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Excitability changes of dorsal root axons following nerve injury: implications for injury-induced changes in axonal Na+ channels
Brain Research 859 Ž2000. 280–285 www.elsevier.comrlocaterbres
Research report
Excitability changes of dorsal root axons following nerve injury: implications for injury-induced changes in axonal Naq channels Tadashi Nonaka a , Osamu Honmou a
a,b,c,)
, Jun Sakai a , Kazuo Hashi a , Jeffery D. Kocsis
b,c
Department of Neurosurgery, Sapporo Medical UniÕersity School of Medicine, Sapporo, Hokkaido 060-8543, Japan b Neuroscience and Regeneration Research Center, VA Medical Center, West HaÕen, CT 06516, USA c Department of Neurology, Yale UniÕersity School of Medicine, New HaÕen, CT 06516, USA Accepted 28 December 1999
Abstract Electrophysiological recordings were obtained from rat dorsal roots in a sucrose gap chamber to study changes in Naq currents following nerve injury. Application of 4-aminopyridine unmasks a prominent and well-characterized depolarization Ždelayed depolarization. following the action potential. In our previous studies, this potential, which is only present in cutaneous afferent axons, has been shown to correlate with activation of a slow Naq current. The delayed depolarization in the dorsal root was reduced 1 week after sciatic nerve ligation, suggesting a reduction in the kinetically slow Naq currents on dorsal root axons wcontrol: 44.2 " 7.3% Ž n s 5.; injury: 7.3 " 4.7% Ž n s 5., P - 0.001x. The refractory period of the action potential was reduced following nerve injury, in agreement with biophysical studies indicating faster ‘‘repriming’’ of fast Naq currents on cutaneous afferent cell bodies. Dorsal root ligation near the spinal cord also results in a reduction in the delayed depolarization. These results indicate that changes in Naq channel organization occur on dorsal root axons following either central or peripheral target disconnection, suggesting trophic support can be derived from either the CNS or the PNS. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Axon; Nerve growth factor; Nerve injury; Sodium channel
1. Introduction We have recently reported that kinetically slow Naq currents are present on both the cell body w16,29,31,34x and the peripheral nerve extensions of cutaneous afferent neurons, but not on muscle afferents w16,38x. Administration of 4-aminopyridine Ž4-AP., which blocks a fast potassium current, to cutaneous afferent axons of large diameter results in a prominent Ca2q-independent depolarization following the action potential w16,38x. This depolarizing potential has been termed the delayed depolarization and is several millivolts in amplitude and tens of milliseconds in duration w23x, and has been shown to result from the activation of a kinetically slow Naq current that is electrophysiologically distinct from the fast Naq current, which gives rise to the action potential w16,38x.
)
Corresponding author. Department of Neurosurgery, Sapporo Medical University School of Medicine, South-1st, West-16th, Chuo-ku, Sapporo, Hokkaido 060-8543, Japan. Fax: q81-11-614-1662; e-mail: [email protected]
The Naq-selective channel has emerged as a kinetically and pharmacologically distinct subclass in mammalian dorsal root ganglion ŽDRG. neurons w34x. Although TTX-resistant Naq spikes of relatively long duration are found in small DRG neurons that give rise to C-type primary afferent fibers w34x, recent evidence has inferred the expression of this form of channel on the large diameter neurons of primary cutaneous afferents w16x. A subpopulation of 44- to 50-mm diameter DRG cells giving rise to the cutaneous afferents are more likely to have a relatively slow Naq channel or more than one population of Naq channels when compared to all size-matched, randomly selected neurons w16x. Voltage clamp studies demonstrate that a disproportionately large distribution of cutaneous afferent neurons express slow Naq currents or both fast and slow Naq currents. In contrast, muscle afferent neurons display primarily a singular fast Naq current w16,29,31,34x. Intraaxonal recordings also indicate that the kinetically slow Naq current is present on rapidly conducting, large-diameter myelinated cutaneous afferent axons and virtually absent on muscle afferent and motor axons w16x.
0006-8993r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 0 0 . 0 1 9 7 9 - X
T. Nonaka et al.r Brain Research 859 (2000) 280–285
Primary afferent neurons located in the dorsal root ganglia are pseudo-unipolar and have two targets: one axonal branch extends to peripheral end organs and the other to the central nervous system ŽCNS.. Although the slow Naq current of cutaneous afferent neurons is substantially reduced on both their cell bodies w29,31,35x and their peripheral nerve extensions w38x following peripheral nerve injury, it is not given that similar changes in ion channel organization observed in the cell body and peripheral processes occur on central processes. Indeed, sciatic nerve ligation results in a reduction in dorsal root GABA-mediated Cly conductance w3,19x, but GABA conductances are increased on cutaneous afferent cell bodies w29,30x. The present study was undertaken to address three primary issues: Ž1. do the central nerve extensions Ždorsal root axons. of afferent neurons display a reduction in the delayed depolarization Žslow Naq currents. after sciatic nerve ligation as do their cell bodies and their peripheral nerve extensions?, Ž2. to determine if the central target has a role in the maintenance of slow Naq current expression on these axons, and Ž3. if the excitability properties of the action potential which is generated by a fast TTX-sensitive Naq channel are altered following axotomy. The results indicate the importance of central target connection as well as peripheral in the maintenance of slow Naq currents on these afferent fibers.
2. Materials and methods Experiments were performed on 80 Wistar rats Ž20 unoperated controls and 60 injured rats.. Rats were deeply anesthetized with sodium pentobarbital Ž50 mgrkg, i.p... The sciatic nerve was exposed in the lower popliteal fossa
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and injured. We adopted four types of the nerve injury paradigm in the present study. First, the sciatic nerve was exposed, ligated, and capped with a blind-ending silicon tube to prevent reconnection with the distal segment Žcomplete separation from the peripheral target.. Second, crush region was achieved by surgically exposing the sciatic nerve and compressing for 20 s with a pair of fine forceps, which results in transection of individual axons, but allows the axons to regenerate into the distal nerve segment. Third, the dorsal root ŽL4. was ligated, and capped with a blind-ending silicon tube to prevent reconnection with the proximal segment Ždorsal rhizotomy., which separated the DRG from the central target. Fourth, the combination of dorsal rhizotomy and sciatic nerve ligationq capping, which separated the DRG from both the central and peripheral targets. The nerve injury was performed 1–3 weeks prior to the electrophysiological recordings. Agematched uninjured rats were used as controls. Electrophysiological experiments were carried out on Wistar rats Ž6 weeks old.. The dorsal roots ŽL4. were removed and placed in a modified Krebs’ solution referred to as a normal electrolyte solution. Only nerves that could be removed and with no apparent disruption were studied. Sucrose gap recordings were performed as previously described w16,38x. Briefly, isolated dorsal roots were positioned across a sucrose gap chamber partitioned into compartments by petroleum jelly seals. The center compartment was continuously washed with isotonic sucrose solution Ž320 mM sucrose., the right compartment with modified Krebs’ solution Žin mM: 124 NaCl, 3.0 KCl, 1.3 NaH 2 PO4 , 2.0 MgCl 2 , 2.0 CaCl 2 , 26.0 NaHCO 3 , and 10.0 dextrose: saturated with 95% O 2 and 5% CO 2 ., and the left compartment with isotonic KCl solution Žin mM: 120 KCl, 7.0 NaCl, 1.3 NaH 2 PO4 , 2.0 MgCl 2 , 2.0 CaCl 2 ,
Fig. 1. CAPs recorded in the sucrose gap chamber from dorsal root ŽA.. ŽB. Bath application of 4-AP Ž1 mmolrl. to a normal dorsal root caused a distinct delayed depolarization Žarrow.. Sciatic nerve ligation elicited a reduction of the delayed depolarization in the dorsal root. Amplitude ŽAmp. of the delayed depolarization in the dorsal root significantly decreased following the sciatic nerve ligation ŽC.. A1: the initial action potential amplitude; A2: the delayed depolarization amplitude.
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26.0 NaHCO 3 , and 10.0 dextrose.. All solutions flowed at 1–2 mlrmin. Test solutions were made by adding appropriate concentrations to the modified Krebs’ solution. The two outer compartments were connected to the inputs of a high-impedance DC-coupled differential electrometer ŽAxoclamp 2A; Axon Instruments. with Ag–AgCl wires embedded in agar bridge electrodes Ž3% agar in 1 M NaCl.. The nerve was stimulated with a bipolar Tefloncoated stainless steel electrode cut flush and placed directly on the nerve segment in the test compartment. Stimulation pulses were delivered through an isolation unit and the timing of the pulses was controlled by a digital timing device. The high extracellular resistance in the middle Žisotonic sucrose. compartment limited signal conduction through the axon cylinder. To allow for relatively homogeneous membrane activation and to minimize temporal dispersion, the number of active nodes was reduced by limiting the length of nerve segment positioned in the test well to 2–4 mm. Experiments were carried out at 208C. All variances represent standard error Ž"S.E.M... Differences among groups were assessed by unpaired twotailed t-test or the Mann–Whitney U-test to identify individual group differences. Differences were deemed statistically significant at P - 0.05.
3. Results A compound action potential ŽCAP. recorded from a normal dorsal root of a 6-week-old rat in the sucrose gap chamber is shown in Fig. 1A. The amplitudes of the action potentials were 22.7 " 9.6 mV Ž n s 5. and the half-width of the responses were 0.77 " 0.15 ms Ž n s 5. Žsee Table 1.. A prominent depolarization in a dorsal root develops following the action potential subsequent to bath application of 1 mM 4-AP ŽFig. 1B.. The peak amplitude of the delayed depolarization was 8.02 " 1.69 mV Ž n s 5. and the duration measured from onset was 32.0 " 0.94 ms Ž n s 5.. Several lines of evidence indicate that the delayed depolarization is unmasked by blocking kinetically fast Kq
Fig. 2. Graphic presentation of amplitude recovery vs. interstimulus interval in control and ligated groups. Peak amplitude Žpercent of control value. of the action potential ŽA. and the delayed depolarization ŽB. of CAP responses in control and ligated groups plotted vs. interstimulus interval.
current with 4-AP, and is generated by a kinetically slow Naq current w6,16,23,38x. The delayed depolarization is reduced 1 week following sciatic nerve ligation wcontrol: 44.2 " 7.3% Ž n s 5.; injury: 7.3 " 4.7% Ž n s 5., P 0.001x ŽFig. 1C.. However, the CAP and DC gap potential ŽGP. of dorsal roots were similar for control and following nerve injury, indicating that nerve injury did not result in appreciable axon loss in the dorsal roots ŽTable 1..
Table 1 Standard measures of axonal membrane excitability Nerve injury did not lead to changes in DC gap potential ŽGP., compound action potential ŽCAP. amplitude, and CAP half-width of dorsal root axons. Post-injury Žweek.
Injury type
GP ŽmV.
n
CAP amplitude ŽmV.
n
CAP half-width Žms.
n
1 2 3 1 2 3 1 1
ligation ligation ligation crush crush crush dorsal rhizotomy rhizotomyq ligation control
y26.3 " 8.3 y31.4 " 7.4 y34.0 " 2.6 y35.4 " 7.4 y33.4 " 4.7 y27.4 " 8.8 y33.6 " 7.2 y34.8 " 4.0 y30.7 " 8.5
5 5 5 5 5 5 5 5 5
21.4 " 11.9 21.4 " 13.6 28.1 " 6.2 21.0 " 11.3 29.2 " 11.9 20.1 " 8.8 25.7 " 8.7 32.3 " 10.3 22.7 " 9.6
5 5 5 5 5 5 5 5 5
0.97 " 0.21 0.89 " 0.15 0.69 " 0.02 0.73 " 0.10 0.80 " 0.04 0.88 " 0.18 0.92 " 0.25 0.88 " 0.18 0.77 " 0.15
5 5 5 5 5 5 5 5 5
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Fig. 3. The delayed depolarizations of dorsal root axons obtained at various times following the peripheral injury. Although a marked reduction of the delayed depolarization was seen 1 week after sciatic nerve ligation and the recovery of the delayed depolarization was not obvious, the response recorded from the crushed group showed a remarkable recovery ŽA.. ŽB. Summary of our nerve injury paradigm in which we studied the role of the peripheral target tissue-derived influences. Comparison of the delayed depolarization among groups is graphically demonstrated. A1: the initial action potential amplitude; A2: the delayed depolarization amplitude.
Recovery properties of the axons were studied using paired pulse stimulation. Paired stimuli were presented at varying interstimulus intervals to examine the refractory period of the action potential as well as the delayed depolarization of dorsal roots in control and the injured group. The refractory period of the delayed depolarization, as measured by the time course of amplitude recovery, was significantly greater than that of the action potential. Sciatic nerve injury shortens the absolute and relative refractory periods of the action potential ŽFig. 2A., and prolongs the recovery of the delayed depolarization in the dorsal roots ŽFig. 2B.. A reduction of the delayed depolarization recorded from dorsal roots was observed 1 week after sciatic nerve ligation, and recovery did not occur even at 3 weeks post-injury ŽFig. 3A.. In contrast, the delayed depolarization was less affected following sciatic nerve crush injury.
Fig. 4. Comparison of changes in the delayed depolarization among the nerve injury groups. The sciatic nerve ligation resulted in the marked reduction of the delayed depolarization in the dorsal roots. Dorsal rhizotomy also affected the delayed depolarization in the dorsal roots. All recordings were performed 1 week after nerve ligation. A1: the initial action potential amplitude; A2: the delayed depolarization amplitude.
While there was a significant reduction at 1 week post-injury, the delayed depolarization progressively recovered by 3 weeks ŽFig. 3A.. These data are summarized in Fig. 3B. Although the reduction of the delayed depolarization recorded from dorsal roots following sciatic nerve ligation was observed, dorsal rhizotomy also markedly reduced the delayed depolarization of the dorsal root ŽFig. 4.. All recordings were performed 1 week after nerve injury. This indicates that the signal from the central target is also necessary to maintain the expression of the delayed depolarization in the dorsal roots.
4. Discussion Although myelinated axons in the mammalian nervous system show similar morphological features at both the light and electron microscopic level w6,14x, a large number of ion channel types are present on myelinated axons and individual channel types are regionally distributed in a non-uniform fashion w2,11,21,22,36x. Different types of myelinated axons demonstrate specialized physiological functions which is subserved by distinct distributions of various ion channels w4x. We have demonstrated the selective presence of kinetically slow voltage-dependent Naq channels on large diameter myelinated cutaneous, but not on muscle afferent axons or motor axons w16,20,38x, which supports the idea that sensory signaling in skin may contribute to slow Naq currents on cutaneous afferents. Peripheral nerve injury modifies expression of regulatory genes, chemical and mechanical receptors in axons proximal to the injury site as well as their neuronal cell bodies of origin, which changes functional phenotypes in cutaneous afferent neurons w7,15,18,19,39x. The kinetically slow Naq currents in 40–50 mm diameter DRG neurons and their peripheral processes Žlarge diameter myelinated axons. are reduced following the peripheral nerve injury
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w29,31,35,38x. The present study clearly demonstrates that the delayed depolarization in dorsal roots decreased after sciatic nerve injury, suggesting that the reduction in the slow Naq current following the peripheral nerve injury also occurs on the central processes as well as their cell bodies and peripheral processes. In addition, the refractory period of the action potential in the dorsal roots was shortened after sciatic nerve injury, even though other standard measures of axonal membrane excitability such as the DC gap potential and the action potential were not significantly changed. Biophysical studies on small C-type nociceptive DRG neurons w7x and on large cutaneous afferent neurons w12x indicate that the fast TTX-sensitive Naq currents ‘‘reprime’’ faster after sciatic nerve injury. Our results showing a reduction in the refractory period of the action potential following injury could be the result of this injury-induced change in fast Naq channel kinetics. Thus, changes in Naq currents of cutaneous afferent axons after nerve injury might influence the patterning of action potential activity. These changes in Naq channel properties following peripheral injury could allow inappropriate sensory signaling to the CNS. The changes in the delayed depolarization of dorsal root axons after two types of sciatic nerve injury provide clues to a possible peripheral signal that regulate these changes. Following axotomy by nerve ligation, which prevents target reinnervation and isolates the cut nerve from the distal nerve segment, the injury-induced decrement in the delayed depolarization was maximal and did not recover with time. However, the changes are considerably ameliorated if the nerve is damaged by crush, which transects the axons but allows re-establishment of peripheral target Ži.e., skin. connections subsequent to injury. These results suggest that in response to peripheral nerve injury of afferent neurons, a component of the peripheral target tissue assumes a significant role in regulating slow sodium channel expression. Nerve growth factor ŽNGF. regulates the expression of TTX-resistant action potentials in cutaneous afferent neurons during development w25,33x, and in adults w1x. Exogenous NGF has also been observed to influence sodium current expression in vitro w8,13,17,37x. Molecular biological studies also support the idea that sodium channel gene expression is regulated by NGF w40,43x. In addition, nerve injury modulates the phenotypic expression of the Naq channel. The expression of type III Naq channels increases and sensory-neuron-specific Naq channel ŽSNS. transcripts decrease following sciatic nerve transection in some DRG neurons w5,9x. Naq channel mRNA expression reverts to an embryonic pattern after axotomy, with the re-emergence of type III Naq channel mRNA that is expressed at high levels embryonically but undetectable in normal adult neurons w41x. Moreover, NGF has recently been shown to regulate slow Naq currents on axotomized large cutaneous afferent neurons w31x, and a-SNS Naq channel expression is rescued in DRG neurons after axotomy by NGF in vivo w9,10x. These arguments strongly
suggest that there is a switch in the mode of Naq channel expression in at least some cutaneous afferent DRG neurons following axotomy, and it seems likely that NGF may modulate axonal Naq channels. Although the interaction between the neuronal cell body and the innervated peripheral field is important for nerve growth and differentiation w24x, the importance of retrograde-transport from the central target is also emphasized during early development w32,42x. Our nerve injury models represent a system in which DRG neurons are deprived of either or both their central and peripheral target-derived trophic support and are therefore convenient for studying the role of the target tissue-derived influences in maintaining the phenotypic expression of sensory neurons. The present study demonstrates that trophic support from the peripheral target as well as the central is important to maintain the slow Naq channel expression in adult DRG neurons, even though most DRG neurons no longer require target-derived trophic support for survival in adults w26– 28x. A distinct trophic factor support may have an important role in maintaining the slow Naq channel expression on the entire afferent in adult mammalians and replenishment of appropriate trophic factor support could be a therapeutic strategy in the treatment of chronic abnormal sensation following the nerve injury.
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Research report
Excitability changes of dorsal root axons following nerve injury: implications for injury-induced changes in axonal Naq channels Tadashi Nonaka a , Osamu Honmou a
a,b,c,)
, Jun Sakai a , Kazuo Hashi a , Jeffery D. Kocsis
b,c
Department of Neurosurgery, Sapporo Medical UniÕersity School of Medicine, Sapporo, Hokkaido 060-8543, Japan b Neuroscience and Regeneration Research Center, VA Medical Center, West HaÕen, CT 06516, USA c Department of Neurology, Yale UniÕersity School of Medicine, New HaÕen, CT 06516, USA Accepted 28 December 1999
Abstract Electrophysiological recordings were obtained from rat dorsal roots in a sucrose gap chamber to study changes in Naq currents following nerve injury. Application of 4-aminopyridine unmasks a prominent and well-characterized depolarization Ždelayed depolarization. following the action potential. In our previous studies, this potential, which is only present in cutaneous afferent axons, has been shown to correlate with activation of a slow Naq current. The delayed depolarization in the dorsal root was reduced 1 week after sciatic nerve ligation, suggesting a reduction in the kinetically slow Naq currents on dorsal root axons wcontrol: 44.2 " 7.3% Ž n s 5.; injury: 7.3 " 4.7% Ž n s 5., P - 0.001x. The refractory period of the action potential was reduced following nerve injury, in agreement with biophysical studies indicating faster ‘‘repriming’’ of fast Naq currents on cutaneous afferent cell bodies. Dorsal root ligation near the spinal cord also results in a reduction in the delayed depolarization. These results indicate that changes in Naq channel organization occur on dorsal root axons following either central or peripheral target disconnection, suggesting trophic support can be derived from either the CNS or the PNS. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Axon; Nerve growth factor; Nerve injury; Sodium channel
1. Introduction We have recently reported that kinetically slow Naq currents are present on both the cell body w16,29,31,34x and the peripheral nerve extensions of cutaneous afferent neurons, but not on muscle afferents w16,38x. Administration of 4-aminopyridine Ž4-AP., which blocks a fast potassium current, to cutaneous afferent axons of large diameter results in a prominent Ca2q-independent depolarization following the action potential w16,38x. This depolarizing potential has been termed the delayed depolarization and is several millivolts in amplitude and tens of milliseconds in duration w23x, and has been shown to result from the activation of a kinetically slow Naq current that is electrophysiologically distinct from the fast Naq current, which gives rise to the action potential w16,38x.
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Corresponding author. Department of Neurosurgery, Sapporo Medical University School of Medicine, South-1st, West-16th, Chuo-ku, Sapporo, Hokkaido 060-8543, Japan. Fax: q81-11-614-1662; e-mail: [email protected]
The Naq-selective channel has emerged as a kinetically and pharmacologically distinct subclass in mammalian dorsal root ganglion ŽDRG. neurons w34x. Although TTX-resistant Naq spikes of relatively long duration are found in small DRG neurons that give rise to C-type primary afferent fibers w34x, recent evidence has inferred the expression of this form of channel on the large diameter neurons of primary cutaneous afferents w16x. A subpopulation of 44- to 50-mm diameter DRG cells giving rise to the cutaneous afferents are more likely to have a relatively slow Naq channel or more than one population of Naq channels when compared to all size-matched, randomly selected neurons w16x. Voltage clamp studies demonstrate that a disproportionately large distribution of cutaneous afferent neurons express slow Naq currents or both fast and slow Naq currents. In contrast, muscle afferent neurons display primarily a singular fast Naq current w16,29,31,34x. Intraaxonal recordings also indicate that the kinetically slow Naq current is present on rapidly conducting, large-diameter myelinated cutaneous afferent axons and virtually absent on muscle afferent and motor axons w16x.
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Primary afferent neurons located in the dorsal root ganglia are pseudo-unipolar and have two targets: one axonal branch extends to peripheral end organs and the other to the central nervous system ŽCNS.. Although the slow Naq current of cutaneous afferent neurons is substantially reduced on both their cell bodies w29,31,35x and their peripheral nerve extensions w38x following peripheral nerve injury, it is not given that similar changes in ion channel organization observed in the cell body and peripheral processes occur on central processes. Indeed, sciatic nerve ligation results in a reduction in dorsal root GABA-mediated Cly conductance w3,19x, but GABA conductances are increased on cutaneous afferent cell bodies w29,30x. The present study was undertaken to address three primary issues: Ž1. do the central nerve extensions Ždorsal root axons. of afferent neurons display a reduction in the delayed depolarization Žslow Naq currents. after sciatic nerve ligation as do their cell bodies and their peripheral nerve extensions?, Ž2. to determine if the central target has a role in the maintenance of slow Naq current expression on these axons, and Ž3. if the excitability properties of the action potential which is generated by a fast TTX-sensitive Naq channel are altered following axotomy. The results indicate the importance of central target connection as well as peripheral in the maintenance of slow Naq currents on these afferent fibers.
2. Materials and methods Experiments were performed on 80 Wistar rats Ž20 unoperated controls and 60 injured rats.. Rats were deeply anesthetized with sodium pentobarbital Ž50 mgrkg, i.p... The sciatic nerve was exposed in the lower popliteal fossa
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and injured. We adopted four types of the nerve injury paradigm in the present study. First, the sciatic nerve was exposed, ligated, and capped with a blind-ending silicon tube to prevent reconnection with the distal segment Žcomplete separation from the peripheral target.. Second, crush region was achieved by surgically exposing the sciatic nerve and compressing for 20 s with a pair of fine forceps, which results in transection of individual axons, but allows the axons to regenerate into the distal nerve segment. Third, the dorsal root ŽL4. was ligated, and capped with a blind-ending silicon tube to prevent reconnection with the proximal segment Ždorsal rhizotomy., which separated the DRG from the central target. Fourth, the combination of dorsal rhizotomy and sciatic nerve ligationq capping, which separated the DRG from both the central and peripheral targets. The nerve injury was performed 1–3 weeks prior to the electrophysiological recordings. Agematched uninjured rats were used as controls. Electrophysiological experiments were carried out on Wistar rats Ž6 weeks old.. The dorsal roots ŽL4. were removed and placed in a modified Krebs’ solution referred to as a normal electrolyte solution. Only nerves that could be removed and with no apparent disruption were studied. Sucrose gap recordings were performed as previously described w16,38x. Briefly, isolated dorsal roots were positioned across a sucrose gap chamber partitioned into compartments by petroleum jelly seals. The center compartment was continuously washed with isotonic sucrose solution Ž320 mM sucrose., the right compartment with modified Krebs’ solution Žin mM: 124 NaCl, 3.0 KCl, 1.3 NaH 2 PO4 , 2.0 MgCl 2 , 2.0 CaCl 2 , 26.0 NaHCO 3 , and 10.0 dextrose: saturated with 95% O 2 and 5% CO 2 ., and the left compartment with isotonic KCl solution Žin mM: 120 KCl, 7.0 NaCl, 1.3 NaH 2 PO4 , 2.0 MgCl 2 , 2.0 CaCl 2 ,
Fig. 1. CAPs recorded in the sucrose gap chamber from dorsal root ŽA.. ŽB. Bath application of 4-AP Ž1 mmolrl. to a normal dorsal root caused a distinct delayed depolarization Žarrow.. Sciatic nerve ligation elicited a reduction of the delayed depolarization in the dorsal root. Amplitude ŽAmp. of the delayed depolarization in the dorsal root significantly decreased following the sciatic nerve ligation ŽC.. A1: the initial action potential amplitude; A2: the delayed depolarization amplitude.
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26.0 NaHCO 3 , and 10.0 dextrose.. All solutions flowed at 1–2 mlrmin. Test solutions were made by adding appropriate concentrations to the modified Krebs’ solution. The two outer compartments were connected to the inputs of a high-impedance DC-coupled differential electrometer ŽAxoclamp 2A; Axon Instruments. with Ag–AgCl wires embedded in agar bridge electrodes Ž3% agar in 1 M NaCl.. The nerve was stimulated with a bipolar Tefloncoated stainless steel electrode cut flush and placed directly on the nerve segment in the test compartment. Stimulation pulses were delivered through an isolation unit and the timing of the pulses was controlled by a digital timing device. The high extracellular resistance in the middle Žisotonic sucrose. compartment limited signal conduction through the axon cylinder. To allow for relatively homogeneous membrane activation and to minimize temporal dispersion, the number of active nodes was reduced by limiting the length of nerve segment positioned in the test well to 2–4 mm. Experiments were carried out at 208C. All variances represent standard error Ž"S.E.M... Differences among groups were assessed by unpaired twotailed t-test or the Mann–Whitney U-test to identify individual group differences. Differences were deemed statistically significant at P - 0.05.
3. Results A compound action potential ŽCAP. recorded from a normal dorsal root of a 6-week-old rat in the sucrose gap chamber is shown in Fig. 1A. The amplitudes of the action potentials were 22.7 " 9.6 mV Ž n s 5. and the half-width of the responses were 0.77 " 0.15 ms Ž n s 5. Žsee Table 1.. A prominent depolarization in a dorsal root develops following the action potential subsequent to bath application of 1 mM 4-AP ŽFig. 1B.. The peak amplitude of the delayed depolarization was 8.02 " 1.69 mV Ž n s 5. and the duration measured from onset was 32.0 " 0.94 ms Ž n s 5.. Several lines of evidence indicate that the delayed depolarization is unmasked by blocking kinetically fast Kq
Fig. 2. Graphic presentation of amplitude recovery vs. interstimulus interval in control and ligated groups. Peak amplitude Žpercent of control value. of the action potential ŽA. and the delayed depolarization ŽB. of CAP responses in control and ligated groups plotted vs. interstimulus interval.
current with 4-AP, and is generated by a kinetically slow Naq current w6,16,23,38x. The delayed depolarization is reduced 1 week following sciatic nerve ligation wcontrol: 44.2 " 7.3% Ž n s 5.; injury: 7.3 " 4.7% Ž n s 5., P 0.001x ŽFig. 1C.. However, the CAP and DC gap potential ŽGP. of dorsal roots were similar for control and following nerve injury, indicating that nerve injury did not result in appreciable axon loss in the dorsal roots ŽTable 1..
Table 1 Standard measures of axonal membrane excitability Nerve injury did not lead to changes in DC gap potential ŽGP., compound action potential ŽCAP. amplitude, and CAP half-width of dorsal root axons. Post-injury Žweek.
Injury type
GP ŽmV.
n
CAP amplitude ŽmV.
n
CAP half-width Žms.
n
1 2 3 1 2 3 1 1
ligation ligation ligation crush crush crush dorsal rhizotomy rhizotomyq ligation control
y26.3 " 8.3 y31.4 " 7.4 y34.0 " 2.6 y35.4 " 7.4 y33.4 " 4.7 y27.4 " 8.8 y33.6 " 7.2 y34.8 " 4.0 y30.7 " 8.5
5 5 5 5 5 5 5 5 5
21.4 " 11.9 21.4 " 13.6 28.1 " 6.2 21.0 " 11.3 29.2 " 11.9 20.1 " 8.8 25.7 " 8.7 32.3 " 10.3 22.7 " 9.6
5 5 5 5 5 5 5 5 5
0.97 " 0.21 0.89 " 0.15 0.69 " 0.02 0.73 " 0.10 0.80 " 0.04 0.88 " 0.18 0.92 " 0.25 0.88 " 0.18 0.77 " 0.15
5 5 5 5 5 5 5 5 5
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Fig. 3. The delayed depolarizations of dorsal root axons obtained at various times following the peripheral injury. Although a marked reduction of the delayed depolarization was seen 1 week after sciatic nerve ligation and the recovery of the delayed depolarization was not obvious, the response recorded from the crushed group showed a remarkable recovery ŽA.. ŽB. Summary of our nerve injury paradigm in which we studied the role of the peripheral target tissue-derived influences. Comparison of the delayed depolarization among groups is graphically demonstrated. A1: the initial action potential amplitude; A2: the delayed depolarization amplitude.
Recovery properties of the axons were studied using paired pulse stimulation. Paired stimuli were presented at varying interstimulus intervals to examine the refractory period of the action potential as well as the delayed depolarization of dorsal roots in control and the injured group. The refractory period of the delayed depolarization, as measured by the time course of amplitude recovery, was significantly greater than that of the action potential. Sciatic nerve injury shortens the absolute and relative refractory periods of the action potential ŽFig. 2A., and prolongs the recovery of the delayed depolarization in the dorsal roots ŽFig. 2B.. A reduction of the delayed depolarization recorded from dorsal roots was observed 1 week after sciatic nerve ligation, and recovery did not occur even at 3 weeks post-injury ŽFig. 3A.. In contrast, the delayed depolarization was less affected following sciatic nerve crush injury.
Fig. 4. Comparison of changes in the delayed depolarization among the nerve injury groups. The sciatic nerve ligation resulted in the marked reduction of the delayed depolarization in the dorsal roots. Dorsal rhizotomy also affected the delayed depolarization in the dorsal roots. All recordings were performed 1 week after nerve ligation. A1: the initial action potential amplitude; A2: the delayed depolarization amplitude.
While there was a significant reduction at 1 week post-injury, the delayed depolarization progressively recovered by 3 weeks ŽFig. 3A.. These data are summarized in Fig. 3B. Although the reduction of the delayed depolarization recorded from dorsal roots following sciatic nerve ligation was observed, dorsal rhizotomy also markedly reduced the delayed depolarization of the dorsal root ŽFig. 4.. All recordings were performed 1 week after nerve injury. This indicates that the signal from the central target is also necessary to maintain the expression of the delayed depolarization in the dorsal roots.
4. Discussion Although myelinated axons in the mammalian nervous system show similar morphological features at both the light and electron microscopic level w6,14x, a large number of ion channel types are present on myelinated axons and individual channel types are regionally distributed in a non-uniform fashion w2,11,21,22,36x. Different types of myelinated axons demonstrate specialized physiological functions which is subserved by distinct distributions of various ion channels w4x. We have demonstrated the selective presence of kinetically slow voltage-dependent Naq channels on large diameter myelinated cutaneous, but not on muscle afferent axons or motor axons w16,20,38x, which supports the idea that sensory signaling in skin may contribute to slow Naq currents on cutaneous afferents. Peripheral nerve injury modifies expression of regulatory genes, chemical and mechanical receptors in axons proximal to the injury site as well as their neuronal cell bodies of origin, which changes functional phenotypes in cutaneous afferent neurons w7,15,18,19,39x. The kinetically slow Naq currents in 40–50 mm diameter DRG neurons and their peripheral processes Žlarge diameter myelinated axons. are reduced following the peripheral nerve injury
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w29,31,35,38x. The present study clearly demonstrates that the delayed depolarization in dorsal roots decreased after sciatic nerve injury, suggesting that the reduction in the slow Naq current following the peripheral nerve injury also occurs on the central processes as well as their cell bodies and peripheral processes. In addition, the refractory period of the action potential in the dorsal roots was shortened after sciatic nerve injury, even though other standard measures of axonal membrane excitability such as the DC gap potential and the action potential were not significantly changed. Biophysical studies on small C-type nociceptive DRG neurons w7x and on large cutaneous afferent neurons w12x indicate that the fast TTX-sensitive Naq currents ‘‘reprime’’ faster after sciatic nerve injury. Our results showing a reduction in the refractory period of the action potential following injury could be the result of this injury-induced change in fast Naq channel kinetics. Thus, changes in Naq currents of cutaneous afferent axons after nerve injury might influence the patterning of action potential activity. These changes in Naq channel properties following peripheral injury could allow inappropriate sensory signaling to the CNS. The changes in the delayed depolarization of dorsal root axons after two types of sciatic nerve injury provide clues to a possible peripheral signal that regulate these changes. Following axotomy by nerve ligation, which prevents target reinnervation and isolates the cut nerve from the distal nerve segment, the injury-induced decrement in the delayed depolarization was maximal and did not recover with time. However, the changes are considerably ameliorated if the nerve is damaged by crush, which transects the axons but allows re-establishment of peripheral target Ži.e., skin. connections subsequent to injury. These results suggest that in response to peripheral nerve injury of afferent neurons, a component of the peripheral target tissue assumes a significant role in regulating slow sodium channel expression. Nerve growth factor ŽNGF. regulates the expression of TTX-resistant action potentials in cutaneous afferent neurons during development w25,33x, and in adults w1x. Exogenous NGF has also been observed to influence sodium current expression in vitro w8,13,17,37x. Molecular biological studies also support the idea that sodium channel gene expression is regulated by NGF w40,43x. In addition, nerve injury modulates the phenotypic expression of the Naq channel. The expression of type III Naq channels increases and sensory-neuron-specific Naq channel ŽSNS. transcripts decrease following sciatic nerve transection in some DRG neurons w5,9x. Naq channel mRNA expression reverts to an embryonic pattern after axotomy, with the re-emergence of type III Naq channel mRNA that is expressed at high levels embryonically but undetectable in normal adult neurons w41x. Moreover, NGF has recently been shown to regulate slow Naq currents on axotomized large cutaneous afferent neurons w31x, and a-SNS Naq channel expression is rescued in DRG neurons after axotomy by NGF in vivo w9,10x. These arguments strongly
suggest that there is a switch in the mode of Naq channel expression in at least some cutaneous afferent DRG neurons following axotomy, and it seems likely that NGF may modulate axonal Naq channels. Although the interaction between the neuronal cell body and the innervated peripheral field is important for nerve growth and differentiation w24x, the importance of retrograde-transport from the central target is also emphasized during early development w32,42x. Our nerve injury models represent a system in which DRG neurons are deprived of either or both their central and peripheral target-derived trophic support and are therefore convenient for studying the role of the target tissue-derived influences in maintaining the phenotypic expression of sensory neurons. The present study demonstrates that trophic support from the peripheral target as well as the central is important to maintain the slow Naq channel expression in adult DRG neurons, even though most DRG neurons no longer require target-derived trophic support for survival in adults w26– 28x. A distinct trophic factor support may have an important role in maintaining the slow Naq channel expression on the entire afferent in adult mammalians and replenishment of appropriate trophic factor support could be a therapeutic strategy in the treatment of chronic abnormal sensation following the nerve injury.
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