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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online September 27, 2004 as doi:10.1096/fj.04-1805fje. |
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,
* Institute of Neuroscience CNR and
Scuola Normale Superiore, Pisa, Italy; and
IRCCS Santa Lucia Foundation, Rome, Italy
1Correspondence: Institute of Neuroscience CNR, Via Moruzzi 1, Pisa, Italy 56100. E-mail: gimmi{at}in.cnr.it
SPECIFIC AIM
Although the transection of the axon of central neurons has dramatic consequences on damaged cells and nerves with important pathophysiological consequences, the acute physiological response to trauma of mammalian neurons is poorly characterized. In this study we provide a coherent scheme to understand the effects of axonal transection on ionic homeostasis and on electrical excitability by monitoring ion concentration and electrophysiological response of central neurons axotomized in vitro.
PRINCIPAL FINDINGS
1. Axotomy caused an increase of intracellular calcium
We used primary cultures of neurons from the rodent cortex plated at low density. Neurons were well isolated and their axons were easily recognizable by morphological criteria. The low density of the culture allowed for sparse and isolated axons that could be sectioned by a computer-controlled micromanipulator during the physiological recordings.
The spatio-temporal dynamic of the calcium response after axotomy is displayed in Fig. 1
A, B. Imaging with the low-affinity indicator fura-ff (Kd=5.5 µM), showed that the maximal calcium load occurred at the tip of the transected axon and propagated to the cell body. The calcium response in most of the cell was due to the activation of voltage-dependent channels, since the presence of TTX (1 µM), a blocker of the voltage-gated sodium channel, caused a reduction of the response in the axon and almost complete suppression at the soma (Fig. 1B-D
). The high-calcium domain at the axonal tip was likely due to a transient leak at the lesion site (Fig. 1E
).
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2. Axotomy was followed by membrane depolarization and action potential firing
Imaging data suggested that the propagation of calcium increase to the soma depended on the firing of action potentials. Neurons recorded in patch clamp responded to lesion with the firing of high frequency spikes sitting on top of a steady depolarization (see Fig. 3C
). Perfusion with TTX entirely abolished the spike activity but had only a minor effect on the steady depolarization.
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3. Calcium influx was triggered by the action potentials
Since TTX did not abolish the sustained depolarization that followed axotomy but had a powerful inhibitory effect on calcium increase, it is likely that calcium influx was induced not by steady depolarization, but by high frequency spiking. It is not possible to monitor directly calcium currents at the time of axotomy. Since most of the total current is carried by sodium and potassium, it is necessary to block their channels to be able to record the calcium component. Of course, this cannot be accomplished without suppressing the spike pattern itself. We circumvented this problem by recording membrane current in the voltage clamp configuration while feeding the cell with the voltage response to axotomy recorded in another neuron. During this particular stimulus, each spike delivered by voltage clamp circuitry elicited a transient inward current (see Fig. 3D, E
). This current was carried by calcium since its amplitude depended on the extracellular calcium concentration and was abolished in presence of calcium channel blockers (cadmium and flunarizine). The injection of a response obtained in TTX did not cause any calcium influx.
4. Axotomy caused sodium load and inversion of Na/Ca exchange
Perfusion with cadmium caused only a relatively modest reduction of the maximum amplitude of calcium load (Fig. 2
A). Presence of a residual calcium load during blockade of voltage-dependent calcium channels shows that there must be another way of entry for calcium ions which must be linked to activation of TTX-sensitive sodium channels. A consequence of the postinjury electrical activity was the gradual build up of a sodium load after axotomy, as demonstrated by imaging with the ratiometric sodium indicator SBFI (Fig. 2B
). As sodium concentration rose after axotomy, its electrochemical gradient became insufficient to drive the extrusion of calcium from the cell through the Na/Ca exchanger. In this condition the Na/Ca exchanger inverts its operation by driving sodium out of cytoplasm and letting in calcium ions. This mode of operation is maintained until energetic equilibrium between sodium and calcium gradients is reached (Fig. 3
A). The kinetic of axotomy-induced calcium increase was much slower in CdCl2 (Fig. 2C
), suggesting that the rapid early phase of the response was mainly due to influx through the V-dependent calcium channels, while inversion of the exchanger contributed at a later time. Activation of V-gated calcium channels and reversal of the Na/Ca exchange accounted for total calcium influx, since simultaneous treatment with CdCl2 and with exchange blocker Bepredil (25 µM) suppressed calcium response after axotomy (Fig. 2C
, orange trace).
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5. Acute response to axotomy and induction of sprouting
Cultured central neurons respond to sectioning of the axon with a sprouting response that is started within 4–6 h of injury and leads to formation of a new axon. We asked whether the physiological response to injury plays some role in activation of this regenerative response by observing morphological changes induced by axotomy in control conditions and after inhibition of electrical activity with TTX. Neurons were cultured on numbered grids for identification and were imaged in transmitted light before and immediately after section of the axon. After 6 h in the incubator, cells were returned to the microscope to visualize the morphological response to axotomy. Only a small fraction of neurons (12%) were lost as a consequence of the injury, and this percentage was not affected by TTX. The large majority of cells axotomized in normal culture medium (61 of 89, 69%) activated a regenerative response (witnessed by regrowth at the lesion site or from the axotomized axon). Addition of 2 µM TTX caused a significant reduction of the fraction of neurons that expressed a regenerative response (21 of 46, 46%, z test, P=0.017).
CONCLUSIONS AND SIGNIFICANCE
It is thought that the sectioning of the axon is signaled to the cell body by a two stage mechanism. Initially neurons are believed to respond to a depolarization back-propagating to the soma from the lesion site. This is followed by biochemical signals such as the loss of factors released by the target and retrogradely transported to the soma and the transport of molecules produced or activated at the lesion. Very little is known about the early phase of this signaling scheme and although it is often speculated that axotomy may cause an acute disruption of ionic regulation, a detailed picture of the physiological events occurring in mammalian central neurons at the time of axotomy was still missing.
In this study we developed an in vitro model of axotomy of neurons isolated from the rat brain to study early phases of physiological response to axotomy by a combination of ion concentration imaging and electrophysiological recording. We observed that axotomy evoked a depolarization of the membrane potential and vigorous spiking activity that brought about an increase of intracellular calcium and sodium. These physiological changes were caused not by the loss of electrochemical gradients due to defective membrane integrity but because of an active and specific response of neurons. This response required activation of the TTX sensitive channel which caused repetitive action potentials and sodium influx. Action potentials triggered opening of voltage sensitive calcium channels and subsequent calcium influx. Sodium load determined inversion of the Na/Ca exchange, providing a second means of entry for calcium. Finally, inhibition with TTX of the physiological response to axotomy significantly reduced the fraction of axotomized neurons that exhibited postinjury regeneration.
In vivo, axotomy causes cell death and degeneration of the lesioned tracts. Calcium accumulation in axons and neurons plays a central role in degenerative processes occurring in a wide variety of pathological scenarios, regardless of specific initiating stimuli and compounds that can prevent calcium influx are actively searched. We found that sodium channel blocker TTX reduced to a large extent the acute response to axotomy by suppressing both the spiking pattern responsible for repetitive opening of calcium channels, and the sodium load responsible for the inversion of the Na/Ca exchange. Blocking of sodium voltage dependent channels with TTX attenuated damage caused by injury to the spinal chord and reduced the pathology of injured axons shortly after the lesion. Efficacy of TTX in preventing axonal degeneration was optimal when the drug was delivered at the time of injury, suggesting that causal events leading to degeneration occur immediately after axotomy.
This study provides the first direct evidence that axotomy triggers a process of electrical signaling to the cell body of mammalian central neurons and that this activity causes a disruption of ionic homeostasis. Another system of signaling involves the retrograde transport of positive-injury protein derived from the lesion site. It has been shown that this protein-based signaling is executed by a mechanism that involves de novo synthesis of importin and the formation of a importin-dynein complex that is transported retrogradely toward the cell body. Although novel experimental designs are required to discriminate between the specific roles of these two processes of injury signaling, our data and those of others suggest that both mechanisms of communication are required for successful regeneration.
Convergence between the two pathways can occur at multiple levels. At the injury site, the calcium signal may be involved in activation of post-translational processes required for preparation of the cargo carried by the importin-dynein complex. A further site of convergence could occur at the cell soma, where calcium has important effects on gene expression. In this scheme, the early episode of calcium-dependent gene expression could somehow prime the cell that will receive (after a certain delay) the injury signal carried by the retrograde transport system.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-1805fje;
This article has been cited by other articles:
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  K.-i. Miyake, M. Yoshida, Y. Inoue, and Y. Hata Neuroprotective Effect of Transcorneal Electrical Stimulation on the Acute Phase of Optic Nerve Injury Invest. Ophthalmol. Vis. Sci., May 1, 2007; 48(5): 2356 - 2361. [Abstract] [Full Text] [PDF]
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20 µM in this cell. B) Perfusion with TTX reduced the amplitude of the calcium change in the axon and strongly inhibited its spread into the cell body (calibration bar 30 µm in panels A, B). C) Quantification of the fluorescence ratio at four locations (see arrowheads in panels A, B for precise localization). D) Amplitude of the calcium response at the site of lesion, at the soma, and at an intermediate location in the axon (Ax). TTX was effective in reducing the amplitude of the response in the axon and soma (t test *P
0.02). E) Amplitude of relative fluorescence (normalized to preaxotomy levels) excited at the calcium insensitive wavelength. Sharp decline of fluorescence at the section site indicates a temporary loss of membrane integrity.
