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Differing alterations of sodium currents in small dorsal root ganglion neurons after ganglion compression and peripheral nerve injury

Differing alterations of sodium currents in small dorsal root ganglion neurons after ganglion... Voltage-gated sodium channels play important roles in modulating dorsal root ganglion (DRG) neuron hyperexcitability and hyperalgesia after peripheral nerve injury or inflammation. We report that chronic compression of DRG (CCD) produces profound effect on tetrodotoxin-resistant (TTX-R) and tetrodotoxin-sensitive (TTX-S) sodium currents, which are different from that by chronic constriction injury (CCI) of the sciatic nerve in small DRG neurons. Whole cell patch- clamp recordings were obtained in vitro from L and/or L dissociated, small DRG neurons following 4 5 in vivo DRG compression or nerve injury. The small DRG neurons were classified into slow and fast subtype neurons based on expression of the slow-inactivating TTX-R and fast-inactivating TTX-S Na currents. CCD treatment significantly reduced TTX-R and TTX-S current densities in the slow and fast neurons, but CCI selectively reduced the TTX-R and TTX-S current densities in the slow neurons. Changes in half-maximal potential (V ) and curve slope (k) of steady-state inactivation of 1/2 Na currents were different in the slow and fast neurons after CCD and CCI treatment. The window current of TTX-R and TTX-S currents in fast neurons were enlarged by CCD and CCI, while only that of TTX-S currents in slow neurons was increased by CCI. The decay rate of TTX- S and both TTX-R and TTX-S currents in fast neurons were reduced by CCD and CCI, respectively. These findings provide a possible sodium channel mechanism underlying CCD- induced DRG neuron hyperexcitability and hyperalgesia and demonstrate a differential effect in the Na currents of small DRG neurons after somata compression and peripheral nerve injury. This study also points to a complexity of hyperexcitability mechanisms contributing to CCD and CCI hyperexcitability in small DRG neurons. play important roles in modulating neural excitability Background Nerve injury produces dorsal root ganglion (DRG) neu- [1,2]. The VGSCs are critically important for electrogene- ron hyperexcitability, which is thought to underlie neuro- sis and nerve impulse conduction, and a target for impor- pathic pain by causing central sensitization. The voltage- tant clinically relevant analgesics. However, mechanisms gated sodium channels (VGSCs) can be dynamically regu- of the VGSCs contributing to hyperexcitability of DRG lated after axonal injury or peripheral inflammation and neurons and neuropathic pain remain unclear and the Page 1 of 15 (page number not for citation purposes) Molecular Pain 2008, 4:20 http://www.molecularpain.com/content/4/1/20 observations are controversial. For instance, inhibition or ment and both feet in control groups did not show signif- specific knock-down of tetrodotoxin-resistant (TTX-R) icant change during the period of time. Data are shown in current Nav1.8 channel can effectively suppress neuro- Fig. 1. All of these rats were used later for electrophysio- pathic pain [3-5], while the Nav1.8 mRNA, protein and logical recordings during 10–14 postoperative days. current are substantially decreased in DRG neurons in axotomized DRG neurons [6-9] or sciatic nerve injury TTX-R and TTX-S Na currents in small neurons from CCD, CCI and control DRGs [10]. The tetrodotoxin-sensitive (TTX-S) current Nav1.7 channel plays a critical role in various pain conditions [1], Majority of the small DRG neurons express both TTX-R but nociceptors specific deletion of Nav1.7 did not elimi- and TTX-S Na currents, which have different activation nate neuropathic pain behavior in mice [11]. Thus, there and inactivation properties [6,8]. Prepulse inactivation, is a need to further investigate roles of the VGSCs in differ- which takes advantage of differences in inactivation prop- ent neuropathic pain conditions. erties of the TTX-R and TTX-S currents, was used to sepa- rate the slow-inactivating TTX-R, and fast-inactivating Different from nerve injury models that produce injury to TTX-S Na currents [6,18]. A current-voltage protocol with the peripheral axons of DRG neurons such as the chronic a 700 ms prepulse to -120 mV followed by respective test constriction injury (CCI) of the sciatic nerve, chronic pulse was applied first at a holding potential of -80 mV to compression of DRG (CCD) is used as an animal model obtain the total Na current. The slow-inactivating, TTX-R that produces injury directly to DRG somata. We have currents were recorded using a prepulse of 700 ms -50 mV shown that CCD treatment produces behavioral hyperal- before the test pulse. This protocol inactivates the TTX-S gesia and allodynia and DRG neuron hyperexcitability in currents while leaving the TTX-R current intact. TTX-S cur- rats [12-14]. However, ionic mechanisms contributing to rents were then obtained by subtracting the TTX-R cur- CCD-induced neural hyperexcitability remain unclear. A rents from the total Na current in the cells. These recent study shows that TTX-R Na currents are upregu- protocols allowed simultaneous measurement of both lated in the cutaneous medium-sized CCD DRG neurons TTX-R and TTX-S currents in each neuron recorded. In [15], which is somewhat different from the findings in some neurons, the TTX-R currents were recorded with axon injury models. The small DRG neurons most are existence of TTX (300 nM, n = 6). The TTX-R and TTX-S nociceptive and play critical roles in neuropathic pain. currents were similar to that recorded by using protocols + + Expression of the Na currents is different between small- and the Na currents were completely blocked by lido- and medium-sized DRG neurons in CCI rats [16]. How- caine (200 μM, n = 6) (data not shown). These data were ever, ionic mechanisms have not been investigated in similar to that described previously [6]. these small neurons after CCD treatment. The purpose of this study was to analyze the effects of CCD on the prop- One hundred and twenty-four small neurons including erties of TTX-R and TTX-S Na currents in the small DRG 53 from control, 38 CCD and 33 CCI DRGs were recorded neurons. Because of the complex and diverse expression and analyzed. Examples of recordings and calculations of of the VGSCs in different neuropathic pain conditions, we the Na currents are shown in Fig. 2. All of the neurons compared alterations of density and kinetic property of analyzed and discussed in this study expressed both slow- + + the TTX-R and TTX-S Na currents in CCD with CCI DRGs inactivating TTX-R, and fast-inactivating TTX-S Na cur- in the same recording condition. This study provides rents, which we refer to as "slow" and "fast" currents, sodium channel mechanisms underlying CCD-induced respectively. Neurons expressing predominantly (>70% of DRG neuron hyperexcitability and behavioral hyperalge- total) TTX-R currents are referred to as "slow neurons", sia and indicates different effects of CCD- and CCI-treat- while neurons expressing predominantly (>70% of total) ment on the TTX-R and TTX-S Na currents. TTX-S currents are referred to as "fast neurons" [6,18,19]. There were 5 controls-, 3 CCD- and 5 CCI- neurons that Preliminary data have been published in an abstract form expressed "mixed currents" (both slow and fast currents [17]. >70% of total) were not included in the analysis of this study. The results showed that CCD treatment increased Results percentage of the small-slow neurons and reduced per- CCD and CCI produced thermal hyperalgesia centage of small-fast neurons, while CCI treatment We began by confirming with earlier demonstrations that decreased percentage of small-slow neurons and increased CCD- or CCI-treatment produced pain and hyperalgesia. that of small-fast neurons. Data are summarized in Table All the CCD- and CCI-treated rats showed behavioral 1. indications of thermal hyperalgesia. Withdrawal latencies of the foot ipsilateral to CCD or CCI treatment decreased significantly from the preoperative values. Withdrawal latencies of the foot contralateral to CCD or CCI treat- Page 2 of 15 (page number not for citation purposes) Molecular Pain 2008, 4:20 http://www.molecularpain.com/content/4/1/20 Control left (10) Control right (10) CCD ipsil (12) CCD contral (12) CCI ipsil (10) CCI contral (10) 2 nA TTX-S 5 ms 40 ms -10 mV -50 mV 70 0 ms 40 ms TTX-R -8 0 mV -10 mV 10 -80 mV ** Total 700 ms -120 mV ** ** ** ** ** ** Representative Na ron fr Figure 2 om a control DRG currents traces recorded in a small neu- Representative Na currents traces recorded in a small neuron from a control DRG. The total current surgery recording was recorded with a 700 ms prepulse to -120 mV followed by the test pulse. The TTX-R Na current was recorded with -3 -1 1 3 5 7 9 11 a 700 ms prepulse to -50 mV followed by a test pulse. The 10-14 TTX-S Na current was obtained by digital subtraction of the Postoperative Day TTX-R current from the total current. All the test pulses were 40 ms to -10 mV pulses. The holding potential was at - Th ra Figure 1 ts ermal hyperalgesia following CCD- or CCI-treatment in 80 mV. Thermal hyperalgesia following CCD- or CCI-treat- ment in rats. Repeated measurements are shown of ther- mal sensitivity of the foot withdrawal response in CCD-, CCI- and control rats. Numbers of rats used in each group pared to control (Fig. 3C and 3D). The TTX-R current den- are indicated in the parentheses. The arrow indicates the sity was significantly reduced by approximately 30% and point of surgery of CCD or CCI performed. The dash line 20% in CCD and CCI DRGs, respectively (Fig. 3C). The above "recording" indicates the period of time that the rats TTX-S current density was reduced by approximately 50% were sacrificed for electrophysiological recordings and the and 25% in CCD and CCI DRGs, respectively. Reduction data were collected at different days of postoperative 10–14. of TTX-S current density by CCD treatment was signifi- **P < 0.01 indicate significant differences between groups of cantly more than that by CCI (p < 0.05) (Fig. 3D). To fur- CCD ipsilateral or CCI ipsilateral to the other groups. ther demonstrate reduction in the current levels, densities were binned and plotted against neuron number (Fig. 3E TTX-R and TTX-S Na current densities are reduced in and 3F). The peak distribution of TTX-R current density small-slow neurons in CCD and CCI DRGs shifted from 400–600 pA/pF in control to the lower levels The TTX-R and TTX-S current densities (peak current of <200 and 200–400 pA/pF in CCD and 200–600 pA/pF amplitude normalized to C ) were examined and com- in CCI (Fig. 3E). The peak distribution of TTX-S current pared in the small-slow neurons among CCD, CCI and density shifted from 200–400 pA/pF in control to the lev- control DRGs. The peak TTX-R and TTX-S current ampli- els of <200 pA/pF in CCD and <200 and 200–400 pA/pF , which could affect the results of tudes were measured with a 40 ms test pulse to -10 mV. in CCI (Fig. 3F). The C Examples of the TTX-R and TTX-S currents from CCD, CCI the current density, was not significantly changed in CCD and control DRG neurons are given in Fig. 3A and 3B. and CCI compared to that in control DRG neurons (Fig. CCD and CCI treatment significantly reduced TTX-R and 3G). TTX-S current densities in the small-slow neurons com- TTX-R and TTX-S Na current densities are reduced in Table 1: Proportion and distribution of the small-slow and small- small-fast neurons in CCD, but not CCI DRGs fast neurons in control, CCD and CCI DRGs. The TTX-R and TTX-S current densities were also examined and compared in the small-fast neurons among CCD, CCI Number of neurons (% of Total) and control DRGs. Examples are given in Fig. 3H and 3I. nC (pF) Small-Slow Small-Fast CCD treatment significantly reduced TTX-R and TTX-S current densities approximately 40% and 30%, respec- 27 (51) 26 (49) tively, in the small-fast neurons compared to control (Fig. CCD 38 25.9 ± 1.2 25 (66)* # 13 (34)* # 3J and 3K). Current densities were again binned and plot- CCI 33 24.6 ± 1.2 13 (39)* 20 (61)* ted against the neuron number as shown in Fig. 3L and 3M. The peak distribution of TTX-R current densities *, #, p < 0.05 indicate significant differences compared with control shifted from 200–400 pA/pF in control neurons to the (*) or CCD (#) groups. Page 3 of 15 (page number not for citation purposes) Mean Latency of Thermal Paw Withdrawal (Sec) Molecular Pain 2008, 4:20 http://www.molecularpain.com/content/4/1/20 Slow Neurons A C E -600 Control CCD -500 60 CCI -400 CCD ** 5 nA CCI 10 ms -300 Control 30 -200 -100 Control CCD CCI 15 <200 200-400 400-600 >600 TTX-R Current Density (pA/pF) B D F -600 70 Control 5 CCD CCD -500 0 Control CCD CCI CCI CCI -400 Control 5 nA -300 10 ms -200 -100 Control CCD CCI <200 200-400 400-600 >600 TTX-S Current Density (pA/pF) Fast Neurons H J L -60 0 70 Control CCD -50 0 CCI CCD -40 0 CCI Control -30 0 30 30 5 nA -20 0 10 ms -10 0 Co nt rol CCD CCI <200 200-400 400-600 >600 TTX-R Current Density (pA/pF) I K M -600 70 Control 5 CCD -500 Control CCI CCD CCI CCD -400 CCI 5 nA -300 10 ms -200 Control -100 0 0 Control CCD CCI <200 200-400 400-600 >600 TTX-S Current Density (pA/pF) Alterat Figure 3 ion of TTX-R and TTX-S Na current densities in slow and fast small DRG neurons after CCD and CCI treatment Alteration of TTX-R and TTX-S Na current densities in slow and fast small DRG neurons after CCD and CCI treatment. A, B, H and I: Examples of the TTX-R and TTX-S currents recorded with the prepulse inactivation protocol with 40 ms to -10 mV test pulse in the slow neurons (A and B) and the fast neurons (H, I) from CCD, CCI and control DRGs, respectively. C, D, J and K: Alterations of the TTX-R and TTX-S current densities in the slow (C and D) and the fast (J and K) neurons. E, F, L and M: Distribution of the TTX-R and TTX-S current densities in the slow (E and F) and the fast (L and M) neu- rons. The densities were binned and plotted against neuron number (%). G and N: Input capacitance (C ) of the slow (G) and fast (N) neurons from control, CCD and CCI groups. *, p < 0.05 and **, p < 0.01 indicate significant differences compared with the control group. #, p < 0.05 indicate significant differences compared with CCI group. Page 4 of 15 (page number not for citation purposes) TTX-S TTX-S TTX-R TTX-R TTX-S Current Density (pA/pF) TTX-S Current Density (pA/pF) TTX-R Current Density (pA/pF) TTX-R Current Density (pA/pF) Fast Neuron Number (%) Slow Neuron Number (%) Slow Neuron Number (%) Fast Neuron Number (%) Fast Neurons C (pF) Slow Neurons C (pF) m m Molecular Pain 2008, 4:20 http://www.molecularpain.com/content/4/1/20 lower levels of <200 pA/pF in CCD (Fig. 3L). The peak dis- prepulse to -120 or -50 mV, followed by a series of test tribution of TTX-S current densities shifted from 200–600 pulse from -70 to +60 mV with +10 mV increments. The total Na current was obtained by using the protocol with pA/pF in control neurons to the lower levels of <200–400 pA/pF in CCD (Fig. 3M). In contrast, CCI treatment failed a prepulse to -120 mV. The TTX-R component was to change the densities of both TTX-R and TTX-S currents recorded by using the protocol with a prepulse to -50 mV (Fig. 3J–M). The membrane capacitance (C ) was not sig- which inactivates TTX-S currents. The TTX-S component nificantly changed in CCD and CCI compared to that in was obtained by digitally subtracting the TTX-R compo- control DRGs (Fig. 3N). nent from the total Na current. Examples of TTX-R and TTX-S currents in the small slow and fast neurons from a Previous studies have shown that nerve injury including control DRG are given in Fig. 5A–D. Plots of normalized CCI do not produce significant change in the TTX-S cur- peak Na current density versus test pulse voltage for the rent [16,20,21]. Here it is shown that CCI does reduce the small slow and fast neurons in control, CCD and CCI TTX-S currents in the small-slow neurons (Fig. 3D), but DRGs are shown in Fig. 5E–H. Activation threshold of the not in the small-fast neurons (Fig. 3K). Interestingly, if the TTX-R currents the control neurons was between -35 and data from the slow and fast neurons are combined, CCI- -30 mV and the maximum inward current fell between -10 induced alteration in TTX-S current density in the slow and 0 mV (Fig. 5E,G). Activation threshold of the TTX-S neurons is hidden, while CCD-induced change in TTX-S currents in the control neurons was detected between -50 current still exhibit clearly (Fig. 4A). Both CCD- and CCI- to -45 mV with maximum inward current at approxi- induced reduction in TTX-R current densities are mately -20 mV (Fig. 5F, H). All currents measured dis- unchanged in this analysis (Fig. 4B). The C was not sig- played a reversal potential (V ) of about 50–55 mV, m rev nificantly different between CCI and control DRGs and corresponding to the calculated equilibrium potential for not different among the groups of CCD, CCI and control sodium ions under these recording conditions (E = 50 Na (see Table 1). mV). The voltage at which 50% of the Na channels were activated (V ) and the slope for activation (k) were 1/2 Voltage dependence of activation of TTX-R and TTX-S obtained from fitting the normalized conductance (G/ Na currents in small-slow and small-fast neurons is not G )-voltage curve with the Boltzmann equation. Effects max altered by CCD and CCI of CCD and CCI on the voltage dependence activation of Current-voltage relationship of the TTX-R and TTX-S Na TTX-R and TTX-S currents were examined and analyzed. currents was measured using a I-V protocol with a 700 ms Data are expressed and summarized in Fig. 5I–L and Table 2. In both small slow and fast neurons, neither CCD nor CCI treatment significantly altered parameters of activa- A B tion of the TTX-R and TTX-S Na currents such as V , k, 1/2 -450 -450 activation threshold, and voltage range of the maximum -400 -400 inward current fell in. These negative results support the -350 -350 findings in the current density by excluding the possibility of changes in the current amplitude caused by alterna- -300 -300 ** tions of the activation properties of Na currents. -250 -250 -200 -200 Voltage-dependence of steady-state inactivation of TTX-R -150 -150 and TTX-S Na currents in small-slow and small-fast -100 -100 neurons is altered by CCD and CCI -50 -50 Steady-state inactivation of TTX-R and TTX-S Na currents 0 was measured with 500 ms prepulse to potentials over the Control CCD CCI Control CCD CCI range of -120 mV to -10 mV with 5 mV increments fol- lowed by, with a 0.8 ms interpulse interval to -80 mV, a - Alterat Figure 4 small DRG neur ion of TTX-R and TTX-S Na ons after CCD and CC cuI treatment rrent densities in 10 mV test pulse. The TTX-R inactivation currents were Alteration of TTX-R and TTX-S Na current densi- measured at the time of the peak current evoked following ties in small DRG neurons after CCD and CCI treat- a -50 mV prepulse. The TTX-S inactivation currents were ment. Data shown here are from Fig. 2C, D, J and 2K and measured at the time of the peak of the maximum current the data from the slow and fast neurons are combined. CCI- evoked following a -120 mV prepulse [18,22]. Examples induced significant alteration in TTX-S current density in the of recordings of steady-state inactivation of the TTX-R and slow neurons (see Fig. 2) is hidden, while CCD-induced TTX-S currents in the slow and fast neurons are shown in change in TTX-S currents still exhibit clearly (A). Both CCD- Fig. 6A and 6B. CCD and CCI treatment significantly and CCI-induced reduction in TTX-R currents density is unchanged in this analysis (B). altered the V and k of TTX-R and TTX-S currents in the 1/2 slow and/or fast neurons as shown in Fig. 6(C–F) and Page 5 of 15 (page number not for citation purposes) TTX-S Current Density (pA/pF) . TTX-R Current Density (pA/pF) . Molecular Pain 2008, 4:20 http://www.molecularpain.com/content/4/1/20 Table 2: Voltage-dependence of activation and steady-state inactivation of the TTX-R and TTX-S Na currents in the slow and fast small-sized control, CCD and CCI DRGs. TTX-R Current TTX-S Current V (mV) k V (mV) k V (mV) k V (mV) k 1/2act act 1/2inact inact 1/2act act 1/2inact inact Slow Neurons Control -17.9 ± 1.1 4.3 ± 0.3 (14) -35.3 ± 0.7 -4.0 ± 0.1 (14) -26.6 ± 1.3 5.6 ± 1.1 (14) -77.6 ± 2.2 -8.0 ± 0.5 (14) (14) (14) (14) (14) CCD -18.7 ± 1.7 4.9 ± 0.4 (15) -38.8 ± 0.8** -4.6 ± 0.2* -26.2 ± 1.4 5.2 ± 0.6 (15) -84.0 ± 1.3* -9.9 ± 0.5* (15) (12) (12) (15) (12) (12) CCI -15.6 ± 1.6 (7) 5.1 ± 0.5 (7) -38.1 ± 0.7* -4.6 ± 0.1* (7) -26.1 ± 1.4 (7) 4.0 ± 0.8 (7) -75.1 ± 5.1 (7) -12.4 ± 1.8** (7) (7) Fast Neurons Control -13.0 ± 1.2 5.6 ± 0.4 (13) -39.4 ± 1.1 -4.4 ± 0.1 (14) -26.2 ± 1.4 4.9 ± 0.6 (13) -84.4 ± 1.9 -8.9 ± 0.4 (14) (13) (14) (13) (14) CCD -14.3 ± 1.7 (9) 5.8 ± 0.7 (9) -37.9 ± 1.3 (7) -5.3 ± 0.2** -23.0 ± 1.8 (9) 5.6 ± 1.1 (9) -75.7 ± 1.5* -9.4 ± 0.8 (7) (7) (7) CCI -12.0 ± 0.8 5.9 ± 0.4 (12) -36.8 ± 1.1 -5.0 ± 0.2* -26.8 ± 1.2 5.4 ± 1.1 (12) -80.4 ± 2.8 -10.1 ± 0.8 (12) (10) (10) (12) (10) (10) V : membrane potential at which activation is half-maximal. k : slope of the activation curve. 1/2act act V : membrane potential at which inactivation is half-maximal. k : slope of the inactivation curve. 1/2inact inact The number in parenthesis indicates the number of neurons. *, p < 0.05, **, p < 0.01 indicate significant differences compared with control group. summarized in Table 2. CCD and CCI produced similar Inactivation of TTX-R and TTX-S Na currents in small- effects on the TTX-R current of the slow neurons, but dif- fast neurons is slowed by CCD and CCI ferent on the TTX-R current of the fast neurons and the To quantitate changes in decay rate of the TTX-R and TTX- S Na currents, we fit the currents with single exponentials TTX-S current of the slow and fast neurons (Table 2). as that previously described [25]. The results showed that The shift in steady-state inactivation affected the window the inactivation of TTX-R currents in the small-fast neu- current, which is the region of overlap between the curves rons was significantly slowed by CCI treatment from 6.09 for the dependence of activation and inactivation. The ± 0.48 ms in control to 8.34 ± 1.12 ms (p < 0.05), but not overlapping activation/inactivation Boltzmann curves by CCD treatment although the inactivation tended to be were used to determine the fraction of sodium channels slower (p > 0.05) (Fig. 8A, Table 3). In contrast, inactiva- activated in the peak of the window current [23,24]. The- tion of TTX-S Na currents in the fast neurons was signifi- oretical analysis of the voltage dependencies presented in cantly slowed by both CCD and CCI treatment (Fig. 8B, Fig. 6 indicates that both CCD and CCI treatment increase Table 3). Neither CCD nor CCI treatment significantly the window current of the TTX-R currents in the fast, but altered inactivation of both TTX-R and TTX-S currents in not slow neurons (Fig. 7A and 7B). In the TTX-R currents the slow neurons (Table 3). in fast neurons at the membrane potential where maximal overlap of inactivation and activation occurred, approxi- Discussion mately 7% of the Na channels in control neurons were in The present study investigated alterations of TTX-R and a non-inactivated state and approximately 7% of the avail- TTX-S Na currents in the small DRG neurons after DRG able channels were activated. This fraction was increased somata compression (CCD treatment) and compared the ~28% and ~56% by CCD and CCI treatment, respectively different effects of DRG somata compression and the (Fig. 7B). The fractions of the window currents of the TTX- axons injury (CCI treatment) on the Na currents. The S currents were increased approximately 110% in the slow principle findings are 1) CCD treatment significantly neurons and 100% in the fast neurons by CCI (Fig. 7C reduces the TTX-R and TTX-S current densities in the and 8D). In contrast, CCD treatment increased window small-slow and small-fast subtypes of DRG neurons; 2) current of the TTX-S only in the fast neurons, but not the CCD alters voltage-dependent steady-state inactivation of slow neurons and the fraction was increased by ~86% the TTX-R and TTX-S currents and increases window cur- (Fig. 7D). rent of the activation and inactivation, but exhibits differ- ent effects on V and k of the TTX-S currents in both slow 1/2 and fast neurons; 3) CCD reduces the decay rates of the TTX-S, but not TTX-R currents inactivation in the fast neu- rons; and 4) CCI shows different effects from CCD in Page 6 of 15 (page number not for citation purposes) Molecular Pain 2008, 4:20 http://www.molecularpain.com/content/4/1/20 Slow Neurons A E I 1.1 0.1 0.9 -0.1 0.7 -0.3 0.5 -0.5 0.3 -0.7 Control 5 nA Control CCD CCD -0.9 0.1 CCI 8 ms CCI -1.1 -0.1 -70 -50 -30 -10 10 30 50 -60 -40 -20 0 20 V (mV) m V (mV) B F J 1.1 0.1 0.9 -0.1 0.7 -0.3 0.5 -0.5 0.3 -0.7 5 nA Control Control CCD CCD -0.9 0.1 8 ms CCI CCI -1.1 -0.1 -70 -50 -30 -10 10 30 50 -70 -50 -30 -10 10 V (mV) V (mV) Fast Neurons F C G K 1.1 0.1 0.9 -0.1 0.7 -0.3 0.5 -0.5 0.3 -0.7 5 nA Control Control CCD CCD 0.1 -0.9 8 ms CCI CCI -1.1 -0.1 -70 -50 -30 -10 10 30 50 -60 -40 -20 0 20 V (mV) V (mV) D H L 1.1 0.1 0.9 -0.1 0.7 -0.3 0.5 -0.5 0.3 -0.7 5 nA Control Control CCD CCD 0.1 -0.9 8 ms CCI CCI -1.1 -0.1 -70 -50 -30 -10 10 30 50 -70 -50 -30 -10 10 V (mV) V (mV) m Th altered by CCD and CCI tre Figure 5 e current-voltage relationships of TTX-R atment and TTX-S Na currents obtained from either slow or fast DRG neurons are not The current-voltage relationships of TTX-R and TTX-S Na currents obtained from either slow or fast DRG neurons are not altered by CCD and CCI treatment. A-D: Representative currents families from the slow (A and B) and fast (C and D) neurons were recorded by using the prepulse inactivation protocol with a series of test pulse ranging from -70 mV to +60 mV (in a +10 mV increments). E-H: Normalized peak current was plotted against test pulse voltage. I-L: The conductance (G) was calculated and plotted against test pulse voltage. Page 7 of 15 (page number not for citation purposes) TTX-S TTX-R TTX-S TTX-R Normalized TTX-S Current Normalized TTX-R Current Normalized TTX-S Current Normalized TTX-R Current TTX-S G/G TTX-R G/G TTX-S G/G TTX-R G/G max max max max Molecular Pain 2008, 4:20 http://www.molecularpain.com/content/4/1/20 Slow Neuron Fast Neuron A B 5 nA 5 ms C D 1.1 1.1 Control Control CCD CCD 0.9 0.9 CCI CCI 0.7 0.7 0.5 0.5 0.3 0.3 0.1 0.1 -0.1 -0.1 -80-70-60-50 -40-30-20-10 -80-70-60-50 -40-30-20-10 V (mV) V (mV) pre pre E F 1.1 1.1 Control Control CCD CCD 0.9 0.9 CCI CCI 0.7 0.7 0.5 0.5 0.3 0.3 0.1 0.1 -0.1 -0.1 -120 -100 -80 -60 -40 -20 -120 -100 -80 -60 -40 -20 V (mV) V (mV) pre pre A and CCI treatm Figure 6 lteration of the steady-state in ent activation of TTX-R and TTX-S Na currents in slow and fast small DRG neurons after CCD Alteration of the steady-state inactivation of TTX-R and TTX-S Na currents in slow and fast small DRG neu- rons after CCD and CCI treatment. A and B: Representative records of the inactivation current from a slow neuron (A) and a fast neuron (B) from a control DRG. The currents were recorded by using a double protocol with a 500 ms prepulse ranging from -120 mV to -10 mV (in 5 mV increments), followed by, with a 0.8 ms interpulse interval to -80 mV, a test pulse to -10 mV. The inter-pulse period was 10 s. The TTX-R inactivation currents were measured at the time of the peak current evoked following a -50 mV prepulse. The TTX-S inactivation currents were measured at the time of the peak of the maximum current evoked following a -120 mV prepulse. Each data set was normalized and fit with a Boltzman equation. The best fitted steady-state inactivation curves were showed in C-F. Page 8 of 15 (page number not for citation purposes) TTX-S Current I / I TTX-R Current I / I max max TTX-S Current I / I TTX-R Current I / I max max Molecular Pain 2008, 4:20 http://www.molecularpain.com/content/4/1/20 A after CCD and CCI treatment Figure 7 lteration of the window current of activation and steady-state inactivation curves in the slow and fast small DRG neurons Alteration of the window current of activation and steady-state inactivation curves in the slow and fast small DRG neurons after CCD and CCI treatment. Boltzmann fits from the data shown in Fig. 4 and 5 are shown in A-D. The small figures inserted in each figure illustrate the overview of the activation and inactivation fit curves. most of the tested properties of the TTX-R and TTX-S cur- Table 3: Inactivation of the TTX-R and TTX-S Na currents of rents. These findings suggest a possible sodium channel small-slow and small-fast neurons evoked with -10 mV test pulse mechanism underlying CCD-induced DRG neuron hyper- in control, CCD and CCI DRGs. excitability and indicate that injuries to DRG somata and n Current inactivation time constant (ms) peripheral axons may result in different alterations of the VGSCs. TTX-R TTX-S The DRG neurons, particularly the nociceptive small neu- Small-Slow Neurons rons, are notable in expressing multiple sodium channel Control 27 5.11 ± 0.42 0.90 ± 0.07 isoforms including Nav1.8 and Nav1.9 contributing to CCD 25 4.82 ± 0.29 1.00 ± 0.08 the TTX-R currents, and Nav1.7, Nav1.6, Nav1.5, Nav1.3, CCI 13 6.65 ± 0.89 1.10 ± 0.12 Nav1.2 and Nav1.1 contributing to the TTX-S currents Small-Fast Neurons Control 26 6.09 ± 0.48 0.92 ± 0.05 [26-31]. Many of these sodium channels can be dynami- CCD 13 7.18 ± 0.54 1.12 ± 0.09* cally regulated after nerve injury and/or inflammation CCI 20 8.43 ± 1.12* 1.17 ± 0.07** and the specific channels may play crucial roles in nocice- ption. Several lines of studies including many with trans- *, p < 0.05, **, p < 0.01 indicate significant differences compared with genic mice lines have clearly implicated Nav1.7, Nav1.8 control group. Page 9 of 15 (page number not for citation purposes) Molecular Pain 2008, 4:20 http://www.molecularpain.com/content/4/1/20 were also found in rats that received CCI treatment [16], indicating that TTX-R Na currents may have different functions in the small- vs the medium-sized DRG neurons after neuron injury, and may contribute to the different 0.2 mechanisms of these neurons in the abnormal neuron excitability. TTX-R Na channel is also found upregulated 0.4 in the sciatic nerve axons at the site of injury [32] and redistributed in the uninjured adjacent axons of DRG neu- CCI 0.6 rons [33]. Consistently, contribution of Nav1.8 currents CCD to neuropathic pain conditions has been demonstrated 0.8 controversial. Another interesting finding in the present Control study is that CCI treatment reduces the TTX-R currents 1 only in the slow, but not the fast neurons, further support- ing that nerve injury can produce different effects on 0 5 10 15 20 25 30 sodium channels in different types of DRG neurons. In Time (ms) addition, the Nav1.9 channel can have profound effects on resting membrane potential (RMP), and thus on excit- ability in DRG neurons [26,27]. Nav1.9 current activates at more negative potentials (-80 mV), differing from the Nav1.8 current activating at potentials close to RMP (-60 0.2 CCI to -70 mV), and generates the persistent TTX-R current identified [26,34,35]. Nav1.9 current plays an important 0.4 CCD role in setting RMP as well as contributing to subthreshold electrogenesis in small DRG neurons [26,27]. Nav1.9 was 0.6 Control not observed in the present study because of ultraslow inactivation at the holding potentials used and at the time 0.8 domain of the pre-pulse 700 ms to -120 mV applied to remove the fast inactivation of TTX-S currents. This yielded an estimation of the sodium current in the cell 02 46 8 10 minus the Nav1.9 current. Time (ms) The TTX-S Nav1.7 and Nav1.3 channels have been identi- Figure 8 small DRG neur Represen tion of TTX-R ( tative recordings showin A ons after ) and TTX-S CCD and CC (B) Na g alterat currents in the fast I treatment ion of the inactiva- fied to play important roles in neural hyperexcitability Representative recordings showing alteration of the inactivation of TTX-R (A) and TTX-S (B) Na cur- and chronic pain [1], but the observations again are con- rents in the fast small DRG neurons after CCD and troversial. Nav1.7, Nav1.6 and Nav1.3 channels are upreg- CCI treatment. ulated [36-39] or down-regulated [25,39-41] in DRG neurons after nerve injury or axotomy. In the present study, CCD and CCI treatment both result in down-regu- and Nav1.9 in inflammatory and probably neuropathic lation of TTX-S currents. However, CCI treatment selec- pain [1,2]. The present study shows that CCD treatment tively reduces density of TTX-S currents in the slow, but significantly down-regulates both the TTX-R and TTX-S not the fast neurons. These results suggest different roles + + Na currents in the small DRG neurons. The TTX-R Na for TTX-S currents in these two subtypes of neurons after currents recorded in our study are predominantly the peripheral nerve injury. This differentiation might in Nav1.8 as identified by the specific protocol [1]. Such some way link to the conflict findings in the experiments alteration of TTX-R Na currents after CCD treatment is that knock-down Nav1.3 leads to decrease [42] or no consistent to majority of the previous findings that change [43] in pain sensitivity. The differential effects in Nav1.8 mRNA, protein and current are substantially the TTX-R and TTX-S Na+ current induced by CCD and decreased in DRG neurons in axotomized DRG neurons CCI may contribute partly to certain differences in neural [6-9] or sciatic nerve injury [10,16]. These observations excitability and behavioral manifestations between the demonstrate a common role for the TTX-R Na channels two models [13]. In addition, it is worthy while to men- in the DRG neurons after injury to either somata or tion that because only a proportion of the L4/5 DRG neu- peripheral axons. A recent study indicates that the cutane- rons were directly injured in the CCI model (due to the ous, medium-sized dissociated CCD DRG neurons exhibit contribution of L4/5 to sciatic nerve in the injured level), an increase in TTX-R Na currents [15]. Such different the neurons under investigation might also include some changes of Na currents in different types of DRG neurons Page 10 of 15 (page number not for citation purposes) Normalized TTX-S Current Normalized TTX-R Current Molecular Pain 2008, 4:20 http://www.molecularpain.com/content/4/1/20 intact uninjured ones [7] and therefore the sensitivity of the slope factor. The window current (overlap between the statistics could be lowered. curves for the dependence of activation and inactivation) represents a voltage region in which sodium channels can How these differential alterations of sodium channels/ continue to open because some channels are activated currents would contribute to neural excitability and neu- and not all of the channels are inactivated. The increased ropathic pain remains unclear [1,2]. A recent study by window current therefore may result in an increase in the Rush et al [44] may provide an explanation to such con- persistent current that can seize activity and may affect troversial and conflict observations. It is shown that a sin- neuronal excitability [23,24]. These alternations of steady- gle sodium channel (Nav1.7) mutation can produce state inactivation curves therefore show a potential to opposing phenotypes (hyperexcitability vs hypoexcitabil- depolarize the resting membrane potential and increase ity) in sensory neurons and sympathetic neurons, respec- the neuronal excitability. A recent study indicates that the tively, and the selective presence of the Nav1.8 channel is CCD treatment hyperpolarizing shifts the activation a major determinant of these opposing effects. Majority of curves of the TTX-S current, but not the steady-state inac- nociceptive DRG neurons express Nav1.8 [29,45], which tivation curves in cutaneous medium-sized DRG neurons contributes most of the current underlying the action [15]. Such a hyperpolarizing shift of the activation curve potential upstroke [9,46]. Because it has depolarized volt- may also increase the window current. Thus, the increased age-dependence of activation and inactivation window current may underlie the neural hyperexcitabil- [6,10,45,47] compared with other sodium channels, ity. In addition, our results show that inactivation of the Nav1.8 permits DRG neurons to generate action poten- TTX-R and TTX-S currents are slowed down by CCD and/ tials sustain repetitive firing when depolarized [9,46]. This or CCI in the small-fast neurons. This may also contribute finding suggests that the physiological coupling of Nav1.8 to the neural hyperexcitability. We hypothesize that alter- and Nav1.7 in the nociceptive DRG neurons may contrib- ations of the sodium channel gating properties associated ute to the phenotypes in the different cell types as well as with down- or up-regulation of the current density may in the different neuropathic conditions. In the same study contribute to the neural hyperexcitability, while redistri- [44], RMP of the sensory and sympathetic neurons is bution of the sodium channels to the adjacent uninjured depolarized following Nav1.7 mutation and such depo- fibers may contribute to the development of neuropathic larization is thought to be a result of increased window pain [33]. This hypothesis may also provide an explana- currents [23,44]. Interestingly, a similar depolarization is tion for the controversial observations. also true in the large- and medium-sized and small DRG neurons after CCD treatment as we demonstrated recently CCD treatment produces local inflammation particularly [14] and the window currents are increased in the small- in the first postoperative week as described in the previous fast neurons after CCD treatment and in the small-slow studies [12,13,48], in addition to producing compression neurons after CCI treatment, while the density of both of the ganglion. In this study, the down-regulation of the TTX-R and TTX-S Na+ currents is downregulated, as TTX-R currents are similar to those observed in the nerve shown in the present study. These findings might support injury model, but not the inflammation model in which a possibility that either upregulation or downregulation up-regulated [1]. In the present study, the DRG neurons of the TTX-R or TTX-S current densities in the injured were isolated from rats 10–14 days after continuous com- medium-sized [15,8] and small DRG neurons, which pression. We noticed that the inflammation during this express both Nav1.8 and Nav1.7 channels, can produce period of time was much less than that in the first week neural hyperexcitability. Such alteration of Na+ channels/ after the rod was initially introduced into the interverte- currents on neural function should also depend on the bral foramen (data not collected), which was described in basis of the cell background in which the alteration is our previous studies [12,13,48]. Such a point may also expressed. Further investigation and analysis are urgently contribute to the difference between our study and Tan et needed to elucidate such complex relationships between al [15], in addition to the different types of DRG neurons neural hyperexcitability and alterations of the sodium recorded. channels after nerve injury. It needs to be pointed out that nerve injury alters the elec- Steady-state inactivation of TTX-R and TTX-S Na currents trophysiological properties of diverse types of primary alters and exhibits different changes in the midpoints (V afferent neurons and triggers a myriad of changes in gene 1/ ) and slopes (k) of inactivation curves in the small-slow expression that affect many proteins, including ion chan- and small-fast neurons after CCD and CCI treatment. It is nels, receptors, and other membrane proteins [49-52]. interesting that both CCD and CCI treatment increase the Such alterations are likely to complicate the changes in window current in both slow and fast neurons by differ- Na currents we observed. These complexities might be ently (depolarizing or hyperpolarizing) shifting the reduced by sampling functionally homogeneous subpop- steady-state inactivation curves midpoint and decreasing ulations or recording from the same neurons before and Page 11 of 15 (page number not for citation purposes) Molecular Pain 2008, 4:20 http://www.molecularpain.com/content/4/1/20 after injury. While this can be done with some inverte- another at L . CCI model was employed to produce brate nociceptors [53,54], it is not yet practical for DRG peripheral nerve injury and the procedure was similar to neurons. that described in the CCI model [56]. The left common sciatic nerve of each rat (n = 10) was exposed at the level Additional ion channel mechanisms may also contribute of mid-thigh. Proximal to the sciatic nerve's trifurcation, to neural hyperexcitability and behavioral hyperalgesia about 7 mm of nerve was freed of adhering tissue and four and allodynia after DRG compression. Recent studies ligatures (4-0 chronic gut) were tied loosely around it with have shown that CCD causes a decrease in fast-inactivat- about 1 mm spacing. The length of affected nerve was ing K current [15] and an increase in expression of a about 5 mm. Another group of rats (n = 10) received nei- hyperpolarization-activated cation current (I ), in addi- ther surgery nor injury and were served as control. A series tion to an increase in TTX-R Na currents in cutaneous, of previous studies in our lab and others has shown that medium-sized DRG neurons [55]. The I current is acti- sham surgery for CCD and CCI treatment did not produce vated during the afterhyperpolarization that follows an significant electrophysiological differences between neu- action potential and leads to a sustained depolarizing cur- rons from previously unoperated versus sham-operated rent, resulting in repetitive firing [55]. controls [13-15,21], therefore, the sham operations were not considered necessary in the present study. Conclusion In summary, this study shows that CCD treatment can Behavioral testing cause profound changes in densities and properties of Thermal hyperalgesia was indicated by a decrease in the inactivation of TTX-R and TTX-S Na currents of the small latency of foot withdrawal evoked by a radiant heat stim- DRG neurons, and that DRG somata compression results ulus as described previously [13,14]. The IITC Model 336 in different alterations of the Na currents from the Analgesia Meter (Life Science, Series 8) providing a heat peripheral nerve injury. The findings also point to a com- source was used in the present study. In brief, each rat was plexity of hyperexcitability mechanisms contributing to placed in a box (22 × 12 × 12 cm) containing a tempera- CCD and CCI hyperexcitability in small DRG neurons, ture-controlled smooth glass floor associated with the Analgesia Meter. The heat source was focused on a portion Methods of the hindpaw, which was flush against the glass, and Animals and surgical procedures delivered until the hindpaw moved or up to 20 sec to pre- Experiments were performed on adult, male Sprague- vent tissue damage. The range of latency of foot with- Dawley rats (n = 32, 200–250 g). The rats were housed in drawal in naïve, control rats was 9–15 sec. Thermal groups of 3–4 in plastic cages (40 × 60 × 30 cm) with soft stimuli were delivered 4 times to each hind paw at 5–6 bedding and free access to food and water under a 12-h min intervals. The rats were tested on each of 2 successive day/12-h night cycle. Under these conditions, they were days prior to surgery (the first test was at 2 days and the kept 3–5 days, before and up to 14 days after surgery and/ second at 2 hours prior to surgery). Postoperative tests or treatment. The animals were divided into groups as were conducted on the day of electrophysiological record- described below (CCD, CCI and Control). All surgeries ing (days 10–14). Thermal hyperalgesia for a given rat was were done under anesthesia induced by intraperitoneal defined as a postoperative decrease of foot withdrawal injection (i.p.) of sodium pentobarbital (40 mg/kg). After latency from the mean preoperative value, with a differ- surgery, the muscle and skin layers were sutured. These ence score ≥ 3 s [14]. Only rats that exhibited thermal procedures were conducted in agreement with the regula- hyperalgesia after CCD or CCI treatment were used for the tions of the ethics committee of the International Associ- electrophysiological studies. ation for the Study of Pain, the National Institute of Dissociation of DRG neurons Health guide for the care and use of Laboratory animals and approved by Parker Research Institute Animal Care DRG neurons were dissociated from L and/or L ganglia 4 5 and Use Committee. taken from 8 CCD, 8 CCI and 8 Control rats. The protocol was the same as that we have described recently [57]. In DRG compression was produced by surgically implanting brief, the excised ganglion was minced using microdissec- stainless steel rods unilaterally into the intervertebral tion scissors, the DRG fragments transferred into 10 ml of foramen at L and L using the procedure for CCD we pre- the buffered solution containing collagenase (type IA, 1 4 5 viously described [12,13]. In brief, the rats (n = 12) were mg/ml, Sigma) and trypsin (0.5 mg/ml, Sigma), and then anesthetized; paraspinal muscles were separated from the incubated for 30 min at 35°C. The DRG fragments were mammillary and transverse processes, and the interverte- removed, rinsed 2–3 times in the buffered solution, and bral foramina of L and L were exposed. One stainless put into the buffered solution (5 ml) containing DNase 4 5 steel L-shaped rod, 4 × 2 mm in length and 0.6 mm in (0.2 mg/ml, Sigma) to prevent possible toxicity from DNA diameter, was implanted into the foramen at L and leaking from ruptured cells. Individual neurons were dis- Page 12 of 15 (page number not for citation purposes) Molecular Pain 2008, 4:20 http://www.molecularpain.com/content/4/1/20 sociated by passing DRG fragments through a set of fire- data for each cell were divided by the respective driving polished glass pipettes with decreasing diameter. force (V - V ), plotted against V , and fit to a Boltzmann m rev m distribution equation of the following form: Voltage-clamp electrophysiology Voltage-clamp recordings were performed in the dissoci- G = G /(1 + exp((V - V )/k)), max 1/2 m ated small DRG neurons with the standard whole-cell patch-clamp configuration. All recordings were conducted Where G is the maximum G, V is the potential at max 1/2 at room temperature (20~22°C) and during 2~8 hrs after which activation is half-maximal, and k is the slope of the dissociation. Fire-polished electrodes were fabricated curve. from 1.5 mm out diameter borosilicate capillary glass (Sutter Instruments, Novato, CA) by using a Sutter P-97 For the analysis of steady-state inactivation kinetics, the puller (Sutter Instruments, Novato, CA), and had a resist- inactivation parameter was fitted to a Boltzmann distribu- ance of 1 – 3 MΩ. The pipette solution contained (in tion equation: mM): 110 CsF, 11 EGTA, 10 NaCl, MgCl 5, and 10 = 1/(1 + exp ((V - V )/k)), HEPES, pH 7.3 with CsOH. Isolated sodium current was I/I max 1/2 pre recorded from the single neuron in the presence of a bath solution that contained (in mM): 65 NaCl, 2.5 KCl, 5 Where I is the maximum sodium current elicited after max MgCl , 0.01 CaCl , 50 Choline-Cl, 20 TEA-Cl, 5 glucose, the most hyperpolarized prepulse, the V is the prepulse 2 2 pre 5 Na-HEPES, and 5 HEPES, pH 7.4 with NaOH. Bath solu- potential, V is the potential at which inactivation is half- 1/2 tion was applied to the recording chamber and removed maximal, and k is the slope factor. via a Peri-Star Pro peristaltic pump (World Precision Instruments, Sarasota, FL). Statistical tests The student t-test was used to examine the differences in Voltage-clamped currents were recorded with an Axo- mean latency of thermal paw withdrawal between preop- patch-200B amplifier (Molecular Devices, Union city, erative (mean value of the two preoperative tests) and CA). Data were acquired on a PC computer with the postoperative on the day of electrophysiological record- Clampex v10.0 software (Molecular Devices), filtered ings. The specific hypotheses about differences between with a low-pass Bessel filter setting of 5 kHz and digitized each treated (CCD or CCI) and the control group for each at a sampling rate of 40 kHz via a Digidata 1440A analog- electrophysiological parameter was examined. Compari- to-digital converter (Molecular Devices). The membrane sons among CCD, CCI and control groups were per- capacitance (C ) was read from the amplifier by software formed with one-way ANOVA followed by Newman- Clampex v10.0 for determining the size of cells and calcu- Keuls tests. X tests were used to identify differences in the lating the current density. Voltage errors were minimized incidence of effects. All data are presented as mean ± SE. by using 80–90% series resistance compensation and the Statistical results are considered significant if p < 0.05. capacitance artifact was canceled by the patch-clamp amplifier. Linear leakage currents were digitally subtracted Abbreviations on-line using hyperpolarizing potential after the test pulse CCD: Chronic compression of dorsal root ganglion; CCI: (P/6 procedure). Data acquisition began 5 min after estab- Chronic constriction injury of the sciatic nerve; DRG: Dor- lishing whole-cell configuration and the holding poten- sal root ganglion; TTX-R: Tetrodotoxin-resistant; TTX-S: tial was at -80 mV. Tetrodotoxin-sensitive; VGSCs: Voltage-gated sodium channels Somata of the small DRG neurons were classified by their diameters (15 ~30 μm) and C (≤ 45 pF). Neurons were Competing interests not considered for analysis if they had high leakage cur- The authors declare that they have no competing interests. rents (holding current >1.0 nA at -80 mV), membrane blebs, total sodium current < 500 pA, or an access resist- Authors' contributions ance > 5 MΩ. Access resistance was monitored throughout XJS and ZJH planned the studies. ZJH conducted the the experiment and data were not used if resistance experiments, analyzed the data and contributed to the changes of >20% occurred. Data were not corrected to writing of the paper. XJS participated in the studies and account for liquid junction potential. The offset potential data analysis and wrote the paper. Both authors approved was zeroed before patching the cells and checked after the final manuscript. each recording for drift. Acknowledgements This study was supported by grants from Parker Research Foundation To analyze the voltage dependence of channel activation, (PCCRF-BSR0501 and PCCRF-BSR0602) and National Natural Science the sodium conductance (G) was calculated. Peak current Foundation of China (NSFC-30628027). Page 13 of 15 (page number not for citation purposes) Molecular Pain 2008, 4:20 http://www.molecularpain.com/content/4/1/20 20. Black JA, Cummins TR, Plumpton C, Chen YH, Hormuzdiar W, Clare References JJ, Waxman SG: Upregulation of a silent sodium channel after 1. Cummins TR, Sheets PL, Waxman SG: The roles of sodium chan- peripheral, but not central, nerve injury in DRG neurons. J nels in nociception: Implications for mechanisms of pain. 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Ungless MA, Gasull X, Walters ET: Long-term alteration of S- type potassium current and passive membrane properties in Publish with Bio Med Central and every aplysia sensory neurons following axotomy. J Neurophysiol scientist can read your work free of charge 2002, 87:2408-2420. 55. Yao H, Donnelly DF, Ma C, LaMotte RH: Upregulation of the "BioMed Central will be the most significant development for hyperpolarization-activated cation current after chronic disseminating the results of biomedical researc h in our lifetime." compression of the dorsal root ganglion. J Neurosci 2003, Sir Paul Nurse, Cancer Research UK 23:2069-2074. 56. Bennett GJ, Xie YK: A peripheral mononeuropathy in rat that Your research papers will be: produces disorders of pain sensation like those seen in man. available free of charge to the entire biomedical community Pain 1988, 33:87-107. 57. Zheng JH, Walters ET, Song XJ: Dissociation of dorsal root gan- peer reviewed and published immediately upon acceptance glion neurons induces hyperexcitability that is maintained by cited in PubMed and archived on PubMed Central increased responsiveness to cAMP and cGMP. J Neurophysiol 2007, 97:15-25. yours — you keep the copyright BioMedcentral Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp Page 15 of 15 (page number not for citation purposes) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Molecular Pain Springer Journals

Differing alterations of sodium currents in small dorsal root ganglion neurons after ganglion compression and peripheral nerve injury

Huang, Zhi-Jiang; Song, Xue-Jun
Molecular Pain , Volume 4 (1) – May 30, 2008
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Springer Journals
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Copyright © 2008 by Huang and Song; licensee BioMed Central Ltd.
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Medicine & Public Health; Pain Medicine; Molecular Medicine; Neurobiology; Neurosciences; Neurology
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1744-8069
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10.1186/1744-8069-4-20
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18513405
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Abstract

Voltage-gated sodium channels play important roles in modulating dorsal root ganglion (DRG) neuron hyperexcitability and hyperalgesia after peripheral nerve injury or inflammation. We report that chronic compression of DRG (CCD) produces profound effect on tetrodotoxin-resistant (TTX-R) and tetrodotoxin-sensitive (TTX-S) sodium currents, which are different from that by chronic constriction injury (CCI) of the sciatic nerve in small DRG neurons. Whole cell patch- clamp recordings were obtained in vitro from L and/or L dissociated, small DRG neurons following 4 5 in vivo DRG compression or nerve injury. The small DRG neurons were classified into slow and fast subtype neurons based on expression of the slow-inactivating TTX-R and fast-inactivating TTX-S Na currents. CCD treatment significantly reduced TTX-R and TTX-S current densities in the slow and fast neurons, but CCI selectively reduced the TTX-R and TTX-S current densities in the slow neurons. Changes in half-maximal potential (V ) and curve slope (k) of steady-state inactivation of 1/2 Na currents were different in the slow and fast neurons after CCD and CCI treatment. The window current of TTX-R and TTX-S currents in fast neurons were enlarged by CCD and CCI, while only that of TTX-S currents in slow neurons was increased by CCI. The decay rate of TTX- S and both TTX-R and TTX-S currents in fast neurons were reduced by CCD and CCI, respectively. These findings provide a possible sodium channel mechanism underlying CCD- induced DRG neuron hyperexcitability and hyperalgesia and demonstrate a differential effect in the Na currents of small DRG neurons after somata compression and peripheral nerve injury. This study also points to a complexity of hyperexcitability mechanisms contributing to CCD and CCI hyperexcitability in small DRG neurons. play important roles in modulating neural excitability Background Nerve injury produces dorsal root ganglion (DRG) neu- [1,2]. The VGSCs are critically important for electrogene- ron hyperexcitability, which is thought to underlie neuro- sis and nerve impulse conduction, and a target for impor- pathic pain by causing central sensitization. The voltage- tant clinically relevant analgesics. However, mechanisms gated sodium channels (VGSCs) can be dynamically regu- of the VGSCs contributing to hyperexcitability of DRG lated after axonal injury or peripheral inflammation and neurons and neuropathic pain remain unclear and the Page 1 of 15 (page number not for citation purposes) Molecular Pain 2008, 4:20 http://www.molecularpain.com/content/4/1/20 observations are controversial. For instance, inhibition or ment and both feet in control groups did not show signif- specific knock-down of tetrodotoxin-resistant (TTX-R) icant change during the period of time. Data are shown in current Nav1.8 channel can effectively suppress neuro- Fig. 1. All of these rats were used later for electrophysio- pathic pain [3-5], while the Nav1.8 mRNA, protein and logical recordings during 10–14 postoperative days. current are substantially decreased in DRG neurons in axotomized DRG neurons [6-9] or sciatic nerve injury TTX-R and TTX-S Na currents in small neurons from CCD, CCI and control DRGs [10]. The tetrodotoxin-sensitive (TTX-S) current Nav1.7 channel plays a critical role in various pain conditions [1], Majority of the small DRG neurons express both TTX-R but nociceptors specific deletion of Nav1.7 did not elimi- and TTX-S Na currents, which have different activation nate neuropathic pain behavior in mice [11]. Thus, there and inactivation properties [6,8]. Prepulse inactivation, is a need to further investigate roles of the VGSCs in differ- which takes advantage of differences in inactivation prop- ent neuropathic pain conditions. erties of the TTX-R and TTX-S currents, was used to sepa- rate the slow-inactivating TTX-R, and fast-inactivating Different from nerve injury models that produce injury to TTX-S Na currents [6,18]. A current-voltage protocol with the peripheral axons of DRG neurons such as the chronic a 700 ms prepulse to -120 mV followed by respective test constriction injury (CCI) of the sciatic nerve, chronic pulse was applied first at a holding potential of -80 mV to compression of DRG (CCD) is used as an animal model obtain the total Na current. The slow-inactivating, TTX-R that produces injury directly to DRG somata. We have currents were recorded using a prepulse of 700 ms -50 mV shown that CCD treatment produces behavioral hyperal- before the test pulse. This protocol inactivates the TTX-S gesia and allodynia and DRG neuron hyperexcitability in currents while leaving the TTX-R current intact. TTX-S cur- rats [12-14]. However, ionic mechanisms contributing to rents were then obtained by subtracting the TTX-R cur- CCD-induced neural hyperexcitability remain unclear. A rents from the total Na current in the cells. These recent study shows that TTX-R Na currents are upregu- protocols allowed simultaneous measurement of both lated in the cutaneous medium-sized CCD DRG neurons TTX-R and TTX-S currents in each neuron recorded. In [15], which is somewhat different from the findings in some neurons, the TTX-R currents were recorded with axon injury models. The small DRG neurons most are existence of TTX (300 nM, n = 6). The TTX-R and TTX-S nociceptive and play critical roles in neuropathic pain. currents were similar to that recorded by using protocols + + Expression of the Na currents is different between small- and the Na currents were completely blocked by lido- and medium-sized DRG neurons in CCI rats [16]. How- caine (200 μM, n = 6) (data not shown). These data were ever, ionic mechanisms have not been investigated in similar to that described previously [6]. these small neurons after CCD treatment. The purpose of this study was to analyze the effects of CCD on the prop- One hundred and twenty-four small neurons including erties of TTX-R and TTX-S Na currents in the small DRG 53 from control, 38 CCD and 33 CCI DRGs were recorded neurons. Because of the complex and diverse expression and analyzed. Examples of recordings and calculations of of the VGSCs in different neuropathic pain conditions, we the Na currents are shown in Fig. 2. All of the neurons compared alterations of density and kinetic property of analyzed and discussed in this study expressed both slow- + + the TTX-R and TTX-S Na currents in CCD with CCI DRGs inactivating TTX-R, and fast-inactivating TTX-S Na cur- in the same recording condition. This study provides rents, which we refer to as "slow" and "fast" currents, sodium channel mechanisms underlying CCD-induced respectively. Neurons expressing predominantly (>70% of DRG neuron hyperexcitability and behavioral hyperalge- total) TTX-R currents are referred to as "slow neurons", sia and indicates different effects of CCD- and CCI-treat- while neurons expressing predominantly (>70% of total) ment on the TTX-R and TTX-S Na currents. TTX-S currents are referred to as "fast neurons" [6,18,19]. There were 5 controls-, 3 CCD- and 5 CCI- neurons that Preliminary data have been published in an abstract form expressed "mixed currents" (both slow and fast currents [17]. >70% of total) were not included in the analysis of this study. The results showed that CCD treatment increased Results percentage of the small-slow neurons and reduced per- CCD and CCI produced thermal hyperalgesia centage of small-fast neurons, while CCI treatment We began by confirming with earlier demonstrations that decreased percentage of small-slow neurons and increased CCD- or CCI-treatment produced pain and hyperalgesia. that of small-fast neurons. Data are summarized in Table All the CCD- and CCI-treated rats showed behavioral 1. indications of thermal hyperalgesia. Withdrawal latencies of the foot ipsilateral to CCD or CCI treatment decreased significantly from the preoperative values. Withdrawal latencies of the foot contralateral to CCD or CCI treat- Page 2 of 15 (page number not for citation purposes) Molecular Pain 2008, 4:20 http://www.molecularpain.com/content/4/1/20 Control left (10) Control right (10) CCD ipsil (12) CCD contral (12) CCI ipsil (10) CCI contral (10) 2 nA TTX-S 5 ms 40 ms -10 mV -50 mV 70 0 ms 40 ms TTX-R -8 0 mV -10 mV 10 -80 mV ** Total 700 ms -120 mV ** ** ** ** ** ** Representative Na ron fr Figure 2 om a control DRG currents traces recorded in a small neu- Representative Na currents traces recorded in a small neuron from a control DRG. The total current surgery recording was recorded with a 700 ms prepulse to -120 mV followed by the test pulse. The TTX-R Na current was recorded with -3 -1 1 3 5 7 9 11 a 700 ms prepulse to -50 mV followed by a test pulse. The 10-14 TTX-S Na current was obtained by digital subtraction of the Postoperative Day TTX-R current from the total current. All the test pulses were 40 ms to -10 mV pulses. The holding potential was at - Th ra Figure 1 ts ermal hyperalgesia following CCD- or CCI-treatment in 80 mV. Thermal hyperalgesia following CCD- or CCI-treat- ment in rats. Repeated measurements are shown of ther- mal sensitivity of the foot withdrawal response in CCD-, CCI- and control rats. Numbers of rats used in each group pared to control (Fig. 3C and 3D). The TTX-R current den- are indicated in the parentheses. The arrow indicates the sity was significantly reduced by approximately 30% and point of surgery of CCD or CCI performed. The dash line 20% in CCD and CCI DRGs, respectively (Fig. 3C). The above "recording" indicates the period of time that the rats TTX-S current density was reduced by approximately 50% were sacrificed for electrophysiological recordings and the and 25% in CCD and CCI DRGs, respectively. Reduction data were collected at different days of postoperative 10–14. of TTX-S current density by CCD treatment was signifi- **P < 0.01 indicate significant differences between groups of cantly more than that by CCI (p < 0.05) (Fig. 3D). To fur- CCD ipsilateral or CCI ipsilateral to the other groups. ther demonstrate reduction in the current levels, densities were binned and plotted against neuron number (Fig. 3E TTX-R and TTX-S Na current densities are reduced in and 3F). The peak distribution of TTX-R current density small-slow neurons in CCD and CCI DRGs shifted from 400–600 pA/pF in control to the lower levels The TTX-R and TTX-S current densities (peak current of <200 and 200–400 pA/pF in CCD and 200–600 pA/pF amplitude normalized to C ) were examined and com- in CCI (Fig. 3E). The peak distribution of TTX-S current pared in the small-slow neurons among CCD, CCI and density shifted from 200–400 pA/pF in control to the lev- control DRGs. The peak TTX-R and TTX-S current ampli- els of <200 pA/pF in CCD and <200 and 200–400 pA/pF , which could affect the results of tudes were measured with a 40 ms test pulse to -10 mV. in CCI (Fig. 3F). The C Examples of the TTX-R and TTX-S currents from CCD, CCI the current density, was not significantly changed in CCD and control DRG neurons are given in Fig. 3A and 3B. and CCI compared to that in control DRG neurons (Fig. CCD and CCI treatment significantly reduced TTX-R and 3G). TTX-S current densities in the small-slow neurons com- TTX-R and TTX-S Na current densities are reduced in Table 1: Proportion and distribution of the small-slow and small- small-fast neurons in CCD, but not CCI DRGs fast neurons in control, CCD and CCI DRGs. The TTX-R and TTX-S current densities were also examined and compared in the small-fast neurons among CCD, CCI Number of neurons (% of Total) and control DRGs. Examples are given in Fig. 3H and 3I. nC (pF) Small-Slow Small-Fast CCD treatment significantly reduced TTX-R and TTX-S current densities approximately 40% and 30%, respec- 27 (51) 26 (49) tively, in the small-fast neurons compared to control (Fig. CCD 38 25.9 ± 1.2 25 (66)* # 13 (34)* # 3J and 3K). Current densities were again binned and plot- CCI 33 24.6 ± 1.2 13 (39)* 20 (61)* ted against the neuron number as shown in Fig. 3L and 3M. The peak distribution of TTX-R current densities *, #, p < 0.05 indicate significant differences compared with control shifted from 200–400 pA/pF in control neurons to the (*) or CCD (#) groups. Page 3 of 15 (page number not for citation purposes) Mean Latency of Thermal Paw Withdrawal (Sec) Molecular Pain 2008, 4:20 http://www.molecularpain.com/content/4/1/20 Slow Neurons A C E -600 Control CCD -500 60 CCI -400 CCD ** 5 nA CCI 10 ms -300 Control 30 -200 -100 Control CCD CCI 15 <200 200-400 400-600 >600 TTX-R Current Density (pA/pF) B D F -600 70 Control 5 CCD CCD -500 0 Control CCD CCI CCI CCI -400 Control 5 nA -300 10 ms -200 -100 Control CCD CCI <200 200-400 400-600 >600 TTX-S Current Density (pA/pF) Fast Neurons H J L -60 0 70 Control CCD -50 0 CCI CCD -40 0 CCI Control -30 0 30 30 5 nA -20 0 10 ms -10 0 Co nt rol CCD CCI <200 200-400 400-600 >600 TTX-R Current Density (pA/pF) I K M -600 70 Control 5 CCD -500 Control CCI CCD CCI CCD -400 CCI 5 nA -300 10 ms -200 Control -100 0 0 Control CCD CCI <200 200-400 400-600 >600 TTX-S Current Density (pA/pF) Alterat Figure 3 ion of TTX-R and TTX-S Na current densities in slow and fast small DRG neurons after CCD and CCI treatment Alteration of TTX-R and TTX-S Na current densities in slow and fast small DRG neurons after CCD and CCI treatment. A, B, H and I: Examples of the TTX-R and TTX-S currents recorded with the prepulse inactivation protocol with 40 ms to -10 mV test pulse in the slow neurons (A and B) and the fast neurons (H, I) from CCD, CCI and control DRGs, respectively. C, D, J and K: Alterations of the TTX-R and TTX-S current densities in the slow (C and D) and the fast (J and K) neurons. E, F, L and M: Distribution of the TTX-R and TTX-S current densities in the slow (E and F) and the fast (L and M) neu- rons. The densities were binned and plotted against neuron number (%). G and N: Input capacitance (C ) of the slow (G) and fast (N) neurons from control, CCD and CCI groups. *, p < 0.05 and **, p < 0.01 indicate significant differences compared with the control group. #, p < 0.05 indicate significant differences compared with CCI group. Page 4 of 15 (page number not for citation purposes) TTX-S TTX-S TTX-R TTX-R TTX-S Current Density (pA/pF) TTX-S Current Density (pA/pF) TTX-R Current Density (pA/pF) TTX-R Current Density (pA/pF) Fast Neuron Number (%) Slow Neuron Number (%) Slow Neuron Number (%) Fast Neuron Number (%) Fast Neurons C (pF) Slow Neurons C (pF) m m Molecular Pain 2008, 4:20 http://www.molecularpain.com/content/4/1/20 lower levels of <200 pA/pF in CCD (Fig. 3L). The peak dis- prepulse to -120 or -50 mV, followed by a series of test tribution of TTX-S current densities shifted from 200–600 pulse from -70 to +60 mV with +10 mV increments. The total Na current was obtained by using the protocol with pA/pF in control neurons to the lower levels of <200–400 pA/pF in CCD (Fig. 3M). In contrast, CCI treatment failed a prepulse to -120 mV. The TTX-R component was to change the densities of both TTX-R and TTX-S currents recorded by using the protocol with a prepulse to -50 mV (Fig. 3J–M). The membrane capacitance (C ) was not sig- which inactivates TTX-S currents. The TTX-S component nificantly changed in CCD and CCI compared to that in was obtained by digitally subtracting the TTX-R compo- control DRGs (Fig. 3N). nent from the total Na current. Examples of TTX-R and TTX-S currents in the small slow and fast neurons from a Previous studies have shown that nerve injury including control DRG are given in Fig. 5A–D. Plots of normalized CCI do not produce significant change in the TTX-S cur- peak Na current density versus test pulse voltage for the rent [16,20,21]. Here it is shown that CCI does reduce the small slow and fast neurons in control, CCD and CCI TTX-S currents in the small-slow neurons (Fig. 3D), but DRGs are shown in Fig. 5E–H. Activation threshold of the not in the small-fast neurons (Fig. 3K). Interestingly, if the TTX-R currents the control neurons was between -35 and data from the slow and fast neurons are combined, CCI- -30 mV and the maximum inward current fell between -10 induced alteration in TTX-S current density in the slow and 0 mV (Fig. 5E,G). Activation threshold of the TTX-S neurons is hidden, while CCD-induced change in TTX-S currents in the control neurons was detected between -50 current still exhibit clearly (Fig. 4A). Both CCD- and CCI- to -45 mV with maximum inward current at approxi- induced reduction in TTX-R current densities are mately -20 mV (Fig. 5F, H). All currents measured dis- unchanged in this analysis (Fig. 4B). The C was not sig- played a reversal potential (V ) of about 50–55 mV, m rev nificantly different between CCI and control DRGs and corresponding to the calculated equilibrium potential for not different among the groups of CCD, CCI and control sodium ions under these recording conditions (E = 50 Na (see Table 1). mV). The voltage at which 50% of the Na channels were activated (V ) and the slope for activation (k) were 1/2 Voltage dependence of activation of TTX-R and TTX-S obtained from fitting the normalized conductance (G/ Na currents in small-slow and small-fast neurons is not G )-voltage curve with the Boltzmann equation. Effects max altered by CCD and CCI of CCD and CCI on the voltage dependence activation of Current-voltage relationship of the TTX-R and TTX-S Na TTX-R and TTX-S currents were examined and analyzed. currents was measured using a I-V protocol with a 700 ms Data are expressed and summarized in Fig. 5I–L and Table 2. In both small slow and fast neurons, neither CCD nor CCI treatment significantly altered parameters of activa- A B tion of the TTX-R and TTX-S Na currents such as V , k, 1/2 -450 -450 activation threshold, and voltage range of the maximum -400 -400 inward current fell in. These negative results support the -350 -350 findings in the current density by excluding the possibility of changes in the current amplitude caused by alterna- -300 -300 ** tions of the activation properties of Na currents. -250 -250 -200 -200 Voltage-dependence of steady-state inactivation of TTX-R -150 -150 and TTX-S Na currents in small-slow and small-fast -100 -100 neurons is altered by CCD and CCI -50 -50 Steady-state inactivation of TTX-R and TTX-S Na currents 0 was measured with 500 ms prepulse to potentials over the Control CCD CCI Control CCD CCI range of -120 mV to -10 mV with 5 mV increments fol- lowed by, with a 0.8 ms interpulse interval to -80 mV, a - Alterat Figure 4 small DRG neur ion of TTX-R and TTX-S Na ons after CCD and CC cuI treatment rrent densities in 10 mV test pulse. The TTX-R inactivation currents were Alteration of TTX-R and TTX-S Na current densi- measured at the time of the peak current evoked following ties in small DRG neurons after CCD and CCI treat- a -50 mV prepulse. The TTX-S inactivation currents were ment. Data shown here are from Fig. 2C, D, J and 2K and measured at the time of the peak of the maximum current the data from the slow and fast neurons are combined. CCI- evoked following a -120 mV prepulse [18,22]. Examples induced significant alteration in TTX-S current density in the of recordings of steady-state inactivation of the TTX-R and slow neurons (see Fig. 2) is hidden, while CCD-induced TTX-S currents in the slow and fast neurons are shown in change in TTX-S currents still exhibit clearly (A). Both CCD- Fig. 6A and 6B. CCD and CCI treatment significantly and CCI-induced reduction in TTX-R currents density is unchanged in this analysis (B). altered the V and k of TTX-R and TTX-S currents in the 1/2 slow and/or fast neurons as shown in Fig. 6(C–F) and Page 5 of 15 (page number not for citation purposes) TTX-S Current Density (pA/pF) . TTX-R Current Density (pA/pF) . Molecular Pain 2008, 4:20 http://www.molecularpain.com/content/4/1/20 Table 2: Voltage-dependence of activation and steady-state inactivation of the TTX-R and TTX-S Na currents in the slow and fast small-sized control, CCD and CCI DRGs. TTX-R Current TTX-S Current V (mV) k V (mV) k V (mV) k V (mV) k 1/2act act 1/2inact inact 1/2act act 1/2inact inact Slow Neurons Control -17.9 ± 1.1 4.3 ± 0.3 (14) -35.3 ± 0.7 -4.0 ± 0.1 (14) -26.6 ± 1.3 5.6 ± 1.1 (14) -77.6 ± 2.2 -8.0 ± 0.5 (14) (14) (14) (14) (14) CCD -18.7 ± 1.7 4.9 ± 0.4 (15) -38.8 ± 0.8** -4.6 ± 0.2* -26.2 ± 1.4 5.2 ± 0.6 (15) -84.0 ± 1.3* -9.9 ± 0.5* (15) (12) (12) (15) (12) (12) CCI -15.6 ± 1.6 (7) 5.1 ± 0.5 (7) -38.1 ± 0.7* -4.6 ± 0.1* (7) -26.1 ± 1.4 (7) 4.0 ± 0.8 (7) -75.1 ± 5.1 (7) -12.4 ± 1.8** (7) (7) Fast Neurons Control -13.0 ± 1.2 5.6 ± 0.4 (13) -39.4 ± 1.1 -4.4 ± 0.1 (14) -26.2 ± 1.4 4.9 ± 0.6 (13) -84.4 ± 1.9 -8.9 ± 0.4 (14) (13) (14) (13) (14) CCD -14.3 ± 1.7 (9) 5.8 ± 0.7 (9) -37.9 ± 1.3 (7) -5.3 ± 0.2** -23.0 ± 1.8 (9) 5.6 ± 1.1 (9) -75.7 ± 1.5* -9.4 ± 0.8 (7) (7) (7) CCI -12.0 ± 0.8 5.9 ± 0.4 (12) -36.8 ± 1.1 -5.0 ± 0.2* -26.8 ± 1.2 5.4 ± 1.1 (12) -80.4 ± 2.8 -10.1 ± 0.8 (12) (10) (10) (12) (10) (10) V : membrane potential at which activation is half-maximal. k : slope of the activation curve. 1/2act act V : membrane potential at which inactivation is half-maximal. k : slope of the inactivation curve. 1/2inact inact The number in parenthesis indicates the number of neurons. *, p < 0.05, **, p < 0.01 indicate significant differences compared with control group. summarized in Table 2. CCD and CCI produced similar Inactivation of TTX-R and TTX-S Na currents in small- effects on the TTX-R current of the slow neurons, but dif- fast neurons is slowed by CCD and CCI ferent on the TTX-R current of the fast neurons and the To quantitate changes in decay rate of the TTX-R and TTX- S Na currents, we fit the currents with single exponentials TTX-S current of the slow and fast neurons (Table 2). as that previously described [25]. The results showed that The shift in steady-state inactivation affected the window the inactivation of TTX-R currents in the small-fast neu- current, which is the region of overlap between the curves rons was significantly slowed by CCI treatment from 6.09 for the dependence of activation and inactivation. The ± 0.48 ms in control to 8.34 ± 1.12 ms (p < 0.05), but not overlapping activation/inactivation Boltzmann curves by CCD treatment although the inactivation tended to be were used to determine the fraction of sodium channels slower (p > 0.05) (Fig. 8A, Table 3). In contrast, inactiva- activated in the peak of the window current [23,24]. The- tion of TTX-S Na currents in the fast neurons was signifi- oretical analysis of the voltage dependencies presented in cantly slowed by both CCD and CCI treatment (Fig. 8B, Fig. 6 indicates that both CCD and CCI treatment increase Table 3). Neither CCD nor CCI treatment significantly the window current of the TTX-R currents in the fast, but altered inactivation of both TTX-R and TTX-S currents in not slow neurons (Fig. 7A and 7B). In the TTX-R currents the slow neurons (Table 3). in fast neurons at the membrane potential where maximal overlap of inactivation and activation occurred, approxi- Discussion mately 7% of the Na channels in control neurons were in The present study investigated alterations of TTX-R and a non-inactivated state and approximately 7% of the avail- TTX-S Na currents in the small DRG neurons after DRG able channels were activated. This fraction was increased somata compression (CCD treatment) and compared the ~28% and ~56% by CCD and CCI treatment, respectively different effects of DRG somata compression and the (Fig. 7B). The fractions of the window currents of the TTX- axons injury (CCI treatment) on the Na currents. The S currents were increased approximately 110% in the slow principle findings are 1) CCD treatment significantly neurons and 100% in the fast neurons by CCI (Fig. 7C reduces the TTX-R and TTX-S current densities in the and 8D). In contrast, CCD treatment increased window small-slow and small-fast subtypes of DRG neurons; 2) current of the TTX-S only in the fast neurons, but not the CCD alters voltage-dependent steady-state inactivation of slow neurons and the fraction was increased by ~86% the TTX-R and TTX-S currents and increases window cur- (Fig. 7D). rent of the activation and inactivation, but exhibits differ- ent effects on V and k of the TTX-S currents in both slow 1/2 and fast neurons; 3) CCD reduces the decay rates of the TTX-S, but not TTX-R currents inactivation in the fast neu- rons; and 4) CCI shows different effects from CCD in Page 6 of 15 (page number not for citation purposes) Molecular Pain 2008, 4:20 http://www.molecularpain.com/content/4/1/20 Slow Neurons A E I 1.1 0.1 0.9 -0.1 0.7 -0.3 0.5 -0.5 0.3 -0.7 Control 5 nA Control CCD CCD -0.9 0.1 CCI 8 ms CCI -1.1 -0.1 -70 -50 -30 -10 10 30 50 -60 -40 -20 0 20 V (mV) m V (mV) B F J 1.1 0.1 0.9 -0.1 0.7 -0.3 0.5 -0.5 0.3 -0.7 5 nA Control Control CCD CCD -0.9 0.1 8 ms CCI CCI -1.1 -0.1 -70 -50 -30 -10 10 30 50 -70 -50 -30 -10 10 V (mV) V (mV) Fast Neurons F C G K 1.1 0.1 0.9 -0.1 0.7 -0.3 0.5 -0.5 0.3 -0.7 5 nA Control Control CCD CCD 0.1 -0.9 8 ms CCI CCI -1.1 -0.1 -70 -50 -30 -10 10 30 50 -60 -40 -20 0 20 V (mV) V (mV) D H L 1.1 0.1 0.9 -0.1 0.7 -0.3 0.5 -0.5 0.3 -0.7 5 nA Control Control CCD CCD 0.1 -0.9 8 ms CCI CCI -1.1 -0.1 -70 -50 -30 -10 10 30 50 -70 -50 -30 -10 10 V (mV) V (mV) m Th altered by CCD and CCI tre Figure 5 e current-voltage relationships of TTX-R atment and TTX-S Na currents obtained from either slow or fast DRG neurons are not The current-voltage relationships of TTX-R and TTX-S Na currents obtained from either slow or fast DRG neurons are not altered by CCD and CCI treatment. A-D: Representative currents families from the slow (A and B) and fast (C and D) neurons were recorded by using the prepulse inactivation protocol with a series of test pulse ranging from -70 mV to +60 mV (in a +10 mV increments). E-H: Normalized peak current was plotted against test pulse voltage. I-L: The conductance (G) was calculated and plotted against test pulse voltage. Page 7 of 15 (page number not for citation purposes) TTX-S TTX-R TTX-S TTX-R Normalized TTX-S Current Normalized TTX-R Current Normalized TTX-S Current Normalized TTX-R Current TTX-S G/G TTX-R G/G TTX-S G/G TTX-R G/G max max max max Molecular Pain 2008, 4:20 http://www.molecularpain.com/content/4/1/20 Slow Neuron Fast Neuron A B 5 nA 5 ms C D 1.1 1.1 Control Control CCD CCD 0.9 0.9 CCI CCI 0.7 0.7 0.5 0.5 0.3 0.3 0.1 0.1 -0.1 -0.1 -80-70-60-50 -40-30-20-10 -80-70-60-50 -40-30-20-10 V (mV) V (mV) pre pre E F 1.1 1.1 Control Control CCD CCD 0.9 0.9 CCI CCI 0.7 0.7 0.5 0.5 0.3 0.3 0.1 0.1 -0.1 -0.1 -120 -100 -80 -60 -40 -20 -120 -100 -80 -60 -40 -20 V (mV) V (mV) pre pre A and CCI treatm Figure 6 lteration of the steady-state in ent activation of TTX-R and TTX-S Na currents in slow and fast small DRG neurons after CCD Alteration of the steady-state inactivation of TTX-R and TTX-S Na currents in slow and fast small DRG neu- rons after CCD and CCI treatment. A and B: Representative records of the inactivation current from a slow neuron (A) and a fast neuron (B) from a control DRG. The currents were recorded by using a double protocol with a 500 ms prepulse ranging from -120 mV to -10 mV (in 5 mV increments), followed by, with a 0.8 ms interpulse interval to -80 mV, a test pulse to -10 mV. The inter-pulse period was 10 s. The TTX-R inactivation currents were measured at the time of the peak current evoked following a -50 mV prepulse. The TTX-S inactivation currents were measured at the time of the peak of the maximum current evoked following a -120 mV prepulse. Each data set was normalized and fit with a Boltzman equation. The best fitted steady-state inactivation curves were showed in C-F. Page 8 of 15 (page number not for citation purposes) TTX-S Current I / I TTX-R Current I / I max max TTX-S Current I / I TTX-R Current I / I max max Molecular Pain 2008, 4:20 http://www.molecularpain.com/content/4/1/20 A after CCD and CCI treatment Figure 7 lteration of the window current of activation and steady-state inactivation curves in the slow and fast small DRG neurons Alteration of the window current of activation and steady-state inactivation curves in the slow and fast small DRG neurons after CCD and CCI treatment. Boltzmann fits from the data shown in Fig. 4 and 5 are shown in A-D. The small figures inserted in each figure illustrate the overview of the activation and inactivation fit curves. most of the tested properties of the TTX-R and TTX-S cur- Table 3: Inactivation of the TTX-R and TTX-S Na currents of rents. These findings suggest a possible sodium channel small-slow and small-fast neurons evoked with -10 mV test pulse mechanism underlying CCD-induced DRG neuron hyper- in control, CCD and CCI DRGs. excitability and indicate that injuries to DRG somata and n Current inactivation time constant (ms) peripheral axons may result in different alterations of the VGSCs. TTX-R TTX-S The DRG neurons, particularly the nociceptive small neu- Small-Slow Neurons rons, are notable in expressing multiple sodium channel Control 27 5.11 ± 0.42 0.90 ± 0.07 isoforms including Nav1.8 and Nav1.9 contributing to CCD 25 4.82 ± 0.29 1.00 ± 0.08 the TTX-R currents, and Nav1.7, Nav1.6, Nav1.5, Nav1.3, CCI 13 6.65 ± 0.89 1.10 ± 0.12 Nav1.2 and Nav1.1 contributing to the TTX-S currents Small-Fast Neurons Control 26 6.09 ± 0.48 0.92 ± 0.05 [26-31]. Many of these sodium channels can be dynami- CCD 13 7.18 ± 0.54 1.12 ± 0.09* cally regulated after nerve injury and/or inflammation CCI 20 8.43 ± 1.12* 1.17 ± 0.07** and the specific channels may play crucial roles in nocice- ption. Several lines of studies including many with trans- *, p < 0.05, **, p < 0.01 indicate significant differences compared with genic mice lines have clearly implicated Nav1.7, Nav1.8 control group. Page 9 of 15 (page number not for citation purposes) Molecular Pain 2008, 4:20 http://www.molecularpain.com/content/4/1/20 were also found in rats that received CCI treatment [16], indicating that TTX-R Na currents may have different functions in the small- vs the medium-sized DRG neurons after neuron injury, and may contribute to the different 0.2 mechanisms of these neurons in the abnormal neuron excitability. TTX-R Na channel is also found upregulated 0.4 in the sciatic nerve axons at the site of injury [32] and redistributed in the uninjured adjacent axons of DRG neu- CCI 0.6 rons [33]. Consistently, contribution of Nav1.8 currents CCD to neuropathic pain conditions has been demonstrated 0.8 controversial. Another interesting finding in the present Control study is that CCI treatment reduces the TTX-R currents 1 only in the slow, but not the fast neurons, further support- ing that nerve injury can produce different effects on 0 5 10 15 20 25 30 sodium channels in different types of DRG neurons. In Time (ms) addition, the Nav1.9 channel can have profound effects on resting membrane potential (RMP), and thus on excit- ability in DRG neurons [26,27]. Nav1.9 current activates at more negative potentials (-80 mV), differing from the Nav1.8 current activating at potentials close to RMP (-60 0.2 CCI to -70 mV), and generates the persistent TTX-R current identified [26,34,35]. Nav1.9 current plays an important 0.4 CCD role in setting RMP as well as contributing to subthreshold electrogenesis in small DRG neurons [26,27]. Nav1.9 was 0.6 Control not observed in the present study because of ultraslow inactivation at the holding potentials used and at the time 0.8 domain of the pre-pulse 700 ms to -120 mV applied to remove the fast inactivation of TTX-S currents. This yielded an estimation of the sodium current in the cell 02 46 8 10 minus the Nav1.9 current. Time (ms) The TTX-S Nav1.7 and Nav1.3 channels have been identi- Figure 8 small DRG neur Represen tion of TTX-R ( tative recordings showin A ons after ) and TTX-S CCD and CC (B) Na g alterat currents in the fast I treatment ion of the inactiva- fied to play important roles in neural hyperexcitability Representative recordings showing alteration of the inactivation of TTX-R (A) and TTX-S (B) Na cur- and chronic pain [1], but the observations again are con- rents in the fast small DRG neurons after CCD and troversial. Nav1.7, Nav1.6 and Nav1.3 channels are upreg- CCI treatment. ulated [36-39] or down-regulated [25,39-41] in DRG neurons after nerve injury or axotomy. In the present study, CCD and CCI treatment both result in down-regu- and Nav1.9 in inflammatory and probably neuropathic lation of TTX-S currents. However, CCI treatment selec- pain [1,2]. The present study shows that CCD treatment tively reduces density of TTX-S currents in the slow, but significantly down-regulates both the TTX-R and TTX-S not the fast neurons. These results suggest different roles + + Na currents in the small DRG neurons. The TTX-R Na for TTX-S currents in these two subtypes of neurons after currents recorded in our study are predominantly the peripheral nerve injury. This differentiation might in Nav1.8 as identified by the specific protocol [1]. Such some way link to the conflict findings in the experiments alteration of TTX-R Na currents after CCD treatment is that knock-down Nav1.3 leads to decrease [42] or no consistent to majority of the previous findings that change [43] in pain sensitivity. The differential effects in Nav1.8 mRNA, protein and current are substantially the TTX-R and TTX-S Na+ current induced by CCD and decreased in DRG neurons in axotomized DRG neurons CCI may contribute partly to certain differences in neural [6-9] or sciatic nerve injury [10,16]. These observations excitability and behavioral manifestations between the demonstrate a common role for the TTX-R Na channels two models [13]. In addition, it is worthy while to men- in the DRG neurons after injury to either somata or tion that because only a proportion of the L4/5 DRG neu- peripheral axons. A recent study indicates that the cutane- rons were directly injured in the CCI model (due to the ous, medium-sized dissociated CCD DRG neurons exhibit contribution of L4/5 to sciatic nerve in the injured level), an increase in TTX-R Na currents [15]. Such different the neurons under investigation might also include some changes of Na currents in different types of DRG neurons Page 10 of 15 (page number not for citation purposes) Normalized TTX-S Current Normalized TTX-R Current Molecular Pain 2008, 4:20 http://www.molecularpain.com/content/4/1/20 intact uninjured ones [7] and therefore the sensitivity of the slope factor. The window current (overlap between the statistics could be lowered. curves for the dependence of activation and inactivation) represents a voltage region in which sodium channels can How these differential alterations of sodium channels/ continue to open because some channels are activated currents would contribute to neural excitability and neu- and not all of the channels are inactivated. The increased ropathic pain remains unclear [1,2]. A recent study by window current therefore may result in an increase in the Rush et al [44] may provide an explanation to such con- persistent current that can seize activity and may affect troversial and conflict observations. It is shown that a sin- neuronal excitability [23,24]. These alternations of steady- gle sodium channel (Nav1.7) mutation can produce state inactivation curves therefore show a potential to opposing phenotypes (hyperexcitability vs hypoexcitabil- depolarize the resting membrane potential and increase ity) in sensory neurons and sympathetic neurons, respec- the neuronal excitability. A recent study indicates that the tively, and the selective presence of the Nav1.8 channel is CCD treatment hyperpolarizing shifts the activation a major determinant of these opposing effects. Majority of curves of the TTX-S current, but not the steady-state inac- nociceptive DRG neurons express Nav1.8 [29,45], which tivation curves in cutaneous medium-sized DRG neurons contributes most of the current underlying the action [15]. Such a hyperpolarizing shift of the activation curve potential upstroke [9,46]. Because it has depolarized volt- may also increase the window current. Thus, the increased age-dependence of activation and inactivation window current may underlie the neural hyperexcitabil- [6,10,45,47] compared with other sodium channels, ity. In addition, our results show that inactivation of the Nav1.8 permits DRG neurons to generate action poten- TTX-R and TTX-S currents are slowed down by CCD and/ tials sustain repetitive firing when depolarized [9,46]. This or CCI in the small-fast neurons. This may also contribute finding suggests that the physiological coupling of Nav1.8 to the neural hyperexcitability. We hypothesize that alter- and Nav1.7 in the nociceptive DRG neurons may contrib- ations of the sodium channel gating properties associated ute to the phenotypes in the different cell types as well as with down- or up-regulation of the current density may in the different neuropathic conditions. In the same study contribute to the neural hyperexcitability, while redistri- [44], RMP of the sensory and sympathetic neurons is bution of the sodium channels to the adjacent uninjured depolarized following Nav1.7 mutation and such depo- fibers may contribute to the development of neuropathic larization is thought to be a result of increased window pain [33]. This hypothesis may also provide an explana- currents [23,44]. Interestingly, a similar depolarization is tion for the controversial observations. also true in the large- and medium-sized and small DRG neurons after CCD treatment as we demonstrated recently CCD treatment produces local inflammation particularly [14] and the window currents are increased in the small- in the first postoperative week as described in the previous fast neurons after CCD treatment and in the small-slow studies [12,13,48], in addition to producing compression neurons after CCI treatment, while the density of both of the ganglion. In this study, the down-regulation of the TTX-R and TTX-S Na+ currents is downregulated, as TTX-R currents are similar to those observed in the nerve shown in the present study. These findings might support injury model, but not the inflammation model in which a possibility that either upregulation or downregulation up-regulated [1]. In the present study, the DRG neurons of the TTX-R or TTX-S current densities in the injured were isolated from rats 10–14 days after continuous com- medium-sized [15,8] and small DRG neurons, which pression. We noticed that the inflammation during this express both Nav1.8 and Nav1.7 channels, can produce period of time was much less than that in the first week neural hyperexcitability. Such alteration of Na+ channels/ after the rod was initially introduced into the interverte- currents on neural function should also depend on the bral foramen (data not collected), which was described in basis of the cell background in which the alteration is our previous studies [12,13,48]. Such a point may also expressed. Further investigation and analysis are urgently contribute to the difference between our study and Tan et needed to elucidate such complex relationships between al [15], in addition to the different types of DRG neurons neural hyperexcitability and alterations of the sodium recorded. channels after nerve injury. It needs to be pointed out that nerve injury alters the elec- Steady-state inactivation of TTX-R and TTX-S Na currents trophysiological properties of diverse types of primary alters and exhibits different changes in the midpoints (V afferent neurons and triggers a myriad of changes in gene 1/ ) and slopes (k) of inactivation curves in the small-slow expression that affect many proteins, including ion chan- and small-fast neurons after CCD and CCI treatment. It is nels, receptors, and other membrane proteins [49-52]. interesting that both CCD and CCI treatment increase the Such alterations are likely to complicate the changes in window current in both slow and fast neurons by differ- Na currents we observed. These complexities might be ently (depolarizing or hyperpolarizing) shifting the reduced by sampling functionally homogeneous subpop- steady-state inactivation curves midpoint and decreasing ulations or recording from the same neurons before and Page 11 of 15 (page number not for citation purposes) Molecular Pain 2008, 4:20 http://www.molecularpain.com/content/4/1/20 after injury. While this can be done with some inverte- another at L . CCI model was employed to produce brate nociceptors [53,54], it is not yet practical for DRG peripheral nerve injury and the procedure was similar to neurons. that described in the CCI model [56]. The left common sciatic nerve of each rat (n = 10) was exposed at the level Additional ion channel mechanisms may also contribute of mid-thigh. Proximal to the sciatic nerve's trifurcation, to neural hyperexcitability and behavioral hyperalgesia about 7 mm of nerve was freed of adhering tissue and four and allodynia after DRG compression. Recent studies ligatures (4-0 chronic gut) were tied loosely around it with have shown that CCD causes a decrease in fast-inactivat- about 1 mm spacing. The length of affected nerve was ing K current [15] and an increase in expression of a about 5 mm. Another group of rats (n = 10) received nei- hyperpolarization-activated cation current (I ), in addi- ther surgery nor injury and were served as control. A series tion to an increase in TTX-R Na currents in cutaneous, of previous studies in our lab and others has shown that medium-sized DRG neurons [55]. The I current is acti- sham surgery for CCD and CCI treatment did not produce vated during the afterhyperpolarization that follows an significant electrophysiological differences between neu- action potential and leads to a sustained depolarizing cur- rons from previously unoperated versus sham-operated rent, resulting in repetitive firing [55]. controls [13-15,21], therefore, the sham operations were not considered necessary in the present study. Conclusion In summary, this study shows that CCD treatment can Behavioral testing cause profound changes in densities and properties of Thermal hyperalgesia was indicated by a decrease in the inactivation of TTX-R and TTX-S Na currents of the small latency of foot withdrawal evoked by a radiant heat stim- DRG neurons, and that DRG somata compression results ulus as described previously [13,14]. The IITC Model 336 in different alterations of the Na currents from the Analgesia Meter (Life Science, Series 8) providing a heat peripheral nerve injury. The findings also point to a com- source was used in the present study. In brief, each rat was plexity of hyperexcitability mechanisms contributing to placed in a box (22 × 12 × 12 cm) containing a tempera- CCD and CCI hyperexcitability in small DRG neurons, ture-controlled smooth glass floor associated with the Analgesia Meter. The heat source was focused on a portion Methods of the hindpaw, which was flush against the glass, and Animals and surgical procedures delivered until the hindpaw moved or up to 20 sec to pre- Experiments were performed on adult, male Sprague- vent tissue damage. The range of latency of foot with- Dawley rats (n = 32, 200–250 g). The rats were housed in drawal in naïve, control rats was 9–15 sec. Thermal groups of 3–4 in plastic cages (40 × 60 × 30 cm) with soft stimuli were delivered 4 times to each hind paw at 5–6 bedding and free access to food and water under a 12-h min intervals. The rats were tested on each of 2 successive day/12-h night cycle. Under these conditions, they were days prior to surgery (the first test was at 2 days and the kept 3–5 days, before and up to 14 days after surgery and/ second at 2 hours prior to surgery). Postoperative tests or treatment. The animals were divided into groups as were conducted on the day of electrophysiological record- described below (CCD, CCI and Control). All surgeries ing (days 10–14). Thermal hyperalgesia for a given rat was were done under anesthesia induced by intraperitoneal defined as a postoperative decrease of foot withdrawal injection (i.p.) of sodium pentobarbital (40 mg/kg). After latency from the mean preoperative value, with a differ- surgery, the muscle and skin layers were sutured. These ence score ≥ 3 s [14]. Only rats that exhibited thermal procedures were conducted in agreement with the regula- hyperalgesia after CCD or CCI treatment were used for the tions of the ethics committee of the International Associ- electrophysiological studies. ation for the Study of Pain, the National Institute of Dissociation of DRG neurons Health guide for the care and use of Laboratory animals and approved by Parker Research Institute Animal Care DRG neurons were dissociated from L and/or L ganglia 4 5 and Use Committee. taken from 8 CCD, 8 CCI and 8 Control rats. The protocol was the same as that we have described recently [57]. In DRG compression was produced by surgically implanting brief, the excised ganglion was minced using microdissec- stainless steel rods unilaterally into the intervertebral tion scissors, the DRG fragments transferred into 10 ml of foramen at L and L using the procedure for CCD we pre- the buffered solution containing collagenase (type IA, 1 4 5 viously described [12,13]. In brief, the rats (n = 12) were mg/ml, Sigma) and trypsin (0.5 mg/ml, Sigma), and then anesthetized; paraspinal muscles were separated from the incubated for 30 min at 35°C. The DRG fragments were mammillary and transverse processes, and the interverte- removed, rinsed 2–3 times in the buffered solution, and bral foramina of L and L were exposed. One stainless put into the buffered solution (5 ml) containing DNase 4 5 steel L-shaped rod, 4 × 2 mm in length and 0.6 mm in (0.2 mg/ml, Sigma) to prevent possible toxicity from DNA diameter, was implanted into the foramen at L and leaking from ruptured cells. Individual neurons were dis- Page 12 of 15 (page number not for citation purposes) Molecular Pain 2008, 4:20 http://www.molecularpain.com/content/4/1/20 sociated by passing DRG fragments through a set of fire- data for each cell were divided by the respective driving polished glass pipettes with decreasing diameter. force (V - V ), plotted against V , and fit to a Boltzmann m rev m distribution equation of the following form: Voltage-clamp electrophysiology Voltage-clamp recordings were performed in the dissoci- G = G /(1 + exp((V - V )/k)), max 1/2 m ated small DRG neurons with the standard whole-cell patch-clamp configuration. All recordings were conducted Where G is the maximum G, V is the potential at max 1/2 at room temperature (20~22°C) and during 2~8 hrs after which activation is half-maximal, and k is the slope of the dissociation. Fire-polished electrodes were fabricated curve. from 1.5 mm out diameter borosilicate capillary glass (Sutter Instruments, Novato, CA) by using a Sutter P-97 For the analysis of steady-state inactivation kinetics, the puller (Sutter Instruments, Novato, CA), and had a resist- inactivation parameter was fitted to a Boltzmann distribu- ance of 1 – 3 MΩ. The pipette solution contained (in tion equation: mM): 110 CsF, 11 EGTA, 10 NaCl, MgCl 5, and 10 = 1/(1 + exp ((V - V )/k)), HEPES, pH 7.3 with CsOH. Isolated sodium current was I/I max 1/2 pre recorded from the single neuron in the presence of a bath solution that contained (in mM): 65 NaCl, 2.5 KCl, 5 Where I is the maximum sodium current elicited after max MgCl , 0.01 CaCl , 50 Choline-Cl, 20 TEA-Cl, 5 glucose, the most hyperpolarized prepulse, the V is the prepulse 2 2 pre 5 Na-HEPES, and 5 HEPES, pH 7.4 with NaOH. Bath solu- potential, V is the potential at which inactivation is half- 1/2 tion was applied to the recording chamber and removed maximal, and k is the slope factor. via a Peri-Star Pro peristaltic pump (World Precision Instruments, Sarasota, FL). Statistical tests The student t-test was used to examine the differences in Voltage-clamped currents were recorded with an Axo- mean latency of thermal paw withdrawal between preop- patch-200B amplifier (Molecular Devices, Union city, erative (mean value of the two preoperative tests) and CA). Data were acquired on a PC computer with the postoperative on the day of electrophysiological record- Clampex v10.0 software (Molecular Devices), filtered ings. The specific hypotheses about differences between with a low-pass Bessel filter setting of 5 kHz and digitized each treated (CCD or CCI) and the control group for each at a sampling rate of 40 kHz via a Digidata 1440A analog- electrophysiological parameter was examined. Compari- to-digital converter (Molecular Devices). The membrane sons among CCD, CCI and control groups were per- capacitance (C ) was read from the amplifier by software formed with one-way ANOVA followed by Newman- Clampex v10.0 for determining the size of cells and calcu- Keuls tests. X tests were used to identify differences in the lating the current density. Voltage errors were minimized incidence of effects. All data are presented as mean ± SE. by using 80–90% series resistance compensation and the Statistical results are considered significant if p < 0.05. capacitance artifact was canceled by the patch-clamp amplifier. Linear leakage currents were digitally subtracted Abbreviations on-line using hyperpolarizing potential after the test pulse CCD: Chronic compression of dorsal root ganglion; CCI: (P/6 procedure). Data acquisition began 5 min after estab- Chronic constriction injury of the sciatic nerve; DRG: Dor- lishing whole-cell configuration and the holding poten- sal root ganglion; TTX-R: Tetrodotoxin-resistant; TTX-S: tial was at -80 mV. Tetrodotoxin-sensitive; VGSCs: Voltage-gated sodium channels Somata of the small DRG neurons were classified by their diameters (15 ~30 μm) and C (≤ 45 pF). Neurons were Competing interests not considered for analysis if they had high leakage cur- The authors declare that they have no competing interests. rents (holding current >1.0 nA at -80 mV), membrane blebs, total sodium current < 500 pA, or an access resist- Authors' contributions ance > 5 MΩ. Access resistance was monitored throughout XJS and ZJH planned the studies. ZJH conducted the the experiment and data were not used if resistance experiments, analyzed the data and contributed to the changes of >20% occurred. Data were not corrected to writing of the paper. XJS participated in the studies and account for liquid junction potential. The offset potential data analysis and wrote the paper. Both authors approved was zeroed before patching the cells and checked after the final manuscript. each recording for drift. Acknowledgements This study was supported by grants from Parker Research Foundation To analyze the voltage dependence of channel activation, (PCCRF-BSR0501 and PCCRF-BSR0602) and National Natural Science the sodium conductance (G) was calculated. Peak current Foundation of China (NSFC-30628027). Page 13 of 15 (page number not for citation purposes) Molecular Pain 2008, 4:20 http://www.molecularpain.com/content/4/1/20 20. Black JA, Cummins TR, Plumpton C, Chen YH, Hormuzdiar W, Clare References JJ, Waxman SG: Upregulation of a silent sodium channel after 1. Cummins TR, Sheets PL, Waxman SG: The roles of sodium chan- peripheral, but not central, nerve injury in DRG neurons. J nels in nociception: Implications for mechanisms of pain. 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Ungless MA, Gasull X, Walters ET: Long-term alteration of S- type potassium current and passive membrane properties in Publish with Bio Med Central and every aplysia sensory neurons following axotomy. J Neurophysiol scientist can read your work free of charge 2002, 87:2408-2420. 55. Yao H, Donnelly DF, Ma C, LaMotte RH: Upregulation of the "BioMed Central will be the most significant development for hyperpolarization-activated cation current after chronic disseminating the results of biomedical researc h in our lifetime." compression of the dorsal root ganglion. J Neurosci 2003, Sir Paul Nurse, Cancer Research UK 23:2069-2074. 56. Bennett GJ, Xie YK: A peripheral mononeuropathy in rat that Your research papers will be: produces disorders of pain sensation like those seen in man. available free of charge to the entire biomedical community Pain 1988, 33:87-107. 57. Zheng JH, Walters ET, Song XJ: Dissociation of dorsal root gan- peer reviewed and published immediately upon acceptance glion neurons induces hyperexcitability that is maintained by cited in PubMed and archived on PubMed Central increased responsiveness to cAMP and cGMP. J Neurophysiol 2007, 97:15-25. yours — you keep the copyright BioMedcentral Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp Page 15 of 15 (page number not for citation purposes)

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Molecular Pain – Springer Journals

Published: May 30, 2008

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