Ultrastructural studies of severed medial giant and other CNS axons in crayfish

Summary The distal stumps of severed medial giant axons (MGAs) and of non-giant axons (NGAs) in the CNS of the crayfish Procambarus clarkii show long-term (5–9 months) survival associated with disorientation of mitochondria and thickening of the glial sheath. However, the morphological responses of the two axonal types differ in that neither the proximal nor the distal stump of severed MGAs ever fills with mitochondria as is observed in some severed NGAs. Furthermore, the adaxonal glial layer never completely encircles portions of MGA axoplasm as occurs in many severed NGAs; in fact, ultrastructural changes in the adaxonal layer around severed MGAs are often difficult to detect. No multiple axonal profiles are ever seen within the glial sheath of the proximal or distal stumps of severed MGAs whereas these structures are easily located within severed NGAs.This work was supported by NIH research grant #NS-14412 and an RCDA#00070 to GDB     

Regeneration of motor axons in crayfish limbs: Distal stump activation followed by synaptic reformation

Summary Servered distal stumps of limb motor axons in the crayfish Procambarus clarkii remain ultrastructurally intact for at least 2–3 ms after being severed from their cell body. Initial regeneration of a motor axon is associated with the appearance of up to 200 small profiles (satellite axons) having no glial sheath adjacent to the large surviving stump for about 1 cm distal to the lesion at 4–5 wks postoperatively. These satellite axons are seen 2–4 cm distally at the target muscles 3–4 ms postoperatively. By 14–15 ms postoperative, the motor sheaths from the lesion site to the target muscles contain small axonal processes having thick glial sheaths. Behavioral tests show that some axons that are reconnected to the CNS at 4–5 wks may not be connected at 14–15 ms, whereas other axons not connected by 3–4 ms may be connected at 14–15 ms when the original distal stumps have degenerated.We suggest that all these data can best be explained by the view that motor axons in crayfish limbs initially regenerate via activation of the surviving distal stump by satellite axons which grow out from proximal stump. In most cases, these satellite axons continue to activate the surviving distal stump as they slowly grow to the target muscle. Eventually the satellite axons reform synapses on the target muscle and the original distal stump degenerates.This work was supported by NSF grants BNS 77-27678 and 80-22248 and an NIH RCDA 00070 to GDB. The authors would like to thank Mr. Martis Ballinger, Mr. Robert Reiss, and Mrs. Mary Raymond for their excellent technical assistance. We would also like to thank Dr. Wesley Thompson and Mr. Douglas Baxter for helpful discussions.     

Ultrastructural changes at gap junctions between lesioned crayfish axons

Summary In crayfish, the severed distal segment of single lateral giant axon (SLGA) often survives for at least 10 months after lesioning if this segment retains a septal region of apposition with an adjacent, intact SLGA. In control (unsevered) SLGAs, this septal region usually contains gap junctions and 50–60 nm vesicles near the axolemma of both SLGAs. From 1–14 days after lesioning, the distal segment of a severed SLGA undergoes obvious ultrastructural changes in mitochondria and neurotubular organization compared to control SLGAs or to adjacent, intact SLGAs in the same animal. Gap junctions are very difficult to locate in severed SLGAs within 24 h after lesioning. From two weeks to ten months after lesioning, the surviving stumps of severed SLGAs often appear remarkably normal except that structures normally associated with the presence of gap junctions remain very difficult to find.These and other data suggest that SLGA distal segments receive trophic support from adjacent, intact SLGAs. The mechanism of this support probably could not be via diffusion across gap junctions between intact and severed SLGAs since gap junctions largely disappear after lesioning. However, trophic maintenance could occur via the exocytotic — pinocytotic action of 50–60 nm vesicles which are always present on both sides of the septum between an intact SLGA and a severed SLGA distal segment.This work was supported by NIH research grant NS-14412 and and RCDA 00070 to G.D.B.     

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.     

Using fluorescence photoablation to study the regeneration of singly cut leech axons

The regeneration of the axons of leech Retzius cells was compared following two different methods of axonal severing: (1) a crush of the whole connective that includes the Retzius axon; and (2) photoablation of a small segment of only the Retzius axon. The photoablation was carried out after filling the Retzius cell with Lucifer Yellow (LY). Several tests were carried out to determine whether the photoablation actually severed the axon. These included (1) using the lipophilic membrane probe DiI as an indicator of membrane severance (2) electron microscopic examination of the photoablated axon after filling it with horseradish peroxidase (HRP); and (3) filling the Retzius cell first with HRP, then photoablating, and looking for the disappearance of the HRP in the photoablated region. These and other observations indicated that the photoablated axon was actually severed. Two differences were seen in the regeneration of the Retzius axon after crush versus after photoablation. First, the sprouting following crush was far more disorganized, and included significantly more lateral spread. Second, after photoablation, over 70% of the axons, upon refilling with LY after 3 days or more, showed the newly introduced LY suddenly extending far down the distal segment, indicating that the proximal and distal segments had become reconnected. This was never seen following a crush. The photoablated axons did not pass HRP into the distal segment, suggesting that the reconnection was not by fusion, but perhaps by a gap junction. The results show that axonal regeneration can take a dramatically different form than it does following a standard crush procedure if, instead, the axon is severed in a way that preserves the structural integrity of the surrounding tissue.     

Endogenous Nmnat2 Is an Essential Survival Factor for Maintenance of Healthy Axons

The molecular triggers for axon degeneration remain unknown. We identify endogenous Nmnat2 as a labile axon survival factor whose constant replenishment by anterograde axonal transport is a limiting factor for axon survival. Specific depletion of Nmnat2 is sufficient to induce Wallerian-like degeneration of uninjured axons which endogenous Nmnat1 and Nmnat3 cannot prevent. Nmnat2 is by far the most labile Nmnat isoform and is depleted in distal stumps of injured neurites before Wallerian degeneration begins. Nmnat2 turnover is equally rapid in injured Wld ^S neurites, despite delayed neurite degeneration, showing it is not a consequence of degeneration and also that Wld^S does not stabilize Nmnat2. Depletion of Nmnat2 below a threshold level is necessary for axon degeneration since exogenous Nmnat2 can protect injured neurites when expressed at high enough levels to overcome its short half-life. Furthermore, proteasome inhibition slows both Nmnat2 turnover and neurite degeneration. We conclude that endogenous Nmnat2 prevents spontaneous degeneration of healthy axons and propose that, when present, the more long-lived, functionally related Wld^S protein substitutes for Nmnat2 loss after axon injury. Endogenous Nmnat2 represents an exciting new therapeutic target for axonal disorders.     

Electron microscopy of severed motor fibers in the crayfish

Summary Cut and crushed crayfish claw nerves were examined with the electron microscope at intervals up to 6 months after lesion. In sections 1 centimeter distal to the lesion there were no signs of degeneration among the giant motor axons even after many months. Swelling of glial wrappings was observed within 48 hours of nerve severance and was particularly notable in the innermost glial layer, the adaxonal layer. Golgi elements, rough endoplasmic reticulum, and mitochondria accumulated in the glia. These changes were perhaps indicative of a greater supportive role required by the severed axons. Regeneration from the proximal stumps of the giant axons began within one week and had proceeded across the lesion gap by 4 weeks. Axon sprouts appeared to travel toward the terminals within the glial sheaths of the distal giant axon segments. Before regeneration was complete, as determined by a simple behaviour test, the regenerating axons occupied increasing proportions of the sheath space. After regeneration was complete occasional degenerations were seen among the sprouts. These degenerations may have occurred in regenerating axons which had grown to the incorrect muscles. The original distal giant axons probably degenerated, as well, after regeneration was complete. There was no evidence of rehealing of proximal and distal segments of the axons.This work was supported by NIH postdoctoral fellowship number 1F2 NB 32, 723 N RB awarded to RHN and grants in aid from the Multiple Sclerosis Society, The American Cancer Society and The National Institutes of Health.     

Fiber composition of the distal accessory flexor muscle in several decapod crustaceans

The fiber composition of the distal accessory flexor muscle (DAFM) and the branching pattern of its excitor axon were compared in several species of crabs, in the lobster and the crayfish. The muscle is composed exclusively of long sarcomere (> 6 μm) fibers and therefore of the slow type. In all the crab species, except one, there is a distal to proximal gradient of fibers with increasing sarcomere lengths; this gradient is reverse in lobsters and crayfish. A proximal to distal gradient of increasing fiber diameters occurs in the DAFM of all crab species but not in the lobster and crayfish, in which all the fibers are approximately equal in diameter. The single excitatory axon traverses the width of the DAFM and gives off primary branches on either side in the lobster and crayfish but on only one side in crabs. The hypothesis that the axonal branching pattern may govern the regional distribution of fibers with differing sarcomere lengths in proposed.     

Chemical degeneration of indolamine axons in rat brain by 5,6-dihydroxytryptamine

Summary Evidence has been obtained by electron microscopy of a direct cytotoxic effect of intraventricularly administered 5,6-dihydroxytryptamine (5,6-DHT) on unmyelinated axons in the rat brain. Ultrastructural signs of axonal damage were observed in areas rich in indolamine nerve terminals as early as 2 hrs after injection. By 6–24 hrs, characteristic and more dramatic signs of degeneration developed, involving coalescence of all axonal constituents—often in combination with a uniform osmiophilic impregnation of the axoplasm—accompanied by engulfment of the dystrophic structures by glial processes. During the next five days, the degenerating axons and axon terminals appeared to be removed by glial cell phagocytosis, whose equivalents were the inclusion of axonal residues into membrane-bound lysosome-like bodies. Concomitantly, there was a progressively increasing number of extremely large and dilated axons in all regions analysed. These axonal swellings, which have an ultramorphology similar to that of dilated stumps of mechanically severed monoamine axons, correspond most probably to proximal, dilated portions of drug-damaged axons.The present results, in combination with biochemical and fluorescence microscopical data, indicate that within a proper dose range the 5,6-DHT-induced degeneration is largely restricted to indolamine axons and axon terminals. However, unselective effects on other unmyelinated axons, on myelin, and on glial cells were observed in narrow subependymal zones close to the lateral ventricles, i.e. close to the injection cannula.Supported by grants from the Deutsche Forschungsgemeinschaft.Supported by grants from the National Institutes of Health, USPHS (NS-06701-06) and from the Swedish Medical Research Council (grants No. B72-14X-712-07B and B72-14X-56-08B).     

Structure of allotransplanted ganglia and regenerated neuromuscular connections in crayfish

In adult crayfish, Procambarus clarkii, motoneurons to a denervated abdominal superficial flexor muscle regenerate long-lasting and highly specific synaptic connections as seen from recordings of excitatory postsynaptic potentials, even when they arise from the ganglion of another crayfish. To confirm the morphological origins of these physiological connections we examined the fine structure of the allotransplanted tissue that consisted of the third abdominal ganglion and the nerve to the superficial flexor muscle (the fourth ganglion and the connecting ventral nerve cord were also included). Although there is considerable degeneration, the allotransplanted ganglia display intact areas of axon tracts, neuropil, and somata. Thus in both short (6–8 weeks) and long (24–30 weeks) term transplants approximately 20 healthy somata are present and this is more than the five axons regenerated to the host muscle. The principal neurite and dendrites of these somata receive both excitatory and inhibitory synaptic inputs, and these types of synaptic contacts also occur among the dendritic profiles of the neuropil. Axon tracts in the allotransplanted ganglia and ventral nerve cord consist largely of small diameter axons; most of the large axons including the medial and lateral giant axons are lost. The transplanted ganglia have many blood vessels and blood lacunae ensuring long-term survival. The transplanted superficial flexor nerve regenerates from the ventral to the dorsal surface of the muscle where it has five axons, each consisting of many profiles rather than a single profile. This indicates sprouting of the individual axons and accounts for the enlarged size of the regenerated nerve. The regenerated axons give rise to normal-looking synaptic terminals with well-defined synaptic contacts and presynaptic dense bars or active zones. Some of these synaptic terminals lie in close proximity to degenerating terminals, suggesting that they may inhabit old sites and in this way ensure target specificity. The presence of intact somata, neuropil, and axon tracts are factors that would contribute to the spontaneous firing of the transplanted motoneurons. © 1996 John Wiley & Sons, Inc.     

Ibotenic acid-induced lesions of striatal target and projection neurons: ultrastructural manifestations in dopaminergic and non-dopaminergic neurons and in glia

The cytological changes elicited by central microinjections of the excitotoxin, ibotenic acid (IBO) were examined in the adult rat striatonigral system using electron microscopic immunocytochemistry. The chemical markers included tyrosine hydroxylase (TH), a biosynthetic enzyme in dopaminergic neurons, and glial fibrillary acidic protein (GFAP). Both short (1-7 day) and long (30-60 days) term effects were evaluated at the site of IBO-injections in the striatum and more distally in the substantia nigra, which both contributes afferents and receives efferents from the striatum. In the neostriatum at every survival period examined, TH-labeled axonal processes appeared equally numerous in the control and IBO-injected hemispheres. However, the TH-labeled axons in the striatum ipsilateral to the IBO-injection were slightly enlarged, and generally lacked synaptic densities. In the early period the remaining neuropil showed signs of edema and contained perikarya and dendrites with vacuolar or dense cytoplasm as well as intact, unlabeled terminals. Numerous astrocytes, and apparently demyelinated axons were more commonly seen at the 7 day period. At 30 and 60 days, bundles of myelinated axons, unlabeled axon terminals, and astrocytes containing a variety of cytosomes and other cytoplasmic inclusions were in close apposition to TH-labeled axon terminals. These results suggest that the dopaminergic terminals may serve neuromodulatory functions with respect to glia or other afferent axons remaining after IBO-injections in the striatum. In the substantia nigra, homolateral to the injection, a dense type of degeneration was seen in a few perikarya and dendrites at 7 days of survival. At this stage, electron dense anterograde degeneration also was seen in terminals contacting both TH-labeled and unlabeled dendrites. The secondary long term changes in nuclear groups located distal to the primary lesion are characteristic of certain types of progressive human neuropathological disorders.     

Retrograde and orthograde axon transport by brief-exposure to nickel chloride: Methodology and parameters for success in the brain-retrocerebral complex of the cockroach Diploptera punctata

Exposure of the intact brain-retrocerebral complex of the cockroach Diploptera punctata to 500 mM NiCl₂ in 0.1% bovine serum albumen for 10 min resulted in the filling of neurones connecting the components of the brain-retrocerebral complex. Following severance of one corpus cardiacum from the brain prior to exposure to NiCl₂, a larger number of neurones filled in the contralateral side of the pars intercerebralis. Reducing exposure time to NiCl₂ to 30s did not result in backfilling of intact preparations. However, if the nervi corporis allati I (NCA 1) were sectioned, then exposed to NiCl₂ for 30s and incubated in culture medium at 4°C for 24 h, neurones of the severed NCA I backfilled to somata originating in the pars intercerebralis and pars lateralis. In addition, the axons distal to the cut also filled with the tracer in the orthograde direction, revealing a complex network of axon terminals investing the corpora allata. Sectioned nervi corporis allati I exposed to the tracer solution, then incubated in culture medium for 0.5 h, rapidly backfilled at a rate of 30–100 mm/day. Rapid backfilling did not occur in the absence of BSA. One per cent fluorescein isothiocyanide-labelled BSA backfilled as rapidly as the $\text{NiCl}{}_2\text{BSA}$ solution. 2,4-Dinitrophenol and cytochalasin B inhibited brief-exposure backfilling whereas colchicine had no effect in short term incubations but partially inhibited backfilling in long term incubations. Backfilling is thus a metabolically mediated event and the rate of transport of the tracer is similar to that of retrograde fast axonal transport of proteins.     

Evidence for the glia-neuron protein transfer hypothesis from intracellular perfusion studies of squid giant axons

Incubation of intracellulary perfused squid giant axons in [3H]leucine demonstrated that newly synthesized proteins appeared in the perfusate after a 45-min lag period. The transfer of labeled proteins was shown to occur steadily over 8 h of incubation, in the presence of an intact axonal plasma membrane as evidenced by the ability of the perfused axon to conduct propagated action potentials over this time-period. Intracellularly perfused RNase did not affect this transfer, whereas extracellularly applied puromycin, which blocked de novo protein synthesis in the glial sheath, prevented the appearance of labeled proteins in the perfusate. The uptake of exogenous 14C-labeled bovine serum albumin (BSA) into the axon had entirely different kinetics than the endogenous glial labeled protein transfer process. The data provide support for the glia-neuron protein transfer hypothesis.     

Potassium homeostasis in the nervous system of cephalopods and crustacea

1. Previous work has shown that nerve activity is associated with a significant release of potassium in the vicinity of the axonal membrane. Several mechanisms are normally present which reduce K+ accumulation in the extra-axonal space. 2. In intact connectives of the crayfish, Procambarus clarkii, repetitive stimulation of the giant axons was associated with an apparent hyperpolarization measured by an interstitial microelectrode, which most probably corresponds to depolarization of the inner face of the perineurial cells by K+ ions leaving the axons. 3. In desheathed connectives of the crayfish, potassium accumulated during long depolarizing voltage-clamp pulses but cleared away very quickly at the end of the pulse. 4. In the small squid, Alloteuthis subulata, repetitive stimulation of giant axons in situ in fresh and well-perfused animals did not result in a large decrease in the positive after potential (undershoot), reflecting the absence of potassium accumulation. A similar absence of accumulation was observed in vitro for carefully and freshly dissected isolated axons from live squids. 5. In both cases, deterioration of the physiological state of the axon was accompanied by a significant potassium accumulation. Potassium accumulation could also be reversibly enhanced by decreasing the osmotic pressure of the bathing medium, whereas hyperosmotic solutions had the opposite effect. These results are compatible with the idea that Schwann cells around the axon play a key role in K+ homeostasis. 6. Experiments on giant axons of the large squid species, Loligo forbesi confirmed the observations made on Alloteuthis in that fresh preparations exhibited little potassium accumulation. Under voltage-clamp conditions, 10 ms depolarizing pulses to various potential levels did not induce any accumulation in these preparations as reflected by the outward tail current. Large accumulation was observed in older axons under similar experimental conditions. 7. A large peri-axonal space associated with healthy glial cells appears to be a prerequisite for efficient K+ homeostasis in both crayfish and squid. Other mechanisms involving specific transport mechanisms across axonal and glial membranes are also likely to be involved.     

Bridging Schwann cell transplants promote axonal regeneration from both the rostral and caudal stumps of transected adult rat spinal cord

Transplantation of cellular components of the permissive peripheral nerve environment in some types of spinal cord injury holds great promise to support regrowth of axons through the site of injury. In the present study, Schwann cell grafts were positioned between transected stumps of adult rat thoracic spinal cord to test their efficacy to serve as bridges for axonal regeneration. Schwann cells were purified in culture from adult rat sciatic nerve, suspended in Matrigel:DMEM (30:70), and drawn into polymeric guidance channels 8mm long at a density of 120×106 cells ml-1. Adult Fischer rat spinal cords were transected at the T8 cord level and the next caudal segment was removed. Each cut stump was inserted 1mm into the channel. One month later, a bridge between the severed stumps had been formed, as determined by the gross and histological appearance and the ingrowth of propriospinal axons from both stumps. Propriospinal neurons (mean, 1064±145 SEM) situated as far away as levels C3 and S4 were labelled by retrograde tracing with Fast Blue injected into the bridge. Near the bridge midpoint there was a mean of 1990±594 myelinated axons and eight times as many nonmyelinated, ensheathed axons. Essentially no myelinated or unmyelinated axons were observed in control Matrigel-only grafts. Brainstem neurons were not retrogradely labelled from the graft, consistent with growth of immunoreactive serotonergic and noradrenergic axons only a short distance into the rostral end of the graft, not far enough to reach the tracer placed at the graft midpoint. Anterograde tracing with PHA-L introduced rostral to the graft demonstrated that axons extended the length of the graft but essentially did not leave the graft. This study demonstrates that Schwann cell grafts serve as bridges that support (1) regrowth of both ascending and descending axons across a gap in the adult rat spinal cord and (2) limited regrowth of serotonergic and noradrenergic fibres from the rostral stump. Regrowth of monoaminergic fibres into grafts was not seen in an earlier study of similar grafts placed inside distally capped rather than open-ended channels. Additional intervention will be required to foster growth of the regenerated axons from the graft into the distal cord tissue.     

Chronic excitotoxin-induced axon degeneration in a compartmented neuronal culture model

Glutamate excitotoxicity is a major pathogenic process implicated in many neurodegenerative conditions, including AD (Alzheimer''s disease) and following traumatic brain injury. Occurring predominantly from over-stimulation of ionotropic glutamate receptors located along dendrites, excitotoxic axonal degeneration may also occur in white matter tracts. Recent identification of axonal glutamate receptor subunits within axonal nanocomplexes raises the possibility of direct excitotoxic effects on axons. Individual neuronal responses to excitotoxicity are highly dependent on the complement of glutamate receptors expressed by the cell, and the localization of the functional receptors. To enable isolation of distal axons and targeted excitotoxicity, murine cortical neuron cultures were prepared in compartmented microfluidic devices, such that distal axons were isolated from neuronal cell bodies. Within the compartmented culture system, cortical neurons developed to relative maturity at 11 DIV (days in vitro) as demonstrated by the formation of dendritic spines and clustering of the presynaptic protein synaptophysin. The isolated distal axons retained growth cone structures in the absence of synaptic targets, and expressed glutamate receptor subunits. Glutamate treatment (100 μM) to the cell body chamber resulted in widespread degeneration within this chamber and degeneration of distal axons in the other chamber. Glutamate application to the distal axon chamber triggered a lesser degree of axonal degeneration without degenerative changes in the untreated somal chamber. These data indicate that in addition to current mechanisms of indirect axonal excitotoxicity, the distal axon may be a primary target for excitotoxicity in neurodegenerative conditions.     

Barrier permeability at cut axonal ends progressively decreases until an ionic seal is formed

After axonal severance, a barrier forms at the cut ends to rapidly restrict bulk inflow and outflow. In severed crayfish axons we used the exclusion of hydrophilic, fluorescent dye molecules of different sizes (0.6-70 kDa) and the temporal decline of ionic injury current to levels in intact axons to determine the time course (0-120 min posttransection) of barrier formation and the posttransection time at which an axolemmal ionic seal had formed, as confirmed by the recovery of resting and action potentials. Confocal images showed that the posttransection time of dye exclusion was inversely related to dye molecular size. A barrier to the smallest dye molecule formed more rapidly (<60 min) than did the barrier to ionic entry (>60 min). These data show that axolemmal sealing lacks abrupt, large changes in barrier permeability that would be expected if a seal were to form suddenly, as previously assumed. Rather, these data suggest that a barrier forms gradually and slowly by restricting the movement of molecules of progressively smaller size amid injury-induced vesicles that accumulate, interact, and form junctional complexes with each other and the axolemma at the cut end. This process eventually culminates in an axolemmal ionic seal, and is not complete until ionic injury current returns to baseline levels measured in an undamaged axon.     

Axonal degeneration within the tympanal nerve of Schistocerca gregaria

This study describes time course and ultrastructural changes during axonal degeneration of different neurones within the tympanal nerve of the locust Schistocerca gregaria. The tympanal nerve innervates the tergit and pleurit of the first abdominal segment and contains the axons of both sensory and motor neurones. The majority of axons (approx. 97%) belong to several types of sensory neurones: mechano- and chemosensitive hair sensilla, multipolar neurones, campaniform sensilla and sensory cells of a scolopidial organ, the auditory organ. Axons of campaniform sensilla, of auditory sensory cells and of motor neurones are wrapped by glial cell processes. In contrast, the very small and numerous axons (diameter <1 microm) of multipolar neurones and hair sensilla are not separated individually by glia sheets. Distal parts of sensory and motor axons show different reactions to axotomy: 1 week after separation from their somata, distal parts of motor axons are invaded by glial cell processes. This results in fascicles of small axon bundles. In contrast, distal parts of most sensory axons degenerate rapidly after being lesioned. The time to onset of degeneration depends on distance from the lesion site and on the type of sensory neurone. In axons of auditory sensory neurones, ultrastructural signs of degeneration can be found as soon as 2 days after lesion. After complete lysis of distal parts of axons, glial cell processes invade the space formerly occupied by sensory axons. The rapid degeneration of distal auditory axon parts allows it to be excluded that they provide a structure that leads regenerating axons to their targets. Proximal parts of severed axons do not degenerate.     

Degeneration and Regeneration in Crustacean Neuromuscular Systems

Crayfish motor neurons seem to repair damage to peripheral axonsby selective fusion of outgrowing proximal stumps with severeddistal processes that can survive morphologically and physiologicallyintact for over 200 days. Survival of isolated motor and CNSgiant axons is associated with much hypertrophy of their glialsheath. The severed stumps of peripheral sensory neurons oftendegenerate within 21 days and their glial sheath does not hypertrophy.Denervation and immobilization produce relatively little changein the morphology and physiology of the opener muscle, whereastenotomy produces much atrophy within 30-60 days. Crayfish motor and CNS giant neurons show no capability forregenerating ablated cell bodies, whereas peripheral sensorysomata regenerate after limb autotomy. An entire opener musclecan be replaced after limb autotomy but the organism shows littleor no ability to redifferentiate an entire muscle in the absenceof body part regeneration. However, a few opener muscle fiberscan be regenerated if the bulk of the muscle mass remains intact.The significance of all these findings are interpreted withrespect to the developmental capabilities and environmentaladaptations of the crayfish together with the evolution of regenerativeabilities in anthropods and vertebrates.     

Differences in synaptic output between excitatory and inhibitory motoneurons in a crayfish muscle

A pair of antagonistic motoneurons, one excitatory and one inhibitory, innervates the distal accessory flexor muscle in the walking limb of the crayfish Procambarus clarkii. The number and size of synapses formed by these two axons on the muscle fibers (neuromuscular synapses) and on each other (axo-axonal synapses) were estimated using thin-section electron microscopy. Although profiles of nerve terminals of the two axons occur in roughly equal proportions, the frequency of occurrence of neuromuscular synapses differed markedly: 73% were excitatory and 27% were inhibitory. However, inhibitory synapses were 4–5 times larger than excitatory ones, and consequently, the total contact areas devoted to neuromuscular synapses were similar for both axons. Axo-axonal synapses were predominantly from the inhibitory axon to the excitatory axon (86%), and a few were from the excitatory axon to the inhibitory axon (14%). The role of the inhibitory axo-axonal synapse is presynaptic inhibition, but that of the excitatory axo-axonal synapse is not known. The differences in size of neuromuscular synapses between the two axons may reflect intrinsic determinants of the neuron, while the similarity in total synaptic area may reflect retrograde influences from the muscle for regulating synapse number.