Flatman, J. A., Engberg, I. & Lambert, J. D. C. (1982) J. Neurophysiol. 48, 419-430. 19. Kuwada, J. Y. (1981) J. Physiol. (London) 317, 463-473. 20. Lombet, A.
Proc. Nati. Acad. Sci. USA Vol. 83, pp. 7966-7970, October 1986 Neurobiology
Sodium-dependent regenerative responses in dendrites of axotomized motoneurons in the cat (Na' spike/partial spike/lumbosacral/QX-314/spinal cord)
EVELYNE SERNAGOR, Y. YAROM, AND R. WERMAN Department of Neurobiology, Institute of Life Sciences, The Hebrew University, Jerusalem 91904, Israel
Communicated by Eric R. Kandel, June 10, 1986
(5) confirmed the changes in monosynaptic EPSPs and the presence of hump-like responses. Shapovalov and Grantyn' (6) investigated brainstem inputs to axotomized motoneurons and found the size and latency of brainstem EPSPs to be greatly reduced, while their rise and decay times were significantly increased. They also observed partial spikes and made the interesting observation that their occurrence depended not on the size of the somatically recorded depolarization but on its source, indicating segregation of sites of partial spike generation on individual dendrites. Kuno and Llinas (7) found that partial spikes were blocked by remote dendritic inhibition produced by stimulation of the bulbar reticular formation but not by somatic inhibition, indicating remote dendritic location of the partial spikes. There was no relation between the size of EPSPs and the occurrence of partial responses, which arose at different levels of somatic depolarization, indicating the presence of multiple trigger zones. It has been suggested on theoretical grounds that partial spikes in motoneurons are the result of Na' currents (8) but the same authors note that Ca2l currents may also participate in generation of partial spikes. It therefore is of interest to investigate the sensitivity of these various depolarizations to materials that either block or promote Na+ and Ca2+ currents. The experiments reported in this paper used intracellular injections of QX-314, a lidocaine derivative that is known to block Na+ channels from the inner surface of the membrane (9) without affecting Ca2' currents in central neurons (10-12), to investigate a Na+ component of the partial spikes.
Ten days after extradural axotomy, partial ABSTRACT spikes are found in >20% of cat L7 motoneurons, while 15-21 days after axotomy the incidence increases to 60%. These responses are produced in excitable (hot) spots in the dendrites by synaptic excitation. Intracellular injection of QX-314, a lidocaine derivative and effective blocker of Na+ channels from within neurons, results in elimination of partial spikes before blocking somadendritic spikes. The action of QX.314 does not depend on changes in passive membrane properties or on changes in synaptic properties. Inijections of Cs' and ClP ions rule out any major role for calcium, potassium, or chloride currents in the production of partial spikes. The partial spikes represent an unusual Na+-dependent dendritic phenomenon induced by axotomy when carried out relatively near the soma. It is reasonable to postulate that the partial spikes result from a higher concentration of Na+ channels in the dendrites. This may be the consequence of a high rate of production of Na+-channel proteins that are intended for the cut end of the axon; alternatively, they may result from the reflection from the cut end of such proteins produced at either a normal or an increased rate. These aberrant channels are inserted into somatic and dendritic membranes in higher concentrations than normal and, as well as producing local dendritic regions of low safety factor responsible for the partial spikes, also produce somadendritic spikes of unusually fast rise time and lower than usual threshold, which are relatively resistant to QX-314.
After section of their axons (axotomy), motoneurons undergo a complex sequence of changes-metabolic, morphological, and physiological (for review, see ref. 1). Electrophysiological studies of axotomized limb motoneurons began with Campbell (2), who demonstrated a slowing of the reflex response to dorsal root stimulation and a reduction in its
MATERIALS AND METHODS Most of our experiments were done 2-3 weeks after sterile partial laminectomy and extradural section of the L7 ventral
amplitude. Downman et al. (3) investigated spinal reflexes 5 days to 8 weeks after extradural section of cat ventral roots. They noted a progressive decrease in monosynaptic reflexes 5-12 days after injury, but brisk polysynaptic reflexes were present, indicating functional preservation of motoneurons. In fact, it appeared that fewer afferents were necessary to fire a motoneuron than in normal cells. Using intracellular electrodes, Eccles et al. (4) found that monosynaptic excitatory postsynaptic potentials (EPSPs) showed significant decreases in both the size and the rate of rise. Frequently found on the EPSPs were spike-like depolarizations, which were abolished by hyperpolarization and which they felt were likely to originate in distal dendrites. The local or dendritic spikes (partial spikes) were not by themselves associated with axonal discharges but could bring the initial segment and the somadendritic membranes to threshold. McIntyre et al.
root at the level of the dorsal root ganglion in adult cats. At the time of the experiment, after sodium pentobarbital anesthesia, tracheotomy, and catheterization of a vein and an artery, ipsilateral hind-limb motor nerves [usually three: posterior biceps-semitendinosus (PBST), lateral gastrocnemius-soleus (LGS), and medial gastrocnemius (MG)] as well as the ipsilateral dorsal L6 and ventral L7 roots were prepared for stimulation. Body and lumbosacral pool temperatures were maintained automatically at 37°C. Recording techniques were routine. The contents of the microelectrodes were either iontophoresed or, less frequently, injected by pressure into the motoneurons. Partial spikes appear rather abruptly '.10 days after axotomy in our hands, are present in 23.6% of 106 neurons 10-14 days after axotomy, and the incidence more than doubles to 60.0% of 90 neurons 15-21 days after axotomy. Initially, a number of experiments were carried out in
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Abbreviations: EPSP, excitatory postsynaptic potential; PBST, posterior biceps-semitendinosus; LGS, lateral gastrocnemiussoleus; MG, medial gastrocnemius. 7966
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conditions that are known (10) to greatly enhance Ca2+ currents, using intracellular injections from electrodes containing 200 mM QX-314 to block Na' currents and 2 M CsCl to block K+ currents. With these injections, partial spikes uniformly and rapidly disappeared, even when K+-channel blockade was expressed by an up to 2-fold increase in membrane input resistance and enhanced Ca2+ currents were expressed as marked increases in the duration of somatically evoked direct spikes. We subsequently examined the effects of QX-314 alone, using it in electrodes filled with 2 M potassium citrate. We found that it was necessary to reduce the concentration of the Na'-channel blocker to obtain enough partial spikes for measurements before they disappeared completely. With 20-50 mM QX-314, partial spikes disappeared completely in 22 of the first 26 motoneurons with partial spikes tested. The other 4 cases were characterized by clear reduction in partial spike amplitude, and in 2 of these cases the incidence of partial spikes evoked by a given synaptic input was also clearly reduced. In these experiments, the somadendritic spike was generally affected much less than the partial spikes by the QX-314 injections and, in 7 of 20 cases in which the somadendritic spike was followed, disappearance of partial spikes occurred with little or no reduction in the amplitude of the somadendritic spike. Partial spikes disappeared after application of QX-314 by current, by pressure, or even, at times, by diffusion from electrode tips.
RESULTS In Fig. 1, responses to three different sources of stimulation are-,shown, each giving rise to partial spikes of different conformation. These are essentially similar to those reported by others. After injection of QX-314 with a positive current (25 nA) for 30 sec from an electrode containing 30 mM Na'-channel blocker, the partial spikes evoked by stimulation of the motor nerves (PBST and LGS) disappeared completely, while the multiple never-failing partial-spike response to dorsal root L6 stimulation was greatly reduced in PBST
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size (Fig. 1B), and even isolated partial spikes frequently failed to appear. At the same time, the direct spike was reduced from 121 mV (Fig. 1D) to 88 mV (Fig. 1E). A second injection of 25 nA for 30 sec completely eliminated partialspike responses to L6 stimulation as well (Fig. 1C), while the direct spike was further reduced to 62 mV (Fig. 1F). In the absence of the Na'-channel blocker, partial spikes can be recorded for hours. We also used carefully controlled injections of QX-314 to examine intermediary changes in partial spikes before they disappeared completely. In Fig. 2, a histogram of the amplitudes (Fig. 2A) of partial spikes from a single source (MG) before QX-314 injection shows a bimodal distribution, a not uncommon finding, as does the histogram of the rate of rise (Fig. 2A'). In general, the responses showing higher rates of rise were larger. The mean amplitude of the control somadendritic spike was 128.8 mV (Fig. 2A Inset). Some of the variability of the partial spikes can be seen in the superimposed records of Fig. 2C. It is of interest to note the slow rise time of the monosynaptic EPSP, characteristic of axotomized motoneurons as already reported by others (4-6, 13, 14) and confirmed by us. The fastest rise times (0.2 msec) of partial spikes are faster than those of normal EPSPs, but the slower rise times of partial spikes overlap those of normal EPSPs. However, it should be emphasized that the rise times of all partial spikes are faster than the rise times of any EPSP in axotomized motoneurons. After injection from a microelectrode containing 30 mM QX-314 with a small current (10 nA) for 3 min, all partial spikes from the same MG stimulus disappeared (Fig. 2D). Although there are reports of reduction of synaptic potentials by extracellular QX-314 (15, 16) and by intracellular QX-222 (17), a closely related compound, QX-222 did not produce any change of EPSP in cat lumbosacral motoneurons (18). Nor did we find any effect of QX-314 on EPSPs in our experiments. In fact, the EPSP in this experiment was insignificantly increased from 5.1 (Fig. 2C) to 5.4 mV (Fig. 2D). Thus, reduction in the EPSP cannot account for the elimination of partial spikes. The orthodromic LGS
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mv FIG. 1. QX-314 blocks partial spikes in an axotomized L7 motoneuron. The columns PBST, LGS, and L6 (dorsal root) in A, B, and C represent the responses to stimulation of these nerves before (A) and after one injection of depolarizing current (25 nA) for 30 sec (B), and after a second iontophoretic injection of the same current and duration (C). Partial spikes were no longer present in response to motor nerve stimulation after the first injection. The responses to L6 dorsal root stimuli were greatly diminished after the first injection and disappeared after the second. Directly evoked spikes are shown before (D), after the first injection (E), and after the second injection (F). Concentration of QX-314 in microelectrode
was
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mean amplitude, there is no way to eliminate the possibility that in this case the stronger stimulation of MG introduced a third population of partial spikes. In other experiments, such as that shown in Fig. 3, it is possible to demonstrate unequivocally that the Na'-channel blocker reduces the amplitude of the partial spikes before it eliminates them completely. In this experiment, partial spikes evoked by PBST stimulation were monomodally distributed (Fig. 3A; mean, 5.0 mV). After injection from an electrode containing 20 mM QX-314 with a current of 10 nA for 65 sec, the partial spikes were still evoked by the same strength stimulus from PBST but were distinctly smaller (mean, 3.9 mV), even though still monomodally distributed (Fig. 3B). It can be seen that the input resistance of this motoneuron was not affected by injection of the Na+-channel blocker (Fig. 3C). Stronger injections of QX-314, which completely eliminated partial spikes, were not associated with a reduc-
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5 msec FIG. 2. QX-314 in small doses reduces the amplitude, rate of rise, and frequency of partial spikes but does not reduce the EPSP size. In this axotomized L7 motoneuron, the mean amplitude of the control orthodromic spike (A, Inset) was 128.8 mV. MG motor nerve stimulation resulted in partial spikes (C; four superimposed traces) whose amplitudes (A) and rates of rise (A') were distributed bimodally. Ordinate: percent Qf observations. After injection of depolarizing current (10 nA) for 3 min, all partial spikes disappeared (D; three traces), even though the mean EPSP size grew from 5.1 ± 0.9 (SD) to 5.4 ± 0.8 mV. When the stimulus strength was increased to give a mean EPSP of 9.0 ± 0.9 mV, partial spikes of an intermediate size (E; three traces) were seen, monomodally distributed in amplitude (B) and in rate of rise (B'). The orthodromic spike (B, Inset) was now 125.2 mV. QX-314 concentration in microelectrode was 30 mM.
spike amplitude was virtually unchanged (125.2 mV; Fig. 2B Inset) despite elimination of the partial spikes. When the strength of the MG stimulus was increased to give a larger EPSP (9.0 mV; Fig. 2E), partial spikes once again appeared. Analysis of these partial spikes in the histograms of Fig. 2 B and B' shows that the new population is smaller and more slowly rising than the larger-amplitude and faster rising population in Fig. 2 A and A' but slightly larger in size and faster in rise time than the smaller-amplitude slower rising group in the control. Although it is attractive to postulate that the QX-314 injection in this experiment eliminated the group of smalleramplitude partial spikes and raised the threshold for evoking the larger-amplitude control group as well as reducing its
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FIG. 3. QX-314 in small doses reduces the amplitude of partial spikes but not the input resistance of an axotomized L7 motoneuron. The amplitudes of partial spikes recorded in response to stimulation of the PBST motor nerve showed a monomodal distribution with a mean (solid circle on abcissa) of 5.0 mV (A). Ordinate: number of observations. After injection of QX-314 with a depolarizing current (10 nA) for 65 sec, the mean amplitude of the partial spikes was reduced to 3.9 mV (B). The input resistance of the motoneuron (C) was identical (1.1 MQl) before (e) and after (o) injection of QX-314 (concentration in microelectrode was 20 mM).
Neurobiology: Semagor et al. tion in motoneuron input resistance. In fact, increases were sometimes observed, as reported in other neurons (12). If, as our evidence indicates, the presence of partial spikes is a reflection of Na' channels inserted into various locations on the dendritic membrane, several questions arise. What is the source of these channels? Why are they more vulnerable to Na'-channel blockade than are those of the somadendritic spike? Are other ionic channels involved in the partial spike? Why are partial spikes not seen in normal motoneurons? A reasonable explanation for the source of the Na' currents is that they reflect manufacture of new Na' channels at the normal or even at an increased rate in response to axotomy (19). These channels are intended for the regenerating axon but are concentrated in all regions above the lesion, particularly near the soma, and are inserted in the somadendritic membranes. A number of observations made by other workers are in accord with this idea. First of all, Na'-channel proteins are apparently manufactured in the neuronal soma and transported to axonal sites where they are inserted (20). Second, when axotomy is performed at the level of the motor nerve, partial spikes do not develop (21). This suggests that the partial spikes result from damming up of Na' channels when the axon is cut relatively close to the cell body. It has been suggested that a simple redistribution of Na' channels, without increased synthesis of channel proteins, might be enough to explain changes in the distribution of excitability found after axotomy (22). This view, although possible, fails to take into account the evidence for greatly increased protein synthesis following axotomy (1, 23) as well as the evidence in invertebrates that protein synthesis is critical for the appearance of axotomy-induced Na' spikes in proximal regions of the neuron where they were formerly absent (19). We therefore prefer explanations that involve Na'-channel protein synthesis. One might expect, according to this view, that excess Na+ channels might be present in the soma as well as in remote dendrites. In fact, a number of observations support this view. A lowering of the somadendritic threshold has been noted by several investigators (4, 5, 7), indicating a possible increase in the density of Na+ channels in the somatic membrane. Although a significant reduction in current is needed to fire motoneurons after axotomy (7), as we have also observed, at least part of this reduction is the result of an increase in motoneuron-input resistance (24, 25), again confirmed by us.
Proc. Natl. Acad. Sci. USA 83 (1986)
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However, there is additional evidence supporting the idea that partial spikes are part of a more general phenomenon, that new Na+ channels are incorporated into somatic as well as remote dendritic membranes following axotomy. In normal motoneurons, the maximal rate of rise of the antidromic somadendritic spike rarely exceeds 200 V/sec, and almost never exceeds 250 V/sec. This is true even in the presence of K+-channel blockers, which both slow spike repolarization as well as decrease resting membrane conductance (10). Antidromic spikes in axotomized motoneurons examined by us routinely show rates of rise above 400 V/sec and may be faster than 700 V/sec. Thus, the antidromic action potential of Fig. 4A has a maximum rate of rise of 688 V/sec. A similar finding can be seen in an earlier paper (see figure 2 in ref. 26). The initial segment-somadendritic break that is normally quite marked in records of antidromically activated spikes is not visible on the voltage traces (Fig. 4A), but it can be seen in the fastest electronically differentiated record (Fig. 4B) and indicates a break at 22 mV. The low somadendritic threshold found by others (4, 5, 7, 27) and confirmed by us (frequently as low as 10 mV) as well as the marked increase in maximal rate of rise of the action potential and the reduction of the normal initial segment-somadendritic delay can best be explained by the presence of a higher density of Na+ channels in the somadendritic membrane. Another finding that also appears to reflect this change is that axotomized motoneurons routinely show an overshoot (positive phase) greater than that of normal motoneurons (28). A similar finding is reported after axotomy of the fish Mauthner cell (22). An interesting and unusual morphological finding has been reported after axotomy (29, 30)-that synapses are physically separated from motoneurons by the insertion of glial processes. This finding has been used to explain the slower rising and smaller postsynaptic potentials found after axotomy. It is not unlikely that separation of synapses from the motoneuron and its dendrites may also provide a more favorable environment in the soma and dendritic membranes for insertion of new Na+ channels. It appears remarkable that partial spikes, which probably originate in remote dendrites (6, 7), are more vulnerable to somatic injections of QX-314 than are the somatic (somadendritic) spikes themselves. It is reasonable, however, to assume that the Na+-channel density in the electrically excitable regions of the dendritic membrane (hot spots) is relatively low and, therefore, that the safety factor for partial spike initiation is not great. In such circumstances, even the small amount of the Na+-channel blocker that diffuses to the
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FIG. 4. Antidromically evoked action potentials in axotomized L7 motoneurons exhibit unusually rapid rates of rise. Different sweep speeds (A) as well as electronically differentiated records (B) of the same antidromically evoked spike are shown. The maximum rate of rise was 688 V/sec. Evidence of an initial segment-somadendritic break at 22 mV depolarization is seen only in the fastest differentiated record. We argue (see text) that the fast rate of rise is evidence of new Na+ channels inserted into the somadendritic membrane.
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hot spots would be sufficient to prevent partial spike initiation by blocking only a few Na' channels. On the other hand, the somadendritic membrane of the axotomized motoneuron appears to be enriched with a higher than normal density of Na' channels (Fig. 4). This enrichment would result in an increased safety factor and explain the relative invulnerability of the somadendritic spike to QX-314. In other experiments in which Cs' ions were injected alone into axotomized motoneurons, we found that partial spikes increased only slightly in amplitude (not shown). The falling phase of the partial spikes was slowed, but never more than twice control values. Similarly, the input resistance of the motoneurons also increased only up to 2-fold (see also ref. 10). On the other hand, the somadendritic spikes were, under the same conditions, prolonged four to several hundred times (see also ref. 10). Furthermore, as mentioned before, partial spikes disappeared completely when Cs' ions were injected together with QX-314. Since Cs' is an effective K+-channel blocker and promotes appearance of Ca2+ spikes, it appears that neither K+ nor Ca2l currents play an essential role in the generation of partial spikes. Injection of chloride ions occasionally resulted in the appearance of new partial spikes (not shown), when inhibitory inputs were converted into depolarizing potentials, but did not affect partial spikes already present. These findings are consistent with the other evidence that Na' currents play a dominant role in the generation of partial spikes.
DISCUSSION If our view of the generation of partial spikes is correct, the absence of partial spikes in normal motoneurons is the result of two factors-a relatively low rate of Na'-channel protein production in the cell body and a preferential axonifugal gradient for Na'-channel protein transport. Axotomy close to the motoneuron would reverse one or both of these factors as well as produce damming up of Na' channels at the cut end and their reflexion toward the soma and would result in Na'-channel insertion into the soma and dendritic membranes in higher concentrations than normal. On the other hand, axotomy at a greater distance might only increase the rate of production but not interfere with the normal gradient and thus explain the absence of partial spikes reported in these circumstances (21). It is of interest that partial spike-like activity is found in the motoneurons of newborn kittens (31) when high rates of Na+-channel protein may still be present. In the Mauthner cell of the fish, axotomy results in growth of a somadendritic Na+ spike (22). Moreover, in insect neurons, which do not normally exhibit somatic spikes of any kind, either axotomy or application of the axonal transport blocking agent, colchicine, results in the induction of spiking in the neuronal soma (32). The ionic mechanism of this new spike has been invetstigated after colchicine treatment (33, 34) and axotomy (34 35); in both cases, the new somatic spike is produced by Na currents. Aithough dendritic Ca2+ spikes have been reported in a number of neurons (36-43), claims of dendritic spiking produced by Na+ are rather rare (44) and have only been reported in a cell that is also known to have predominant Ca2' dendritic spikes (37, 38). The dendritic Na+ spike found in axotomized motoneurons and described here appears to be the result of dynamic changes in neuronal function induced by pathology. We thank Mr. Hanoch Meiri for his unfailing technical assistance. This work was supported in part by grants from the United States-Israel Binational Science Foundation and the Amyotrophic Lateral Sclerosis Society of America.
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