Vesicular fast axonal transport rates in young and old rat axons.

An isolated sciatic nerve preparation was used to measure the transport rates of more than 18,000 vesicles in 72 axons from young (3-4 months of age) and old (24-26 months of age) rats from two strains (Harlan Sprague-Dawley and Fisher-344). Average anterograde and retrograde vesicle transport rates were significantly slower in the older animals. The amount of slowing of anterograde vesicles was twice as great as the slowing of retrograde vesicles. Age-related slowing of vesicle transport was inversely proportional to vesicle speed, with the result that transport of the slowest and largest vesicles may essentially be blocked in older axons. One possible explanation for these data is that long-lived axonal cytoskeletal proteins are subject to age-related changes that impede vesicle transport.

A crustacean neuronal cytoskeletal protein with characteristics of neurofilaments and microtubule-associated proteins.

The purpose of this study was to characterize further a unique protein that is a component of the cytoskeleton of crayfish neurons. This protein, referred to as P600, is unique because it is unusually large (Mr greater than 600 kD), and because it has characteristics in common with both mammalian microtubule-associated proteins and neurofilaments. Immunohistochemical techniques have shown that P600 colocalizes with microtubules and is a component of the fibrous side-arms that extend from microtubules (Weaver and Viancour, Brain Res. 544:49, 1991). We have developed a method for obtaining purified P600 by using gel filtration techniques. When viewed by negative staining electron microscopy, P600 obtained by that method produced 11 nm-wide beaded filaments. The number of filaments was strictly related to the P600 concentration in a column fraction. A small amount of P600 consistently copurified with taxol-stabilized microtubules. The proportion copurifying with microtubules was increased by using apyrase to deplete ATP, or by using a nonhydrolyzable ATP analogue to compete with ATP. Immunogold labeling localized P600 near the ends of a subset of the fibrous side-arms extending from endogenous axonal microtubules. Several polyclonal antibodies against mammalian microtubule-associated proteins were tested for P600 labeling on immunoblots, and positive labeling was obtained with an antiserum directed against a region of microtubule-associated protein 1B that has microtubule binding activity. Epitope homology between P600, mammalian microtubule-associated protein 1B, and the mammalian mid-molecular weight neurofilament subunit is discussed in the context of possible evolutionary relationships among these cytoskeletal proteins.

The crayfish neuronal cytoskeleton: an investigation of proteins having neurofilament-like immunoreactivity.

We have evaluated the possibility that proteins similar to mammalian neurofilament proteins (NFPs) are present in crustacean neurons. A panel of monoclonal antibodies (mAbs), raised against mammalian NFP, was used to identify candidate proteins. The degree to which these proteins are similar to mammalian NFPs was further evaluated using the following criteria: tissue specificity, recognition by the neurofilament-specific Bodian silver strain, recognition by the intermediate filament-specific Pruss mAb, and insolubility following detergent-extraction. Three candidate polypeptides were identified by mAb screening: a very high molecular weight polypeptide (Mr greater than 300 kDa), a 40 kDa polypeptide, and a group of 4 bands at Mr = 66-84 kDa. Although all of these polypeptides were recognized by one or more anti-NFP mAb, not one of them was found exclusively in neuronal tissue, not one was stained by the NFP-specific Bodian method, and all were soluble under conditions in which mammalian NFPs are insoluble. As a result of this thorough evaluation, we conclude that crayfish neurons do not contain neurofilament-like proteins. Although not closely related to mammalian neurofilaments, the very high molecular weight crayfish polypeptide which was strongly labeled by a commercially available anti-NF-M mAb (clone NN18) during the mAb screening, may be a novel cytoskeletal protein. The evidence for this conclusion comes from immunocytochemical labeling experiments. Indirect immunofluorescence labeling of this protein differentially labeled axons, such that labeling intensity of specific axons was proportional to the relative concentration of cytoskeletal organelles in those axons. Labeling of neuronal cell bodies delineated a fibrous network throughout the cytoplasm, and intensely labeled microtubule-rich regions of cytoplasm which are characteristic of larger neuronal somata. Immunogold labeling and electron microscopic analysis of the distribution of this protein revealed that the NN18-clone antibody bound to an antigen located on microtubule side-arms.

Organelle flux in intact and transected crayfish giant axons.

The flux of organelles moving by fast axonal transport in distal segments of severed crayfish medial giant axons (MGAs) and lateral giant axons (LGAs) was measured for survival times of up to 35 days (MGAs) or 60 days (LGAs). The response to transection occurred in 4 phases: (1) Organelle fluxes remained nearly normal for the first 24 h. (2) Fluxes then declined continuously until day 6 or 7. (3) A rebound toward normal levels lasted until day 21 (MGAs) or longer (LGAs). (4) During the final phase, fluxes declined either to zero (MGAs) or plateaued at a level which was a significant percentage of normal flux (LGAs). Changes in anterograde and retrograde flux were identical. The distribution of various size classes of translocating vesicles in distal segments of these axons was normal until day 4, with small and medium size, rapidly moving vesicles predominating. Afterwards, larger, slower vesicles predominated. During long-term survival, the axons remained physiologically intact, and cytoskeletons appeared to be normal, retaining intact microtubules which remained normally oriented with positive ends pointing distally. The evidence suggests that the two initial phases of the response to transection represent clearance from distal segments of organelle traffic which normally moves between axon and cell body. The rebound phase may be trauma induced, possibly a transient phase of cytoplasmic degeneration resulting from the loss of trophic support from the cell body. Differences between LGAs and MGAs with respect to organelle flux during prolonged survival, i.e. during the 4th phase of the response to transection, are consistent with different mechanisms of long-term survival which have been proposed for these axons.

Protein transport between crayfish lateral giant axons.

Segmental lateral giant axons (SLGAs) in crayfish were used to determine whether functionally intact proteins can move between axons under physiological conditions. Horseradish peroxidase (HRP) was chosen as the tracer protein because its localization requires intact enzymatic activity and because it can be localized in living cells using a non-cytotoxic procedure. Following iontophoretic injection of HRP in a single SLGA, HRP often transferred to adjacent SLGAs. HRP transferred from an injected SLGA to a caudal SLGA with greater frequency than HRP transferred to a rostral SLGA. When HRP transferred between SLGAs, it was ultrastructurally associated with vesicles on both sides of septate junctions between adjacent SLGAs and was also seen in the perijunctional extracellular space. There was no difference between the electrical resistance of synapses at which HRP transferred and those synapses where HRP did not transfer. HRP transfer was significantly reduced when axons were bathed in reduced calcium saline. These and other observations indicate that axon-to-axon transport in this system is accomplished by exocytosis of HRP from injected axons followed by its endocytotic uptake by adjacent, non-injected axons. Similar transfer of endogenous proteins may contribute to the long-term survival for months to years of distal stumps of severed SLGAs.

Organization of axoplasm in crayfish giant axons.

Distributions of subcellular organelles in medial giant axons (MGAs) and segmental lateral giant axons (SLGAs) of crayfish were evaluated as part of an ongoing effort to understand and explain differences in distal stump survival following axonotomy. Both axons were able to endocytose tracer proteins placed extracellularly, and horseradish peroxidase injected by cannulation into MGAs could transfer into adaxonal glial cells. Concentrations of tubulovesicular organelles near axonal cell membranes were measured as a possible index of the relative level of axonal endo- and exocytosis, and concentrations in MGAs were found to be twice those in SLGAs. In both axons, microtubule concentrations were highest near the axolemma and lowest in the central core of axoplasm. In thoracic and abdominal regions of MGAs, microtubules and other organelles were located only in a thin layer of subaxolemmal axoplasm. Overall, MGAs contained fewer microtubules per cross-section than did SLGAs, although MGAs are five to ten times as long as SLGAs and support more synapses. Total numbers of microtubules per cross-section varied with distance from the cell body of an MGA, whereas microtubule numbers were similar in proximal and distal cross-sections of SLGAs. In addition to a layer of subaxolemmal mitochondria which was observed in MGAs and SLGAs and which is characteristic of crayfish axons, mitochondria were also concentrated in the central core of SLGA axoplasm.

Polarity orientations of microtubules in squid and lobster axons.

Polarity orientations of microtubules in periaxolemmal and internal regions of squid and lobster axons were determined in order to test the hypothesis that regional differences in particle transport are produced by differentially distributed microtubule subclasses. Over 95% of the microtubules in all regions of the axons investigated were oriented with plus ends located distally, pointing away from axonal somata, and there were no significant differences in orientation ratios in periaxolemmal and internal axoplasm. In axonal sheath glial cells of lobsters, microtubules were found to be oriented parallel to axonal microtubules and to have approximately equally mixed polarities. The results for axonal microtubules did not support the possibility of subclasses of axonal microtubules.

Peripheral electrosense physiology: a review of recent findings.

Advances, since 1974, in understanding the physiology of electroreceptors are reviewed. In brief: 1. In fish that produce a weak electric discharge with electric organs, the tuberous electroreceptors are generally most sensitive to stimulus frequencies near the species', individual's, and even local, waveform of the electric organ discharge; there is a good match between receptor sensitivity and the normal stimulus. 2. The ability of tuberous electroreceptors to detect field distortions produced by reasonably sized objects is limited; an object must be closer than a body-length to be detected, and the afferent response is a negative power function of object distance. 3. The second major electroreceptor class, the ampullary electroreceptors, is sensitive to low frequency, low intensity electric fields, and this acute sensitivity results in the ability of the receptors in marine species to detect magnetic fields on the order of the Earth's. 4. The calcium ion is essential for normal functioning of ampullary electroreceptors.