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The conduction velocity of the squid giant axon can reach 30 m/s. Is there any known example of an even faster conducting unmyelinated axons?
The conductance velocity in the unmyelinated axon has been calculated and measured to be proportional to the square root of the axon diameter (see for example: Rushton, 1951). Since the giant axon is, well, giant, it conducts much faster than others. AFAIK, all other large neurons studied are myelinated. Maybe try to find bigger squids! ;)
J.Z. AND THE DISCOVERY OF SQUID GIANT NERVE FIBRES
J.Z., to give him the title by which he was universally known, initially acquired an interest in cephalopods when working in Naples with Enrico Sereni in 1932 on the axons in the mantle connectives and stellar nerves of octopus. This led him to further studies at the Plymouth Marine Laboratory of some structures in the mantles of squid that he tentatively identified as giant nerve fibres (Young, 1936). In the summer of 1936 he visited Woods Hole in Massachusetts, determined to prove that these `curious structures' were in fact motor axons. With F. O. Schmitt and R. Bear, he successfully examined the axoplasm of axons from the mantle of the squid Loligo pealii with polarized light, but failed in attempts with Ralph Gerard, Detlev Bronk and Keffer Hartline to make any oscilloscope recordings of action potentials from single fibres. However, he and Hartline did better one day when they found that application of a solution of sodium citrate to one end of the supposed axons generated a rhythmic discharge at the other, showing that they were indeed nerve fibres. He then made a careful study of the anatomy of the mantles, and in his classical paper on `The functioning of the giant nerve fibres of the squid'(Young, 1938), he showed that the third order giant axons served to bring about the precisely coordinated contraction of the mantle causing expulsion of a powerful jet of water propelling the animals rapidly backwards or forwards according to the position of the funnel, sometimes accompanied by a slug of `ink' to assist the animal's escape.
Having confirmed that the squid giant axons did conduct action potentials,and having with R. J. Pumphrey in 1938(Young and Pumphrey, 1938)looked at the effect of their diameter on the rate of conduction, the only respect in which J.Z. subsequently involved himself in research on the ionic basis of conduction was to measure their electrolyte content(Young and Webb, 1945). He did, nevertheless, devote many years to an important series of observations at the Zoological Station in Naples on the mechanism of memory in octopus. And always interested in the animal as a whole he was working vigorously in the laboratory till the very end of his life on a wide range of problems. He will also be remembered as a teacher of great distinction, and as the author of two outstandingly wise and well-written textbooks on vertebrates and invertebrates.
It was, however, the introduction of giant nerve fibres by J.Z. that enabled the biophysics and biochemistry of excitable membranes to be properly studied in depth, which was said by Alan Hodgkin in 1973 to have done more for axonology than any other single advance in technique during the previous 40 years. J.Z. neatly summed up the impact that the discovery of giant motor axons would have on the field when he wrote `on account of their enormous size [the squid's giant nerve fibres] provide unique opportunities for study of the functioning of single neuromuscular units'(Young, 1938).
The first step in the exploitation of squid axons was taken in 1938 at Woods Hole by Kacy Cole and H. J. Curtis(Cole and Curtis, 1939) when they showed using external electrodes that during the passage of an impulse there was a rise and fall of the membrane conductance whose time course was very similar to that of the action potential. Then in the summer of 1939, both Curtis and Cole (1940, 1942) at Woods Hole, and Alan Hodgkin and Andrew Huxley (Hodgkin and Huxley, 1939) at the Laboratory of the Marine Biological Association in Plymouth, succeeded in slightly different ways in pushing long glass tubes, 0.1 mm in diameter and filled with K + solutions, for some distance into the axons and thus recording the potential internally from an undamaged part of the membrane. To their great surprise they found that at the peak of the conducted impulse the membrane potential did not, as was expected, fall close to zero, but was in fact substantially reversed.
After the end of six years of war that had interrupted biological research,the problem of accounting for the reversal of potential at the peak of the spike still remained unsolved. In writing up their 1939 experiments at greater length, Hodgkin and Huxley(1945) presented four elegantly argued alternative explanations, in none of which it was obvious that they had any faith. But then Alan Hodgkin dared to suggest that the permeability of the membrane to Na + ions might undergo a transient increase. Working with squid giant axons at Plymouth in 1947, he and Bernard Katz were able to establish that the sodium theory was sound(Hodgkin and Katz, 1949). As has been described vividly by Hodgkin in his autobiography Chance &Design (Hodgkin, 1992),the great experimental triumph that came next was his and Huxley's development at Plymouth of the voltage-clamp technique for the quantitative analysis of the relationship between current and voltage in an excitable membrane(Hodgkin and Huxley,1952).
There followed a series of research projects on related questions, for example the measurement of the net movements during the nerve impulse of sodium and potassium by Keynes and Lewis(1951) the establishment by Hodgkin and Keynes (1955a) of the existence of the sodium pump studies by Caldwell, Hodgkin, Keynes and Shaw (1960) on the dependence of the sodium pump on a supply of phosphate-bond energy from ATP and arginine phosphate the discovery of Hodgkin and Keynes(1955b) in Cambridge, using cuttlefish giant axons, of the manner in which K + ions diffused in single file through the voltage-gated potassium channels in nerve membranesand to crown Hodgkin's direct participation in experiments on squid axons, the development of a method for perfusing them with a variety of solutions after squeezing out the axoplasm as described by Baker, Hodgkin and Shaw(1962), in order to carry out further rigorous tests of the ionic theory.
During the 1960s and 1970s, experiments on squid giant fibres continued to occupy many axonologists, an advance of particular interest being the records made for the first time independently at Woods Hole by Armstrong and Bezanilla(1974) and at Plymouth by Keynes and Rojas (1974), of the sodium gating current. The existence of such currents generated by the transmembrane movements of the charged gating particles had been predicted by Hodgkin, but they had not previously been recorded because of their small size relative to the ion currents. Then in 1984 Numa and his colleagues in Kyoto had succeeded, as described by Noda et al.(1984), in cloning the sodium channel gene of the electric eel, and soon the primary amino acid sequences of the voltage-gated sodium, potassium and calcium channels in a great many animals were known. What is more, it turned out that the channel proteins could readily be expressed in Xenopus oocytes, where their properties could conveniently be examined by the patch-clamping techniques first developed by Neher and Sakmann(1976). Research on these lines is now being vigorously pursued in many laboratories all over the world on the properties of ion channels gated not only by membrane potential, but also by other agents.
Lowly squid's behavior may yield clues to human brain
The only time most of us think about this strange-looking sea creature is when it is served grilled, fried or basted in its own ink.
But the lowly squid is actually an intelligent invertebrate capable of learning complex behavior at a very young age.
A new study reveals that newborn squid actually learn through the process of trial and error, much like humans do, and that these early-life experiences can physically change a squid's nervous system in ways that may be permanent.
Photo: Hopkins researchers study the species Loligo opalescens, a common squid found in the Pacific Ocean off California. Adult squid like the one shown here are usually four to seven inches long. Courtesy: William Gilly.
These results also could provide new insight into how learning transforms the human brain, says William F. Gilly, a professor of cell and developmental biology at Stanford's Hopkins Marine Station.
Gilly and former postdoctoral fellow Thomas Preuss describe their latest findings on squid behavior in the January issue of The Journal of Experimental Biology.
"The squid is a mollusk -- an animal closely related to a clam," says Gilly, "but it has an amazingly rich behavioral repertoire. Its brain is probably as complicated as that of some mammals."
He points out that the squid is an ideal species for conducting neurological research, because its elaborate brain is connected to a set of giant axons -- the largest nerve cells in the animal kingdom (see illustration below).
When a newborn squid is frightened, its brain sends an electrical signal through the giant axons, causing the mantle muscles to automatically contract and discharge a jet of water. To gain voluntary control of its jet propulsion, the adult brain fires the small axon network first, bypassing the giant axons. Courtesy: William Gilly.
A giant axon can grow to be a millimeter wide, and its large size makes it much easier to measure electrical signals to and from the brain while the squid is carrying out various behaviors.
It is a well-known fact, write Gilly and Preuss, that a startled squid will release a powerful jet of water that propels its body forward or backward so it can escape predators.
This "startle-escape response" is similar to a reflex action and is triggered by the network of giant axons that connects the squid's brain to the muscles in its mantle -- the part of the body many of us like to eat.
When a squid is frightened, its brain sends an electrical signal through the giant axons in less than a tenth of a second -- an "all-or-nothing" impulse that causes the mantle muscles to involuntarily contract and discharge a jet of water.
Every squid is born with this startle-escape reflex, but to be successful in the wild, an animal must be able to voluntarily control and operate its jet propulsion system. That means preventing the giant axon network from automatically firing an all-or-nothing impulse.
And that's just what young squid start doing as soon as they are hatched.
According to Gilly and Preuss, the brain of a newborn squid quickly develops the ability to bypass the giant axons in favor of a parallel nerve network made up of small axons --- narrower neurons that control a different set of muscles in the mantle.
By the time it becomes an adult, a squid is able to regulate the force of its escape jet by simply activating the small axons first, then firing the giant axon network a fraction of a second later.
But is this ability to suppress the giant axon network genetically programmed in every squid, or is it a skill that each animal has to learn through experience?
To answer that question, Gilly and Preuss decided to focus on another important squid behavior that does depend on learning: the ability to hunt and capture prey.
Wild squid love to eat tiny crustaceans called copepods. But copepods are difficult to catch because they can detect and outswim a pursuing squid -- plus, copepods are covered with sharp, lobster-like spines (see drawing below).
Through the process of trial-and-error, a young squid learns that the best way to capture a copepod is not to chase it but to remain still, spread its eight tentacles like a net, then quickly grab the crustacean and bite into it.
But Gilly observed that, when a juvenile squid grasps its first copepod, it often releases the spiny crustacean and jets backward in a classic startle-escape response.
Perhaps the copepod's needlelike exoskeleton irritates and startles the young squid, triggering an all-or-nothing signal through its giant axons and causing it to involuntarily spurt water.
With practice, novice squid eventually learn to hold onto copepods without automatically jetting in reverse -- an observation that led Gilly and Preuss to suspect that a squid's control of its escape reflex goes hand-in-hand with the development of its hunting skills.
Speedy and slow hunters
To find out, the researchers set up an experiment using newly hatched eggs from squid collected in Monterey Bay.
Newborn animals were divided into two groups. One received a diet that included speedy copepods. The other was fed only slow-moving brine shrimp larvae, which are much easier to catch.
When a newly hatched squid sees a potential meal, its first reaction is to lunge at the prey as quickly as possible -- a strategy that worked well for the group that was given brine shrimp.
In fact, two months into the experiment, the majority of shrimp-eaters were still pouncing on their slow-moving prey instead of developing more subtle hunting techniques.
But a different strategy developed among copepod-fed squid. Despite repeated attempts to pounce on their prey, these young squid were never fast enough to capture the swift crustaceans.
After several weeks of trial and error, they finally became adept copepod hunters. They stopped involuntarily jetting around and learned instead to approach copepods stealthily and then grab them -- a technique none of the shrimp-fed squid ever developed.
Clearly, the two experimental groups had learned different styles of hunting. To determine if the animals' escape reflex had also changed, Gilly and Preuss wired each squid's nervous system to miniature electrodes to compare how copepod-eaters and shrimp-eaters would respond to a very brief electrical shock.
Electrode analysis revealed that, after just two weeks, most copepod-fed squid were indeed firing their small axons first, enabling them to control their automatic escape response. Without this important skill, a wild squid would continue to unintentionally dart backward every time it tried to grab a meal, greatly reducing its ability to capture prey.
It was a different story for the shrimp-fed squid.
Electro-analysis showed that, after eight weeks, most shrimp-eaters were still firing their giant axons first, much like newly hatched squid. They had not learned to control the involuntary escape response and were probably using this infantile reflex to lunge at their prey.
"Furthermore," say the authors, "when switched to a copepod diet, these animals show no sign of developing the suppression of jetting that is necessary for captures" -- evidence that voluntary control of jet propulsion is indeed a behavior that must be learned at an early age.
"The inability of shrimp-fed squid to master copepod capture later in life implies that there is a short window of opportunity during the first weeks after birth in which benefit can be derived from trial-and-error experience," adds Gilly.
"If the squid does not learn to control its startle-escape reflex during that critical period, it seems to lose the ability to program its nervous system in a way that allows it to perform the sophisticated hunting skills that are necessary to survive in the wild."
This suggests that the process of learning by trial and error causes actual physical changes in the squid's neurons, says Gilly.
Similar findings have been made in vertebrates, including birds, cats and humans.
For example, research on newborn cats and monkeys has shown that sensory visual deprivation early in life leads to the loss of specific neurons in the brain that would normally respond to the visual images missing during development.
These experiments revealed that a critical time period exists when the effects of learned experience are beneficial.
But, Gilly notes, if the experience comes too late, it may do no good at all.
"This body of work also strongly supports the idea that a rich sensory environment is important for normal brain development in humans," Gilly points out.
He says that discovering exactly how a particular experience acts to modify specific neurons and guarantee their survival is one of the major challenges in neuroscience today.
And it's the unique anatomy of the squid that could allow a breakthrough in our understanding of how learning causes physical alterations in the brain.
"The simplicity of the squid's giant axon system will be advantageous in identifying the genes and chemicals involved in causing and maintaining these cellular changes -- even in people," Gilly predicts.
"In this way, the delectable calamari may actually help unlock the secret of how our own brain cells are modified by early childhood experiences and help explain why we are who we are." SR
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Conduction failures at moderately elevated temperatures in cerebellum and hippocampus
To detect conduction failures, we recorded compound action potentials (cAPs) from cerebellar parallel fibers with two recording electrodes positioned at different distances along the axonal path (Fig. 1A). Fast synaptic signals were blocked in all experiments presented in this article (see Methods). Elevation of the bath temperature from 33 to 42°C reduced the cAP amplitudes at both electrodes (Fig. 1B). The cAPs increased again as temperature was reduced to 33°C, showing that the amplitude drop was not caused by irreversible damage (Fig. 1C).
The observation that the cAP almost disappeared at the highest temperatures is likely due to failure to activate or failure to propagate the spikes. To distinguish those two possibilities, we compared the reduction of the proximal and distal cAP (cAP-p and cAP-d, respectively). The cAPs were measured as the SD of the response in a time window that bracketed the cAP at all temperatures (gray area in Fig. 1B and G), and noise was subtracted, detailed in Methods. Because cAP-p was usually larger than cAP-d, we normalized them to their average values <37°C. We could then see that cAP-d dropped more than cAP-p at temperatures >37°C (displayed as logarithmic values from a typical experiment in Fig. 1D). The measurement of cAP as peak-to-peak amplitude (Fig. 1E) within the gray window gave similar results as SD at high amplitudes, but because of better performance at low amplitudes, as explained in Methods, we used SD for the remaining experiments. The bigger drop in amplitude of cAP-d, relative to cAP-p, at high temperatures was also illustrated by normalizing both cAPs to cAP-p (so that cAP-p has the peak-to-peak value 1.0) at 35, 37, and 40°C (Fig. 1F). Myelinated fibers in alveus (Fig. 1G) did not show a selective drop in cAP-d, when compared with cAP-p (Fig. 1H). This suggests that the temperature conduction failures were specific for thin, unmyelinated axons.
The temperature-sensitive drop of cAP-d found in unmyelinated fibers of cerebellum was confirmed by comparing cAP-p and cAP-d in 78 experiments from rats 10–25 days old (average 17.9 days) at different temperatures. Increasing temperature reduced all cAPs to very small values (Fig. 2A), displayed in Figure 2B as logarithmic values. Similar to the example in Figure 1D, we normalized cAP-p and cAP-d to their average temperatures <37°C (Fig. 2C). Visual inspection of cAP-p and cAP-d from all experiments suggested that increasing temperature reduced cAP-d more than cAP-p (Fig. 2C).
To evaluate if cAP-d dropped more than cAP-p, we calculated the ratio cAP-d/cAP-p in a range of temperatures (Fig. 2D). The average of these ratios showed that cAP-d dropped more than cAP-p at temperatures >39°C (first temperature bin significantly < 0, P = 0.013, was the bin 39–40°C). This preferential distal-recording (cAP-d) drop contrasted with the proportional drop of cAP-p and cAP-d at temperatures <39°C (values around zero in the logarithmic plot in Fig. 2D) and strongly suggested that some spikes failed to propagate between the two recording electrodes at temperatures >39°C.
The effect was not isolated to the youngest animals because the groups 10–14 and 17–25 days old, both showed significant drops in cAP-d/cAP-p ratio at high temperatures (Fig. 2D, right), although the youngest group was slightly more temperature sensitive because their cAP-d/cAP-p ratio dropped significantly >39°C while the oldest group dropped significantly >40°C.
The temperature-sensitive conduction failures may be typical for gray matter axons because such axons in hippocampus also showed preferential drop at the distal recording electrode, at temperatures >38°C (Fig. 2E, average age 20.5 days, n = 25), although of smaller magnitude than in the parallel fibers in cerebellum. Furthermore, thin, unmyelinated CNS axons may be uniquely temperature sensitive because myelinated CNS axons (hippocampal alveus fibers) did not show signs of failures at comparable temperatures (Fig. 2F, average age 21.4 days, n = 16).
Block of voltage-sensitive K + channels reduced temperature-induced failures
One of the hallmarks of conduction failures is that spike broadening, either by reducing temperature or by drugs, help overcome some of the failures (Bostock et al. 1978 Westerfield et al. 1978 Swadlow et al. 1980 ). We tested this idea using 1 mmol/L TEA in the extracellular solution, shown to efficiently block fast Kv3 channels (Rudy and McBain 2001 ) and also known to broaden the spike in cerebellar parallel fibers (Sabatini and Regehr 1997 Matsukawa et al. 2003 Pekala et al. 2014 ). Figure 3A presents two typical experiments with approximately 2°C higher threshold for conduction failures when exposed to TEA.
On average, there were fewer conduction failures at 39.5 and 40.5°C with TEA than without (Fig. 3B). The average may underestimate the TEA effect because the drop in cAP-d/cAP-p ratio occurred at slightly different temperatures in different experiments (like the two illustrated in Fig. 3A). We therefore calculated the difference between log (cAP-d/cAP-p) with and without TEA in the individual experiments. The average of these pairwise differences confirmed that TEA significantly increased the threshold for conduction failures at 39.5 and 40.5°C (Fig. 3C). At higher temperature (41.5°C), the variability prevented clear significance (P = 0.053), which may be due to the fact that cAP magnitude was very small, and also that conditions for spike conduction deteriorated beyond rescue.
The finding that 1 mmol/L TEA reduced the preferential distal-electrode drop supports the hypothesis that voltage-sensitive potassium channels (Kv) contributed to the conduction failures observed at high temperatures, although TEA may have reduced failures caused by other mechanisms too.
Do spikes improve the conduction fidelity of the following spikes?
Many thin, unmyelinated CNS axons have a depolarizing after-potential (DAP) that offers an explanation of the hyper-excitable period following individual spikes in such axons (Gardner-Medwin 1972 Wigstrom and Gustafsson 1981 Palani et al. 2012 ). We therefore tested if the DAP could help spikes propagate at high temperatures.
We repeated the parallel fiber stimulus four times with 20 msec intervals, which is within the duration of the DAP (Palani et al. 2012 Pekala et al. 2014 ) and within the range of granule cell firing frequencies in vivo (Eccles et al. 1966 Chadderton et al. 2004 Jorntell and Ekerot 2006 ). With increasing temperature, all four cAPs declined. The first response at the proximal and distal electrodes were analyzed previously (Figs. 1 and 2) and showed that cAP-d fell more than cAP-p at temperatures >38.5°C, interpreted as conduction failures. Interestingly, during the train of stimuli, we observed that the last cAP (cAP4) in the train declined less than the first cAP at elevated temperatures (cAP1, shown at 36 and 40°C in Fig. 4A from a typical experiment).
The reduced drop of cAP4 relative to cAP1 was quantified similarly to the analysis of cAP-p and cAP-d (details in Methods). First, we normalized cAP1 and cAP4 to their average values <37°C, showing that cAP1 fell more than cAP4 at temperatures >38°C (Fig. 4B, same experiment as in A).
Example traces from 37, 38, and 40°C, normalized to the cAP1 peak-to-peak values (Fig. 4C) show that relative to cAP1, cAP4 was much larger at 38 and 40°C, meaning that cAP4 fell less than cAP1 at those temperatures. Similarly, the cAP4/cAP1 ratio showed that cAP4 declined less than cAP1 at temperatures >37°C (Fig. 4D). On average, the cAP4/cAP1 ratios increased significantly at temperatures from 37.5 to 41.5°C (Fig. 4E). A possible explanation for this is that there were fewer conduction failures at cAP4 compared to cAP1 at the high temperatures because this was in the temperature range in which conduction failures were detected by the cAP-d/cAP-p ratio (Fig. 2). If so, larger values of cAP4/cAP1 would be expected to be associated with the smaller values of cAP-d/cAP-p, and more so with increasing temperature, exactly as seen in Figure 4F (n = 78).
Theoretically, excitability may have increased during the four-stimulus train due to accumulation of extracellular K + . If more K + accumulated at high than low temperature, it may offer an explanation for the temperature-sensitive correlation shown in Figure 4F. However, based on the results described in the following section, we find that unlikely.
Latency changes suggested an excitability increase of constant time course and magnitude following each spike
A spike with a DAP would be expected to speed up conduction of the spike that follows at its tail. This was exactly what we observed, the latencies of the second (Lat2), third (Lat3), and fourth (Lat4) cAPs were shorter than the latency of first (Lat1) cAP (interval = 20 msec, at 37°C, Fig. 5A and B). The reduced latency already at Lat2 confirmed that the axons were more excitable after being activated once (Gardner-Medwin 1972 Wigstrom and Gustafsson 1981 Soleng et al. 2004 ). This effect did not accumulate during the train, observed as overlap of the three last responses (Fig. 5A, right) and was confirmed by similar reduction of Lat2-4 in 20 experiments (at 37°C, Lat2-4 were: 85 ± 2%, 84 ± 1%, and 84 ± 1% of Lat1, respectively, Fig. 5B).
Although the latency to each cAP was shorter at higher temperature, the relative reductions in Lat2-4 were similar (Fig. 5C). At 40°C, Lat2-4 were 89 ± 3%, 91 ± 3%, and 89 ± 3% of Lat1, respectively (all P < 0.003). Lat2-4 were not significantly different from each other (all P > 0.4, n = 20, data not shown).
Such a very stable reduction of Lat2-4 is unlikely to be due to factors that accumulate with repeated spikes, such as extracellular K + , unless the maximal velocity was already reached at Lat2 (a “ceiling effect”). To test for a ceiling effect, we used different stimulus intervals (10, 20, and 30 msec). We found that the maximal velocity was not reached because the different intervals resulted in different latency changes. Average values of Lat2-4 were different, 82 ± 1%, 84 ± 1%, and 89 ± 2% of Lat1, for 10, 20, and 30 msec stimulus intervals, respectively (Fig. 5D).
At the longest intervals (30 msec), the latency reduction was not only the smallest but also had the lowest variability between Lat2-4 (Fig. 5E, coefficient of variation 3.2 ± 0.8%, 2.0 ± 0.7%, 0.8 ± 0.2% at 10, 20, and 30 msec intervals, respectively). This is opposite of what would be expected by a ceiling effect, which would occur at the shortest latency when the axon could not conduct faster. We therefore conclude that the reduced latency was due to an excitability-increasing process of constant time course and magnitude, relatively activity independent.
The DAP in parallel fibers helps propagate spikes
Our hypothesis was that the axonal DAP increased excitability after individual spikes and helped conduction in the temperature range where some axons failed. To test this, we electrically activated the parallel fibers while recording antidromic spikes from the granule cell soma. The recorded cells were assumed to be granule cells based on their location in stratum granulosum, activation from stratum moleculare with a latency, and their high resistance to somatic current injections (Fig. 6A, mean = 2.10 GΩ, SD = 0.72 GΩ, n = 14, similar to e.g., Diwakar et al. 2009 ). The average latency between stimulus and the peak of the spike was 2.00 msec (SD = 1.39 msec, n = 14, Fig. 6B, green traces).
50% invasion failures at the first response in the train of four stimuli. In the presented example, seven consecutive stimuli of equal strength gave five failures and two successes at the first stimulus. (D) The same traces as in C, but vertically separated so that the individual four-stimulus train can be seen. When spike was detected (full or attenuated, in this example, attenuated), the following stimuli always elicited a spike that propagated far enough to be detected at the soma. (E) The average success fraction at the first stimulus in 16 experiments (open bar). When there was a failure at first, second, or third stimulus, the success rate for the following stimulus did not increase (red bars). However, when a successful spike was detected at the soma at first, second, or third stimulus (blue bars), a success followed almost always (only two failures after totally 1784 successes).
At moderately hyperpolarized potentials (−72 mV, Fig. 6B, red traces) compared to rest (−66 mV, Fig. 6B, green traces), the fast component of the spike disappeared, probably because spike conduction failed at some distance from the soma and the fast component was attenuated by the axonal cable (Sheffield et al. 2011 ). At even more hyperpolarized potentials, also the slow component disappeared (−75 mV, Fig. 6B, gray traces). We adjusted the somatic potential to give
50% failures. The responses to identical stimuli showed spike-like all-or-none behavior (red and gray traces).
50% failures at a constant stimulus strength, we repeated the stimulus four times and observed that if the axon conducted one spike, the following stimulus always resulted in a successfully conducted spike (Fig. 6C and D). By counting failures and spikes (including attenuated spikes, like the red traces in B), we observed 1216 failures immediately after a failure, but only two failures immediately after 1784 successfully propagated spikes (16 experiments). This confirms that a successful spike (full or attenuated) increased the chance of successful conduction for the following spikes.
Unless a successful conduction occurred, the success rate did not increase during the train (red bars Fig. 6E). This means that extracellular K + or other excitability-increasing effects from adjacent axons or other cells had minor effects on the excitability compared to the strong effect of a successfully propagated spike which abolished almost all failures at the following stimulus.
A likely explanation for the failure-reducing effect of a single spike is that residual depolarization (the DAP) left from the preceding spike helped overcome regions of low excitability. The DAP may therefore offer an explanation for the larger cAP amplitudes at the fourth stimulus compared to the first in the temperature range that induced failures (Fig. 4).
Constant amplitude and waveform of the DAP
For the DAP to be an acceptable explanation for the very similar latency reduction at all three cAPs (Lat2-4, Fig. 5), the DAP would need to have a relatively constant amplitude at a given interval after each spike, which we will address in this section. The intracellular DAPs of the antidromic spikes, measured on the decay of responses 1–4 immediately before the following stimulus (arrows in Fig. 7A) were very similar during the train of four stimuli (Fig. 7B, −57.0 ± 2.6, −56.7 ± 1.9, −54.7 ± 2.5, and 56.6 ± 2.7 mV). The SD of the membrane potential of those four DAPs in the individual experiments was only 0.75 ± 0.11 mV on average for 14 experiments. That low variability in the membrane potential may explain the similar latency reductions at cAP2–4 (Fig. 5).
Because the initial segment of the axon has other membrane currents, axial resistance, and input resistance than more distal parts (Kole and Stuart 2012 ) which theoretically could influence our somatically recorded DAPs, we also recorded the waveform of the axonal spikes far away from the soma using a miniaturized grease-gap method (Palani et al. 2012 ).
Grease-gap recordings give information about the temporal changes of the transmembrane voltage, but not the true voltage. Therefore, to compare intracellular and grease-gap recordings, we used a measure we could obtain from both recording techniques, namely the difference between DAP amplitude and baseline measured before the first stimulus (arrows in Fig. 7A and C). To compare the DAP variability recorded by different techniques on a similar scale, we normalized the amplitudes to the value of the first DAP amplitude, both in intracellular and grease-gap recordings (Fig. 7D).
The DAP variability between responses 1–4, measured as coefficient of variation (SD/mean) was 0.095 ± 0.019 (n = 14) and 0.090 ± 0.011 (n = 23, P = 0.84, data not shown) for the intracellular and grease-gap measurements, respectively. This similarity between the variation of the DAPs measured intrasomatically and by the grease-gap technique supports the hypothesis that both those techniques detect features of the axonal DAP. The fact that the variability was similar also suggests that the axons may have a DAP variability of approximately 0.75 mV, as recorded at the soma, also further away from the soma. Additionally, these data show that the axonal DAP was relatively resistant to moderate activity (the repetition of four spikes).
Influence of temperature on action potential and membrane potential
When Hodgkin and Katz ( 1949 ) described the temperature effects on the propagating spike in the squid giant axon, they noted that increasing temperature caused a small depolarization of the resting membrane potential, but a large amplitude- and width-reducing effect on the spike. This led them to conclude that the spike failure was not simply due to loss of membrane potential, but depended on spike-specific mechanisms.
Since it has not yet been possible to measure membrane potential directly in thin unmyelinated cortical axons, we measured steady-state membrane potential at the soma using tight-seal recordings. However, fast voltage changes in the axon, like the spike, are probably not well represented at the soma. For example, Kv10.1 is located almost exclusively to the axon, and most likely influences spike repolarization, but no effects of this could be detected by somatic recordings (Mortensen et al. 2015 ). We therefore measured cAP amplitude from the parallel fibers using a grease-gap method (Palani et al. 2012 ). Somatic membrane potential and grease-gap recorded cAP will be influenced by axonal membrane potential and spike amplitude, respectively, although further qualifications will be given in the Discussion.
The results were qualitatively very similar to Hodgkin and Katz ( 1949 ) (Fig. 8A). Membrane potential depolarized by 3% (0.97 ± 0.02, n = 8) at 40°C compared to its value at 37°C (average membrane potential before normalization at 37°C = −66.0 ± 3.4 mV). In contrast, grease-gap peak cAP decreased by 37% (0.63 ± 0.06, n = 13) at 40°C compared to its value at 37°C.
If the DAP helps to reduce failures of spike propagation during elevated temperatures, as we propose, it should be present also at the temperatures at which axons start to fail. This was indeed the case, as shown by the average shape of potentials from nine grease-gap recordings at 36 and 40°C, temporally aligned at their peak amplitude (Fig. 8B).
This activity is suitable for high school students and above who have a basic conceptual understanding of what an axon and an action potential are. Since electrophysiology is novel for many students and the methods are sensitive to electromagnetic noise that can easily confuse and frustrate students, close observation and help by the teacher during experiments (especially during data collection for the first worm) is essential (see Troubleshooting below). A teacher may find doing a quick demonstration of one conduction velocity reading at the beginning of class to be helpful.
The Basis of Cellular Compartmentation
Role of cis-acting elements in RNA targeting
The prevailing view now is that structural and functional asymmetries of various cell phenotypes are determined by the distribution of RNAs to subcellular domains. Numerous examples of the importance of translation of localized specific mRNAs to local function have been described in cells ranging from fibroblasts to neurons (see 8 ). As one of the most highly polarized cell, the translation of mRNAs in neuronal specific subcellular domains appears to confer its characteristic regional functionality. Nucleotide sequences, referred to as cis-acting elements, located in the 3′ or 5′ UTR of mRNAs are responsible for targeting to specific cellular domains ( 26 ). Although there is little or no homology between such sequences, trans-acting factor (TAF) proteins have been found to bind cis-acting regions of mRNAs, and thereby form RNPs. They have been found associated with molecular motor proteins, which suggest that the latter may translocate them over cytoskeletal transport pathways 7-9 ). Such a mechanism may mediate the targeting of tau mRNA and β-actin mRNA to immature axons. The cis-acting region of tau mRNA has a 240 bp sequence localized in the 3′UTR, which is bound by HuD, a TAF protein ( 27 ). The cis acting 54 nt sequence in the 3 ′UTR of β-actin, referred to as the ‘zipcode’, is absent in other actin isoforms and is responsible for specific targeting to distal growing axons by binding to ZBP1 ( 28 ). The targeting of β-actin mRNA and local translation in growth cones is believed to account for actin enrichment needed for growth cone motility during axon elongation ( 28, 29 ). A 65 nt sequence in the 5′UTR of BC1 RNA, an untranslated neuronal cytoplasmic RNA specific to rodent brain, has been shown to be sufficient for targeting to the Mauthner axon ( 30 ), despite the fact that it is a heterologous transcript. Interestingly, BC1 RNA has been shown to act as a repressor of translation ( 9 ).
Role of trans-acting factors in RNA targeting
Ribonucleoprotein particles vary in composition and include multiple mRNAs, ribosomes, proteins, other yet uncharacterized components, as well as molecular motor proteins. As such they are considered metabolically silent ( 31 ). Among mRNA binding proteins that have been described are mStaufen, Hu family of proteins (considered mRNA stabilizers), FMRP (Fragile X Mental Retardation Protein), and ZBP1 (for review, see 8 ). TAF proteins, such as Pur α, mStaufen and FMRP, were associated with ribosomes, KIF5A (kinesin I) and myosin Va molecular motor proteins, and ER in the same particle ( 32 ). Tau mRNA, HuD, and KIF3A (a subunit of kinesin II) were associated with microtubules in P19 neurons in culture, and Tau mRNA with HuD in PC-12 cells, both depending on an uracyl-rich element in the 3′UTR ( 27, 33 ). To date, molecular motor proteins such as Kinesins I and II ( 27, 34 ), and myosin II and V ( 32, 35 ) have been proposed to mediate RNA trafficking in neurons, and other cell types. However, the only experimental demonstration that a motor protein binds mRNA via adaptor proteins was made in yeasts ( 12 ). In the latter case, binding protein She2p recruits the Myo4p-She3p complex to ASH1 mRNA. The mechanism whereby adaptor proteins bind to mRNAs and interact with appropriate molecular motor proteins in neurons needs yet to be elucidated. Given the variety of molecular motor protein isoforms and their adaptor molecules, it is likely that more possible combinations will be discovered that could transport RNA cargo.
Is the squid giant axon the fastest conducting unmyelinated axon known? - Biology
An axon (from Greek ἄξων ''áxōn'', axis), or nerve fiber (or nerve fibre: see spelling differences), is a long, slender projection of a nerve cell, or neuron, in vertebrates, that typically conducts electrical impulses known as action potentials away from the nerve cell body. The function of the axon is to transmit information to different neurons, muscles, and glands. In certain sensory neurons (pseudounipolar neurons), such as those for touch and warmth, the axons are called afferent nerve fibers and the electrical impulse travels along these from the periphery to the cell body, and from the cell body to the spinal cord along another branch of the same axon. Axon dysfunction has caused many inherited and acquired neurological disorders which can affect both the peripheral and central neurons. Nerve fibers are classed into three types – group A nerve fibers, group B nerve fibers, and group C nerve fibers. Groups A and B are myelinated, and group C are unmyelinated. These groups include both sensory fibers and motor fibers. Another classification groups only the sensory fibers as Type I, Type II, Type III, and Type IV. An axon is one of two types of cytoplasmic protrusions from the cell body of a neuron the other type is a dendrite. Axons are distinguished from dendrites by several features, including shape (dendrites often taper while axons usually maintain a constant radius), length (dendrites are restricted to a small region around the cell body while axons can be much longer), and function (dendrites receive signals whereas axons transmit them). Some types of neurons have no axon and transmit signals from their dendrites. In some species, axons can emanate from dendrites known as axon-carrying dendrites. No neuron ever has more than one axon however in invertebrates such as insects or leeches the axon sometimes consists of several regions that function more or less independently of each other. Axons are covered by a membrane known as an axolemma the cytoplasm of an axon is called axoplasm. Most axons branch, in some cases very profusely. The end branches of an axon are called telodendria. The swollen end of a telodendron is known as the axon terminal which joins the dendron or cell body of another neuron forming a synaptic connection. Axons make contact with other cells—usually other neurons but sometimes muscle or gland cells—at junctions called synapses. In some circumstances, the axon of one neuron may form a synapse with the dendrites of the same neuron, resulting in an autapse. At a synapse, the membrane of the axon closely adjoins the membrane of the target cell, and special molecular structures serve to transmit electrical or electrochemical signals across the gap. Some synaptic junctions appear along the length of an axon as it extends—these are called ''en passant'' ("in passing") synapses and can be in the hundreds or even the thousands along one axon. Other synapses appear as terminals at the ends of axonal branches. A single axon, with all its branches taken together, can innervate multiple parts of the brain and generate thousands of synaptic terminals. A bundle of axons make a nerve tract in the central nervous system, and a fascicle in the peripheral nervous system. In placental mammals the largest white matter tract in the brain is the corpus callosum, formed of some 200 million axons in the human brain.
thumb|right|A dissected human brain, showing grey_matter_and_[[white_matter_.html" style="text-decoration: none"class="mw-redirect" title="white_matter.html" style="text-decoration: none"class="mw-redirect" title >grey matter and [[white matter ">white_matter.html" style="text-decoration: none"class="mw-redirect" title >grey matter and [[white matter Axons are the primary transmission lines of the [[nervous system]], and as bundles they form [[nerve]]s. Some axons can extend up to one meter or more while others extend as little as one millimeter. The longest axons in the human body are those of the [[sciatic nerve]], which run from the base of the spinal cord to the big toe of each foot. The diameter of axons is also variable. Most individual axons are microscopic in diameter (typically about one micrometer (µm) across). The largest mammalian axons can reach a diameter of up to 20 µm. The squid giant axon, which is specialized to conduct signals very rapidly, is close to 1 millimetre in diameter, the size of a small pencil lead. The numbers of axonal telodendria (the branching structures at the end of the axon) can also differ from one nerve fiber to the next. Axons in the central nervous system (CNS) typically show multiple telodendria, with many synaptic end points. In comparison, the cerebellar granule cell axon is characterized by a single T-shaped branch node from which two parallel fibers extend. Elaborate branching allows for the simultaneous transmission of messages to a large number of target neurons within a single region of the brain. There are two types of axons in the nervous system: myelinated and unmyelinated axons. Myelin is a layer of a fatty insulating substance, which is formed by two types of glial cells: Schwann cells and oligodendrocytes. In the peripheral nervous system Schwann cells form the myelin sheath of a myelinated axon. In the central nervous system oligodendrocytes form the insulating myelin. Along myelinated nerve fibers, gaps in the myelin sheath known as nodes of Ranvier occur at evenly spaced intervals. The myelination enables an especially rapid mode of electrical impulse propagation called saltatory conduction. The myelinated axons from the cortical neurons form the bulk of the neural tissue called white matter in the brain. The myelin gives the white appearance to the tissue in contrast to the grey matter of the cerebral cortex which contains the neuronal cell bodies. A similar arrangement is seen in the cerebellum. Bundles of myelinated axons make up the nerve tracts in the CNS. Where these tracts cross the midline of the brain to connect opposite regions they are called ''commissures''. The largest of these is the corpus callosum that connects the two cerebral hemispheres, and this has around 20 million axons. The structure of a neuron is seen to consist of two separate functional regions, or compartments – the cell body together with the dendrites as one region, and the axonal region as the other.
The axonal region or compartment, includes the axon hillock, the initial segment, the rest of the axon, and the axon telodendria, and axon terminals. It also includes the myelin sheath. The Nissl bodies that produce the neuronal proteins are absent in the axonal region. Proteins needed for the growth of the axon, and the removal of waste materials, need a framework for transport. This axonal transport is provided for in the axoplasm by arrangements of microtubules and intermediate filaments known as neurofilaments.
The axon hillock is the area formed from the cell body of the neuron as it extends to become the axon. It precedes the initial segment. The received action potentials that are summed in the neuron are transmitted to the axon hillock for the generation of an action potential from the initial segment.
The axonal initial segment (AIS) is a structurally and functionally separate microdomain of the axon. One function of the initial segment is to separate the main part of an axon from the rest of the neuron another function is to help initiate action potentials. Both of these functions support neuron cell polarity, in which dendrites (and, in some cases the soma) of a neuron receive input signals at the basal region, and at the apical region the neuron's axon provides output signals. The axon initial segment is unmyelinated and contains a specialized complex of proteins. It is between approximately 20 and 60 µm in length and functions as the site of action potential initiation. Both the position on the axon and the length of the AIS can change showing a degree of plasticity that can fine-tune the neuronal output. A longer AIS is associated with a greater excitability. Plasticity is also seen in the ability of the AIS to change its distribution and to maintain the activity of neural circuitry at a constant level. The AIS is highly specialized for the fast conduction of nerve impulses. This is achieved by a high concentration of voltage-gated sodium channels in the initial segment where the action potential is initiated. The ion channels are accompanied by a high number of cell adhesion molecules and scaffolding proteins that anchor them to the cytoskeleton. Interactions with ankyrin G are important as it is the major organizer in the AIS.
The axoplasm is the equivalent of cytoplasm in the cell. Microtubules form in the axoplasm at the axon hillock. They are arranged along the length of the axon, in overlapping sections, and all point in the same direction – towards the axon terminals. This is noted by the positive endings of the microtubules. This overlapping arrangement provides the routes for the transport of different materials from the cell body. Studies on the axoplasm has shown the movement of numerous vesicles of all sizes to be seen along cytoskeletal filaments – the microtubules, and neurofilaments, in both directions between the axon and its terminals and the cell body. Outgoing anterograde transport from the cell body along the axon, carries mitochondria and membrane proteins needed for growth to the axon terminal. Ingoing retrograde transport carries cell waste materials from the axon terminal to the cell body. Outgoing and ingoing tracks use different sets of motor proteins. Outgoing transport is provided by kinesin, and ingoing return traffic is provided by dynein. Dynein is minus-end directed. There are many forms of kinesin and dynein motor proteins, and each is thought to carry a different cargo. The studies on transport in the axon led to the naming of kinesin.
In the nervous system, axons may be myelinated, or unmyelinated. This is the provision of an insulating layer, called a myelin sheath. The myelin membrane is unique in its relatively high lipid to protein ratio. In the peripheral nervous system axons are myelinated by glial cells known as Schwann cells. In the central nervous system the myelin sheath is provided by another type of glial cell, the oligodendrocyte. Schwann cells myelinate a single axon. An oligodendrocyte can myelinate up to 50 axons. The composition of myelin is different in the two types. In the CNS the major myelin protein is proteolipid protein, and in the PNS it is myelin basic protein.
Nodes of Ranvier (also known as ''myelin sheath gaps'') are short unmyelinated segments of a myelinated axon, which are found periodically interspersed between segments of the myelin sheath. Therefore, at the point of the node of Ranvier, the axon is reduced in diameter. These nodes are areas where action potentials can be generated. In saltatory conduction, electrical currents produced at each node of Ranvier are conducted with little attenuation to the next node in line, where they remain strong enough to generate another action potential. Thus in a myelinated axon, action potentials effectively "jump" from node to node, bypassing the myelinated stretches in between, resulting in a propagation speed much faster than even the fastest unmyelinated axon can sustain.
An axon can divide into many branches called telodendria (Greek–end of tree). At the end of each telodendron is an axon terminal (also called a synaptic bouton, or terminal bouton). Axon terminals contain synaptic vesicles that store the neurotransmitter for release at the synapse. This makes multiple synaptic connections with other neurons possible. Sometimes the axon of a neuron may synapse onto dendrites of the same neuron, when it is known as an autapse.
Most axons carry signals in the form of action potentials, which are discrete electrochemical impulses that travel rapidly along an axon, starting at the cell body and terminating at points where the axon makes synaptic contact with target cells. The defining characteristic of an action potential is that it is "all-or-nothing" — every action potential that an axon generates has essentially the same size and shape. This all-or-nothing characteristic allows action potentials to be transmitted from one end of a long axon to the other without any reduction in size. There are, however, some types of neurons with short axons that carry graded electrochemical signals, of variable amplitude. When an action potential reaches a presynaptic terminal, it activates the synaptic transmission process. The first step is rapid opening of calcium ion channels in the membrane of the axon, allowing calcium ions to flow inward across the membrane. The resulting increase in intracellular calcium concentration causes synaptic vesicles (tiny containers enclosed by a lipid membrane) filled with a neurotransmitter chemical to fuse with the axon's membrane and empty their contents into the extracellular space. The neurotransmitter is released from the presynaptic nerve through exocytosis. The neurotransmitter chemical then diffuses across to receptors located on the membrane of the target cell. The neurotransmitter binds to these receptors and activates them. Depending on the type of receptors that are activated, the effect on the target cell can be to excite the target cell, inhibit it, or alter its metabolism in some way. This entire sequence of events often takes place in less than a thousandth of a second. Afterward, inside the presynaptic terminal, a new set of vesicles is moved into position next to the membrane, ready to be released when the next action potential arrives. The action potential is the final electrical step in the integration of synaptic messages at the scale of the neuron. Extracellular recordings of action potential propagation in axons has been demonstrated in freely moving animals. While extracellular somatic action potentials have been used to study cellular activity in freely moving animals such as place cells, axonal activity in both white and gray matter can also be recorded. Extracellular recordings of axon action potential propagation is distinct from somatic action potentials in three ways: 1. The signal has a shorter peak-trough duration (
150μs) than of pyramidal cells (
250μs). 2. The voltage change is triphasic. 3. Activity recorded on a tetrode is seen on only one of the four recording wires. In recordings from freely moving rats, axonal signals have been isolated in white matter tracts including the alveus and the corpus callosum as well hippocampal gray matter. In fact, the generation of action potentials in vivo is sequential in nature, and these sequential spikes constitute the digital codes in the neurons. Although previous studies indicate an axonal origin of a single spike evoked by short-term pulses, physiological signals in vivo trigger the initiation of sequential spikes at the cell bodies of the neurons. In addition to propagating action potentials to axonal terminals, the axon is able to amplify the action potentials, which makes sure a secure propagation of sequential action potentials toward the axonal terminal. In terms of molecular mechanisms, voltage-gated sodium channels in the axons possess lower threshold and shorter refractory period in response to short-term pulses.
The development of the axon to its target, is one of the six major stages in the overall development of the nervous system. Studies done on cultured hippocampal neurons suggest that neurons initially produce multiple neurites that are equivalent, yet only one of these neurites is destined to become the axon. It is unclear whether axon specification precedes axon elongation or vice versa, although recent evidence points to the latter. If an axon that is not fully developed is cut, the polarity can change and other neurites can potentially become the axon. This alteration of polarity only occurs when the axon is cut at least 10 μm shorter than the other neurites. After the incision is made, the longest neurite will become the future axon and all the other neurites, including the original axon, will turn into dendrites. Imposing an external force on a neurite, causing it to elongate, will make it become an axon. Nonetheless, axonal development is achieved through a complex interplay between extracellular signaling, intracellular signaling and cytoskeletal dynamics.
The extracellular signals that propagate through the extracellular matrix surrounding neurons play a prominent role in axonal development. These signaling molecules include proteins, neurotrophic factors, and extracellular matrix and adhesion molecules. Netrin (also known as UNC-6) a secreted protein, functions in axon formation. When the UNC-5 netrin receptor is mutated, several neurites are irregularly projected out of neurons and finally a single axon is extended anteriorly. Neuroglia and pioneer neurons express UNC-6 to provide global and local netrin cues for guiding migrations in ''C. elegans'' The neurotrophic factors – nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NTF3) are also involved in axon development and bind to Trk receptors. The ganglioside-converting enzyme plasma membrane ganglioside sialidase (PMGS), which is involved in the activation of TrkA at the tip of neutrites, is required for the elongation of axons. PMGS asymmetrically distributes to the tip of the neurite that is destined to become the future axon.
During axonal development, the activity of PI3K is increased at the tip of destined axon. Disrupting the activity of PI3K inhibits axonal development. Activation of PI3K results in the production of phosphatidylinositol (3,4,5)-trisphosphate (PtdIns) which can cause significant elongation of a neurite, converting it into an axon. As such, the overexpression of phosphatases that dephosphorylate PtdIns leads into the failure of polarization.
The neurite with the lowest actin filament content will become the axon. PGMS concentration and f-actin content are inversely correlated when PGMS becomes enriched at the tip of a neurite, its f-actin content is substantially decreased. In addition, exposure to actin-depolimerizing drugs and toxin B (which inactivates Rho-signaling) causes the formation of multiple axons. Consequently, the interruption of the actin network in a growth cone will promote its neurite to become the axon.
Growing axons move through their environment via the growth cone, which is at the tip of the axon. The growth cone has a broad sheet-like extension called a lamellipodium which contain protrusions called filopodia. The filopodia are the mechanism by which the entire process adheres to surfaces and explores the surrounding environment. Actin plays a major role in the mobility of this system. Environments with high levels of cell adhesion molecules (CAMs) create an ideal environment for axonal growth. This seems to provide a "sticky" surface for axons to grow along. Examples of CAM's specific to neural systems include N-CAM, TAG-1—an axonal glycoprotein— —and MAG, all of which are part of the immunoglobulin superfamily. Another set of molecules called extracellular matrix-adhesion molecules also provide a sticky substrate for axons to grow along. Examples of these molecules include laminin, fibronectin, tenascin, and perlecan. Some of these are surface bound to cells and thus act as short range attractants or repellents. Others are difusible ligands and thus can have long range effects. Cells called guidepost cells assist in the guidance of neuronal axon growth. These cells that help axon guidance, are typically other neurons that are sometimes immature. When the axon has completed its growth at its connection to the target, the diameter of the axon can increase by up to five times, depending on the speed of conduction required. It has also been discovered through research that if the axons of a neuron were damaged, as long as the soma (the cell body of a neuron) is not damaged, the axons would regenerate and remake the synaptic connections with neurons with the help of guidepost cells. This is also referred to as neuroregeneration. Nogo-A is a type of neurite outgrowth inhibitory component that is present in the central nervous system myelin membranes (found in an axon). It has a crucial role in restricting axonal regeneration in adult mammalian central nervous system. In recent studies, if Nogo-A is blocked and neutralized, it is possible to induce long-distance axonal regeneration which leads to enhancement of functional recovery in rats and mouse spinal cord. This has yet to be done on humans. A recent study has also found that macrophages activated through a specific inflammatory pathway activated by the Dectin-1 receptor are capable of promoting axon recovery, also however causing neurotoxicity in the neuron.
Axons vary largely in length from a few micrometers up to meters in some animals. This emphasizes that there must be a cellular length regulation mechanism allowing the neurons both to sense the length of their axons and to control their growth accordingly. It was discovered that motor proteins play an important role in regulating the length of axons. Based on this observation, researchers developed an explicit model for axonal growth describing how motor proteins could affect the axon length on the molecular level. These studies suggest that motor proteins carry signaling molecules from the soma to the growth cone and vice versa whose concentration oscillates in time with a length-dependent frequency.
Different sensory receptors innervate different types of nerve fibers. Proprioceptors are innervated by type Ia, Ib and II sensory fibers, mechanoreceptors by type II and III sensory fibers and nociceptors and thermoreceptors by type III and IV sensory fibers.
In order of degree of severity, injury to a nerve can be described as neurapraxia, axonotmesis, or neurotmesis. Concussion is considered a mild form of diffuse axonal injury. Axonal injury can also cause central chromatolysis. The dysfunction of axons in the nervous system is one of the major causes of many inherited neurological disorders that affect both peripheral and central neurons. When an axon is crushed, an active process of axonal degeneration takes place at the part of the axon furthest from the cell body. This degeneration takes place quickly following the injury, with the part of the axon being sealed off at the membranes and broken down by macrophages. This is known as Wallerian degeneration. Trauma and Wallerian Degeneration
, University of California, San Francisco Dying back of an axon can also take place in many neurodegenerative diseases, particularly when axonal transport is impaired, this is known as Wallerian-like degeneration. Studies suggest that the degeneration happens as a result of the axonal protein NMNAT2, being prevented from reaching all of the axon. Demyelination of axons causes the multitude of neurological symptoms found in the disease multiple sclerosis. Dysmyelination is the abnormal formation of the myelin sheath. This is implicated in several leukodystrophies, and also in schizophrenia. A severe traumatic brain injury can result in widespread lesions to nerve tracts damaging the axons in a condition known as diffuse axonal injury. This can lead to a persistent vegetative state. It has been shown in studies on the rat that axonal damage from a single mild traumatic brain injury, can leave a susceptibility to further damage, after repeated mild traumatic brain injuries. A nerve guidance conduit is an artificial means of guiding axon growth to enable neuroregeneration, and is one of the many treatments used for different kinds of nerve injury.
German anatomist Otto Friedrich Karl Deiters is generally credited with the discovery of the axon by distinguishing it from the dendrites. Swiss Rüdolf Albert von Kölliker and German Robert Remak were the first to identify and characterize the axon initial segment. Kölliker named the axon in 1896. Louis-Antoine Ranvier was the first to describe the gaps or nodes found on axons and for this contribution these axonal features are now commonly referred to as the nodes of Ranvier. Santiago Ramón y Cajal, a Spanish anatomist, proposed that axons were the output components of neurons, describing their functionality. Joseph Erlanger and Herbert Gasser earlier developed the classification system for peripheral nerve fibers, based on axonal conduction velocity, myelination, fiber size etc. Alan Hodgkin and Andrew Huxley also employed the squid giant axon (1939) and by 1952 they had obtained a full quantitative description of the ionic basis of the action potential, leading to the formulation of the Hodgkin–Huxley model. Hodgkin and Huxley were awarded jointly the Nobel Prize for this work in 1963. The formulae detailing axonal conductance were extended to vertebrates in the Frankenhaeuser–Huxley equations. The understanding of the biochemical basis for action potential propagation has advanced further, and includes many details about individual ion channels.
Action potential stimulation and velocity
We evoked an action potential by simulating a current injection into one end of the model axon. Generally, injections of about 10 μA for 100 μs were sufficient, although for some values of the biophysical parameters larger injections were necessary. After a brief initial phase, the properties of the resulting action potentials do not depend on the details of their production.
Simulated action potential velocities were determined from the times at which a particular polarization of the wavefront passed two different points along the model axon. We chose these points to be 5 and 8 cm from the simulated current injection site, by which time all the action potentials investigated had long since stabilized in shape and velocity.
Materials and Methods
Experiments were conducted on adult male locusts (Locusta migratoria) aged 2–5 weeks past their final molt. They were raised in a crowded colony maintained at Queen's University with a 12 h:12 h photoperiod. Animals were fed wheat grass and a mixture of bran, skim milk powder and yeast daily. Cage temperatures were elevated above room temperature (25°C) to 30°C using 60-W incandescent bulbs during the light cycle.
Locusts were dissected as previously described (Robertson and Pearson 1982 ). Briefly, the animals were pinned open to expose the meso- and metathoracic ganglia and a metal plate was placed under the thoracic ganglia to improve stability. Standard locust saline bathed the thoracic ganglia and was composed of (in mmol/L): 147 NaCl, 10 KCl, 4 CaCl2, 3 NaOH, and 10 HEPES. Nerve roots were cut close to the mesothoracic ganglion to allow for drug delivery and a chlorided silver wire was placed in the abdomen and grounded (Fig. 1A).
For temperature-controlled experiments, we continuously flowed heated saline into the thoracic cavity using a peristaltic pump (Peri-star) and a proportional temperature controller (Scientific Systems Design). A BAT-12 thermometer (PhysiTemp) was connected to a type T thermocouple wire of diameter
0.3 mm and was used to monitor the saline temperature by placing the thermocouple near the mesothoracic ganglion.
Glass suction electrodes were placed on the dorso-medial surface of the connective between the pro- and mesothoracic ganglia and between the meso- and metathoracic ganglia. Signals were amplified and filtered using an AM-Systems AC Differential Amplifier model 1700 and digitized with an Axon Instruments Digidata 1440A digitizer. Recordings were made with AxoScope 10.3 (Molecular Devices) and were analyzed using the Clampfit module of pClamp 10.2 (Molecular Devices) software. The DCMD activity was easily distinguished from background spiking in the connective by its large spike amplitude and its robust response to visual stimuli (extracellular recording in Fig. 1B).
Intracellular electrodes were pulled from borosilicate glass and filled with 3 mol/L KCl, giving them a tip resistance between 20 and 40 MΩ. Recordings were amplified and filtered with an AM-Systems Neuroprobe Amplifier model 1600 and digitized at 83 kHz by a Digidata 1440A. The amplifier's DC offset was zeroed relative to the bath before penetration of DCMD and the penetration was made just caudal to the mesothoracic ganglion on the dorso-medial surface (Fig. 1A). The temperature was maintained at 35°C and the resting membrane potential was fixed to −65 mV, near the natural resting membrane potential for the DCMD at 35°C (Money et al. 2005 ) allowing for comparisons of AP parameters.
All chemicals used were obtained from Sigma-Aldrich Canada. For cadmium and nickel experiments, 10 mmol/L of CdCl2 or NiCl2 in standard locust saline was applied for 50 min or until conduction failed. We chose such a high concentration of the divalent cations to ensure timely delivery, as the cut nerve roots that facilitate drug delivery are small and reside on the periphery of the ganglion, whereas the DCMD traverses the medial surface. Longer bath applications are not ideal, particularly with intracellular recordings which last only
15–20 min when the preparation is exposed to divalent cations.
We used two types of calcium-free saline both were standard locust saline without calcium, however, one contained an additional 4 mmol/L of MgCl2 to maintain the divalent cation concentration. High calcium saline had an additional 4 mmol/L of CaCl2 added to standard saline to double the calcium concentration. In all experiments, an initial recording of the DCMD activity was made before exposure and every 5 min thereafter.
In TTX (tetrodotoxin) experiments, TTX was dissolved in 1% acetic acid buffer with a pH of 4.75 to produce a concentration of 300 nmol/L.
Analysis of extracellular measurements included the instantaneous frequency of APs determined by the interspike interval, and the CV, which is proportional to the reciprocal of the time taken to travel from the anterior to the posterior electrode. CVs were calculated relative to the first AP in the recording to control for slight variation in the distance between the electrodes. Metrics used to analyze the intracellular recordings were extracted by custom Python scripts using the StimFit library (Guzman et al. 2014 ).
When comparing multiple groups we used a one-way ANOVA with Holm–Sidak pairwise multiple comparisons or one-way ANOVA on Ranks with Student–Neuman–Keuls method pairwise multiple comparisons. When comparing multiple groups over time we used a two-way ANOVA or a two-way repeated measures (RM) ANOVA with Holm–Sidak pairwise multiple comparisons. Statistical tests were completed with SigmaPlot 11.0 (Systat Software) and significance was defined as P < 0.05. Data are reported as either mean ± standard error (SE) for normally distributed data or median (Mdn) and interquartile range (IQR) for nonparametric data.
Statistical significance (P < 0.05) in plots is denoted by a letter. Columns with different letters indicate statistically different groups, whereas columns with the same letter are not significantly different.
We used a basic model whose current kinetics were described by Wang ( 1998 ) and which has been used previously to describe the LGMD axon (Peron and Gabbiani 2009 ). We modified the model by including only the transient sodium, delayed-rectifying potassium and leakage currents. We changed the reversal potential for the sodium current (VNa) to 45 mV to better fit the experimental data and changed the leakage conductance 0.3 mS/cm 2 as explained in the results section. We also temporally evolved the sodium activation gate , where m is the activation gate, and τm = 1/(αm + βm) as opposed to Wang ( 1998 ) which assumed . Functions for αm and βm are identical to those in Wang ( 1998 ).
We included a persistent sodium current where GNap is the persistent sodium conductance set to either 0.2 or 0.3 mS/cm 2 , Vm is the membrane potential, and mp is the activation gate that evolved similar to the transient sodium current mentioned earlier. It was governed by the activation gate's steady-state value (Smith et al. 1998 ) and time constant tp = 1 msec (Chatelier et al. 2010 ).
We also included simulations where we added a resurgent sodium current to our base model (no persistent sodium current) INar = GNar mr hr(Vm − VNa) where GNar is the resurgent sodium conductance set to 1.0 or 1.5 mS/cm 2 , mr and hr are the activation and inactivation gate, respectively. Kinetics for the activation and inactivation gate are from D'Angelo et al. ( 2001 ).(1)
To test frequency-dependent fidelity, the single compartment was stimulated with a 150 μA/cm 2 current for 0.2 sec for 100 spikes.
We stimulated the first compartment in the model with a current injection of 500 μA/cm 2 for 0.2 msec to elicit APs. The timing pattern for the stimulus was produced from the DCMD activity of 8 locusts in response to a looming visual stimulus.
1. When a squid giant axon is voltage clamped to 0 mV, a brief inward current is required to maintain the membrane potential. An outward current is then necessary for the duration of the clamping. What ions, moving in what directions, are responsible for such behavior?
2. Why would one observe no initial inward current when a squid giant axon is voltage clamped to 50 mV? Also, why is outward current observed prior to the outward current expected due to delayed potassium efflux when the same axon is clamped to 75 mV?
3. In figure 3.8, why is sodium's conductance peak sooner, shorter-lived, and greater than that of potassium?
4. l=Ö[rm/(r0+ri)] is the equation for determining the length constant of an axon. Define each of the variables and identify the properties of axons that affect the values of the variables rm and ri.
5. The voltage-gated channels present in myelinated axons are found at the nodes of Ranvier. Are any voltage-gated channels found in the myelinated portions of the axons? Why or why not? In this context, how might multiple sclerosis affect the axon's ability to propagate action potentials?
6. Why is action potential propagation unidirectional?
7. Inside-out and outside-out recordings with patch clamps allow a researcher to observe the properties of different domains of a membrane channel protein. To what regions are these two domains typically exposed? Why must one understand the properties of both the exterior and interior pores of a channel protein?
8. From figure 4.1, why are the patch clamp currents into the sodium channels so short-lived and soon after clamp application? On the other hand (figure 4.2), why are the outward currents associated with potassium channels prolonged and delayed?
9. What is the conformational difference between a closed voltage-gated sodium channel and an inactivated voltage-gated sodium channel? What causes a closed channel to open? What causes an inactivated channel to be once again ready for activity?
10. What is the region of a channel protein that determines what ion it will transport called? Why can an ion with a larger ionic radius not pass through this region? Furthermore, why will an ion of smaller ionic radius not pass through?
11. What characteristic of the voltage sensor domain of a voltage-gated channel protein allows the channel to respond to changes in membrane potential? What responses, in terms of the voltage sensor domain and in terms of the channel's pore, are elicited as a result of membrane depolarization?
12. What is the defect in axonal voltage-gated sodium channels that leads to seizures in patients afflicted with GEFS? Why does such a defect cause seizures?