Department of Neurology, Yale University School of Medicine, New Haven 06510; and Department of Veterans Affairs, Neuroscience Research Center, West Haven, Connecticut 06516
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ABSTRACT |
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Baccei, Mark L. and
Jeffery D. Kocsis.
Voltage-Gated Calcium Currents in Axotomized Adult Rat Cutaneous
Afferent Neurons.
J. Neurophysiol. 83: 2227-2238, 2000.
The effect of sciatic nerve injury on the
somatic expression of voltage-gated calcium currents in adult rat
cutaneous afferent dorsal root ganglion (DRG) neurons identified via
retrograde Fluoro-gold labeling was studied using whole cell
patch-clamp techniques. Two weeks after a unilateral ligation and
transection of the sciatic nerve, the L4-L5
DRG were dissociated and barium currents were recorded from cells 3-10
h later. Cutaneous afferents (35-50 µm diam) were classified as type
1 (possessing only high-voltage-activated currents; HVA) or type 2 (having both high- and low-voltage-activated currents). Axotomy did
not change the percentage of neurons exhibiting a type 2 phenotype or
the properties of low-threshold T-type current found in type 2 neurons.
However, in type 1 neurons the peak density of HVA current available at
a holding potential of 60 mV was reduced in axotomized neurons
(83.9 ± 5.6 pA/pF, n = 53) as compared with
control cells (108.7 ± 6.9 pA/pF, n = 58, P < 0.01, unpaired t-test). A
similar reduction was observed at more negative holding potentials,
suggesting differences in steady-state inactivation are not responsible
for the effect. Separation of the type 1 cells into different size
classes indicates that the reduction in voltage-gated barium current
occurs selectively in the larger (capacitance >80 pF) cutaneous
afferents (control: 112.4 ± 10.6 pA/pF, n = 30; ligated: 72.6 ± 5.0 pA/pF, n = 36;
P < 0.001); no change was observed in cells with
capacitances of 45-80 pF. Isolation of the N- and P\Q-type components of the HVA current in the large
neurons using
-conotoxin GVIA and
-agatoxin TK suggests a
selective reduction in N-type barium current after nerve injury, as the
density of
-CgTx GVIA-sensitive current decreased from 56.9 ± 6.6 pA/pF in control cells (n = 13) to 31.3 ± 4.6 pA/pF in the ligated group (n = 12;
P < 0.005). The HVA barium current of large
cutaneous afferents also demonstrates a depolarizing shift in the
voltage dependence of inactivation after axotomy. Injured type 1 cells exhibited faster inactivation kinetics than control neurons, although the rate of recovery from inactivation was similar in the two groups.
The present results indicate that nerve injury leads to a
reorganization of the HVA calcium current properties in a subset of
cutaneous afferent neurons.
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INTRODUCTION |
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The plasticity of calcium channel characteristics
after axotomy has been previously demonstrated in sympathetic ganglion
B-cells of the bullfrog as a reduction in peak barium current and
accelerated inactivation kinetics (Jassar et al. 1993).
Voltage-gated calcium channels (VGCC) are generally classified into
high-voltage-activated (HVA; N, L, P, Q, and R-type) and
low-voltage-activated (T-type) groups that possess distinct kinetic
and pharmacological properties (for review see Dolphin
1995
; Llinas et al. 1992
; Stea et al. 1995
). The changes in sympathetic B-cells predominantly
involved N-type channels because ~90% of the voltage-gated calcium
current in these neurons consists of N-type current (Elmslie et
al. 1992
; Jassar et al. 1993
; Jones and
Jacobs 1990
; Jones and Marks 1989a
). Mounting
evidence suggests that cellular functions may be selectively regulated
by particular calcium channel subtypes, such as the modulation of
neurotransmitter release by N- and P\Q-type channels (Tsien et al. 1988
) and the role of T-type current in
burst firing (White et al. 1989
). Calcium influx via
VGCC is known to regulate a variety of neuronal processes such as gene
transcription, intracellular Ca2+ release, and
neurite outgrowth (Ghosh and Greenberg 1995
). In addition, Ca2+ modulates membrane excitability in
many neurons via the activation of Ca2+-dependent
K+ conductances (Sah 1996
). Thus
an injury-induced change in the relative proportions of various calcium
channel subtypes expressed by neurons may have significant functional
implications. Dorsal root ganglion (DRG) neurons possess a diversity of
voltage-gated calcium channels (Fox et al. 1987
;
Mintz et al. 1992
). This heterogeneity of channel
subtypes found in DRG neurons provides an opportunity to determine
whether axotomy preferentially targets a specific subtype of VGCC.
The present study examined the effect of sciatic nerve transection on
the biophysical properties of voltage-gated calcium currents in medium
and large-sized (35-50 µm diam) cutaneous afferent DRG neurons of
the adult rat. Injury-induced alterations in the electrophysiological
properties of these neurons have been implicated in pathophysiological
events following nerve injury such as tactile allodynia (Gracely
et al. 1992). These cells have previously been shown to alter
the expression of GABAA receptors (Oyelese
and Kocsis 1996
), Na+ (Oyelese et
al. 1997
; Rizzo et al. 1995
) and
K+ (Everill and Kocsis 1999
)
currents after injury. Although the properties of the low-threshold
T-type current were unchanged by axotomy in this class of DRG neuron,
the inactivation kinetics of the HVA current were accelerated after
nerve injury. Moreover, in the largest cutaneous afferents there was a
reduction in the peak density of HVA barium current after axotomy due
to a selective reduction in N-type current, and a shift in the voltage
dependence of inactivation. These alterations in voltage-gated calcium
currents, along with previously documented changes in
Na+ and K+ currents,
suggest that all three major classes of voltage-gated ion channels
undergo changes after nerve injury in a subset of cutaneous afferent neurons.
A portion of this work has been previously reported in abstract form
(Baccei and Kocsis 1999).
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METHODS |
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Identification of cutaneous afferent DRG neurons
The somata of cutaneous afferents were identified via retrograde
labeling with hydroxy-stilbamidine (Fluoro-gold) (Honmou et al.
1994; Oyelese and Kocsis 1996
). Female Wistar
rats (140-160 g) were anesthetized with an intraperitoneal injection
of ketamine (40 mg/kg) and xylazine (2.5 mg/kg). A 4% solution of
Fluoro-gold (Fluorochrome, Englewood, CO) mixed in distilled water was
injected subcutaneously into the lateral aspect of the foot and ankle
(innervated by the sural nerve). Cutaneous afferents could be
distinguished in vitro by fluorescence on brief exposure to ultraviolet light.
Nerve ligation and cell culture techniques
One week after the injection of Fluoro-gold, the female rats
were again anesthetized as described above. The sciatic nerve was
exposed and ligated (with 4-0 silk suture) unilaterally near the
sciatic notch (Kocsis et al. 1984) and subsequently
transected. To prevent regeneration and promote the development of a
neuroma, the proximal nerve stump was sutured into a silicon cap, and a 10- to 15-mm section of the nerve was removed distally as described previously (Oyelese and Kocsis 1996
). Cutaneous afferent
neurons from the contralateral (unoperated) side were used as controls.
Although transection of the sciatic nerve will not axotomize the entire population of DRG neurons, cells in the DRG that send axons into peripheral nerves other than the sciatic nerve are unlikely to innervate the skin of the lateral foot or ankle. As a result, the neurons spared by the sciatic nerve injury will not be labeled with Fluoro-gold and subsequently sampled in these experiments. Thus on the side ipsilateral to the axotomy, the Fluoro-gold was used as a marker of axotomized cutaneous afferent neurons that originally innervated a selective area of the skin.
Two to four weeks after axotomy, the rats (180-240 g) were deeply
anesthetized with an intraperitoneal injection of pentobarbitol sodium
(60 mg/kg) and exsanguinated. The L4 and
L5 DRG were excised and dissociated
(Oyelese et al. 1995). Cells were plated on
polyornithine-treated coverslips and maintained at 37°C until use.
Our analysis focused on the medium to large diameter cutaneous
afferents (35-50 µm), which possess myelinated axons in vivo
(Harper and Lawson 1985
).
Electrophysiological techniques and analysis
The neurons were studied 3-10 h after dissociation to minimize
neurite outgrowth and subsequent space-clamp problems. Coverslips were
placed in a recording chamber (~0.5 ml volume) on the stage of an
inverted phase-contrast microscope (Nikon Diaphot) and rinsed with a
solution consisting of (in mM) 140 NaCl, 3 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES
(315-320 mosM, pH 7.3-7.4) at a rate of 1-3 ml/min. Patch electrodes
were constructed from thin-walled, single-filamented borosilicate glass
capillaries (World Precision Instruments) using a micropipette puller
(Model P-97, Sutter Instruments) and fire-polished with a Narishige MI
83. Pipette resistances ranged from 1.5 to 2.5 M; seal resistances
were >1 G
.
Whole cell patch-clamp recordings (Hamill et al. 1981)
were obtained using an Axopatch 1-D amplifier equipped with a low-gain headstage (CV-4 0.1/100). Currents were filtered at 5 kHz through a
3
dB, 4-pole low-pass Bessel filter, then digitally sampled at 1.5-5 kHz
and stored on a computer (Gateway 2000 486/33C) using a commercially
available data acquisition system (TL-1 DMA Interface with pClamp
software, Axon Instruments). Subtraction of capacitive and leak
currents were performed using a P-P/6 protocol. Series resistance
compensation of 60-90% was generally achieved to reduce voltage
error. Measurements of cell capacitance were obtained by fitting a
single exponential curve to the uncompensated current trace resulting
from a voltage-clamp step from
60 to
70 mV. Recordings were
obtained at room temperature (20-22°C).
Solutions
To isolate Ca2+ currents, the extracellular solution contained (in mM) 160 tetraethylammonium chloride (TEA-Cl), 10 HEPES, 1 BaCl2, 1 MgCl2, 0.0002 tetrodotoxin (RBI; 315-325 mosM with sucrose, pH 7.4 with TEA-OH). Patch electrodes were filled with a solution consisting of (in mM) 90 CsCl, 30 TEA-Cl, 20 HEPES, 5 BAPTA, 5 MgATP, 0.4 Na2GTP (300-305 mosM with sucrose, pH 7.4 with CsOH). The whole cell calcium current was abolished by addition of 100 µM CdCl2 to the above perfusion solution.
An accurate quantification of the inhibition of HVA
Ca2+ currents by antagonists or neuromodulators
requires consideration of the rundown of current amplitudes that
results from cell dialysis and a process of slow inactivation
(Fenwick et al. 1982; Forscher and Oxford
1985
). At a stimulus frequency of 0.10 Hz, current amplitudes
generally decayed monoexponentially and became relatively stable 5-10
min after patch rupture, after which various antagonists could be
applied to block selective subtypes of Ca2+
current. Once the rate of decay had attenuated, the rate of rundown in
the period 40-60 s before drug application was calculated, and this
rate was used to correct the drug effects to reflect the approximate
extent of current rundown during drug application. To achieve block of
N-type calcium channels, the flow of the bath was stopped, and a
10-µM solution of
-conotoxin GVIA (Sigma; prepared on the day of
the experiment from a 0.5-mM stock in distilled H20) was pipetted into the bath at 1:10 to give a
final concentration of ~0.9 µM. N-type current was defined as the
fraction of peak current blocked by this concentration of
-CgTx GVIA
(Scroggs and Fox 1992
). Once a stable current amplitude
was observed in the presence of
-CgTx GVIA, a 10 µM solution of
the P\Q-type antagonist
-agatoxin TK (Sigma,
prepared from a 0.2-mM stock in distilled H20 on
day of experiment) was pipetted into the bath at 1:9 to obtain a final
concentration of ~1.0 µM. The fraction of HVA current blocked by
this concentration of
-AgaTx TK was classified as
P\Q-type for the purposes of this study.
Data analysis and statistics
Data were analyzed using pClamp software (Clampfit, Axon Instruments). Unless otherwise stated, independent two-tailed t-tests assuming unequal variances were utilized to test for levels of significant difference between groups. Data are expressed as means ± SE.
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RESULTS |
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Classification of cutaneous afferent neurons
Patch-clamp recordings were obtained from identified cutaneous
afferents between 35 and 50 µm diam, which constituted medium to
large neurons in the DRG cell population. Smaller DRG neurons (<35
µm diam) were not examined in the present study. Measurements of cell
capacitance were used to classify cutaneous afferents into medium
(Cin = 45-80 pF) and large
(Cin > 80 pF) groups. Neurons were
also categorized as "type 1" or "type 2" based on the absence (Fig. 1A) or the presence
(Figs. 1B, 8) of a low-threshold barium current that
inactivated rapidly ( = 25-50 ms) and resembled the T-type
current previously characterized in DRG neurons (Carbone and Lux
1984
, 1987
; Schroeder et al.
1990
). In addition to exhibiting multiple types of HVA
currents, type 2 cells displayed T-type current that is evident by a
shoulder present on the current-voltage (I-V) curve at
negative test depolarizations (Fig. 1C). In contrast, in
type 1 neurons only HVA current was observed as indicated by the
absence of a rapidly inactivating current component (Fig. 1A) and the lack of a shoulder at negative potentials of the
I-V curve (Fig. 1C). In control cutaneous
afferents, 72.5% (58/80) of all cells examined exhibited a type 1 phenotype, whereas 27.5% (22/80) had significant low-threshold current
characteristic of type 2 neurons. Axotomy of the sciatic nerve had no
effect on the frequency of the different subtypes, because 82.8%
(53/64) and 17.2% (11/64) of injured neurons demonstrated the type 1 and type 2 phenotype, respectively (Fig. 1D,
P > 0.05,
2 test).
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Reduction in peak barium current density in large cutaneous afferents after axotomy
Current-voltage relationships in control and axotomized neurons
were examined by the application of 150-ms voltage steps to various
test potentials from a holding potential of 60 mV. From this
relatively depolarized holding potential, cells could be reliably
classified as type 1 or type 2 because a small component of the T-type
current was available from Vh =
60
mV in type 2 neurons (see Fig. 8D). Peak inward barium
current levels were measured and expressed as peak current density
(pA/pF) to account for variations in cell size. As shown in Fig.
2A, peak current density was
greater in type 1 cutaneous afferents than in type 2 neurons for both
control (P < 0.0001, 2-tailed t-test
assuming unequal variances) and injured (P < 0.005)
neurons. In type 2 neurons, there was no significant effect of axotomy
on the peak density of barium current. Uninjured type 2 cutaneous
afferents showed a peak density of 49.0 ± 3.0 (SE) pA/pF
(n = 22), whereas axotomized type 2 cells had a maximum
of 55.4 ± 6.3 pA/pF (n = 11; see Fig.
2A). However, transection of the sciatic nerve resulted in a
significant reduction in peak current density in type 1 neurons from
108.7 ± 6.9 pA/pF (n = 58) in control neurons to
83.9 ± 5.6 pA/pF (n = 53) in the injured group
(P < 0.01). Further separation of the type 1 neurons
into medium (45-80 pF) and large (>80 pF) size classes reveals that
the downregulation of barium current occurs selectively in the larger
cutaneous afferents (Fig. 2B). Axotomy decreased peak
current density in this size group from 112.4 ± 10.6 pA/pF
(n = 30) in control neurons to 72.6 ± 5.0 pA/pF (n = 36) in the injured group (P < 0.001). In the medium-sized type 1 neurons, the peak current densities
were similar between the two groups (control: 104.8 ± 9.0 pA/pF,
n = 28; ligated: 107.9 ± 12.3 pA/pF,
n = 17).
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Effect of N-type and P\Q-type antagonists in control and axotomized cutaneous afferents
The selective Ca2+ channel antagonists
-conotoxin GVIA (N-type) and
-agatoxin TK
(P\Q-type) were utilized to determine whether certain
subtypes of calcium channels were preferentially downregulated in large
cutaneous afferents after nerve injury, or whether the composition of
the HVA current remained unaltered in axotomized neurons. The HVA
current of large (>80 pF) type 1 neurons was dissected into its
composite subtypes by repetitively stepping from a holding potential of
80 mV to a test potential of
10 mV at a frequency of 0.10 Hz (Fig.
3A). On observing a stable current amplitude in response to the voltage step, the bath flow was
stopped and 0.9 µM
-CgTx GVIA was applied via micropipetting a
stock solution near the cell of interest (see METHODS),
which resulted in a rapid, largely irreversible block of N-type barium current (Fig. 3, A and B). Once the peak effect
of
-CgTx GVIA had been reached, the P\Q-type
component of the HVA current was isolated via application of 1 µM
-agatoxin TK (see METHODS). Subsequent application of
100 µM CdCl2 resulted in the complete abolition
of barium influx (Fig. 3A).
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The effect of axotomy on the relative distribution of N- and
P\Q-type currents in large type 1 cutaneous afferents
is depicted in Fig. 3C. The peak current density of total
HVA barium influx available from a holding potential of 80 mV was
99.1 ± 8.0 pA/pF (n = 13) in control neurons and
68.1 ± 7.7 pA/pF (n = 12) in axotomized cutaneous
afferents (P < 0.01, 1-tailed t-test). The
density of
-CgTx GVIA-sensitive current was significantly reduced in
the ligated group (31.3 ± 4.6 pA/pF, n = 12) as
compared with control neurons (56.9 ± 6.6 pA/pF,
n = 13; P < 0.005), suggesting an
injury-induced reduction in N-type calcium current. In contrast, the
density of P\Q-type current was not significantly
changed by axotomy, because 1 µM
-agatoxin TK blocked 25.6 ± 8.5 pA/pF of current in control neurons (n = 11) and
19.9 ± 5.1 pA/pF (n = 11) in the ligated group
(Fig. 3C). There was a relatively small contribution of
L-type channels to HVA barium influx in large type 1 cutaneous afferents as <10% of HVA barium current was blocked by 2 µM
nimodipine in both control and axotomized neurons (data not shown).
Effect of axotomy on the properties of voltage-dependent inactivation in type 1 cutaneous afferents
To determine whether the axotomy of type 1 neurons altered the
inactivation kinetics of the HVA current or the rate at which the
channels recovered from the inactivated state, a 1-s prepulse to 10
mV was applied from a holding potential of
80 mV followed by a test
step back to
10 mV after various intervals (Fig.
4A). In control neurons, the
HVA current showed 60.79 ± 1.25% inactivation (n = 20) during the 1-s prepulse, which was not significantly different
from the 58.40 ± 1.62% decrease (n = 17)
observed in ligated cutaneous afferents (Table
1). The decay phases of the prepulse
currents were fit with a sum of two exponentials (plus an offset) and
averaged to produce the results detailed in Table 1. The kinetics
describing the "fast" component of inactivation were more rapid in
injured neurons (
1 = 49.13 ± 1.71 ms,
n = 17) than in control cells
(
1 = 56.76 ± 2.60 ms, n = 20; P < 0.05), although the contribution of the fast
component to the total HVA current amplitude was similar in the two
groups (control: 26.42 ± 0.86%; ligated: 25.70 ± 1.28%;
P > 0.05). The time constant derived from the second
"intermediate" component of inactivation was also smaller in
injured neurons (
2 = 510.25 ± 15.35 ms)
compared with uninjured neurons (
2 = 573.90 ± 15.08 ms; P < 0.01). Axotomy also
reduced the percentage of peak current amplitude attributable to this
component from 39.40 ± 0.75% in control cutaneous afferents to
34.92 ± 1.38% in the ligated group (P < 0.01).
The kinetics of an additional "slow" component of inactivation were
not analyzed in detail.
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The kinetics of recovery from inactivation at different holding
potentials are illustrated in Fig. 4, B-D. The recovery
process showed a marked voltage dependence with faster kinetics
occurring at more negative holding potentials in both control and
axotomized type 1 neurons. After observing the kinetics of recovery at
Vh = 60 mV, a second trial from
Vh =
100 mV was conducted in some neurons to ensure reversibility of the voltage-dependent recovery kinetics (Jones and Marks 1989b
). Reversibility was
observed in all cells tested (not shown). The fractional recovery data
from each neuron was fit with a single exponential function to compare the rates of recovery from inactivation between the control and axotomized populations of cutaneous afferents. At each holding potential, the two groups showed no significant difference in the time
constant describing the recovery process (P > 0.05, unpaired t-test).
To investigate the possibility that the reduced levels of barium
current in large axotomized type 1 cells resulted from differences in
steady-state inactivation properties, the current-voltage relationship was examined from two different holding potentials in a subset of
cutaneous afferent neurons. In these experiments, currents were
recorded at various test potentials as described above from either
Vh = 60 mV or
Vh =
100 mV. To adjust for current
rundown, which may obscure the true dependence of peak current on
holding potential, after an I-V relationship was obtained at
each holding potential the trials were repeated in reverse order and
the currents were averaged before analysis. As illustrated in Fig.
5, the observed reduction in barium
current density in ligated cutaneous afferents is not dependent on the
choice of holding potential. From Vh =
60 mV, ligation reduced peak current levels in type 1 neurons from
108.0 ± 8.2 pA/pF (n = 21) to 67.3 ± 6.0 pA/pF (n = 22; P < 0.001). A similar
reduction was observed at Vh =
100 mV as control neurons averaged 124.4 ± 8.8 pA/pF compared
with 74.1 ± 7.7 pA/pF in the axotomized group (P < 0.001).
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The voltage dependence of the "fast" inactivation of HVA current
was examined using a 500-ms conditioning pulse to various potentials
followed by a 75-ms test pulse to 10 mV (Fig.
6A). To adjust for possible
current rundown that may occur during the trial presentation, the data
from each cell results from the average of two trials in which the
order of prepulse voltage steps was reversed during the second trial
(Jassar et al. 1993
; Jones and Marks
1989b
). In the example illustrated in Fig. 6B,
maximal inactivation occurs at a similar prepulse potential that
produces maximal Ba2+ influx during the prepulse,
and progressively larger depolarizations lead to a partial relief of
the inactivation. These characteristics can often indicate the presence
of a current-dependent form of calcium channel inactivation
(Eckert and Chad 1984
). However, if data such as that
exhibited in Fig. 6B are normalized and replotted as the
fraction of maximal prepulse current and the fraction of maximal
inactivation (Jones and Marks 1989b
), it becomes clear that significant inactivation occurs at prepulse potentials that produce no measurable Ba2+ current during the
prepulse. This relationship between maximal current activation and
maximal inactivation, illustrated in Fig. 7A for a population of control
(n = 21) cutaneous afferents, suggests the inactivation
occurs via voltage-dependent mechanisms. Other support for a
voltage-dependent process arises from the use of Ba2+ as the main charge carrier and
the inclusion of 5 mM BAPTA in the recording pipette, which should
limit the process of Ca2+-dependent inactivation
(Cox and Dunlap 1994
; Eckert and Chad 1984
).
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Fractional inactivation data such as that illustrated in Fig.
7A were obtained from control and ligated type 1 cutaneous
afferents, and the data from each cell were fit with a Boltzman
function. If the entire population of type 1 neurons is considered, the voltage dependence of inactivation (for HVA current) was not
significantly different after axotomy. Control cutaneous afferents
exhibited a midpoint of inactivation
(V1/2) of 45.8 ± 0.9 mV
(n = 21) and ligated neurons possessed a
V1/2 of
41.8 ± 1.0 mV
(n = 19; 0.05 < P < 0.10). The
slopes (k) of the Boltzman curves were similar in the two
groups (k = 14.4 ± 0.80 in control;
k = 13.2 ± 0.85 for ligated cells). However, if
the large (>80 pF) type 1 cells are considered separately (Fig.
7B), the inactivation curve for axotomized neurons is
significantly shifted to more depolarized potentials. Large control
type 1 neurons demonstrate half-maximal inactivation at
47.2 ± 1.0 mV (n = 11), whereas large ligated cells showed a
V1/2 of
40.8 ± 2.2 mV
(n = 13; P < 0.05). The slopes of the
inactivation curves in these two groups showed no significant
differences (control: k = 14.3 ± 1.4; ligated:
k = 14.3 ± 1.0).
Properties of the low-threshold barium current in control and axotomized type 2 neurons
As previously shown in Fig. 1D, the relative frequency
of cutaneous afferents expressing low-threshold T-type current (i.e., type 2 neurons) was unaltered by nerve injury. Additional experiments were performed to determine whether axotomy influenced the
characteristics of T-type barium current within this group of neurons.
Voltage steps (of 500 ms duration) to various potentials from a holding potential of 100 mV were applied (Fig.
8A), and
peak T-type current levels were measured at the test step to
40 mV (a
potential likely to produce maximal T-type current in isolation from
HVA subtypes). The voltage dependence of activation of the T-type
current was evaluated by normalizing the current amplitudes to the peak
amplitude observed at
40 mV. Steady-state inactivation of the
low-threshold current was examined through the application of a test
step to
40 mV from different holding potentials (Fig. 8B).
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A comparison of these parameters in control and axotomized type 2 cutaneous afferents is summarized in Fig. 8, C and
D. Peak T-type current levels in control and injured neurons
were not significantly different, with control neurons possessing a
peak low-threshold current amplitude of 2,186 ± 583 pA
(n = 20) and ligated neurons exhibiting an average
T-type current amplitude of 2,492 ± 870 pA (n = 10, Fig. 8C). Adjusting for variability in cell size did not
change the results, because there was no significant difference between
the two groups in the peak density of T-type current (control:
28.7 ± 7.8 pA/pF; ligated: 32.3 ± 5.7 pA/pF). There was
also no significant effect of axotomy on the voltage dependence of
activation or inactivation of the T-type current (Fig. 8D).
Boltzman fits to the data indicated that the low-threshold current in
control neurons showed a similar midpoint of activation
(V1/2 = 54.1 ± 1.0 mV) and
slope (k = 5.4 ± 0.4, n = 7) as
the ligated cutaneous afferents (V1/2 =
53.6 ± 1.4 mV; k = 5.0 ± 0.1, n = 5). There was a slight shift in the voltage dependence of inactivation of the low-threshold current as half-maximal inactivation (V1/2) occurred at
66.9 ± 1.2 mV (n = 12) in control type 2 neurons and
70.1 ± 1.4 mV (n = 10) in
axotomized cells (Fig. 8D), but this difference was not
significant (0.05 < P < 0.10). The slopes of the
inactivation curves were similar between the two groups (control:
k = 6.1 ± 0.4; ligated: k = 6.3 ± 0.8). Finally, the inactivation kinetics of the T-type
current (measured with a monoexponential fit to the current decay
during a test step to
40 mV) were not significantly altered by nerve
injury (not shown).
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DISCUSSION |
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Previous work has demonstrated changes in
GABAA receptor-mediated conductance
(Oyelese et al. 1997), Na+
(Oyelese et al. 1997
; Rizzo et al. 1995
),
and K+ (Everill and Kocsis 1999
)
current expression in cutaneous afferent DRG neurons after axotomy of
the sciatic nerve. The current findings suggest that the effects of
axotomy extend to the expression of voltage-gated calcium currents in
this class of sensory neuron, because a significant reduction in the
density of whole cell barium current was observed in large (>80 pF)
type 1 cutaneous afferents after nerve injury. It should be noted that
the presence of cesium and tetraethylammonium (TEA) in the pipette
solution prevented the association of the type 1/type 2 phenotypes of
this study with other physiological parameters, such as the shape of
the action potential waveform, often used to classify sensory neurons in previous reports. However, it is interesting that the documented changes in Na+ and K+
current expression were also found in the largest diameter cutaneous afferents, suggesting a widespread reorganization of somatic
electrophysiological properties in this population. The underlying
mechanisms for the reduction in whole cell barium current in type 1 cutaneous afferents are not known. Our data suggest that the decrease
observed at Vh =
60 mV cannot be
attributed to increased steady-state inactivation in ligated neurons
because the use of a more negative holding potential
(Vh =
100 mV) increased peak
barium currents to a similar degree in both groups (Fig. 5). This
differs from the effect of axotomy in bullfrog sympathetic ganglion
B-cells, in which a decrease in barium current levels was accompanied
by increased steady-state inactivation at depolarized holding
potentials (Jassar et al. 1993
). A possible explanation
for the reduction in barium current in cutaneous afferents is that
axotomy decreases the density of calcium channels in the somatic
membrane. This could occur via alterations in channel synthesis or
degradation, the rate of transport of the channels to more distal
locations, or posttranslational modifications affecting the insertion
of the channel into the membrane. Alternatively, changes in the
single-channel properties of somatic calcium channels may occur. The
present study does not provide detailed information on the time course
of onset or the duration of the axotomy-induced changes, thus future
experiments examining the properties of voltage-gated calcium currents
at a greater range of time points following the nerve injury would be useful.
Given the variety of VGCC found in DRG neurons and the emerging
evidence that different subtypes can regulate distinct cellular functions (Ghosh and Greenberg 1995), it was of interest
to determine whether nerve injury modulates a specific subtype of
calcium current, or whether all subtypes were reduced in an equal
manner after axotomy. Pharmacological dissection of the HVA current
with
-CgTx GVIA and
-AgaTx TK in large type 1 cutaneous
afferents suggests that ligated neurons undergo a reduction in N-type
calcium current, whereas levels of P\Q-type current are
not significantly altered (Fig. 3). Previous studies have indicated
that sympathetic neurons, in which ~90% of the calcium current is
sensitive to
-CgTx GVIA (Elmslie et al. 1992
;
Jones and Jacobs 1990
; Jones and Marks
1989a
), also demonstrate a decrease in N-type current after
axotomy (Jassar et al. 1993
). Given the heterogeneity of
channel subtypes found in DRG neurons, the present experiments add
further support to the possibility that the N-type channel is the
predominant target of injury-induced modulation of calcium currents in
peripheral neurons.
The inactivation properties of the HVA current in type 1 neurons were
also influenced by ligation of the sciatic nerve. Although control and
ligated cells showed a similar degree of inactivation during a 1-s
depolarization, the kinetics of current decay
(fast,
intermediate)
were significantly faster in injured neurons (Table 1). In addition,
the voltage dependence of the fast component of inactivation was
shifted to more depolarized potentials in axotomized large cutaneous
afferents (Fig. 7B). There are several possibilities to
account for the altered inactivation properties observed after injury.
One is that injury alters the state of phosphorylation of somatic
calcium channels. Treatment of sympathetic neurons with the phosphatase
inhibitors okadaic acid or calyculin A enhances the rate of
inactivation of the N-type calcium current (Werz et al.
1993
). Mounting evidence indicates that the N-type channels
possess significant diversity in terms of the degree and kinetics of
inactivation (Aosaki and Kasai 1989
; Boland and Dingledine 1990
; Hirning et al. 1988
). In fact,
single-channel recordings have provided examples of
-CgTx
GVIA-sensitive channels that switch from an inactivating to a
noninactivating mode (Plummer et al. 1989
). This
cumulative evidence suggests that a single N-type channel can
inactivate via different pathways possibly depending on the state of
channel phosphorylation (Werz et al. 1993
). Because
-CgTx GVIA-sensitive current still exists in axotomized cutaneous
afferents, an increase in the level of phosphorylation of the remaining
N-type calcium channels could lead to the acceleration of inactivation
kinetics observed in our experiments.
An alternate explanation for the change in inactivation properties is
that nerve injury induces a reorganization in the subunit structure of
the channel. Studies coexpressing various combinations of the cloned
Ca2+ channel subunits in heterologous systems
have revealed a significant role for the cytoplasmic subunit (1-4)
in regulating the function of the pore-forming
subunits (A-E) (see
Isom et al. 1994
). For example, coexpression of the
subunit increases the amplitude of calcium current conducted through
channels containing the
1A (P\Q-type),
1B (N-type),
1D (L-type), and
1E
subunits (Ellinor et al. 1993
; Sather et al.
1993
; Stea et al. 1993
; Williams et al.
1992
) in addition to modulating the voltage dependence and inactivation kinetics of N-type Ca2+ channels in
Xenopus oocytes (Stea et al. 1993
).
Expression studies have demonstrated that any of the four
subunits
can form a functional channel complex with a given
subunit, which
may be due to the presence of a highly conserved region in the I-II
loop of all
subunits that interacts with the
subunit (De
Waard et al. 1994
; Pragnell et al. 1994
).
However, the different subunits varied in their effect on the function
of the pore-forming subunit (Castellano et al. 1993
;
Singer et al. 1991
; Welling et al. 1993
).
The exact combination of
subunits expressed in cutaneous afferents
is not yet known, but given the ability of these auxillary subunits to
modulate a diversity of calcium channel properties, it is possible that
a shift in the levels or combination of
subunits expressed after
axotomy contributes to the observed changes in the density and
inactivation properties of the whole cell barium current.
Although T-type calcium current is reportedly absent in sympathetic
neurons (Elmslie et al. 1992; Jassar et al.
1993
; Jones and Jacobs 1990
; Jones and
Marks 1989a
), numerous studies have documented the existence of
this low-threshold current in DRG neurons (Carbone and Lux
1984
, 1987
; Schroeder et al.
1990
). Thus an injury model involving sensory neurons provides
an opportunity to examine the effects of axotomy on the characteristics
of the T-type calcium current. The present study has found no evidence that axotomy alters the density, kinetics, or voltage-dependent properties (Fig. 8) of the T-type current in type 2 neurons, and the
percentage of cutaneous afferents classified as type 2 is also
unchanged after ligation (Fig. 1D). This suggests that the high-voltage-activated class of voltage-gated calcium channels, particularly N-type channels, are preferentially modulated by axonal
injury on large cutaneous afferents.
Although exposure to neurotrophins is not required for the survival of
adult sensory neurons (Lindsay 1988), it has been
suggested that these peripheral influences are necessary for the
maintenance of the differentiated neuronal phenotype (Carroll et
al. 1992
; Lindsay 1996
). DRG neurons have been
shown to retrogradely transport nerve growth factor (NGF),
brain-derived-neurotrophic-factor (BDNF), and neurotrophin-3 (NT-3)
(DiStefano et al. 1992
) and express the corresponding
tyrosine kinase receptors Trk A, Trk B, and TrkC (McMahon et al.
1994
). Thus it is possible that the previously described
alterations in the properties of whole cell calcium currents in
cutaneous afferents result from the loss of peripheral trophic support
after injury. There is mounting evidence that voltage-gated calcium
channels are regulated by neurotrophins in other cell types as NGF
up-regulates calcium currents in PC12 cells (Usowicz et al.
1990
), basal forebrain neurons (Levine et al.
1995
), and SK-N-SH neuroblastoma cells (Lesser and Lo
1995
). In addition, subcutaneous injections of NGF antisera
decreased the total Ba2+ conductance
(gBa) of bullfrog sympathetic ganglion
B neurons (Lei et al. 1997
). It is interesting that this
study also reported a decrease in the inactivation of the voltage-gated
barium current after injections of NGF (Lei et al.
1997
). Future experiments could determine whether in vivo
application of NGF to the transected ends of cutaneous afferent neurons
via osmotic pumps can prevent the observed changes in the density and
inactivation properties of voltage-gated barium currents.
It is not known whether a reduction in the somatic density of N-type
voltage-gated calcium current in a selected group of cutaneous
afferents contributes to increased membrane excitability in these
neurons after nerve injury. N-type channels have been linked to the
activation of Ca2+-activated
K+ channels in hippocampal (Marrion and
Tavalin 1998), lamprey spinal (Wikstrom and El Manira
1998
), and otic ganglion (Callister et al. 1997
)
neurons. A decrease in ICa may lead to
a concominant reduction in the somatic
Ca2+-dependent K+
conductances, which could play a role in the increased membrane excitability reported after injury. In support of this hypothesis, some
studies report alterations in the amplitude and duration of the
afterhyperpolarization (AHP) in sensory neurons after axotomy (Titmus and Faber 1990
), although these observations
could also be explained by a direct effect of axotomy on
gK, Ca. Although Ca2+-activated K+
channels have been described in small (C- and A
-type) neurons of the
rat DRG (Scholz et al. 1998
), a characterization of the Ca2+-dependent K+ currents
found in large cutaneous afferents has not yet been performed. The
effect of nerve injury on ICa, K and
the role these currents play in regulating the firing properties of
large cutaneous afferent neurons thus remain unanswered.
It is of interest that the large cutaneous afferents of the present
study likely correspond to myelinated A afferents (Cameron et
al. 1986
; Harper and Lawson 1985
), which convey
information from low-threshold mechanoreceptors in the periphery
(Brown 1981
; Djouhri et al. 1998
;
Shortland et al. 1989
). A
fibers normally terminate
in laminae III and IV of the spinal cord (Brown et al. 1977
; Shortland et al. 1989
), but exhibit
extensive sprouting in the dorsal horn after peripheral nerve injury
(Koerber et al. 1994
; Shortland and Woolf
1993
; Woolf et al. 1992
). The central processes
of injured A
afferents form ectopic synapses with neurons in the
substantia gelatinosa (lamina II) (Koerber et al. 1995
; Shortland and Woolf 1993
; Woolf et al.
1995
), which normally receive nociceptive input from A
and
C-fibers (Willis and Coggeshall 1991
). Evidence suggests
that these new synaptic connections are functional as monosynaptic
A
-mediated excitatory postsynaptic potentials and currents are
observed in substantia gelatinosa neurons after nerve injury
(Kohama et al. 1998
; Okamoto et al. 1996
). In contrast, smaller primary afferents retract synaptic projections to lamina II after nerve injury (Barbut et al.
1981
; Castro-Lopes et al. 1990
; Knyihar
and Csillik 1976
). Although the functional significance of a
selective reduction in somatic N-type current on axotomized A
neurons is not clear, one possibility to account for the present
observations is that axotomy increases the demand for N-type channels
at the nerve terminals to accomodate the newly elaborated synaptic
arbors in the dorsal horn.
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ACKNOWLEDGMENTS |
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We are grateful to H. F. Mi for the preparation of the cutaneous afferent DRG cultures and to B. Toftness for computer assistance.
This work was supported in part by the Medical Research Service of the Department of Veterans Affairs and National Institute of Neurological Disorders and Stroke Grant NS-06208.
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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), VAMC, 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 30 August 1999; accepted in final form 10 January 2000.
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REFERENCES |
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