Long‐term survival followed by degradation of neurofilament proteins in severed Mauthner axons of goldfish

Abstract

The morphology and protein composition of intact and severed Mauthner axons (M-axons) from goldfish were examined on electron micrographs, sodium dodecyl sulfate gels, and immunoblots. Neurofilaments were the most common cytoskeletal element on electron micrographs, and neurofilament proteins (NFPs) were the most intensely silver-stained bands in M-axoplasm microdissected from control M-axons. NFPs at about 235, 145, 123, 105, 80, and 60 kD in M-axoplasm were identified with four monoclonal and three polyclonal antibodies. Similar immunoblots of samples of the M-axon myelin sheath (M-sheath) showed no reactivity to antibodies against NFPs. For up to 62 days following spinal cord severance in goldfish maintained at 15 degrees C, the ultrastructure, protein banding pattern, and anti-NFP immunoreactivity of several distal segments of M-axons did not change compared with control M-axons. At 62 to 81 days after severance, novel bands appeared in many silver-stained gels and anti-NFP immunoblots of distal M-axons. NFP bands completely disappeared from distal M-axon segments of some M-axons as early as 72 days after severance. However, NFP bands persisted in some distal segments for up to 81 days after severance. The degradation of NFPs occurred equally along the entire length of a distal M-axon segment, that is, there was no indication of a proximal-to-distal or distal-to-proximal sequence of NFP degradation in distal segments of severed M-axons. These biochemical data were consistent with morphological data that showed little change in the diameter or ultrastructure of severed M-axons held at 15 degrees C for about 2 months followed by a rapid collapse of the entire distal segment at 72 to 85 days postseverance.

Medium weight neurofilament mRNA in goldfish Mauthner axoplasm

“…This assumption is supported by data showing that a distal axonal segment severed from its cell body in mammals typically degenerates within hours to days [10]. However, when axons are severed from their cell bodies in invertebrates [2] or lower vertebrates such as the goldfish [18], the distal axonal segment often survives for months to years. Proteins in an axon isolated from its cell body could be maintained only through some combination of (1) Although often regarded as a particularly unlikely mechanism to maintain axonai proteins [2,16], axoplasmic protein synthesis has been reported for squid giant axons [7] and goldfish Mauthner axons (M-axons) [14].…”

“…In addition to published data on the presence of all basic components of the translational machinery, other benefits of the goldfish M-axon as an experimental system to examine protein synthetic capabilities in a vertebrate axon include (1) its long length (5-8 cm) and large diameter (50-80μm) provide a large amount of axoplasm per cell for biochemical analysis; (2) its high axonal viscosity and large axonal diameter permit a clean separation of its axoplasm (M-axoplasm) from its surrounding glial sheath (M-sheath) [14,18]; and (3) the availability of goldfish cDNA sequences from distinctly neuronal (NF-M) mRNA transcripts and distinctly glial mRNA transcripts (glial filament acidic protein; GFAP) permits sensitive PCR-based contamination controls to verify clean axonal isolation.…”

“…Goldfish (Carassius auratus) brain, M-sheath, and M-axoplasm were isolated as previously described [14,18]. M-axoplasm was rinsed in several changes of ice-cold calcium-free saline and then lysed by vortexing in an extraction buffer containing 4 M guanidinium thiocyanate, 25 mM sodium citrate, 0.5% N-lauroyl-sarkosinate, 0.1 M β-mercaptoethanol, and 20 μg/ml 5S r-RNA from Escherichia coli (Boehringer Mannheim) added as a carrier.…”

“…Gels containing samples of goldfish M-axoplasm and M-sheath were either silver-stained or analyzed via immunoblotting [18] with monoclonal antibodies directed against GFAP, a glialspecific protein [10]. Sodium dodecyl sulfate (SDS) gels of M-axoplasm produced six prominent silver stained bands at 235, 145, 123, 105, 80, and 60 kDa (Fig.…”

“…3, lane 1, lines to left of figure). These bands have been identified as neurofilament proteins according to their biochemical and immunological characteristics [18]. SDS gels of M-sheath produced more silver-stained bands with a much broader range of molecular weights than the silver-staining bands for M-axoplasm (Fig.…”