Crayfish Motor Axons Research Articles

Inhibitory modulation of action potentials in crayfish motor axons by fluoxetine.

The goal of this report is to explore how K2P channels modulate axonal excitability by using the crayfish ventral superficial flexor preparation. This preparation allows for simultaneous recording of motor nerve extracellular action potentials (eAP) and intracellular excitatory junctional potential (EJP) from a muscle fiber. Previous pharmacological studies have demonstrated the presence of K2P-like channels in crayfish. Fluoxetine (50µM) was used to block K2P channels in this study. The blocker caused a gradual decline, and eventually complete block, of motor axon action potentials. At an intermediate stage of the block, when the peak-to-peak amplitude of eAP decreased to ∼60%-80% of the control value, the amplitude of the initial positive component of eAP declined at a faster rate than that of the negative peak representing sodium influx. Furthermore, the second positive peak following this sodium influx, which corresponds to the after-hyperpolarizing phase of intracellularly recorded action potentials (iAP), became larger during the intermediate stage of eAP block. Finally, EJP recorded simultaneously with eAP showed no change in amplitude, but did show a significant increase in synaptic delay. These changes in eAP shape and EJP delay are interpreted as the consequence of depolarized resting membrane potential after K2P channel block. In addition to providing insights to possible functions of K2P channels in unmyelinated axons, results presented here also serve as an example of how changes in eAP shape contain information that can be used to infer alterations in intracellular events. This type of eAP-iAP cross-inference is valuable for gaining mechanistic insights here and may also be applicable to other model systems.

Effects of Osmolarity on Ultrasound-Induced Membrane Depolarization in Isolated Crayfish Motor Axon.

We have previously identified a novel non-selective membrane conductance (g_US) opened by focused ultrasound (FUS) in crayfish motor axons. In the work described here, we studied g_US properties further by comparing FUS-evoked depolarization (FUSD) in control and hypotonic saline with 75% of control osmolarity. The FUS was a train of 20 FUS bursts (2.1 MHz and 50 µs per burst) delivered at 1 kHz. The amplitude, onset latency, frequency of occurrence and duration of FUSD were compared in a 15-min time window before and after switching to hypotonic saline. Significant increases were observed for amplitude (p < 0.001) and frequency of occurrence (p < 0.01) while the onset latency exhibited a significant decrease (p < 0.001). FUSD duration did not significantly differ. These results support predictions based on our hypothesis that g_US is mediated by opening of nanopores in the lipid bilayer and that stretching of axonal membrane caused by swelling at low osmolarity should increase the probability of nanopore formation under FUS. The FUSD parameters, in addition, exhibited time-dependent trends when the window of observation was expanded to 45 min in each saline. The statistical significance of amplitude and duration differed between 15- and 45-min time windows, indicating the presence of adaptive responses of axonal membrane to osmotic manipulation.

Infrared inhibition and waveform modulation of action potentials in the crayfish motor axon.

The infrared (IR) inhibition of axonal activities in the crayfish neuromuscular preparation is studied using 2 µm IR light pulses with varying durations. The intracellular neuronal activities are monitored with two-electrode current clamp, while the IR-induced temperature changes are measured by the open patch technique simultaneously. It is demonstrated that the IR pulses can reversibly shape or block locally initiated action potentials. Suppression of the AP amplitude and duration and decrease in axonal excitability by IR pulses are quantitatively analyzed. While the AP amplitude and duration decrease similarly during IR illumination, it is discovered that the recovery of the AP duration after the IR pulses is slower than that of the AP amplitude. An IR-induced decrease in the input resistance (8.8%) is detected and discussed together with the temperature dependent changes in channel kinetics as contributing factors for the inhibition reported here.

Focused ultrasound transiently increases membrane conductance in isolated crayfish axon.

We report a novel phenomenon produced by focused ultrasound (US) that may be important for understanding its effects on cell membranes. When a US burst (2.1 MHz, 1-mm focal diameter, 0.1-1 MPa) was focused on a motor axon of the crayfish neuromuscular junction, it consistently produced a fast hyperpolarization, which was followed or superseded by subthreshold depolarizations or action potentials in a stochastic manner. The depolarization persisted in the presence of voltage-gated channel blockers [1 µM TTX ( INa), 50 µM ZD7288 ( Ih), and 200 µM 4-aminopyridine ( IK)] and typically started shortly after the onset of a 5-ms US burst, with a mean latency of 3.35 ± 0.53 ms (SE). The duration and amplitude of depolarizations averaged 2.13 ± 0.87 s and 10.1 ± 2.09 mV, with a maximum of 200 s and 60 mV, respectively. The US-induced depolarization was always associated with a decrease in membrane resistance. By measuring membrane potential and resistance during the US-induced depolarization, the reversal potential of US-induced conductance ( gus) was estimated to be -8.4 ± 2.3 mV, suggesting a nonselective conductance. The increase in gus was 10-100 times larger than the leak conductance; thus it could significantly influence neuronal activity. This change in conductance may be due to stimulation of mechanoreceptors. Alternatively, US may perturb the lateral motion of phospholipids and produce nanopores, which then increase gus. These results may be important for understanding mechanisms underlying US-mediated modulation of neuronal activity and brain function. NEW & NOTEWORTHY We report a specific increase in membrane conductance produced by ultrasound (US) on neuronal membrane. When a 5-ms US tone burst was focused on a crayfish motor axon, it stochastically triggered either depolarization or a spike train. The depolarization was up to 60 mV in amplitude and 200 s in duration and therefore could significantly influence neuronal activity. Depolarization was still evoked by US burst in the presence of Na+ and Ca2+ channel blockers and had a reversal potential of -8.4 ± 2.3 mV, suggesting a nonselective permeability. US can be applied noninvasively in the form of a focused beam to deep brain areas through the skull and has been shown to modulate brain activity. Understanding the depolarization reported here should be helpful for improving the use of US for noninvasive modulation and stimulation in brain-related disease.

Effects of Deltamethrin on crayfish motor axon activity and neuromuscular transmission

Deltamethrin (DM) is a widely used pesticide known to target sodium channels. Although this compound has been studied extensively at molecular and behavioral levels, the detailed action of DM on cellular and synaptic function is less well documented. In this report, we show that DM at nanomolar concentrations can silence tonic motor output of the crayfish ventral superficial flexor (VSF) within ∼10min. Action potential (AP) amplitude was consistently reduced before silencing occurred, whereas AP duration and AP firing frequency did not change. In some synapses EPSP amplitude and synaptic delay were modified by DM, but the direction of change was not consistent. In order to better understand these diverse effects, intracellular recordings from motor axons of the crayfish opener were used for a detailed analysis. DM caused an initial, slow depolarization of resting membrane potential (Vm), which was accompanied by reduced AP amplitude but not AP duration. Resting Vm then underwent a step depolarization of ∼20mV, which we propose corresponds to the onset of the depolarization block. In addition, DM shifted the AP initiation site in some opener axons during prolonged firing. This shift occurred concomitantly with a reduction in synaptic delay. A similar reduction in synaptic delay was also detected at some VSF axons, and can be attributed to the same mechanism. Results reported here suggest that DM at low concentrations result in: (i) depolarization block of motor axons before changes in network output can be detected, (ii) variable effects on synaptic transmission, with this variability presumably due to the diverse morphology and excitability of motor axons.

The cytoplasmic domain of rat synaptotagmin I enhances synaptic transmission.

Synaptotagmin, an integral membrane protein of synaptic vesicles, functions as a calcium sensor in the temporal control of neurotransmitter release. Although synaptotagmin facilitates lipid membrane fusion in biochemical experiments, overexpression of synaptotagmin inhibits neurotransmission. A facilitatory effect of synaptotagmin on synaptic transmission was never observed. To determine whether synaptotagmin may accelerate synaptic transmission in vivo, we injected the cytoplasmic domain of rat synaptotagmin I (CD-syt) into crayfish motor axons and tested the effect of CD-syt on synaptic response. We confirmed that CD-syt accelerates neuromuscular transmission. The injected preparation had larger synaptic potentials with shorter rise time. Experiments with varying calcium concentrations showed that CD-syt increased the maximum synaptic response of the neuromuscular synapses. Further tests on short-term plasticity of neuromuscular synapses revealed that CD-syt increases the release probability of the release-ready vesicles.

An antibody to synaptotagmin I facilitates synaptic transmission

Proper functioning of the nervous system requires precise control of neurotransmitter release. Synaptotagmin, a synaptic vesicle protein, is crucial for the temporal control of neurotransmitter release. The mechanism of synaptotagmin function is still under debate. To investigate the mechanism by which synaptotagmin controls neurotransmitter release, we injected an antibody of rat synaptotagmin I into a crayfish motor axon. We found that the antibody enhanced synaptic transmission at crayfish neuromuscular junctions by increasing the amplitude of the evoked synaptic response. This effect was antibody-dose dependent. The antibody also reduced the rise time of the synaptic potentials. These effects were accompanied by a reduction in the Hill coefficient for Ca(2+)-dependence of synaptic transmission. Our findings support the hypothesis that synaptotagmin inhibits neurotransmitter release in the absence of Ca(2+).

Regenerating crayfish motor axons assimilate glial cells and sprout in cultured explants.

Phasic and tonic motor nerves originating from crayfish abdominal ganglia, in 2-3-day-old cultured explants, display at their transected distal ends growth zones from which axonal sprouts arise. The subcellular morphology of this regenerative response was examined with thin serial-section electron microscopy and reveals two major remodeling features. First, the external sprouts that exit the nerve are a very small part of a much more massive sprouting response by individual axons comprising several orders of internal sprouts confined to the nerve. Both internal and external sprouts have a simple construction: a cytoskeleton of microtubules and populations of mitochondria, clear synaptic vesicles, membranous sacs, and extrasynaptic active zone dense bars, features reminiscent of motor nerve terminals. Close intermingling of the sprouts of several axons give rise to a neuropil-like arbor within the nerve. Thus, extensive sprouting is an intrinsic response of crayfish motor axons to transection. Second, an equally dramatic remodeling feature is the appearance of nuclei, which resemble those of adjacent glial cells, within the motor axons. These nuclei often appear where the adjoining membranes of the axon and glial cell are disrupted and where free-standing lengths of the double membrane are present. These images signify a breakdown of the dividing membranes and assimilation of the glial cell by the axon, the nucleus being the most visible sign of such assimilation. Thus, crayfish motor axons respond to transection by assimilating glial cells that may provide regulatory and trophic support for the sprouting response.

Ca2+ clearance at growth cones produced by crayfish motor axons in an explant culture.

Intracellular free Ca2+ concentration ([Ca2+]i) plays an important role in the regulation of growth cone (GC) motility; however, the mechanisms responsible for clearing Ca2+ from GCs have not been examined. We studied the Ca2+-clearance mechanisms in GCs produced by crayfish tonic and phasic motor axons by measuring the decay of [Ca2+]i after a high [K+] depolarizing pulse using fura-2AM. Tonic motor axons regenerating in explant cultures develop GCs with more rapid Ca2+ clearance than GCs from phasic axons. When Na/Ca exchange was blocked by replacing external Na+ with N-methyl-d-glucamine (NMG), [Ca2+]i decay was delayed in both tonic and phasic GCs. Tonic GCs appear to have higher Na/Ca exchange activity than phasic ones since reversal of Na/Ca exchange by lowering external Na+ caused a greater increase in [Ca2+]i for tonic than phasic GCs. Application of the mitochondrial inhibitors, Antimycin A1 (1 microM) and CCCP (10 microM), demonstrated that mitochondrial Ca2+ uptake/release was more prominent in phasic than tonic GCs. When both Na/Ca exchange and mitochondria were inhibited, the plasma membrane Ca2+ ATPase was effective in extruding Ca2+ from tonic, but not phasic GCs. We conclude that Na/Ca exchange plays a prominent role in extruding large Ca2+ loads from both tonic and phasic GCs. High Na/Ca exchange activity in tonic GCs contributes to the rapid decay of [Ca2+]i in these GCs; low rates of Ca2+ extrusion plus the release of Ca2+ from mitochondria prolongs the decay of [Ca2+]i in the phasic GCs.

Ca2+Clearance at Growth Cones Produced by Crayfish Motor Axons in an Explant Culture

Intracellular free Ca2+concentration ([Ca2+]i) plays an important role in the regulation of growth cone (GC) motility; however, the mechanisms responsible for clearing Ca2+from GCs have not been examined. We studied the Ca2+-clearance mechanisms in GCs produced by crayfish tonic and phasic motor axons by measuring the decay of [Ca2+]iafter a high [K+] depolarizing pulse using fura-2AM. Tonic motor axons regenerating in explant cultures develop GCs with more rapid Ca2+clearance than GCs from phasic axons. When Na/Ca exchange was blocked by replacing external Na+with N-methyl-d-glucamine (NMG), [Ca2+]idecay was delayed in both tonic and phasic GCs. Tonic GCs appear to have higher Na/Ca exchange activity than phasic ones since reversal of Na/Ca exchange by lowering external Na+caused a greater increase in [Ca2+]ifor tonic than phasic GCs. Application of the mitochondrial inhibitors, Antimycin A1 (1 μM) and CCCP (10 μM), demonstrated that mitochondrial Ca2+uptake/release was more prominent in phasic than tonic GCs. When both Na/Ca exchange and mitochondria were inhibited, the plasma membrane Ca2+ATPase was effective in extruding Ca2+from tonic, but not phasic GCs. We conclude that Na/Ca exchange plays a prominent role in extruding large Ca2+loads from both tonic and phasic GCs. High Na/Ca exchange activity in tonic GCs contributes to the rapid decay of [Ca2+]iin these GCs; low rates of Ca2+extrusion plus the release of Ca2+from mitochondria prolongs the decay of [Ca2+]iin the phasic GCs.

Intracellular ionic concentration by calibration from fluorescence indicator emission spectra, its relationship to the Kd, Fmin, Fmax formula, and use with Na-Green for presynaptic sodium

The emission spectra calibration curves for a fluorescence indicator and the F min, F max, and K d formula were shown to be related. Using the known calibrated fluorescence emitted by Sodium Green (Na-Green) and photo-multiplier-tube quantum efficiency, we calculated the detection signal over a range of sodium concentrations. The calculated calibration curves were compared for optical filters passing a narrow band, medium band or full spectrum. We found that a method based on the full emission spectrum was the most appropriate. Given a known resting concentration of intracellular sodium, calibrated readings can be converted to concentration values. This method is applicable to any fluorescence indicator when curves for emission spectra over a range of concentrations are available. We measured sodium concentration changes during trains of action potentials (APs) at a crayfish motor axon's presynaptic terminals injected with Na-Green. During low frequency AP trains, net sodium increases asymptotically with frequency. Average net Na-flux per AP decreases for increasing terminal size. The terminals of crayfish motor axon have surface area to volume ratio which is 7700 times larger than for squid. Thus, in comparison to squid, crayfish terminals exhibit a larger change in [Na +] i during equivalent AP activity.

Elimination of sensory inputs induces growth and synaptic changes in crayfish motor axons

Damage to motor neurons induces regeneration processes including axonal growth and change of synaptic properties. Sensory axons that run along the motor axons are also damaged, but their possible role in the motor neuron's regeneration is generally ignored. Here, the effect of eliminating some sensory inputs from intact motor axons on the motor axon's properties was studied. Micro-dissecting one of the segmental, bilateral, sensory stretch receptor pairs of the crayfish abdomen induced the deep extensor abdominal motor axons to grow and changed their synaptic properties. The results demonstrate directly, probably for the first time, that change in sensory neuron activity can induce motor axons to grow, form new synapses, and change their synaptic properties.

Synaptic physiology and mitochondrial function in crayfish tonic and phasic motor neurons.

Phasic and tonic motor neurons of crustaceans differ strikingly in their junctional synaptic physiology. Tonic neurons generally produce small excitatory postsynaptic potentials (EPSPs) that facilitate strongly as stimulation frequency is increased, and normally show no synaptic depression. In contrast, phasic neurons produce relatively large EPSPs with weak frequency facilitation and pronounced depression. We addressed the hypothesis that mitochondrial function is an important determinant of the features of synaptic transmission in these neurons. Mitochondrial fluorescence was measured with confocal microscopy in phasic and tonic axons and terminals of abdominal and leg muscles after exposure to supravital mitochondrial fluorochromes, rhodamine-123 (Rh123) and 4-diethylaminostyryl-N-methylpyridinium iodide (4-Di-2-Asp). Mitochondria of tonic axons and neuromuscular junctions had significantly higher mean Rh123 and 4-Di-2-Asp fluorescence than in phasic neurons, indicating more accumulation of the fluorochromes. Mitochondrial membrane potential, which is responsible for Rh123 uptake and is related to mitochondrial oxidative activity (the production of ATP by oxidation of metabolic substrates), is likely higher in tonic axons. Electron microscopy showed that tonic axons contain approximately fivefold more mitochondria per microm2 cross-sectional area than phasic axons. Neuromuscular junctions of tonic axons also have a much higher mitochondrial content than those of phasic axons. We tested the hypothesis that synaptic fatigue resistance is dependent on mitochondrial function in crayfish motor axons. Impairment of mitochondrial function by uncouplers of oxidative phosphorylation, dinitrophenol or carbonyl cyanide m-chlorophenylhydrazone, or by the electron transport inhibitor sodium azide, led to marked synaptic depression of a tonic axon and accelerated depression of a phasic axon during maintained stimulation. Iodoacetate, an inhibitor of glycolysis, and chloramphenicol, a mitochondrial protein synthesis inhibitor, had no significant effects on either mitochondrial fluorescence or synaptic depression in tonic or phasic axons. Collectively, the results provide evidence that mitochondrial oxidative metabolism is important for sustaining synaptic transmission during maintained stimulation of tonic and phasic motor neurons. Tonic neurons have a higher mitochondrial content and greater oxidative activity; these features are correlated with their greater resistance to synaptic depression. Conversely, phasic neurons have a lower mitochondrial content, less oxidative activity, and greater synaptic fatigability.

Quantal release at visualized terminals of a crayfish motor axon: Intraterminal and regional differences

Synaptic transmission was measured at visualized terminal varicosities of the motor axon providing the sole excitatory innervation of the “opener” muscle in walking legs of crayfish (Procambarus clarkii Girard). Two questions were addressed: 1) How uniform is quantal emission at different locations along terminals innervating a single muscle fiber, and 2) can differences in quantal emission account for the different excitatory postsynaptic potential (EPSP) amplitudes generated by terminals localized in defined regions of the muscle? Extracellular “macropatch” electrodes were placed over individual varicosities, viewed after brief exposure to a fluorescent dye, and synaptic currents were recorded to determine quantal content of transmission. Along terminals supplying a single muscle fiber, nonuniform release was found: Varicosities closer to the point of origin of the terminal branch released more transmitter than those located more distally. Quantal content was higher for varicosities of the muscle's proximal region (where large EPSPs occur) than for varicosities of the central region (where small EPSPs occur). The probability of transmitter release per synapse is estimated to be greater for the proximal varicosities. At low frequencies of stimulation, quantal content per muscle fiber is two to four times larger in the proximal region. Taken in conjunction with a twofold higher mean input resistance for the proximal muscle fibers, the difference in quantal content can account for a four- to eightfold difference in EPSP amplitude. The observed mean EPSP amplitude is at least eight times larger in the proximal region. We discuss factors contributing to differences in EPSP amplitudes. © 1996 Wiley-Liss, Inc.

Quantal release at visualized terminals of a crayfish motor axon: intraterminal and regional differences.

Synaptic transmission was measured at visualized terminal varicosities of the motor axon providing the sole excitatory innervation of the "opener" muscle in walking legs of crayfish (Procambarus clarkii Girard). Two questions were addressed: 1) How uniform is quantal emission at different locations along terminals innervating a single muscle fiber, and 2) can differences in quantal emission account for the different excitatory postsynaptic potential (EPSP) amplitudes generated by terminals localized in defined regions of the muscle? Extracellular "macropatch" electrodes were placed over individual varicosities, viewed after brief exposure to a fluorescent dye, and synaptic currents were recorded to determine quantal content of transmission. Along terminals supplying a single muscle fiber, nonuniform release was found: Varicosities closer to the point of origin of the terminal branch released more transmitter than those located more distally. Quantal content was higher for varicosities of the muscle's proximal region (where large EPSPs occur) than for varicosities of the central region (where small EPSPs occur). The probability of transmitter release per synapse is estimated to be greater for the proximal varicosities. At low frequencies of stimulation, quantal content per muscle fiber is two to four times larger in the proximal region. Taken in conjunction with a twofold higher mean input resistance for the proximal muscle fibers, the difference in quantal content can account for a four- to eightfold difference in EPSP amplitude. The observed mean EPSP amplitude is at least eight times larger in the proximal region. We discuss factors contributing to differences in EPSP amplitudes.

Quantal currents evoked by graded intracellular depolarization of crayfish motor axon terminals.

1. Quantal transmitter release was examined at nerve terminals of the excitatory motor axon of the crayfish opener muscle. The magnitude of synaptic currents, recorded with macro-patch electrodes at a nerve terminal, served as a measure of quantal size. Transmitter release was initiated by pulses of depolarizing current applied intracellularly to the axonal terminals after application of tetrodotoxin. Quantal release was altered by a variety of methods and the resulting quantal output and quantal size were measured. 2. Amplitude distributions of quantal events were obtained during experimental manipulations which altered the rate of quantal release by up to 25-fold. These manipulations consisted of: varying pulse amplitude or pulse duration; facilitating the release by prolonged depolarization; and application of a potassium channel blocker, 4-aminopyridine. 3. The amplitude of quantal events is impervious to marked changes in presynaptic depolarization and is not affected by experimental procedures which promote accumulation of calcium ions in the terminals. The vesicular mechanism of release, in which transmitter substance is prepackaged in vesicles which individually undergo exocytosis at a release zone, could account for the observed results.

Correlation of presynaptic and postsynaptic events during establishment of long-term facilitation at crayfish neuromuscular junction

Repetitive stimulation (10-20 Hz) of the motor axon supplying the opener muscle in the crayfish leg produces long-lasting enhancement of excitatory postsynaptic potentials. This long-term facilitation (LTF) was investigated by recording simultaneously from the presynaptic nerve terminal and from the innervated muscle fiber with intracellular microelectrodes. On cessation of stimulation, the facilitated postsynaptic potential declines in amplitude when monitored with low-frequency test stimuli. A rapid decline (phase I) occurs over the first 30 s and is succeeded by a more gradual decline lasting several minutes (phase II). Finally, a residual potentiation with a very slow decay (phase III) persists for several hours. Simultaneous pre- and postsynaptic recordings were made during induction of LTF with stimuli delivered at 20 Hz for 10 min. During the tetanus, excitatory postsynaptic potentials were enhanced 20-fold, while action potentials in the presynaptic terminal declined in amplitude from 108.6 to 97.2 mV, and the presynaptic membrane became hyperpolarized by 6.4 mV. The Na+ pump inhibitor ouabain (0.5-1.0 mM) abolished the hyperpolarization, indicating that the latter resulted from activation of an electrogenic Na+ pump. The reduction in amplitude of the presynaptic action potential was consistent with a reduced transmembrane concentration gradient for Na+. Thus, it is suggested that a significant accumulation of Na+ occurs during repetitive stimulation of crayfish motor axons. Decay of phase II of LTF, but not of phases I or III, had approximately the same time course as the decay of Na+ accumulation in the terminals, monitored by changes in the presynaptic action potential. Thus it is probable that in crayfish this phase of LTF is linked to an increased intraterminal Na+ concentration. Injection of Na+ from a microelectrode into the presynaptic terminal produced enhancement of the excitatory postsynaptic potential lasting for many minutes, as well as changes in presynaptic membrane potential and action potential similar to those seen during repetitive stimulation. The results provide the first direct measurements of electrical and ionic changes in axonal terminals during prolonged periods of activity leading to LTF, and support the hypothesis that accumulation of intraterminal Na+ is associated with one phase of LTF.

Protein metabolism in transected peripheral nerves of the crayfish

The significance of the protein metabolism in crayfish peripheral nerve was studied in relation the ability of crayfish motor axons to survive for over 200 days following axotomy. In contrast to frog peripheral nerves, the crayfish nerves appear to more closely resemble ganglia in their profiles of synthesis expressed on sodium dodecyl sulfate (SDS) gels, and have higher incorporation rates of [3H]leucine into protein than ganglia. Since anisomycin inhibits over 95% of protein synthesis in crayfish peripheral nerve, it was concluded that this local protein synthesis was dependent upon a eukaryotic ribosomal mechanism. Radioautography of isolated nerves reveals newly synthesized proteins in glial sheaths, and also within the axoplasm of large motor fibers. Based upon the data available at present, a hypothesis that the glia surrounding the axons are responsible for the local protein synthesis, and that some of these newly synthesized proteins are transported into the axon, is presented. Transection of crayfish peripheral nerves proximal to the neuron cell bodies produced a more than two-fold increase in [3H]leucine incorporation, but no significant changes in labeling profiles of the proteins on SDS gels. The data suggest that while an active local protein synthesis may be necessary for the maintenance of several crayfish motor axons, it is not a sufficient condition.

Distribution of ultrastructural tracers in crustacean axons.

Ruthenium red and horseradish peroxidase were used to compare the uptake of exogenous molecules into crayfish motor axons and their sheaths in severed and intact peripheral nerves. Both tracers penetrated the axonal sheath and were subsequently seen lining small vesicles and tubules in the axoplasm. Tracer appeared to enter the axon via pinocytotic vesicles. There were no perceptible quantitative or qualitative differences in ruthenium red uptake between intact and severed axons. However, counts of tracer-filled vesicles in axons exposed to peroxidase showed that at least three times as much tracer penetrated the severed as opposed to the intact axons.

Effects of temperature on the ultrastructure of severed crayfish motor axons.

AbstractNerves containing opener and closer motor axons in the right claws of crayfish were severed and the animals maintained in three groups at 16, 22 or 28°C. After selected intervals ranging from 5 to 80 days the nerves were prepared for electron microscopy and examined 1.5 to 2 cm distal to the cut. The findings of our earlier study (Nordlander and Singer, '72) that motor axons of animals kept at 16°C showed no morphological evidence of degeneration was confirmed. At the elevated temperatures, however, 30% of the animals showed motor axon degeneration. There was no significant difference in the occurrence of degeneration between the 22 and 28° groups. In early stages degenerating axons were characterized by a central core of amorphous material in which some microtubules were embedded, a surrounding area with mitochondria and assorted vesicles, and often an interrupted or totally lacking axolemma. By two weeks after cutting the axons had completely degenerated. In some animals the axonal sheath later degenerated but in others it persisted for up to eight weeks. Motor axons which failed to degenerate at the higher temperatures showed reactive changes like those occurring at 16°C except that the changes progressed more rapidly; the glial wrappings were loosely arranged; glial cytoplasm swelled with organelles including mitochondria, endoplasmic reticulum and Golgi elements; and in advanced stages, regenerating axons appeared within the glial wrappings.