INFLUENCE OF TEMPERATURE ON THE VELOCITY AND ON THE ISOTOPE PROFILE OF SLOWLY TRANSPORTED LABELED PROTEINS

Abstract— Slow intra-axonal flow of [³H]leucine labeled proteins has been studied in the garfish olfactory nerve. Because of the homogeneity of the nerve a very well defined peak of slowly transported radioactivity is observed. The velocity of slow flow increases linearly with temperature. Between 14 and 28°C, the rate of the peak apex increases from 0.26 to 1.57 mm/day and the rate of the leading edge of the wavefront from 0.54 to 2.75 mm/day. Extrapolation of the rate-temperature function indicates that slow flow should stop at 11°C. However, a velocity of 0.1 mm/day was determined for experiments conducted at 10°C. Between 15 and 25°C a Q ₁₀ of 3.7 was determined for the peak apex and of 3.3 for the leading edge of the wavefront. The Q₁₀'s are significantly larger than the value of 2.2 found for fast transport (G ross & B eidler , 1975) and support the possibility of at least partial differences between the mechanisms of fast and slow transport. A very small peak was found to migrate in front of the main peak. The positioning of this peak seems to be similar to one found by L asek & H offman (1976) in rat ventral motor neurons.
A temperature dependent exponential decrease of the slow moving peak height was measured and it can be estimated that only 1% of the slowly transported radioactivity reaches the synapses. Most of the slow radioactivity appears to remain in the axon behind the peak. The plateau height was also found to decrease exponentially with time. The rate of disappearance greatly affects the profile determined by the slowly transported labeled proteins along the nerve.     

Degeneration and regeneration of olfactory cells induced by ZnSO4 and other chemicals

The necrotic effect of various salt solutions was tested on the catfish olfactory mucosa. Only zinc cations were able to induce an extensive degeneration of the olfactory cells. Two different modes of irrigation of the mucosa with zinc sulfate were investigated. (1) The olfactory cavity is flushed with the chemical for not more than a few seconds. At concentrations above 30 mM, the resulting damage is very reproducible, largely concentration independent and almost completely specific for the olfactory receptor cells. The non-sensory respiratory cells are unaffected, the sustentacular cells surrounding the receptor cells are affected mainly by a loss of microvilli. The olfactory receptor cells, on the contrary, start to degenerate within a few hours and by day 4 only 20% of the original receptor population remains. Division of the mucosal basal cells increases during days 3 and 4 on and day 6 olfactory receptor cells reach the bare surface of the lamella. After day 7, the receptor population reaches a level of more than 80% of its original value. Because of the absence of sustentacular processes covering the olfactory cell's knobs on day 6, it has been possible to confirm that each of the two types of olfactory receptor cells previously characterized are concentrated on each half of the mucosa. (2) The salt is maintained in contact with the tissue for several days. After this treatment most of the lamellae are irreversibly destroyed, some regeneration occurs in limited areas of the mucosa. In these small areas, indifferent respiratory cells reappear first between 20 and 35 days. It is only when the structure of the olfactory tissue is completely reorganized that the new receptor cells reappear between days 45 and 55. Regeneration is not completed before 60–65 days.     

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.     

Axonal Transport in the Garfish Optic Nerve: Comparison with the Olfactory System

Fast and slow axonal transports were studied in the optic nerve of the garfish and compared with previous studies on the olfactory nerve. The composition of fast-transport proteins was very similar in the two nerves. Although the velocity of fast transport was slightly lower in the optic nerve, there was a linear increase in velocity with temperature in both nerves. As in the olfactory nerve, only a single wave of slow-transport protein radioactivity moves along the nerve. The velocity of slow transport also increased linearly with temperature, but the coefficient was less than in the olfactory system. The composition of slow transport in the optic nerve was significantly different from that in the olfactory nerve, a finding reflecting the different cytoskeletal constituents of the two types of axons. The slow wave could be differentiated into several subcomponents, with the order of velocities being a 105-kilodalton protein and actin greater than tubulins and clathrin greater than fodrin much greater than neurofilaments. It can be concluded that the temperature dependence of fast and slow axonal transport in different nerves reflects the influence of temperature on the individual polypeptides constituting the various transport phases. The garfish optic nerve preparation may be advantageous for studies of axonal transport in retinal ganglion cell axons, because its great length avoids the complications of having to study transport in the optic tract or in material accumulating at the tectum.     

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.     

Influence of a detergent on the catfish olfactory mucosa

The olfactory mucosa of the catfish (Ictulurus punctatus) has been briefly exposed to various concentrations of the non-ionic detergent Triton X-100. At high concentrations (1–4%) the upper layer of cells constituting the sensory and non-sensory areas of the lamellae is extensively damaged and new receptor cells do not appear in significant number before 2 months after treatment. Respiratory cells regenerate first followed by sustentacular and olfactory receptors. The regenerative process is very similar to that described previously after prolonged contact between the mucosa and ZnSO₄. Low detergent concentrations 0.03 – 0.1% affect only the sensory area. Olfactory and sustentacular microvilli and cilia, are immediately severed by the chemical. Regeneration occurs within the next 4 days. The cellular membranes appear also to be affected. From anatomical, electrophysiological and biochemical studies both in vivo and in vitro, it can be hypothesized that receptors involved in the transduction process are solubilized by the detergent but reappear at a level corresponding to 50–60% of their original activity within 2 h.Proteins, having an amino acid binding effectiveness correlated to the amino acid electrophysiological activities measured in vivo, can be isolated from the solubilized material. Further studies will be necessary to confirm that some of these molecules are involved in the olfactory transduction mechanism.     

Receptor cells of the catfish olfactory mucosa

In the present study, it has been demonstrated that catfisholfactory cells, having knobs characterized by a crown of longmicrovilli, are bipolar neurons. These cells, found almost exclusivelyon the dorso-medial half of the sensory area of the olfactorylamellae, most probably function as olfactory receptor cells.They appear to be different from microvillous tufted cells describedin several other fishes as well as in the catfish.     

SDS GEL ELECTROPHORESIS OF RAPIDLY TRANSPORTED PROTEINS IN GARFISH OLFACTORY NERVE

Abstract— Proteins undergoing rapid axonal transport in the garfish olfactory nerve were examined by sodium dodecyl sulphate gel electrophoresis. The distribution of polypeptides and the extent of their labeling by transported molecules was determined in several nerve subfractions including: total particulate, total membrane, mitochondrial and two membrane subfractions rich in axolemma. The polypeptide composition of the various fractions was found to be relatively similar, with each showing a major protein with an estimated MW of 58,000. Specific differences in the concentrations of certain proteins were noted between fractions, including differences between the lower and higher density axolemma rich subfractions. Axonally transported radioactivity was predominantly localized among high molecular weight proteins, with all fractions, except mitochondrial pellet, displaying a major peak of radioactivity centered at 126,000-MW. Several major proteins including the 58,000-MW band were labeled by rapid transport to a much smaller extent. Certain labeled peaks were found to be concentrated in individual fractions, particularly a polypeptide (MW 35,000) more predominantly found in the lower density axolemma rich fraction.
Systemic labeling of the nerve is found to give a general distribution of radioactivity on gels, which is clearly different from the pattern obtained after axonal transport labeling.     

Isolation and Characterization of the Olfactory Epithelial Cells of the Catfish

A preparation enriched in olfactory receptor cells has beenobtained from the olfactory mucosa of the catfish (Ictaluruspunctatus). The tissue was treated successively with trypsin,DNase, trypsin inhibitor, EDTA in Ca^{+ +} , Mg^{+ +} free mediumaccording to a method derived from that of Cohen, et al.⁽¹⁾.After mechanical disruption of the isolated olfactory lamellae,the cells were isolated by centrifugation on a Ficoll gradient.Each type of cell was morphologically identified by comparingin situ and in vitro preparations by SEM. Small round cellswere collected on 10% Ficoll. The nature of these cells is notknown but part of them are certainly basal cells which havebeen shown⁽²⁾ to be the precursors of the constantly regeneratingolfactory neurons. Respiratory cells settled mainly on 20% Ficoll.A fraction containing 60% sustentacular cells was collectedon 33% Ficoll. Olfactory cells characterized by an axon, a dendriteand several cilia, were found on 37% Ficoll. This fraction alsocontains up to 40% sustentacular cells. A yield of 20% was measuredfor olfactory cell isolation. Vital staining and ability tosynthesize RNA indicate a viability of the final preparationof 70% to 80%. Further identification of the cells was performedby measuring the binding activity of a series of amino acidsto a preparation enriched in olfactory cells. A good correlationwas determined between the extent of the binding and the reportedelectrophysiological activities of these amino acids recordedin vivo. Although the final olfactory cell suspension is notpure, it constitutes the first step in the study of the olfactoryreceptor sites.