a) in regenerating nerves the rate of slow flow is multiplied by a factor of 3.3 as compared to the rate characteristic of an intact nerve. A similar increase was measured in every regenerating phase regardless of the specific elongation velocity of the growing axons. This accelerated rate of slow flow has been measured previously in degenerating axons detached from their perikarya: therefore the rate increase appears to be triggered by the injury rather than the needs of the neuron; (b) protein synthesis represents one of the main steps controlling axonal elongation. Neurons with the most active level of protein synthesis grow with the highest elongation velocity; (c) the somal releasing times decrease exponentially with temperature but are identical to the values measured in intact neurons; (d) finally the height of the slow flow peak decreases exponentially as a function of the distance, as seen previously in intact nerves, but a significantly larger amount of radioactivity reaches the end of the nerve. It can be concluded that slow flow is intimately linked with the regeneration process. The rate of slow flow and more specifically of Scb represents the upper limit that can be reached by the elongation velocities of regenerating fibers when all the growth conditions are maximal. When the regenerating conditions are not at their peak, the axonal elongation proceeds at velocities which can be very significantly slower than the rate of slow flow and might be determined by the level of protein synthesis in the perikarya. Axonal elongation, however, can never proceed at a rate faster than slow flow.

Proximodistal degeneration of C-fibers detached from their perikarya.

Degeneration was followed in the garfish olfactory nerve after removal of the mucosa containing the cell bodies. Degeneration, as measured by a decrease in the weight of consecutive 3-mm nerve segments, spreads at constant velocity from the site of injury toward the synaptic area. The proximodistal degeneration is temperature dependent and progresses from 0.3 mm/d at 10 degrees C to 13.0 mm/d at 35 degrees C. Between 14 and 35 degrees C, the velocity increases linearly with temperature. At all the temperatures investigated, these proximodistal degeneration velocities are identical to the rates of slow intraaxonal flow measured in axons detached from their cell bodies, or to the rates measured in regenerating fibers, and, except at 10 degrees C, are 3.3 times faster than the rate of slow flow in intact nerves. These results were confirmed by light and electron microscopy. We hypothesize that the collapse and subsequent degeneration of the axons is the result of a proximodistal depletion of cytoskeletal elements no longer provided by the cell body to the axon by slow intraaxonal flow. A significant number of axons disappeared rapidly from the nerve before the arrival of the slow degenerative wave. From studies by other groups, this rapid degeneration may be the result of a lack of rapidly transported, mainly membranous components.

Characterization of axonally transported glycoproteins in regenerating garfish olfactory nerve.

This study examined changes in composition and concanavalin A (Con A) binding of axonally transported glycoproteins and their pronase-generated glycopeptides in regenerating garfish olfactory nerve. A previous study had demonstrated a regeneration-related increase in the proportion of [3H]glucosamine label in lower-molecular-weight Con A-binding glycopeptides derived from transported glycoproteins. Further analysis of carbohydrate composition shows that these molecules resemble mannose-rich oligosaccharides in composition and are increased in absolute amount in regenerating nerve. Subcellular analysis shows that the Con A-binding glycopeptides are enriched in membrane subfractions, particularly in a high-density fraction that morphologically resembles isolated cell surface coat. Regeneration-related changes in intact axonally transported glycoproteins were also detected. Sodium dodecyl sulfate gel electrophoresis of transport-labeled glycoproteins disclosed growth-correlated increases in radioactivity associated with 180-200K, 105-115K, and 80-90K components, while a 150-160K molecular weight class of glycoproteins was diminished in relative labeling. Intact glycoproteins displaying an affinity for Con A were also augmented in regenerating nerve, the increases occurring primarily in molecules in the 50-140K range.

Axonal transport of glycoproteins in regenerating olfactory nerve: enhanced glycopeptide concanavalin A-binding

The size and concanavalin A-binding characteristics of glycopeptides derived from axonally transported glycoproteins were studied in regenerating garfish olfactory nerve. A regeneration related increase was observed in the proportion of total glycopeptide radioactivity associated with the lower molecular weight, dialyzable fraction. There was also a 3–5-fold increase in the axonal transport of low molecular weight concanavalin A-binding glycopeptides in regenerating nerve. These results suggest a shift to an enhanced synthesis and axonal transport of glycoproteins containing low molecular weight, concanavalin A-binding carbohydrate chains in regenerating nerve.

Partial purification and characterization of (Na+ + K+)-ATPase from garfish olfactory nerve axon plasma membrane.

The (Na+ + K+)-ATPase of garfish olfactory nerve axon plasma membrane was purified about sixfold by treatment of the membrane with sodium dodecyl sulfate followed by sucrose density gradient centrifugation. The estimated molecular weights of the two major polypeptide components of the enzyme preparation on sodium dodecyl sulfate gels were 110,000 and 42,000 daltons, which were different from those of the corresponding peptides of rabbit kidney (Na+ + K+)-ATPase. No carbohydrate was detected in the 42,000-dalton component either by the periodic acid-Schiff reagent or by the more sensitive concanavalin A-peroxidase staining procedure. The molecular properties of the garfish (Na+ + K+)-ATPase, such as the Km for ATP, pH optimum, energies of activation, Na and K ion dependence and vanadium inhibition, were, however, similar to those of the kidney enzyme. The partially purified garfish (Na+ + K+)-ATPase was reconstituted into phospholipid vesicles by a freeze-thaw-sonication procedure. The reconstituted enzyme was found to catalyze a time and ATP dependent 22Na+ transport. The ratio of 22Na+ pumped to ATP hydrolyzed was about 1; under the same reconstitution and assay conditions, eel electroplax (Na+ + K+)-ATPase, however, gave a 22Na+ pumped to ATP hydrolyzed ratio of nearly 3.

Rate of movement and composition of rapidly transported proteins in regenerating olfactory nerve.

In a previous study, three successive groups of regenerative fibers, growing initially at 5.8, 2.1, and 0.8 mm/day, were observed in the regenerating garfish olfactory nerve. In the present study, fast axonal transport in the most rapidly regenerating axons (phase I and II) has been examined. Rapid transport in phase I fibers occurs at a velocity of 208 +/- 9 mm/day at 23 degrees, a rate identical to that measured in intact nerves. This first phase of regenerating fibers represents only 3 to 5% of the original axonal population, but each fiber appears to contain 6 to 16 times more transported radioactivity than an axon in an intact nerve. Subcellular distribution of rapidly moving material in phase I and II fibers was closely related to the distribution obtained in intact nerves. Small but significant differences indicate a shift of the transported radioactivity from a heavier to a light axonal membranous fraction. This shift might be characteristic of the immature membrane of a growing axon. The polypeptide distribution of transported radioactivity was also very similar to that of a normal nerve, with most of the radioactivity associated with high-molecular-weight polypeptides.

Study of regeneration in the garfish olfactory nerve.

Previous studies of the olfactory nerve, mainly in higher vertebrates, have indicated that axonal injury causes total degeneration of the mature neurons, followed by replacement of new neuronal cells arising from undifferentiated mucosal cells. A similar regeneration process was confirmed in the garfish olfactory system. Regeneration of the nerve, crushed 1.5 cm from the cell bodies, is found to produce three distinct populations of regenerating fibers. The first traverses the crush site 1 wk postoperative and progresses along the nerve at a rate of 5.8 +/- 0.3 mm/d for the leading fibers of the group. The second group of fibers traverses the crush site after 2 wk postcrush and advances at a rate of 2.1 +/- 0.1 mm/d for the leading fibers. The rate of growth of this group of fibers remains constant for 60 d but subsequently falls to 1.6 +/- 0.2 for the leading population of fibers. The leading fibers in the third group of regenerating axons traverse the crush site after 4 wk and advance at a constant rate of 0.8 +/- 0.2 mm/d. The multiple populations of regenerating fibers with differing rates of growth are discussed in the context of precursor cell maturity at the time of nerve injury and possible conditioning effects of the lesion upon these cells. Electron microscopy indicates that the number of axons decreases extensively after crush. The first two phases of regenerating axons represent a total of between 6 and 10% of the original axonal population and are typically characterized by small fascicles of axons surrounded by Schwann cells and large amounts of collagenous material. The third phase of fibers represents between 50 and 70% of the original axonal population.

Polypeptide components of the (Na+K)-ATPase of garfish olfactory nerve axon plasma membrane

A partially purified (Na+K)-ATPase preparation was obtained from garfish olfactory nerve axon plasma membrane. On SDS polyacrylamide gel electrophoresis, the enzyme preparation showed the presence of two major polypeptides. One of the major polypeptides was identified by phosphorylation with γ [ 32P]-ATP as the large catalytic subunit of (Na+K)-ATPase. This polypeptide had a molecular weight of about 110, 000 and was separable from the catalytic subunit of rabbit kidney (Na+K)-ATPase when coelectrophoresed. The other major polypeptide had a molecular weight of about 42,000 and was not positive to either PAS or concanavalin A-peroxidase stain indicating that the small subunit of garfish enzyme had a significantly less carbohydrate content, if present at all, than that for other small subunits analysed.

The initial heat production in garfish olfactory nerve fibres.

A study has been made of the temperature changes associated with the passage of a single impulse in the non-myelinated fibres of the garfish olfactory nerve: and the time course of these temperature changes has been compared with the time course of the electrical events during the action potential. As in other non-myelinated nerves studied the observed temperature changes result from a biphasic initial heat production consisting of a transient evolution of heat (the positive heat) followed by a rapid heat reabsorption (referred to as the negative heat). There is no evidence of any additional phases of initial heat production. At 0 degrees C the measured positive initial heat is 224 mucal/g impulse (937 muJ/g impulse); and the corresponding negative initial heat is 230 mucal/g impulse (962 muJ/g impulse). The residual initial heat is very small, being about -6 mucal/g impulse (-25 muJ/g impulse). In the range 0-10 degrees C there is no significant effect of temperature on the magnitude of either the positive or the negative phases of heat production. The experimental thermal records were analysed to determine the true time course of the temperature changes in the nerve undistorted by the recording system. The time course of the temperature changes does not fit with that of the transmembrane voltage change as represented by the monophasic compound action potential recorded externally from the same point on the nerve. A better fit is obtained if the temperature changes are compared with the square of the voltage change in accordance with the view that the heat derives almost wholly from free energy changes and entropy changes in the membrane capacity. The best fit is obtained if it is assumed that the membrane potential does not discharge to zero during the action potential but that at the peak of the action potential the charge (and hence the p.d.) across the membrane capacity retains about 24% of its resting value.

Axonal transport of glycoproteins in the garfish olfactory nerve: isolation of high molecular weight glycopeptides labeled with [3H]fucose and [3H]glucosamine.

Axonal transport of glycoproteins in the garfish olfactory nerve: isolation of high molecular weight glycopeptides labeled with [3H]fucose and [3H]glucosamine.

The recovery heat production in non-myelinated garfish olfactory nerve fibres.

1. The recovery heat production of the non-myelinated fibres of garfish olfactory nerve has been measured. 2. At about 20 degrees C the total recovery heat was 381 +/- 26 microcal g-1 impulse-1 at a stimulation frequency of 2 sec-1. 3. The time constant of decay of the recovery heat production after a brief period of stimulation was 78.7 +/- 3.1 sec at about 20 degrees C. 4. Changing the temperature (by +/- 5 degress C) had little effect on the total recovery heat produced. 5. However, lowering the temperature reduced both the rate of rise, and the maximum rate of recovery heat production whereas the time constant of decay was increased. Raising the temperature produced corresponding changes in the opposite direction. 6. the recovery heat production measured in the present experiments is consistent with the previously measured oxygen consumption in the same preparation.