Axotomy- and Autotomy-Induced Changes in Ca2+and K+ Channel Currents of Rat Dorsal Root Ganglion Neurons

Fuad A. Abdulla2 and Peter A. Smith1

 1Department of Pharmacology and Division of Neuroscience, University of Alberta, Edmonton, Alberta T6G 2H7, Canada; and  2Department of Physical Therapy, Tennessee State University, Nashville, Tennessee 37209


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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
REFERENCES

Abdulla, Fuad A. and Peter A. Smith. Axotomy- and Autotomy-Induced Changes in Ca2+and K+ Channel Currents of Rat Dorsal Root Ganglion Neurons. J. Neurophysiol. 85: 644-658, 2001. Sciatic nerve section (axotomy) increases the excitability of rat dorsal root ganglion (DRG) neurons. The changes in Ca2+ currents, K+ currents, Ca2+-sensitive K+ current, and hyperpolarization-activated cation current (IH) that may be associated with this effect were examined by whole cell recording. Axotomy affected the same conductances in all types of DRG neuron. In general, the largest changes were seen in "small" cells and the smallest changes were seen in "large" cells. High-voltage-activated Ca2+-channel current (HVA-IBa) was reduced by axotomy. Although currents recorded in axotomized neurons exhibited increased inactivation, this did not account for all of the reduction in HVA-IBa. Activation kinetics were unchanged, and experiments with nifedipine and/or omega -conotoxin GVIA showed that there was no change in the percentage contribution of L-type, N-type, or "other" HVA-IBa to the total current after axotomy. T-type (low-voltage-activated) IBa was not affected by axotomy. Ca2+-sensitive K+ conductance (gK,Ca) appeared to be reduced, but when voltage protocols were adjusted to elicit similar amounts of Ca2+ influx into control and axotomized cells, IK,Ca(s) were unchanged. After axotomy, Cd2+-insensitive, steady-state K+ channel current, which primarily comprised delayed rectifier K+ current (IK), was reduced by about 60% in small, medium, and large cells. These data suggest that axotomy-induced increases in excitability are associated with decreases in IK and/or decreases in gK,Ca that are secondary to decreased Ca2+-influx. Because IH was reduced by axotomy, changes in this current do not contribute to increased excitability. The amplitude and inactivation of IBa in all cell types was changed more profoundly in animals that exhibited self-mutilatory behavior (autotomy). The onset of this behavior corresponded with significant reduction in IBa of large neurons. This finding supports the hypothesis that autotomy, that may be related to human neuropathic pain, is associated with changes in the properties of large myelinated sensory neurons.


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INTRODUCTION
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DISCUSSION
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The spontaneous activity that is induced in sensory nerves following peripheral nerve injury may be related to the development of "neuropathic" pain in humans (Kauppila 1998; Millan 1999). Because some of this activity arises from the dorsal root ganglia (DRG) (Babbedge et al. 1996; Wall and Devor 1983), there is considerable interest in understanding how nerve injury might influence the electrical properties of sensory neuron cell bodies. In rats, damage to the sciatic nerve increases the excitability of L4 and L5 DRG neurons. This is seen as an increased incidence of spontaneously active cells and lowering of threshold (Study and Kral 1996) as well as a reduction in rheobase (Kim et al. 1998). We have also shown that sciatic nerve section (axotomy) increases the number of action potentials (APs) fired in response to sustained depolarizing current (Abdulla and Smith 2001); i.e., it decreases accommodation and increases "gain." For those experiments, DRG neurons were divided into four categories on the basis of size and/or AP shape (Abdulla and Smith 2001). Most nociceptors were assumed to be contained within the "small" cell population (Bessou and Perl 1969; Gallego and Eyzaguirre 1978), and "large" cells were presumed to be primarily nonnociceptive. We also defined "medium" cells that were intermediate in size and spike width between small and large cells. "AD"-cells were defined as those in which the AP was followed by an afterdepolarization (ADP) (White et al. 1989) rather than by an afterhyperpolarization (AHP). In general, sciatic nerve section affected small cells more than medium or AD-cells, and these were affected more than large cells. In addition to decreasing accommodation and increasing gain, axotomy decreased rheobase and produced significant increases in spike height (AP amplitude) in small, medium, and AD-cells. It also produced a significant increase in spike width (AP duration) in small cells (Abdulla and Smith 2001).

Decreased accommodation of AP discharge and increased gain of DRG neurons is also seen following suppression of Ca2+ channel currents with neuropeptide Y (NPY), omega -conotoxin GVIA (omega -CNTX GVIA), Cd2+, or norepinephrine (Abdulla and Smith 1997, 1999). Similar effects are seen following suppression of Ca2+-sensitive K+ conductances (gK,Ca) with blockers of BKCa channels such as iberiotoxin (Scholz et al. 1998) or charybdotoxin (Abdulla and Smith 1999). Other work has shown that blockade of slowly inactivating K+ conductances with 4-aminopyridine or dendrotoxin increases the excitability of visceral afferent neurons in rat nodose ganglion (Stansfeld et al. 1986). Since these pharmacologically induced increases in excitability resemble those invoked by chronic sciatic nerve section (Abdulla and Smith 2001), we examined the effects of axotomy on the properties of Ca2+ currents, Ca2+-sensitive K+ currents, and various K+ currents in rat DRG neurons. We have also examined the effect of axotomy on the hyperpolarization-activated cation conductance (IH) (Mayer and Westbrook 1983). To relate the results to injury-induced changes in excitability and AP shape of various types of DRG neuron, we have classified cells as small, medium, AD-, or large cells according to criteria established in our current-clamp studies (Abdulla and Smith 2001).

The effects induced in DRG neurons by axotomy were intensified in animals that exhibit a self-mutilatory behavior known as autotomy (Coderre et al. 1986; Wall et al. 1979). Further increases in excitability were seen, and there was an especially clear difference between the properties of large cells from axotomized animals that did not exhibit autotomy and from those that did (Abdulla and Smith 2001). In fact, the onset of autotomy seemed to correlate more with a shift of the properties of large cells than with a shift in the properties of small, putative nociceptive cells. A second aspect of the present work was to examine which changes in ionic conductance accompanied this change in properties of large neurons.

A preliminary report of part of this work has appeared (Abdulla and Smith 1995).


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All experimental procedures were in concordance with the recommendations of the International Association for the Study of Pain (IASP). Protocols were reviewed and approved by the University of Alberta Animal Welfare Committee that maintains standards set forth by the Canadian Council for Animal Care. As in the accompanying paper (Abdulla and Smith 2001), adult male Sprague-Dawley rats weighing 120-170 g before surgery were housed 2 per cage with free access to food and water under an alternating 12-h light and dark cycle at 23°C. The rats were allowed to adapt to their home cages for 1 wk before use. Rats were anesthetized with pentobarbital sodium (50-55 mg/kg ip), and the sciatic nerve was sectioned proximal to its bifurcation into the tibial and the peroneal divisions. A 5- to 10-mm segment of nerve was removed to prevent regeneration. Some of the control animals underwent nerve exploration alone. Two to 7 wk later, control rats or operated rats were killed by decapitation and neurons enzymatically dissociated from L4 and L5 DRG. In a similar fashion to our previous current-clamp study (Abdulla and Smith 2001), data were pooled from axotomized animals 2-7 wk postoperatively. Axotomized animals were divided into two groups: those that exhibited "autotomy" (Coderre et al. 1986; Wall et al. 1979) and those that did not. Autotomy was scored according to the scale devised by Wall et al. (1979). Although the maximum score permitted under IASP guidelines is 11, all of our animals were killed before they attained a score of 8. Whole cell recordings (at 22°C) were made using a single-electrode voltage-clamp amplifier (AXOCLAMP 2A) as described previously (Abdulla and Smith 1997). With low-resistance patch electrodes (2-5 MOmega ), it was possible to use high switching frequencies >30 kHz with clamp gains as high as 30 mV/nA. The fidelity of the clamp was confirmed by examining voltage recordings. Recordings from cells where the voltage trace was slow to rise or distorted were discarded. Data were acquired using PCLAMP 5.5 (Axon Instruments, Foster City, CA) and analyzed using PCLAMP 6, 7, or 8. Final data records were produced using ORIGIN 5.0 (Microcal, Northampton, MA). Input capacitance (Cin) was calculated from the membrane time constant and input resistance or by integration of the capacitative transient generated by a 10-mV voltage jump (for details see Abdulla and Smith 1997). In some experiments, APs were recorded in bridge-balance current-clamp mode before switching to single-electrode voltage clamp. APs were generated using a 2-ms depolarizing current pulse. Spike width (AP duration) was measured at 50% maximum amplitude. For recording APs, instantaneous current-voltage (I-V) relationships or IH, external solution contained (in mM) 150 NaCl, 5 KCl, 2.5 CaCl2, 1 MgCl2, 10 HEPES-NaOH (pH 7.4), and 10 D-glucose (osmolarity 330-340 mOsm). Internal (pipette) solution contained (in mM) 130 KGluconate, 2 Mg-ATP, 0.3 Na-GTP, 11 EGTA, 10 HEPES-KOH (pH 7.2), and 1 CaCl2 (osmolarity 310-320 mOsm). Ca2+ channel currents (ICa) were measured using Ba2+ as a charge carrier (IBa). For these experiments, external "Ba2+" solution contained (in mM) 160 TEA-Cl, 10 HEPES, 2 BaCl2, 10 D-glucose, and 200 nM TTX, adjusted to pH 7.4 with TEA-OH, and internal solution contained (in mM) 120 CsCl, 5 Mg-ATP, 0.4 Na2-GTP, 10 EGTA, and 20 HEPES-CsOH (pH 7.2). For recording K+ currents, external solution contained (in mM) 145 N-methyl-D-glucamine (NMG)Cl (pH 7.4), 10 KCl, 2.5 CaCl2, 10 HEPES, 1.0 MgCl2, and 10 D-glucose, and internal solution contained (in mM) 100 K gluconate, 40 NMG-Cl (pH 7.2), 2 Mg-ATP, 0.3 Na2GTP, 11 EGTA, 10 HEPES, and 1.0 CaCl2. [Predicted K+ equilibrium potential (EK) = -58 mV (at 20°C).]

The total volume of fluid in the recording dishes was about 1 ml. They were superfused with external solutions at a flow rate of 2 ml/min. Drugs were applied by superfusion. Nifedpine solutions were administered under subdued lighting conditions from light-proof reservoirs. The drug was dissolved in DMSO (dimethyl sulfoxide) or polyethylene glycol to make 10-mM stock solutions. These were diluted 1 in 5,000 in external solution for application to the DRG neurons. omega -CNTX GVIA was added directly to the bath pipette near the neuron under study after bath flow was halted, achieving a final concentration of approximately 1 µM.

Clear-cut differences in the Cin provided a criterion for classification of DRG cells (see Fig. 3 in Abdulla and Smith 2001). Cin was always >90 pF for large neurons, 70-90 pF for medium neurons, and always <70 pF for small neurons. In experiments where AP solutions were used, classification of control neurons could be verified by examining AP shape; large neurons had AP duration <3 ms with no deflection in the falling phase of AP, medium neurons had AP duration 3-5 ms with a deflection on the falling phase, and small neurons had AP duration more than 5 ms. AD-neurons were identified by the presence of an ADP in current-clamp recordings or by the presence of a predominant T-type Ca2+ channel current (ICa,T or IBa,T) (Scroggs and Fox 1992; White et al. 1989) following a voltage step from -90 to -40 mV when studied in Ba2+ external solution. The AD-cell population likely includes the "type 2 cutaneous afferent neurons" recently defined by Baccei and Kocsis (2000). The identification of AD-cells was precluded in the solutions used to study K+ currents. Since their size fell mainly within the medium cell range (see Scroggs and Fox 1992), it is presumed that AD-cells make up some of the medium cell category investigated under these conditions. All data are presented as means ± SE. In graphs where no error bars are visible, the error bars are smaller than the symbols used to designate the data points.


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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Calcium channel currents

Ca2+ channel currents (IBa) were evoked by an incremental series of 50-ms depolarizing voltage commands (Vc) from holding potentials (Vh) of -90, -60, or -40 mV. Figure 1, A-D, illustrates typical recordings of currents evoked at -40 and at -10 mV or -15 mV in small, medium, large, and AD-cells from Vh = -90 mV. AD-cells are distinguished from medium cells by the presence of a low-voltage-activated (LVA) current (T-current, ICa,T, or IBa,T) with small depolarizing commands to -40 mV (Fig. 1D) (Fox et al. 1987; Scroggs and Fox 1992; White et al. 1989). No current is evoked in small, medium, or large cells at this potential (Fig. 1, A-C).



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Fig. 1. All top records: typical recordings of IBa from a holding potential Vh of -90 mV. A-C: superimposed recordings of currents evoked at Vc (command potential) = -10 and -40 mV for control small, medium, and large. D: superimposed recordings of currents evoked at -15 and -40 mV for control afterdepolarization (AD)-cells. Note that only AD-cells display inward current (IBa,T) at -40 mV. E-H: sample recordings from axotomized cells; note slight attenuation and increased inactivation of HVA IBa (recorded at -10 or -15 mV) but persistence of IBa,T at -40 mV in AD-cells. I-L: recordings from cells in the autotomy group. Note further attenuation and additional inactivation of HVA IBa (recorded at -10 or -15 mV) and persistence of IBa,T at -40 mV in AD-cells. Ten-nanoampere calibration in A refers to all small cell recordings, 10-nA calibration in B refers to all medium cell recordings, 20-nA calibration in C refers to all large cell recordings, and 10-nA calibration in D refers to all AD-cell recordings. The variable amount of noise on the traces reflects the use of a switching amplifier; with this system noise is a function of electrode resistance, electrode capacitance, and bath fluid level. All bottom records: recordings of voltages used to evoke the corresponding currents in top traces (-10 and -40 mV from Vh = -90 mV for A-C, E-G, and I-K and -15 and -40 mV for D, H, and L).

Families of I-V plots averaged from 20 to 68 neurons of each type are shown in Fig. 2, A-D. The three lines in each graph represent IBa evoked from Vh = -40, -60, or -90 mV. Small cells (Fig. 2A) have the smallest currents, medium-sized cells, and AD-cells have intermediate-sized currents (Fig. 2, B and D), and large cells have the largest currents (Fig. 2C). The presence of IBa,T shows up as shoulder on the I-V plot at voltages between -60 and -30 mV. This is marked with an asterisk in Fig. 2D (see also Fox et al. 1987).



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Fig. 2. Current-voltage (I-V) relationships for IBa from holding potentials (Vh) of -40, -60, and -90 mV. A-D: relationships for control small, medium, large, and AD-cells. E-H: relationships for axotomized cells. I-L: relationships for cells from the autotomy group. Numbers of neurons studied in each category range from n = 68 for control small cells from Vh = -90 mV to n = 20 for axotomized AD-cells from Vh = -60 mV. Errors bars indicate mean ± SE. Asterisks in D, H, and L indicate shoulder on I-V relationship reflecting the presence of IBa,T in AD-cells. Note attenuation of all currents after axotomy and further attenuation of currents recorded from the autotomy group. Also, changing Vh from -90 to -60 mV has little effect on maximal currents recorded from control neurons but generally larger effects on axotomized and autotomy group neurons.

Effects of axotomy on IBa amplitude

Axotomy reduced HVA-IBa in all types of DRG neuron. Table 1 presents a statistical comparison of HVA IBa peak amplitudes (recorded at -10 mV from Vh = -90 mV) in control cells and axotomized cells. The reduction in IBa produced by axotomy was significant in small and medium neurons but not in large and AD-neurons (Table 1). These effects are clearly seen by comparing the I-V plots from control small and medium neurons in Fig. 2, A and B, with those from small and medium axotomized neurons in Fig. 2, E and F. By contrast, there is little difference between the I-V plots for control large and AD-cells (Fig. 2, C and D) and those from axotomized large and AD-cells (Fig. 2, G and H).


                              
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Table 1. Axotomy- and autotomy-induced changes in HVA IBa

LVA-IBa (IBa,T) in AD-cells was unchanged by axotomy. The current, recorded at -50 mV to minimize contamination by HVA IBa in control AD-cells was 2.04 ± 0.18 nA (mean ± SE; n = 25) and in axotomized cells was 1.9 ± 0.19 (n = 24; P > 0.6). The absence of an effect on IBa,T is further illustrated by the persistence of the inward current shoulder on the I-V plot for axotomized AD-cells between -60 and -30 mV (asterisk in Fig. 2H).

Representative data records from axotomized neurons are shown in Fig. 1, E-H. HVA IBa (at -10 or -15 mV) in the axotomized cells is consistently smaller than that in control cells (Fig. 1, A-D). There is, however, little difference in the amplitude of IBa,T recorded at -40 mV from the typical control (Fig. 1D) and from the axotomized AD-cell (Fig. 1H).

IBa amplitude in cells from animals that exhibited autotomy

The effects of axotomy on HVA-IBa in all cell types were intensified in animals that exhibited autotomy. This is clear from the numerical values that are summarized in Table 1 and from the I-V plots shown in Fig. 2, I-L. The profound attenuation of HVA-IBa in cells from the autotomy group (Fig. 1, I-L) compared with axotomized neurons (Fig. 1, E-H) and to those from the control group (Fig. 1, A-D) is also clear from the typical records shown in Fig. 1. There was, however, no difference between the amplitude of LVA-IBa,T, recorded at -50 mV, in axotomized AD-cells (1.9 ± 0.19 nA; n = 24) and in those from animals that exhibited autotomy (1.83 ± 0.18 nA; n = 24; P > 0.7). There was no difference between this value and the amplitude of current seen in control cells (2.04 ± 0.18 nA; n = 25, P > 0.4). The persistence of a robust IBa,T in AD-cells from the autotomy group is illustrated in the data records of Fig. 1L. There is also an obvious difference between the averaged I-V plot evoked from -90 mV and that evoked from -60 mV for this type of cell (marked with an asterisk in Fig. 2L). This reflects the relatively large contribution that IBa,T makes to the total current under these conditions.

Voltage dependence of gBa activation

The voltage dependence of gBa activation was examined to gain further insights into the effects of axotomy and autotomy on Ca2+ channel properties. A series of 50-ms voltage commands was applied from a Vh of -90 mV and tail currents recorded at -40 mV. Activation curves were constructed from normalized tail current amplitudes. These were measured 0.5 ms after the termination of the command voltage pulse. After axotomy, the activation curves of small and medium cells were shifted by about +3 mV (data not shown). In the autotomy group, the activation curves for large and AD-cells were shifted by +3 to +5 mV relative to control. It is difficult to conclude, however, that these modest shifts reflect bona fide alterations in voltage dependence of HVA-gBa. This is because the observed changes in tail current amplitude may reflect differences in inactivation during a 50-ms pulse rather than a change in voltage dependence of activation.

Effects of axotomy and autotomy on inactivation of gBa

Baccei and Kocsis (2000) recently demonstrated that sciatic nerve axotomy increased the inactivation of gBa in cutaneous afferent neurons of rat DRG. An effect of axotomy and autotomy on gBa inactivation also is apparent in the data records from small, medium, large, and AD-cells shown in Fig. 1. In the records chosen for illustration, gBa inactivation at -10 or -15 mV in control cells (Fig. 1, A-D) is less than that in axotomized cells (Fig. 1, E-H), which in turn is less than that in cells from the autotomy group (Fig. 1, I-L). Table 2 shows numerical values for percentage inactivation of IBa in response to 50-ms pulses to -10 mV for small, medium, large, and AD-cells. These percentages were calculated from the ratio of peak to end-of-pulse current recorded at -10 mV. While axotomy is associated with a relatively modest increase in inactivation in large cells (from 4.1 to 8.0%), a much greater effect is seen in small cells (from 7.1 to 34.7%). By contrast, the appearance of autotomy, in axotomized animals, is associated with a further large change in inactivation in large cells (from 8.0 to 32.3%) but with only a modest change in inactivation in small cells (from 34.7 to 52.0%). This is an important point, as it implies that the transition of axotomized animals to the autotomy state coincides with changes in the properties of large rather than small neurons.


                              
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Table 2. Effects of axotomy and autotomy on IBa inactivation

The consequences of these increases in inactivation are seen in the families of I-V plots obtained from different values of Vh (Fig. 2). It is clear that changing Vh has much greater effects on cells from the axotomy or autotomy groups than on control cells. For example, changing Vh from -90 to -60 mV has minimal effect on maximum IBa in all types of control cell (Fig. 2, A-D) but has pronounced effects on IBa amplitude in neurons from animals that exhibited autotomy (Fig. 2, I-L). Thus for control small cells (Fig. 2A), shifting Vh from -90 to -60 mV changes maximum IBa (at -10 mV) by about 14% (from 7.96 ± 0.86 nA to 6.88 ± 0.73 nA, P > 0.3). The same manipulation on small cells in the axotomy group changes the current by about 33% (from 4.98 ± 0.51 to 3.38 ± 0.37 nA; Fig. 2E, P < 0.003).

The effects of changing Vh on peak IBa in typical control and axotomized small cells are further illustrated in Fig. 3A. Figure 3, A1 and A2, shows recordings of currents evoked at -10 mV from three different values of Vh. Figure 3A1 shows that altering Vh from -90 to -60 mV in a control cell has no effect on the current evoked at -10 mV; the two currents exactly superimpose. In the axotomized small cell, however (Fig. 3A2), peak IBa is attenuated by about 30% by shifting Vh from -90 to -60 mV. Also, further reduction of Vh from -90 to -40 mV in the axotomized cell attenuates the current by about 85% (Fig. 3A2), whereas in the control cell (Fig. 3A1), only a 35% attenuation is seen.



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Fig. 3. Effects of holding potential (Vh) on IBa amplitude. A1, top record: currents recorded at -10 mV from a small control neuron from Vh = -40, -60, or -90 mV. Currents evoked from -60 and -90 mV superimpose exactly. Bottom records: voltage records corresponding to current records in top trace. A2: currents recorded from a small axotomized neuron from Vh = -40, -60, or -90 mV. Note attenuation of current recorded at -10 mV from Vh = -60 mV compared with that recorded from -90 mV. Only a small current is evoked when Vh = -40 mV. B-D: graphs of IBa amplitudes evoked at -10 mV vs. Vh for small, medium, and large control (), axotomized (open circle ) and autotomy group () neurons. Note attenuation of IBa in small and medium cells after axotomy and slight further decrease in autotomy group neurons. IBa in large cells is little changed by axotomy but is drastically reduced in autotomy group neurons. Dotted lines show values of Vh required to secure identical IBa and hence Ca2+ influx into control and axotomized cells. E-G: graphs of normalized IBa amplitudes evoked at -10 mV vs. Vh for small, medium, and large control (), axotomized (open circle ) and autotomy group () neurons. Numbers of neurons studied in each category range from n = 68 for small control cells from Vh = -90 to n = 23 for medium cells from Vh = -40 mV in the autotomy group. Error bars indicate mean ± SE.

Effects of Vh on peak HVA-IBa from small, medium, and large control, axotomized, and autotomy group neurons are summarized in Fig. 3, B-D. AD-neurons, that exhibit IBa,T, were excluded from this analysis. Two points emerge. First, currents evoked from Vh = -90 mV in all cell types under all conditions are close to maximal. This implies that the attenuation of the amplitude of IBa seen in axotomy and/or autotomy (Figs. 1 and 2) cannot be accounted for by increased inactivation alone. In other words, axotomy and autotomy reduce IBa evoked from voltages where inactivation is largely removed. Second, IBa, and hence Ca2+ influx evoked in axotomized small cells from a Vh of -90 mV is about the same as the Ca2+ influx invoked in control small cells from Vh = -44 to -51 mV. The dashed lines in Fig. 3B show that about 5 nA of inward current is seen under both conditions. Similarly, Ca2+ influx invoked in axotomized medium cells from a Vh of -80 mV is about the same as that seen in control medium cells from a Vh about of -45 mV (about 6 nA in both cases, dashed lines in Fig. 3C). This point is revisited below during the discussion of Ca2+-dependent K+ currents. For large cells, the same amount of inward current is seen in both control and axotomized cells from all values of Vh (Fig. 3D). Much less current is seen in cells from the autotomy group.

A more traditional illustration of the effects of inactivation are shown in Fig. 3, E-G. Here, peak current amplitudes have been normalized. For small neurons, there is a large difference in the voltage dependence of inactivation between the control and axotomy group and less of a difference between the axotomy and autotomy groups (Fig. 3E). Little difference is seen for medium neurons (Fig. 3F). For large neurons (Fig. 3G), the voltage dependence of inactivation is not much altered by axotomy but is changed significantly in the autotomy group. These data are again consistent with the idea that the transition of axotomized animals to the autotomy state coincides with changes in the properties of large neurons.

Pharmacology of IBa in control, axotomized, and autotomy group neurons

The classical L-, N-, and T-subtypes of ICa were originally defined in DRG cells by Fox et al. (1987). It was subsequently noted that not all types of ICa fit into these categories (Scroggs and Fox 1992), and the presence of P- and Q-type ICa in DRG neurons was demonstrated by Rusin and Moises (1995). Because N-type Ca2+ channel current (ICa,N or IBa,N) inactivates more rapidly than L-type current (ICa,L or IBa,L) (Fox et al. 1987), we asked whether the increased inactivation seen in axotomized neurons might result from altered expression of channel subtypes. For simplicity, HVA-IBa was divided into IBa,N, IBa,L, or "other" (IBa,other) on the basis of the effects of 1 µM omega -CNTX GVIA or 1 µM nifedipine.

A typical experiment on a small control cell is illustrated in Fig. 4. The control current of 1.8 nA was reduced to 0.95 nA by nifedipine and reduced further to 0.4 nA by omega -CNTX GVIA. This procedure was used to calculate the percentage of IBa,N, IBa,L, and IBa,other, in each neuron studied. The results are shown in Table 3. As has been observed by others (Cardenas et al. 1995; Scroggs and Fox 1992), IBa,L is more prevalent in small cells than in other cell types. The prevalence of IBa,other in AD-cells reflects the presence of IBa,T (Scroggs and Fox 1992; White et al. 1989). Despite the changes in the absolute amplitude and inactivation characteristics of IBa seen after axotomy (Figs. 1-3), there seems to be no obvious change in relative proportions of channel types seen after axotomy or in neurons from animals that exhibited autotomy. Data were obtained from six to nine cells of each type under each experimental condition.



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Fig. 4. Pharmacological properties of IBa in control, axotomized, and autotomy group neurons. Top record: sample record of IBa evoked in a small control cell at -10 mV from Vh of -60 mV. Superimposed sweeps of control current, current recorded in 1 µM nifedipine, and in 1 µM nifedipine plus 1 µM omega -CNTX GVIA. Bottom records: voltage recordings corresponding to current traces shown in top records.


                              
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Table 3. Pharmacology of IBa

K+ channel currents

McFarlane and Cooper (1991) described three types of K+ current in sensory neurons: fast and slow A-currents (IAf and IAs) and a noninactivating delayed rectifier (IK). More recently, Gold et al. (1996a) further subdivided the currents into six different types; they recognized IAf and IAs as well as a high-threshold A-current (IAht). Sustained current was divided into three components: IKi, IKlt, and IKn. The differential distribution of K+ channel types in different classes of DRG neurons is now well-established (Everill and Kocsis 1999; Everill et al. 1998; Gold et al. 1996a), and this must contribute to the diversity in AP shape seen in these cells (Abdulla and Smith 2001; Kim et al. 1998; Koerber et al. 1988; Rose et al. 1986; Stebbing et al. 1999; Villière and McLachlan 1996). The studies of McFarlane and Cooper (1991), Gold et al. (1996a), Everill et al. (1998), and Everill and Kocsis (1999) were done in solutions containing Co2+ or Cd2+ to block Ca2+ channel currents as well as Ca2+-dependent conductances such as Ca2+-dependent K+ conductances (gK,Ca). Both voltage-sensitive large conductance (BKCa) and low conductance gK,Cas (gAHP) have been described in DRG neurons (Akins and McCleskey 1993; Fowler et al. 1985; Gold et al. 1996b; Gurtu and Smith 1988; Scholz et al. 1998). Since suppression of gK,Ca either directly (Scholz et al. 1998) or indirectly, via inhibition of gCa (Abdulla and Smith 1997, 1999; Akins and McCleskey 1993), decreases spike frequency accommodation in DRG neurons, we studied effects of axotomy on both Ca2+-insensitive and Ca2+-sensitive K+ currents. The latter experiments are particularly important in the light of the observed attenuation of ICa (IBa) in axotomized neurons (see Figs. 1-3) (see also Baccei and Kocsis 2000).

Outward currents were studied in a solution designed to isolate K+ currents in the absence of Na+ currents while preserving Ca2+ currents. Because this solution contained NMG rather than Na+, it was not feasible to distinguish AD-cells by observing an ADP under current-clamp conditions for this series of experiments. Most AD-cells were presumably included within the medium cell population (Abdulla and Smith 2001; Scroggs and Fox 1992). At least in the presence of Cd2+, all outward currents recorded in the voltage range we employed are carried by K+ (Everill et al. 1998).

We initially divided K+ channel currents into noninactivating currents and those that displayed some degree of inactivation during a 50-ms voltage command. Both types of current were seen in small, medium, and large cells. Inactivating currents were seen in 33/47 (70%) small cells, 18/30 (60%) medium cells, or 34/62 (55%) large cells studied from Vh of -90 or -80 mV (Table 4).


                              
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Table 4. Properties of K+ channel currents in control and axotomized cells

Typical recordings from a small cell in which outward current inactivated are shown in Fig. 5A. Inactivation of the total outward current probably does not reflect inactivation of the delayed rectifier K+ current (IK) (McFarlane and Cooper 1991) but may rather reflect the presence of IAf, IAs, and IAht (Gold et al. 1996a; IAs may correspond to the ID defined by Everill et al. 1998). Alternatively, Ca2+-sensitive K+ currents may decline in amplitude with time as a consequence of the inactivation the Ca2+ conductances responsible for their activation. This mechanism appears to be in effect in the neuron illustrated in Fig. 5A as the apparent inactivation is removed when the current is recorded in the presence of 500 µM Cd2+ (Fig. 5A2). This blocks Ca2+ influx and removes the contribution of gK,Ca to the total conductance. The remaining current, which does not exhibit appreciable inactivation during the 50-ms voltage command, presumably reflects IK. Figure 5A3 illustrates the subtraction of the Cd2+-resistant current shown in Fig. 5A2 from the total current shown in Fig. 5A1. This yields the Cd2+-sensitive portion of the current that presumably reflects IK,Ca.



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Fig. 5. Examples of K+ channel currents in control small neurons. A1, top records: family of outward currents evoked by an incremental series of voltage command (Vc) protocols. Bottom record: recordings of voltages obtained. In this cell, the total outward current displayed noticeable inactivation during the voltage command. A2, top record: responses of the same neuron to the same series of Vc protocols in the presence of 500 µM Cd2+. Note loss of inactivation in the presence of Cd2+. Bottom records: recordings of voltages corresponding to currents in top record. A3: current records obtained by subtracting records in A2 from those in A1. This yields the Cd2+-sensitive current that corresponds to IK,Ca. B, right hand record: examples of noninactivating currents recorded from another control small cell. Left hand record: recordings of voltages used to generate the currents shown in the right hand records.

Figure 5B shows recordings from another small neuron in which no inactivation of outward current was seen.

Inactivation often appeared to abate in the presence of Cd2+. For example, from Vh = -80, Cd2+ removed the apparent inactivation in 15/16 small control cells, 4/5 medium control cells, and 8/13 large cells (Table 4). This implies that gK,Ca contributes to the outward conductances in all neuron types and that it is especially important in small and medium cells. The inactivation that persisted in the presence of Cd2+ was usually quite slow and presumably reflects the presence of IAs/ID (Everill et al. 1998; Gold et al. 1996a; McFarlane and Cooper 1991). Slowly inactivating conductances were more characteristic of large cells than of small cells. Table 4 shows that 5 of 13 large cells exhibited Cd2+-resistant inactivation, whereas this was seen in only 1 of 16 small cells.

Effects of axotomy on K+ currents

In view of the complexity and variability of K+ currents in DRG neurons (see Akins and McCleskey 1993; Gold et al. 1996a), we performed a fairly simple analysis of the effects of axotomy. We initially concentrated on its effects on total, steady-state outward currents evoked from a Vh of -80 mV. The effects of axotomy were examined by constructing I-V plots from control and axotomized small, medium and large cells (n = 11-44; Fig. 6). Steady-state current amplitude was recorded at the end of a 50-ms voltage command. Because the large amplitudes of the currents may have compromised the voltage control attained by the clamp, measured voltages were plotted against measured currents. Figure 6 shows that axotomy promotes the greatest attenuation of total steady-state outward current in small cells (62.4% at approximately +65 mV, Fig. 6A), less attenuation in medium cells (41.5% at approximately +65 mV, Fig. 6B), and the least attenuation in large cells (22.3% at approximately +65 mV, Fig. 6C).



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Fig. 6. A-C: I-V relationships (evoked current vs. recorded voltage) for total, steady-state outward current recorded from control and axotomized small, medium, and large neurons from Vh = -80 mV. For small control neurons, n = 31; for small axotomized neurons, n = 44; for medium control neurons, n = 11; for medium axotomized neurons, n = 10; for large control neurons, n = 31; for large axotomized neurons, n = 22. D-F: steady-state I-V relationships for Cd2+-insensitive IK recorded from control and axotomized small, medium, and large neurons from Vh = -80 mV. Cd2+-insensitive IK is assumed to represent delayed rectifier current (IK; see text). G-I: I-V relationships for Cd2+-sensitive IK (IK,Ca) for control and axotomized small, medium, and large neurons from Vh = -80 mV obtained by subtraction of data from each cell in D-F from the same cells in A-C. Horizontal and vertical error bars represent SE for voltage and current recordings, respectively. For both Cd2+-insensitive currents (D-F) and Cd2+-sensitive (G-I), n = 16 for small control neurons, n = 8 for small axotomized neurons, n = 4 for medium control neurons, n = 3 for medium axotomized neurons, n = 11 for large control neurons, and n = 2 for large axotomized neurons.

Effects of axotomy on Cd2+-insensitive, steady-state K+ current (mainly IK)

The steady-state I-V plots shown in Fig. 6, D-F, illustrate the attenuation of Cd2+-insensitive outward current by axotomy. These plots were obtained from the steady-state currents that were evoked by a series of voltage commands from Vh = -80 mV in the presence of 500 µM or 1 mM Cd2+. Axotomy produces a similar effect in all three cell types; at approximately +65 mV, current is attenuated by 56.4% in small cells, 64.8% in medium cells, and 60.0% in large cells. This is atypical, as axotomy characteristically exerts its strongest effects on ionic currents in small cells and its weakest effects on currents in large cells (see Figs. 1-3 and 6) (see also Abdulla and Smith 2001). Although Cd2+-insensitive inactivation was seen in 1/16 small cells, 1/5 medium cells, and 5/13 large cells (Table 4), the steady-state current flowing at the end of a 50-ms command in the presence of Cd2+ should be dominated by IK because IAs (at +40 mV) is about 80% inactivated at this time (McFarlane and Cooper 1991). It is therefore suggested that the data presented in Fig. 6, D-F, corresponds closely to the effect of axotomy on IK, the delayed rectifier.

Effects of axotomy on gK,Ca/IK,Ca

To examine the effects of axotomy on IK,Ca, steady-state I-V plots obtained in the presence of Cd2+ (Fig. 6, D-F) were subtracted from the total I-V plots (Fig. 6, A-C) to yield the plots for the Cd2+-sensitive component of the current (Fig. 6, G-I). As expected from its effects on ICa, axotomy attenuated steady-state IK,Ca. The effect was stronger in small and medium cells (53.1 and 55.3% attenuation, respectively, at approximately +65 mV; Fig. 6, G and H) and weak in the large cells (13.0% attenuation at approximately +65 mV; Fig. 6I).

Measurements of the ratios of Cd2+-sensitive to Cd2+-insensitive current from the graphs in Fig. 6 also show that at approximately +65 mV, IK,Ca comprises 46.6% of total outward current in small control cells, 50.8% in medium control cells, and 37.6% in large control cells.

Axotomy-induced attenuation of IK,Ca could reflect the attenuation of ICa described above (Figs. 1-3) with or without an additional effects on intracellular Ca2+ buffering and/or on gK,Ca channels themselves. To test whether attenuation of ICa could completely account for IK,Ca attenuation, we compared currents evoked in control and axotomized cells under conditions where Ca2+ influx was unchanged. This was done by using different values of Vh. The experiments illustrated in Fig. 3B show that IBa evoked from Vh of about -50 mV (actually -44 to -51 mV) in control small cells is similar in amplitude to IBa evoked from -90 mV in axotomized cells (about 5 nA in both cases at -10 mV). Figure 7A shows that the steady-state IK,Ca recorded from Vh = -90 mV in axotomized cells is of similar amplitude to that recorded from Vh = -50 mV in control small cells. We also showed in Fig. 3C, that for axotomized medium cells, a Vh of -80 mV permitted about 6 nA of IBa to be evoked -10 mV. A similar current amplitude was seen using a Vh of about -46 mV in control medium cells. Figure 7B shows the effect of activating gK,Ca from close to these two values of Vh in axotomized and control medium cells. IK,Ca recorded from -80 mV in axotomized medium cells was statistically indistinguishable from that seen from a Vh of -50 mV in control medium cells. These results show that when changes in Ca2+ channel properties are accounted for, axotomized and control cells express similar amounts of gK,Ca. This implies that axotomy-induced changes in IK,Ca reflect ICa attenuation alone and that gK,Ca channels are not directly affected by nerve injury.



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Fig. 7. A and B: steady-state I-V relationships for Cd2+-sensitive IK (IK,Ca) recorded from control and axotomized small and medium neurons from different values of Vh. For small control neurons Vh = -50 mV, n = 8; for small axotomized neurons Vh = -90 mV, n = 5; for medium control neurons, Vh = -50 mV, n = 4; for medium axotomized neurons, Vh = -80 mV, n = 5. Horizontal and vertical error bars represent SE for voltage and current recordings, respectively. Note overlap of records from both cell types in both cases.

The properties of K+ channel currents in animals that exhibited autotomy were not examined.

Effect of axotomy on H-current (IH)

DRG neurons exhibit a voltage-dependent rectification that produces a sag in voltage responses to hyperpolarizing current pulses (Abdulla and Smith 2001; Czeh et al. 1977; Gallego et al. 1987; Gurtu and Smith 1988). This slow sag in the voltage trajectory reflects activation of the hyperpolarization-activated cation current, IH (Mayer and Westbrook 1983; Scroggs et al. 1994). Because sciatic nerve section attenuates the sag ("rectification") seen in current clamp (Abdulla and Smith 2001; Czeh et al. 1977; Gallego et al. 1987), we examined the effect of axotomy on IH. The current was elicited by a series of 1,200-ms hyperpolarizing voltage commands (incremental -10-mV steps to -130 mV) from a Vh of -50 mV. The inward current consisted of an initial instantaneous response (IInst, see below), followed by a slow inward relaxation that became larger and that developed more rapidly at more negative voltages. A typical recording of IH in a control large neuron is shown in Fig. 8A1. IH relaxations were seen in 49/49 large cells, 33/37 medium cells, 27/30 AD-cells, and 14/24 small cells; a distribution that parallels that of the voltage-dependent sag seen under current clamp (Abdulla and Smith 2001). Moreover, IH amplitude was greatest in large cells and least in small cells. Absolute values are listed in Table 5. The table also shows that IH was significantly reduced compared with control in all four cell types after axotomy, and further attenuation was seen in small and large cells from animals that exhibited autotomy.



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Fig. 8. Effects of axotomy and autotomy on membrane currents activated by hyperpolarization (IH and IInst). A: typical responses of a control (A1) and an axotomized (A2) large neuron to hyperpolarizing voltage commands (Vc) from Vh = -50 mV. Top records: currents. Bottom records: recorded voltages. Note reduced amplitude of slow inward relaxations (IH) in the axotomized cell compared with the control neuron. B: diagram to show percentages of small, medium, large, and AD-cells exhibiting IH from the control, axotomy, and autotomy groups. C: plots of Vc vs. IH measured from relaxation amplitudes as indicated in inset. Four panels show data for small, medium, large, and AD-cells from control, axotomy, and autotomy groups. n ranges from 49 for control large cells to 8 for AD-cells from the autotomy group. D: plots of Vc vs. IInst measured from instantaneous current amplitude as indicated in inset. Four panels show data for small, medium, large, and AD-cells from control, axotomy, and autotomy groups. n ranges from 49 for control large cells to 11 for AD-cells from the autotomy group.


                              
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Table 5. Axotomy- and autotomy-induced changes in IH

Figure 8A2 shows typical recordings of the attenuated IH relaxations seen in axotomized large cells. Figure 8B shows the percentages of large, AD-, medium, and small cells in the control, axotomy, and autotomy groups that display IH. The graph illustrates the two general trends; the larger the cell, the more likely it is to display IH, and axotomy reduces the frequency of occurrence of IH. Figure 8C shows IH versus VC plots for the four different cell types in the three different situations. The reduction in current amplitude after axotomy is clearly apparent.

Effects on other K+ currents

Figure 8D illustrates the effect of axotomy and the development of autotomy on the instantaneous current (IInst) evoked in small, medium, large, and AD-cells in response to a series of Vc from Vh = -50 mV. These data were obtained from the same experiments that were used to study IH and that are illustrated Fig. 8, A or B. Axotomy reduced input conductance for all cell types for the -120- to -50-mV range. Further decreases occurred in the autotomy group. Since these effects are seen at relatively negative potentials, this implies that axotomy decreases leak conductance. While the instantaneous I-V relationships for control small and large cells are close to linear over the -120- to -50-mV range, there is a progressive increase in slope with increasing hyperpolarization in the I-V relationships of medium and AD-cells (Fig. 8D). This presumably reflects the pseudo-instantaneous, inwardly rectifying K+ current (IIR) that is seen more frequently in medium cells than in small or large DRG cells (Scroggs et al. 1994). Because the extent of the contribution of IIR to IInst is not easily determined using our experimental conditions, it cannot be stated with certainty whether IIR is altered by axotomy.

M-current (IM) is a small, voltage-sensitive, noninactivating K+ current that is activated at potentials positive to -70 mV in autonomic neurons (Adams et al. 1982). Cells that express M-conductance (gM) exhibit a slow inward relaxation of membrane current (due to gM deactivation) when the membrane potential is stepped to -70 mV from a holding potential of -30 mV. Because IM may appear in amphibian DRG cells maintained in tissue culture (Tokimasa and Akasu 1990) and it is enhanced in bullfrog sympathetic ganglion B-neurons following axotomy (Jassar et al. 1994), we used voltage steps to -70 mV from a Vh of -30 mV to see whether IM relaxations occurred in control rat DRG neurons or whether the current was present in the axotomy or autotomy groups. No evidence for gM was found in any of the cell types under any of the conditions used in the present study.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The effects of axotomy on K+ and Ca2+ channel properties in identified cutaneous afferent DRG neurons have recently been described by Everill and Kocsis (1999) and by Baccei and Kocsis (2000). In the present work, we seek to extend the findings of these two studies to the whole DRG population, to incorporate effects on Ca2+-sensitive K+ channels and other conductances activated by membrane hyperpolarization and to relate changes in ion channel properties to the development of autotomy. Using this approach, we find qualitatively similar changes in specific ionic conductances in small, medium, large, and AD-cells; axotomy affects the same types of K+ and Ca2+ channels in all types of DRG neuron. The observation that the electrical properties of small cells are more perturbed by axotomy than large cells (Abdulla and Smith 2001) therefore reflects quantitative differences in the effects of axotomy on K+ and Ca2+ channels in different cell types and from the differential distribution of these channel types in small, medium, AD-, and large neurons (Cardenas et al. 1995; Scholz et al. 1998; Scroggs and Fox 1992; Scroggs et al. 1994).

The main findings were as follows: 1) HVA-IBa, and hence HVA-ICa, was reduced by axotomy, whereas LVA-IBa (ICa,T) was unaffected; 2) although HVA-IBa displayed increased inactivation in axotomized cells, this did not completely account for the observed in decrease in current; 3) the activation kinetics of gBa were unchanged and the relative contribution of IBa,N, IBa,L, and IBa,other to the total current was unchanged after axotomy; 4) the apparent reduction in gK,Ca seen after axotomy was fully attributable to changes in HVA-ICa; 5) IK and IH were reduced by axotomy; 6) changes in HVA-ICa, and hence gK,Ca were largest in small cells, less in medium and AD-cells, and least in large cells, whereas a comparable decreases in IK and IH were seen across all cell types; 7) ICa,L and gK,Ca were preferentially expressed in small DRG cells, whereas IH and inactivating K+ conductances (IA and/or ID) were seen most frequently in large cells; 8) in general, the changes in ionic currents seen in animals exhibiting autotomy were more intense than those seen in axotomized animals; and 9) the onset of autotomy was associated with a substantial change in the properties of large neurons rather than with a change in the properties of small, putative nociceptive neurons.

Several of these observations are in good agreement with those seen by others. For control cells, the preferential expression of ICa,L in small cells was previously noted by Scroggs and Fox (1992), and the preferential expression of IH in large cells was previously described by Scroggs et al. (1994). Also, Scholz et al. (1998) have shown that high conductance gK,Ca (BKCa channel current) is seen only in small cells that exhibited broad APs. McFarlane and Cooper (1991) described the predominance of inactivating K+ currents in large cells. Axotomy-induced increases in the inactivation of HVA ICa have recently been described in identified cutaneous afferent neurons of rat DRG by Baccei and Kocsis (2000). Increased inactivation of gBa may therefore be a characteristic consequence of axotomy as it is also seen in frog sympathetic neurons (Jassar et al. 1993) and in rat facial motoneurons (Umemiya et al. 1993). Everill and Kocsis (1999) have also shown that IK is reduced by axotomy in identified cutaneous afferent DRG neurons.

Changes in Ca2+ channel currents

IBa activated from Vh = -90 mV is close to maximal in all cell types, but the maxima are smaller in axotomized cells (Fig. 3, B-D). In other words, currents from a Vh of -90 mV, where most of the inactivation is removed, are still attenuated by axotomy. This implies that the axotomy-induced increase in inactivation of gBa cannot account for all of the observed reduction in current. A similar conclusion was reached by Baccei and Kocsis (2000) in their recent study of effects of axotomy on identified cutaneous afferent neurons.

Although there appear to be small changes in the voltage dependence of gBa activation after axotomy, this likely reflects increased inactivation because gBa was measured from tail currents that followed 50-ms depolarizing voltage commands. Currents invoked by this pulse length display noticeable inactivation (see Fig. 1 and Table 2). This would reduce tail current amplitude and yield an underestimate of the amount of gBa that would be available at a given voltage. Thus when inactivation is increased after axotomy, more attenuation of tail currents would occur and gBa, at a given voltage, would appear smaller than control. If it is accepted that axotomy does not alter the voltage dependence of activation, this rules out the possibility that axotomy alters opening probability of Ca2+ channels. It is therefore suggested that axotomy reduces the expression of functional Ca2+ channels and that those that are expressed exhibit increased inactivation. This again confirms the findings of Baccei and Kocsis (2000) in cutaneous afferent neurons.

Because IBa,N inactivates more rapidly than IBa,L (Fox et al. 1987), we considered the possibility that axotomy alters the relative contribution of IBa,N to the total current. Although this hypothesis seemed to be excluded by experiments with nifedipine and omega -CNTX GVIA that showed that the ratio of IBa,L to IBa,N to IBa,other was preserved after axotomy (see Table 3), slightly different results were reported in the recent study by Baccei and Kocsis (2000). These authors reported that IBa,N may be selectively attenuated in identified cutaneous afferent neurons, which are included within the large and medium neurons of this study. The reasons for these differences are not yet clear.

We were also particularly interested in knowing how ICa,T might be altered by axotomy as this current is responsible for the ADP that is the defining feature of AD-cells (White et al. 1989). Moreover, the ADP can promote discharge of multiple APs in response to a brief stimulus and is therefore a potential source of repetitive discharge in DRG neurons (Abdulla and Smith 2001). In confirmation of the findings of Baccei and Kocsis (2000), however, IBa,T of AD-cells was unaffected by axotomy (see Figs. 1 and 2). This lack of effect goes along with the observation that AD-cells showed no greater tendency to discharge multiple spikes in axotomized animals (Abdulla and Smith 2001).

Changes in K+ channel currents

We have used 500 µM to 1 mM Cd2+ to eliminate Ca2+-dependent components of K+ conductances. One potential problem with this approach is that divalent cations can interfere with gating and inactivation of Ca2+-insensitive K+ currents (Mayer and Sugiyama 1988). While these authors demonstrated effects of Mn2+, Cd2+, and other divalent cations on A-currents (IA; presumably IAf and IAs) in cultured rat DRG neurons, IK was unaffected by Mn2+ (up to 10 mM). Because Cd2+ is about 3 times as effective as Mn2+ in altering K+ currents (Mayer and Sugiyama 1988), IK should be unaffected by up to 3.3 mM Cd2+. Since we used only 0.5-1 mM Cd2+, the Cd2+-resistant current recorded in our experiments likely reflects relatively pure IK. The effects of Cd2+ on IA are complex; it affects both the activation curve and the inactivation curve for the underlying conductance (gA) (Mayer and Sugiyama 1988). Although we used low concentrations of Cd2+ (500 µM to 1 mM), small alterations in the properties of IA could complicate the assumption that the amplitude of IK,Ca may be deduced from the difference between total outward current and Cd2+-resistant current. Complex interactions of Cd2+ with IA may therefore have affected the detailed time course of some of the currents recorded. Despite this, effects of Cd2+ are unlikely to have affected our interpretation of I-V data as these were obtained from steady-state currents flowing at the end of a 50-ms pulse. At this time, the slowest components of IA are largely inactivated (McFarlane and Cooper 1991).

Unlike changes in HVA-IBa that are small in large cells and large in small cells, the extent of axotomy-induced reduction of IK was independent of cell type. About a 60% reduction was seen in small, medium, and large cells. Everill and Kocsis (1999) also reported large decreases in IA but little change in slowly inactivating K+ current (ID) following axotomy. We have insufficient data to confirm this observation in large and medium cells that likely include the population of cutaneous afferent neurons identified by these authors. Although Cd2+-insensitive inactivation occurred in only 1 of 16 control small cells, it was present in 8 of 15 axotomized small cells (Table 4). This raises the possibility that IA and/or ID increase in small cells but not in large cells after axotomy (Everill and Kocsis 1999). This possibility remains to be tested.

Because gK,Ca links changes in Ca2+ flux to changes in spike frequency accommodation (Abdulla and Smith 1997, 1999; Scholz et al. 1998), we were interested in knowing how axotomy-induced changes in Ca2+ currents might affect gK,Ca. This conductance was reduced by axotomy in small and medium cells (Fig. 6, G and H) but not in large cells (Fig. 6I). We conclude, however, that the gK,Ca channels themselves are unaffected and that the observed reduction in IK,Ca is a straightforward consequence of the decrease in Ca2+ influx produced by axotomy. This argument is supported by the data shown in Fig. 7, where Ca2+ influx in control and axotomized cells was "balanced" by using different values of Vh. Figure 3B shows that small axotomized cells held at -90 mV allow the same amount of Ca2+ influx as control cells held at about -50 mV. Using these two different values of Vh, similar amounts of IK,Ca are seen in control and axotomized small cells (Fig. 7A). A similar "balancing" of Ca2+ fluxes in medium cells produced the same amount of IK,Ca before and after axotomy (Fig. 7B).

Although some types of DRG neuron exhibit a long AHP when studied with microelectrodes (Fowler et al. 1985; Gold et al. 1996b; Gurtu and Smith 1988) and these may reflect the presence of low-conductance, voltage-insensitive gK,Ca channels (IAHP channels) (Pennefather et al. 1985), such channels are not readily recorded in dissociated cells with patch electrodes (Jassar et al. 1994; Zhang et al. 1994). We therefore conclude that most of the effects observed under our experimental conditions devolved from high-conductance, voltage-sensitive (BKCa) channels. This idea is supported by observation that iberiotoxin (Scholz et al. 1998) and/or charybdotoxin (Abdulla and Smith 1999) increase the number of APs evoked by a depolarizing current in dissociated DRG neurons studied with patch electrodes, whereas apamin has little or no effect (M. P. Stebbing and P. A. Smith, unpublished observations).

Changes in other currents

The observation that IH is reduced by axotomy matches our findings with current clamp, where we observed a parallel pattern of axotomy-induced changes in the "sag" in the voltage responses to hyperpolarizing currents (Abdulla and Smith 2001). Because axotomy-induced reductions in IH and attenuation of sag would result in impairment of a depolarizing response in unclamped neurons, this change is unlikely to be relevant to axotomy-induced increases in excitability. In fact, the voltage range for IH activation (Mayer and Westbrook 1983) is probably too negative for it to profoundly influence the excitability of DRG neuron cell bodies.

Relationship between current-clamp and voltage-clamp data

The same criteria that were used to define small, medium, AD-, and large cells in this study were used to define the cell types in a previous current-clamp study (Abdulla and Smith 2001). This approach was adopted to establish the direct relationship between changes in ionic currents and changes in excitability and AP characteristics in each cell type and to compare the magnitude of changes across the whole DRG population. The changes in HVA-gCa, IK, and gK,Ca invoked by axotomy as well as the decrease in leak conductance (Fig. 8D) are in a direction that would be expected to increase excitability. Decreases in ICa, which are manifest as decreases in gK,Ca would tend to reduce spike frequency adaptation as does decreased IK. In fact, decreased IK alone (Fig. 6F) may be responsible for increased excitability of large cells as gK,Ca in these neurons is little altered by axotomy (Fig. 6I).

Although we have too little data to say much about effects on Cd2+-insensitive slowly inactivating currents (ID or IA,S) in large or medium cells, data from another laboratory suggests that these currents are unchanged (Everill and Kocsis 1999).

While this paper has concentrated on changes in Ca2+, K+, and Ca2+-sensitive K+ currents, axotomy-induced changes in voltage-dependent Na+ conductances (gNa) must also play an important role in increased excitability (Zhang et al. 1997). They are also likely to be involved in axotomy-induced increases in spike width and height and in decreases in rheobase (Abdulla and Smith 2001). Although our preliminary data suggest that Na+ currents are increased by axotomy (unpublished observations), the issue of effects of axotomy is complex because several different types of Na+ channels are expressed in DRG neurons (Caffrey et al. 1992; Elliot and Elliot 1993; Ikeda et al. 1986; Rush et al. 1998), and these may be differentially affected by axotomy. For example, there are now several reports that axotomy impairs TTX-resistant, slowly inactivating INa (Cummins and Waxman 1997; Rizzo et al. 1995) as well as TTX-resistant, persistent INa (Sleeper et al. 2000). By contrast, TTX-sensitive current was either increased (Rizzo et al. 1995) or more readily "reprimed" (i.e., it recovered more rapidly from inactivation) (Cummins and Waxman 1997). There is also evidence that new types of Na+ channels appear after axotomy (Waxman et al. 1994). The exact relationship between changes in different types of gNa and axotomy-induced changes in spike shape, rheobase and excitability therefore remain to be elucidated.

Relationship to pain mechanisms

The axotomy-induced reduction in amplitude of HVA-IBa and the increased inactivation was greatest in small cells, less in medium and AD-cells, and least in large cells (Figs. 1-3, Tables 1 and 2). This is reflected by a similar pattern of decreases in gK,Ca and in the excitability changes in unclamped neurons (Abdulla and Smith 2001). By contrast, IK and IH are attenuated by similar amounts in all cell types. One possible explanation for this difference is that the changes in IBa reflect loss of retrograde availability of nerve growth factor (NGF), whereas decreases in IK and IH reflect some other consequence of sciatic nerve section. This argument is supported by the fact that TrkA receptors are preferentially expressed on nociceptive DRG cells (Lewin 1996) and that IBa and the inactivation of IBa are controlled by retrograde availability of target-derived NGF in other peripheral neurons (Lei et al. 1997).

As was previously seen with excitability changes (Abdulla and Smith 2001), the transition of axotomized animals to the autotomy state coincides with large changes in IBa inactivation in large cells (Fig. 3D, Table 2) but not in small and medium cells (Fig. 3, B and C, Table 2). This goes along with the idea that the induction of autotomy is more a function of alterations in the properties of neurons with myelinated axons than a function of altered properties of nociceptors (Coderre et al. 1986; Kajander and Bennett 1992; Nagy et al. 1986). According to some authors, alterations in the properties of myelinated fibers may also contribute to the generation of human neuropathic pain (Campbell et al. 1988; Kauppila 1998; Nystrom and Hagbarth 1981; Woolf et al. 1992).

Since cell body ICa is attenuated by axotomy, this raises the possibility that ICa may also be altered at primary afferent terminals in the spinal cord. Since gK,Ca channels also reside in nerve terminals (Sun et al. 1999), it is possible that axotomy increases the excitability of primary afferent terminals. Terminals might even become a source of spontaneous activity. Alternatively, decreased Ca2+ influx may reduce neurotransmitter release from primary afferent fibers. These interesting possibilities remain to be investigated.


    ACKNOWLEDGMENTS

We thank P. Stemkowski for help with data analysis.

This work was supported by the Medical Research Council of Canada. F. A. Abdulla received postdoctoral fellowships from the Rick Hansen Man-in-Motion/Alberta Paraplegic Foundation and from the Alberta Heritage Foundation for Medical Research. The work was also supported by start-up funds provided by the Office of the Dean, School of Allied Health Professions and the Office of the Academic Vice President, Tennessee State University.


    FOOTNOTES

Address for reprint requests: P. A. Smith, Dept. of Pharmacology, University of Alberta, 9.75 Medical Sciences Bldg., Edmonton, Alberta T6G 2H7, Canada (E-mail: Peter.A.Smith{at}UAlberta.ca).

Received 23 May 2000; accepted in final form 18 October 2000.


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0022-3077/01 $5.00 Copyright © 2001 The American Physiological Society