Department of Neurology, Yale University School of Medicine, New Haven 06510; and Neuroscience Research Center, Department of Veterans Affairs Medical Center, West Haven, Connecticut 06516
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ABSTRACT |
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Everill, Brian and Jeffery D. Kocsis. Reduction in Potassium Currents in Identified Cutaneous Afferent Dorsal Root Ganglion Neurons After Axotomy. J. Neurophysiol. 82: 700-708, 1999. Potassium currents have an important role in modulating neuronal excitability. We have investigated the effects of axotomy on three voltage-activated K+ currents, one sustained and two transient, in cutaneous afferent dorsal root ganglion (DRG) neurons. Fourteen to 21 days after axotomy, L4 and L5 DRG neurons were acutely dissociated and were studied 2-8 h after plating. Whole cell patch-clamp recordings were obtained from identified cutaneous afferent neurons (46-50 µm diam); K+ currents were isolated by blocking Na+ and Ca2+ currents with appropriate ion replacement and channel blockers. Separation of the current components was achieved on the basis of sensitivity to dendrotoxin or 4-aminopyridine and by the response to variation in conditioning voltage. Both control and injured neurons displayed qualitatively similar complex K+ currents composed of distinct kinetic and pharmacological components. Three distinct K+ current components, a sustained (IK) and two transient (IA and ID), were identified in variable proportions. However, total peak current was reduced by 52% in the axotomized cells when compared with control cells. Two current components were reduced after ligation, IA by 60%, IK by over 65%, compared with control cells. ID appeared unaffected after acute ligation. These results indicate a large reduction in overall K+ current, resulting from reductions in IK and IA, on large cutaneous afferent neurons after nerve ligation and have implications for excitability changes of injured primary afferent neurons.
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INTRODUCTION |
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Dorsal root ganglion (DRG) neurons express three
distinct classes of K+ current in varying
quantities (Everill et al. 1998). These consist of a
relatively large dominant sustained current (K), and two transient
currents (A and D). The importance of the different K+ current components in neuronal function is
recognized widely (Albert and Nerbonne 1995
;
Ficker and Heinemann 1992
; Foehring and Surmeier
1993
; McFarlane and Cooper 1991
; Wu and
Barish 1992
). Work by Connor and Stevens
(1971a
,b
) with molluscan ganglion cells demonstrated that fast
transient K+ currents (A current) could be
instrumental in transducing graded stimulating currents into graded
firing rates. Since these initial studies other workers
(Pallotta and Wagoner 1992
; Rogawski
1985
; Rudy 1988
; Storm 1988
) have
described transient K+ current in numerous other
types of neurons and excitable cells. A slower inactivating current,
termed D current, has also been described and is reported
to differ from the faster inactivating A current in so far
as it has slower inactivation rates, steady-state properties at
different voltages, and enhanced sensitivity to 4-aminopyridine (4-AP),
dendrotoxin (DTx), and mast cell degranulating peptide (Castle
et al. 1989
; Dolly 1988
; Moczydlowski et
al. 1988
; Strong 1990
). Foehring and
Surmeier (1993)
, reported that D current is, in effect, a
residue of a number of transient currents other than A.
Cutaneous afferent neurons display hyperexcitability and ectopic
impulse generation after nerve injury (Devor 1994;
Rasminsky 1978
; Scadding 1981
;
Wall and Devor 1978
) and changes in sodium current
properties (Oyelese et al. 1995
, 1997
; Rizzo et
al. 1995
). While neurons undergo distinct changes in response
to nerve injury, it is not clear how these changes may contribute to
the abnormal impulse generation of injured nerve. It has been suggested
that a reduction in K+ currents after injury
contributes to neuronal excitability (Devor 1983
, 1994
).
Other workers have indicated that, in addition to changes in
excitability of injured axons, DRG neurons themselves may become
generators of ectopic impulses (Burchiel 1984
;
Desantis and Duckworth 1982
; Devor and Wall
1990
; Kajander et al. 1992
). It has been
demonstrated that a selective reduction in slow
Na+ currents (Rizzo 1997
;
Rizzo et al. 1995
) and a faster repriming of kinetically
fast Na+ currents (Cummins and Waxman
1997
) takes place after axotomy in these neurons. Given that
K+ currents are important in regulating the
firing properties of neurons, it is essential to determine whether
changes in K+ currents are taking place after
axotomy in cutaneous afferent DRG neurons. Data obtained regarding
K+ currents in these axotomized DRG neurons have
implications for understanding the mechanisms of hyperexcitability
after injury.
In the present study we examined the effects of sciatic nerve ligation
on the two types of inactivating current, and the dominant sustained
current, in large cutaneous afferents. Our results indicate a selective
and large reduction in IK and
IA in axotomized cutaneous afferent
neurons. The neurons studied are of a size suggesting that they give
rise to myelinated axons likely to include A fibers, which are
involved functionally in tactile sensation of the skin. Much work now
indicates that abnormal firing properties of A
fibers contribute to
neuropathic pain either by increased peripheral excitability
(Devor 1994
) or by plastic changes in dorsal horn innervation (Woolf et al. 1995
). It is possible that the
large reduction in the K+ currents reported here
could contribute to the injury-induced increases in excitability in
these neurons.
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METHODS |
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Cell identification and culture techniques
Fluoro-gold labeling (Schmued and Fallon 1986)
was used routinely to identify cutaneous afferent DRG neurons
(Honmou et al. 1994
). Five to 7 days before sciatic
nerve section cutaneous afferents were labeled via fluoro-gold
injections (2-4% in distilled water) into the lateral plantar region
of the rat's foot. This technique has been shown to reveal cutaneous
afferent neurons with distinctly different kinetic and pharmacological
properties to those of muscle afferents (Oyelese and Kocsis
1996
; Oyelese et al. 1995
). The sciatic nerve
was exposed and transected (silk suture) near the sciatic notch
bilaterally (Kocsis et al. 1984
). To prevent
regeneration to peripheral targets, the nerve was sectioned immediately
distal to the ligature site; a 10- to 15-mm section of the distal nerve was removed, and the distal stump was retracted. A silicone cap was
sutured to the end of the proximal stump. Although most of the neurons
in the L4, L5 DRG are
axotomized by this procedure, as many 30% of the DRG neurons at
L4 and L5 may remain
unaffected because their axons leave the nerve above the ligature site
(Himes and Tesslar 1989
).
At 2-3 wk postligation, the adult Wistar rats (180-240 g) were
exsanguinated under pentobarbital sodium anesthesia (60 mg/kg ip), and
lumbar ganglia (L4, L5)
were excised and prepared for dissociation and plating on glass
coverslips (see Honmou et al. 1994; Oyelese et
al. 1995
). Results are reported in this study from 93 identified adult cutaneous afferent DRG neurons taken from 38 rats on
72 separate coverslips. Our analysis was limited to relatively large
(46-50 mm diam) cutaneous afferents, which correspond to medium-size
neurons of the entire DRG neuronal population.
Electrophysiological techniques and analysis
To avoid neurite outgrowth, which could cause variations in
expressed types and amounts of current, and to circumvent space clamp
problems, the neurons were studied 2-8 h after plating. With our
culture conditions 10 h was the maximum time after plating for
injured cells before processes start to develop. Short-term culture was
essential because the axotomized neurons sprout neurites more rapidly
than controls in culture (Lankford et al. 1997). Coverslips plated with the DRG neurons were rinsed with normal bath
solution (see: Table 1; E2), placed in a
recording chamber on the stage of an inverted phase-contrast microscope
(Nikon Diaphot), and perfused with solution. Recording electrodes were
fabricated from thin-walled, single-filamented, borosilicate glass
tubing (Warner Instrument), with a micropipette puller (Model P-97,
Sutter Instruments), and fire-polished with the use of a Narashige MI 83 microforge, to a resistance of 1-2 M
; seal resistances were
1
G
. All recordings were made at room temperature (20-22°C).
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Voltage-clamp recordings were made in the whole cell patch-clamp
configuration (Hamill et al. 1981) using an EPC 9 amplifier driven by the "Pulse" program (Heka-Electronik,
Lambrecht, Germany). The neurons were voltage-clamped and initially
held at
80 mV before application of voltage-clamp protocols. Capacity
and leakage subtraction were performed using a p/±6
subtraction protocol. Cells used in this series of experiments were of
90 ± 5 pF capacitance to reduce overall variability in cell size
when comparing between groups. Series resistance compensation of
60-75% was employed routinely to reduce voltage error. Cells in which
series resistance compensation was possible but <60% were discounted
from the study. Current was sampled at 50 kHz, low-pass filtered
(4-pole Bessel) at 10 kHz unless otherwise stated and initially
digitized stored and analyzed on computers (Macintosh, Quadra 700, and
PowerBook 1400c). Outward potassium currents were elicited by stepping
to a conditioning voltage of either
40 or
120 mV from a holding potential of
80 mV; the membrane then was depolarized to
40 mV and
on up to +50 mV in increments of 10 mV (Fig.
1); +50 mV produced the largest peak
current in each recording. Activation of the currents in the standard
solution was rapid and decay only partial during a 300-ms
depolarization pulse. Amplitudes and rates of rise in absolute current
increased with increasing depolarization. The complement of currents
that may have been manifest in any of the cells being examined
initially was probed using the pulse protocols. This allowed
determination of which type of pharmacological protocol followed when
attempting to dissect out the various types of current. If a particular
current, or currents, was thought to be absent in any one cell,
presentation of relevant pharmacological blockers could confirm
generally the absence or presence of particular currents.
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Heka analysis programs (Adams and List, Westbury, New York), IgorPro
(WaveMetrics, Lake Oswego, OR), and Excel (Microsoft, Redmon, WA) were
used to analyze data. Neurons used in the analysis were those held
stable for 10 min to facilitate diffusion of the electrode solution
into the neuron, ensuring more stable and uniform responses. Where
necessary, currents were zero-subtracted (DC leak current based on the
data points corresponding to the 1st stored segment are subtracted to
0). Exponential fits via the Heka analysis program for Macintosh, which
uses the root mean square (RMS) deviation between fit and data, were
applied to estimate inactivation times. The RMS value is used as
residual for the simplex fit algorithm (see: Heka Patch Clamp EPC9,
Pulse Fit Manual 8.0 1997). Statistical analysis was by way of a paired
two sample Student's t-test for mean.
Solutions
Na+ ions were replaced with tetramethylammonium chloride (TMA), a nonpermeant ion; tetrodotoxin (TTX) and CdCl2 were used to block Na+ and Ca2+ current, thus allowing selective recording of K+ current. The standard bath solution contained (in mM) 140 TMA, 3 KCl, 10 HEPES, 1 CaCl2, and 1 MgCl2 (see Table 1). TTX (1 µM) and CdCl2 (100 µM) were added to block Na+ and Ca2+ currents, respectively. Osmolarity was adjusted to 305-310 mosmol using glucose, and pH was titrated to 7.4 using TMA hydroxide. To reduce current amplitude and in an attempt to reduce errors caused by series resistance artifacts, TMA was used as the primary nonpermeant monovalent cation. Electrodes were filled with (in mM) 100 TMA, 40 KCl, 10 EGTA, and 1 MgCl2; osmolarity was adjusted to 300-305 mosmol with glucose, and the pH was adjusted to 7.2. A preliminary investigation showed no detectable effect on outward currents of either 100 µM (n = 6) or 200 µM (n = 6), CdCl2.
Pharmacological tools by which the individual currents could be isolated were limited. 4-AP, which was used to isolate "A" type currents, was chosen because the cells remain relatively stable and it could, to some extent, be washed out, proving the effect was real. DTx was used to isolate "D" type current, and although it was impossible to wash it out, it is virtually the only compound that can isolate this current type. Drugs were presented by changing the chamber perfusate via a four-way delivery tube attached to a single vent in the chamber. The recording chamber (volume 1.0 ml) was perfused continuously at 0.75-1.25 ml/min. An effective concentration was established for 4-AP (applied in the perfusion solution) by presenting 1, 10, 50, 100, and 200 µM and 1, 2, 4, and 6 mM sequentially. Dendrotoxin, from the African green mamba Dendroaspis angusticeps (Research Biochemicals International) was presented at 1 and 2 µM.
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RESULTS |
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Potassium currents were recorded from relatively large (46-50 mm
diam) cutaneous afferents that correspond to medium-sized neurons of
the entire DRG neuronal population. These neurons give rise to
myelinated axons relating to skin mechanoreceptors; however, we were
unable to distinguish between high (HTMR)- and low (LTMR)-threshold mechanoreceptors. Smaller nociceptive neurons were not examined. Results are reported in this study from 93 identified adult cutaneous afferent DRG neurons. With symmetrical K+
concentrations (40 mM inside and outside; see Table 1, E1) the I-V curve shifted to the right, when compared with
asymmetrical K+ concentrations, and the
K+ equilibrium potential was close to the
predicted reversal potential of 0 mV (Everill et al.
1998) (n = 5). This is commensurate with K+ being the principal charge carrier.
Figure 1 shows a comparison of K+ currents
recorded from an uninjured neuron (Ai and Aii)
with those from an injured (axotomized) neuron (Bi and
Bii). The records were obtained by holding the resting
potential at 80 mV. Activation of these currents was rapid and decay
only partial during a 300-ms depolarization pulse. Ai and
Bi were recorded after a 500-ms conditioning prepulse
potential of
120 mV and those of Aii and Bii
using a conditioning prepulse potential of
40 mV; outward currents
were elicited by stepping from
40 mV in increments of 10 up to +50 mV
(see the pulse protocol representations on the far right of
Fig. 1). These pulse protocols were used in Figs. 1-4. Current
sensitive to the conditioning voltage was exposed by subtraction of the
40-mV protocol current from the
120 mV protocol (Fig. 1,
Ai
Aii and Bi
Bii). Note the reduction in amplitude of the overall current
in the injured neuron (Fig. 1, Bi and Bii) and
the apparent lack of inactivating currents (see also Table
2).
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In many uninjured cells, A current could be dissected from the
sustained current using two different prepulse voltages
(Vc) with identical stimulation pulse
protocols (Vp) (see Everill et al. 1998 for indepth analysis and description of seperation of currents); however, this was dependent on whether the slower D-type current was present. When D current was present, along with A current,
it was difficult to separate the two currents without further
pharmacological protocols. In this example, the small amount of fast
inactivating A current present in this cell can be seen to be
contaminated with the slower inactivating D current in subtraction
Ai
Aii. In the Bi
Bii subtract, the much reduced levels of voltage-activated
currents commonly recorded in injured neurons, as compared with
uninjured neurons (Ai
Aii), is clearly visible.
Figure 2 shows a comparison of uninjured
(Ai and Aii) with injured (Bi and
Bii) neurons after presentation of 1 µM DTx.
Foehring and Surmeier (1993) reported that DTx blocked
virtually all slowly inactivating current while sparing the initial
fast transient A current in rat cortical neurons, an effect confirmed
in these DRG neurons (Everill et al. 1998
). Currents in
Fig. 2 are recorded from the same cells as those in Fig. 1. These are
typical current configuration recordings observed in the injured and
uninjured cell groups obtained within 2 min of presentation of 1 µM
DTx (n = 10; Table 2). DTx is seen to reduce peak
currents in the uninjured cell by as much as 28% (n = 15) (compare Fig. 1, Ai and Aii with Fig. 2,
Ai and Aii) in this example. DTx in the injured cell appeared to have no significant effect on peak currents, indicating that in this cell, as in some other cells, little, if any,
slowly inactivating current was present. DTx, at this concentration,
did not wash out after 20 min; beyond this time the input resistance
decreased, causing recordings to become more unstable as a function of
time.
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A variety of pharmacological and kinetic techniques traditionally have
been used to identify fast transient outward currents (A currents) in
both mammalian and nonmammalian neurons (Thompson 1977).
Criteria for their identification include rapid activation and
inactivation, dependence on the holding potential, and sensitivity to
both 4-AP and DTx (Wu and Barish 1992
). Figure
3 shows the effects of 6 mM 4-AP on
uninjured cutaneous afferent DRG neurons. Uninjured cells generally
expressed large fast inactivating current, or A current, that can be
seen in subtraction Ai
Aii. This fast inactivating current is abolished by high doses (6 mM) of 4-AP (subtract Bi
Bii) and shows some recovery
on washout (Ci
Cii).
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The effects of 6 mM 4-AP on an injured cutaneous afferent DRG neuron is
shown in Fig. 4. High doses of 4-AP (6 mM) completely removed any fast inactivating A current present and also
the slower inactivating D-type current (n = 20). The
injured (axotomized) cell, in this example, shows slowly inactivating
current but little, if any, fast inactivating current (Fig. 4, subtract
Ai Aii; see Table 2). Comparison of
current subtracts in uninjured (Fig. 3) and injured (Fig. 4) neurons,
gives an indication of the differences in the complement of currents
between the two groups. The effects of high concentrations (6 mM) of
4-AP sometimes required up to
40 min for an effective washout
(n = 4). Figure 4 shows a cell expressing slowly
inactivating current with little if any fast inactivating current being
in evidence (Ai
Aii), which also can be
removed completely by a high concentration of 4-AP (Bi
Bii), and partially restored after washout
(Ci
Cii). This series of experiments
demonstrated the large difference between the amounts of "pure"
sustained K current that uninjured cells (Fig. 3, Bi and
Bii) have over injured (Fig. 4, Bi and
Bii) cells and also the relatively large amount of A current
found in the uninjured cells (Fig. 3, subtract Ai
Aii) when compared with the injured cells (Fig. 4, subtract
Ai
Aii; see Table 2).
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Inactivation characteristics of A and D currents in uninjured
(Ai) and injured (Bi) DRG neurons are shown in
Fig. 5. Currents were recorded at a test
potential of +80 mV after 80-ms conditioning depolarizations to
voltages rising from 110 to
10 mV in 10-mV steps. In Fig. 5,
A2 and B2, inactivating currents are isolated by
subtraction of the current recorded after the conditioning depolarization of
10 mV (initiating K current) from all other current
traces in that current family (in A1 and B1).
Figure 5C shows a comparison of conductance between
A2 and B2. This example shows that the rate of
inactivation of the K+ channels in both the
injured and control groups is almost identical. The cells selected for
analysis here have currents approximating those around the mean for
cells in the two experimental groups (control and injured).
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The relative contributions of the three K-current components for the population of neurons studied are compared in Fig. 6 for control and axotomized cells. Ai demonstrates the typical complement of currents in control cells where all three currents are manifest. Aii shows a smaller group of control cells that expressed only A and K current. Bi shows axotomized cells expressing A, K, and D current. Bii shows axotomized cells in which only A and K current could be detected. Overall, injured cells (B, i and ii) show greatly reduced quantities of voltage-activated currents than uninjured cells (A, i and ii). However, current sensitive to DTx in both uninjured and injured cells is manifest in similar quantities. The mean sustained current is >60% smaller in injured neurons: 7.11 nA compared with uninjured neurons, 18.38 nA (see Table 2). Standard error bars on the mean for each current are shown. Statistical comparisons of cells from the axotomized group with those from the group without ligation, matching currents, are also shown (*P > 0.02, **P > 0.05).
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DISCUSSION |
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Primary afferent neurons have a diversity of
Na+ (Caffrey et al. 1992;
Cummins and Waxman 1997
; Honmou et al.
1994
; Kostyuk et al. 1981
; Rizzo et al.
1994
; Roy and Narahashi 1992
) and
K+ (Everill et al. 1998
;
Gold et al. 1996
) currents the relative distribution of
which varies in different functional classes and within a given
functional class. Recently interest has focused on changes in the
relative distribution DRG Na+ currents in normal
and axotomized neurons. It is now clear that kinetically slow
TTX-resistant Na+ currents are reduced on both
nociceptive (Cummins and Waxman 1997
) and larger
cutaneous afferent neurons (Oyelese et al. 1997
; Rizzo et al. 1995
) after nerve ligation. Moreover the
kinetically fast Na+ current, although not
reduced after nerve injury, shows kinetic changes in that it
"reprimes" faster (Cummins and Waxman 1997
). DRG
neurons can express a large number of different sodium channel alpha-subunit mRNAs (Black et al. 1996
), therefore,
sodium channel subunit configuration might change after injury. It
recently has been demonstrated that axotomy induces an increase in the
level of type-III and a decrease in SNS sodium channel mRNA in DRG
neurons (Dib-Hajj et al. 1998
; Waxman et al.
1994
). Although not definitive, these changes in DRG neuronal
Na+ currents after injury of their axons have
been suggested to play a role in the hyperexcitability properties of
injured cutaneous afferent neurons (Cummins and Waxman
1997
; Honmou et al. 1994
; Oyelese et al.
1997
; Rizzo et al. 1995
); reduction in the slow current and more rapid repriming of the fast currents could provide an
environment that would allow higher impulse frequency generation and
abnormal sensory signaling.
In addition to the important role of Na+ currents in defining the firing characteristics of a neuron, K+ currents are also important. The hyperpolarizing effects of K+ currents can significantly alter and shape the frequency-response properties of neurons. Yet little is know of plastic changes in K+ currents of cutaneous afferent DRG neurons in response to nerve injury. In the present study, we demonstrated that specific K+ current components on injured adult cutaneous afferent neurons of relatively large size are reduced several weeks after nerve ligation. This result has an additional element to be considered for explaining the hyperexcitability of injured cutaneous afferents, i.e., a reduction in the hyperpolarizing effects of K+ current that could contribute to increased or ectopic impulse firing.
Specific changes in DRG K+ current components after nerve ligation
Three primary K+ current components have
been identified on large cutaneous afferent DRG neurons, a dominant
sustained current (K current) and two transient currents (A and D
current) (Everill et al. 1998). With regard to
amplitude, the K current is the dominant current. Early studies on DRG
K currents concluded that transient currents were not present
(Robertson and Taylor 1986
). Subsequent work reveals
that transient A and D currents are present but masked by the dominance
of the K current (Everill et al. 1998
; Gold et al. 1996
). In the present study, potassium concentrations in
both external and internal solutions were decreased to reduce the
dominance of the K current to make the study of the smaller amplitude
transient currents more tractable. After nerve ligation, the sustained
K current is reduced by nearly half in the large cutaneous afferent neurons. Functionally, the large primary afferent K current has been
suggested to limit repetitive firing by holding the membrane potential
near EK. Indeed, DRG neurons
(Oyelese and Kocsis 1996
; Oyelese et al.
1995
) and their axons (Birch et al. 1991
;
Kocsis et al. 1983
) show very rapid accommodation to an
applied sustained depolarization whereby only a single or a small group
of action potentials is generated. It will be interesting to determine
if the accommodation properties of injured DRG neurons are altered because of the reduction in K current after axotomy. Fast transient potassium currents (A current) in adult DRG neurons, as in other types
of cell, are thought to modulate the repolarization of single action
potentials, the time required to reach the threshold to fire an action
potential and to influence repetitive firing (Budde et al.
1992
; Hammond and Crepal 1992
; Kocsis et
al. 1986
; Numann et al. 1987
; Storm 1988
,
1990
; Wu and Barish 1992
). Thus these attributes could be altered in cells showing reductions in these currents and could result in abnormal firing properties.
In this study, A current was relatively large in control cells and
reduced by ~50% in axotomized cells. One might predict that the
action potential of the axotomized DRG neuron would broaden because of
the reduction in IA. However, there is
evidence to suggest that the action potential of these neurons actually
narrows (Oyelese et al. 1997). The narrowing is
attributed to a reduction in slow Na+ current
accompanied by a reduction in Ca2+ current in
axotomized neurons, the activation of which gives rise to an inflection
on the falling phase of the action potential, thus broadening the spike
(Oyelese et al. 1997
).
Slow-inactivating current (or D current) has been identified or
observed in a number of CNS and PNS neuronal types (Albert and
Nerbonne 1995; Ficker and Heinemann 1992
;
Foehring and Surmeier 1993
; Gean and
Schinnick-Gallager 1989
; Halliwell et al. 1986
; Stansfeld and Feltz 1988
; Stansfeld et al. 1986
,
1987
, 1991
; Storm 1988
; Surmeier et al.
1989
, 1991
; Wu and Barish 1992
). Other studies have reported only one class of transient potassium current in a
variety of neuronal types (Ahmed 1988
; Cull-Candy
et al. 1989
; Numann et al. 1987
; Zona et
al. 1988
; Segal and Barker 1984
;
Takahashi 1990
). Slowly inactivating transient current
is distinct from A current, showing differences in activation and
inactivation at different voltages (inactivating more slowly during
depolarizing voltage steps) and showing an enhanced sensitivity to 4-AP
and DTx (Castle et al. 1989
; Dolly 1988
;
Moczydlowski et al. 1988
). This suggests that the
channels responsible for the slowly inactivating current are similar to
Foehring and Sumeier's (1993)
proposed K3 channel population in rat neocortical neurons,
which also shows similar sensitivities. Because many of the reports of
this type of current are in fast-conducting axons and their cell
bodies, it has been postulated that this could reflect their
requirement of rapid firing and secure conduction (Brew and
Forsythe 1995
). Although this slowly inactivating current is
variable from cell to cell and relatively difficult to isolate, we did
not observe changes between control and axotomized neurons.
Functional consequences of injury-induced reduction in K+ currents
In the present study, we limited our analysis to the larger
identified cutaneous afferents that represent medium-sized neurons for
the entire DRG population. Many of these larger cutaneous afferents are
likely A neurons, which normally transmit tactile information and
terminate primarily in lamina III of the dorsal horn. However, after
nerve injury there is synaptic reorganization in the spinal cord, and
the A
fibers sprout and have more extensive terminations in lamina
II, the normal synaptic site for nociceptive fibers (Woolf et
al. 1995
). Therefore changes in K+
currents observed in the larger cutaneous afferents studied here, after
nerve injury, have implications for mechanisms underlying the
pathophysiology of neuropathic pain. For example, if the reduction in
overall K+ current renders the neurons more
excitable, A
neurons could increase the afferent input to new
ectopic synaptic sites in lamina II resulting in abnormal sensations.
The reduction in K+ currents on DRG neurons also
has implications for potential changes in K+
currents and firing properties of injured axons. It is well established that axons entering a neuroma become hyperexcitable (Devor and Wall 1990), and some work indicates that
K+ currents are reduced (Devor
1994
). In normal cutaneous afferent axons,
IA has been shown to be important in
preventing activation of a kinetically slow Na+
current (Honmou et al. 1994
; Kocsis et al.
1993
); when IA is blocked by
4-aminopyridine, axonal action potentials observed with intra-axonal
recordings give rise to a delayed depolarization that has been
attributed to activation of a kinetically slow
Na+ current not present on muscle afferents of
the same cell body or axon size (Honmou et al. 1994
).
The delayed depolarization often gives rise to multiple spike discharge
from a single stimulus (Kocsis et al. 1983
). However,
cutaneous afferent DRG neurons lose the kinetically slow
Na+ current after axotomy (Rizzo et al.
1995
). The altered kinetics of the remaining
Na+ current that lead to faster repriming of the
sodium currents (Cummins and Waxman 1997
) and the
reduced IA provide an environment that
could allow higher frequency discharge leading to inappropriate sensory signaling.
Finally it has been shown that nerve growth factor (NGF) applied to the
cut ends of axotomized neurons reduces the loss in slow
Na+ current (Oyelese et al. 1997)
and the reduction in the SNS Na+ channel subunit
mRNA observed after axotomy (Dib-Hajj et al. 1998
). In
contrast, changes in cutaneous afferent DRG neuronal GABAA receptors induced by injury are reduced by
nerve application of brain-derived neuronotropic factor (BDNF)
(Oyelese et al. 1997
). These effects of the NGF and BDNF
are specific to the cutaneous afferents and not to muscle afferent
neurons. It will be interesting to determine if the reduction in
cutaneous afferent K+ current is modulated by
these or other neurotrophins which may be lost when the axon is
disconnected from skin.
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ACKNOWLEDGMENTS |
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We thank Drs. Marco A. Rizzo and Theodore R. Cummins for helpful discussions of data acquisition and analysis and H.-F. Mi for the preparation of neuronal cultures.
This work was supported in part by the Medical Research Service of the Department of Veterans Affairs and by the National Institutes of Health.
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FOOTNOTES |
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Address for reprint requests: J. D. Kocsis, Yale University School of Medicine, Neuroscience Research Center (127A), Department of Veterans Affairs Medical Center, West Haven, CT 06516.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 16 December 1998; accepted in final form 30 March 1999.
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