alpha -SNS Produces the Slow TTX-Resistant Sodium Current in Large Cutaneous Afferent DRG Neurons

M. Renganathan, T. R. Cummins, W. N. Hormuzdiar, and S. G. Waxman

Department of Neurology and Paralyzed Veterans Association/Eastern Paralyzed Veterans Association Neuroscience Research Center, Yale Medical School, New Haven 06510; and Rehabilitation Research Center, Veterans Affairs Connecticut Healthcare Center, West Haven, Connecticut 06516


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Renganathan, M., T. R. Cummins, W. N. Hormuzdiar, and S. G. Waxman. alpha -SNS Produces the Slow TTX-Resistant Sodium Current in Large Cutaneous Afferent DRG Neurons. J. Neurophysiol. 84: 710-718, 2000. In this study, we used sensory neuron specific (SNS) sodium channel gene knockout (-/-) mice to ask whether SNS sodium channel produces the slow Na+ current ("slow") in large (>40 µm diam) cutaneous afferent dorsal root ganglion (DRG) neurons. SNS wild-type (+/+) mice were used as controls. Retrograde Fluoro-Gold labeling permitted the definitive identification of cutaneous afferent neurons. Prepulse inactivation was used to separate the fast and slow Na+ currents. Fifty-two percent of the large cutaneous afferent neurons isolated from SNS (+/+) mice expressed only fast-inactivating Na+ currents ("fast"), and 48% expressed both fast and slow Na+ currents. The fast and slow current densities were 0.90 ± 0.12 and 0.39 ± 0.16 nA/pF, respectively. Fast Na+ currents were blocked completely by 300 nM tetrodotoxin (TTX), while slow Na+ currents were resistant to 300 nM TTX, confirming that the slow Na+ currents observed in large cutaneous DRG neurons are TTX-resistant (TTX-R). Slow Na+ currents could not be detected in large cutaneous afferent neurons from SNS (-/-) mice; these cells expressed only fast Na+ current, and it was blocked by 300 nM TTX. The fast Na+ current density in SNS (-/-) neurons was 1.47 ± 0.14 nA/pF, approximately 60% higher than the current density observed in SNS (+/+) mice (P < 0.02). A low-voltage-activated TTX-R Na+ current ("persistent") observed in small C-type neurons is not present in large cutaneous afferent neurons from either SNS (+/+) or SNS (-/-) mice. These results show that the slow TTX-R Na+ current in large cutaneous afferent DRG is produced by the SNS sodium channel.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Different repertoires of Na+ currents appear to endow different functional classes of dorsal root ganglion (DRG) neurons with distinct encoding and/or transducing properties. Thus it is important to establish the molecular identities of the channels responsible for each of the Na+ currents in various classes of DRG neuron. It is now well established that, in addition to fast-activating and -inactivating ("fast") tetrodotoxin (TTX)-sensitive (TTX-S) Na+ current, DRG neurons express slow ("slow") TTX-resistant (TTX-R) Na+ currents (Caffrey et al. 1992; Elliott and Elliott 1993; Kostyuk et al. 1981; Roy and Narahashi 1992; Scholz et al. 1998). More recently, a novel persistent TTX-R Na+ current has been demonstrated in small C-type DRG neurons (Cummins et al. 1999; Dib-Hajj et al. 1999a). To date, 11 distinct sodium channel alpha -subunits, encoded by different genes, have been cloned (Plummer and Meisler 1999). Five of the sodium channel alpha -subunits are expressed in DRG neurons (Black et al. 1996). These include two distinct TTX-R sodium channel alpha -subunits that are preferentially expressed in DRG neurons, the first termed sensory neuron specific (SNS) (Akopian et al. 1996) or PN3 (Sangameswaran et al. 1996) and the second termed NaN (Dib-Hajj et al. 1998) or SNS2 (Tate et al. 1998). SNS encodes a slow TTX-R Na+ current when expressed in oocytes (Akopian et al. 1996; Sangameswaran et al. 1996). Development of a transgenic mouse model, in which the SNS gene has been knocked out, has made it possible to establish definitively that the SNS channel produces the slow TTX-R Na+ current in one of the normal host cells in which it is expressed, i.e., in small C-type DRG neurons (Akopian et al. 1999). Cummins et al. (1999) have demonstrated the presence of the persistent TTX-R Na+ current, presumably encoded by NaN channels, in C-type DRG neurons in SNS knockout mice.

To date, studies on the physiological role of specific TTX-R sodium channel isoforms (SNS and NaN) have focused on small C-type DRG neurons. However, it is well established that a slow TTX-R Na+ current is also present in some subsets of large DRG neurons. In particular, a slow TTX-R Na+ current is seen in large cutaneous afferent DRG neurons, although it is not present in muscle afferent neurons (Honmou et al. 1994). While a one-to-one correspondence with a specific channel isoform has been accomplished for the slow and persistent TTX-R Na+ currents in small C-type DRG neurons (Akopian et al. 1999; Cummins et al. 1999), the molecular identity of the channel producing the slow TTX-R Na+ current in large cutaneous afferent neurons has not yet been established.

In this study, we used SNS knockout (-/-) mice to determine the identity of the channel that is responsible for the slow TTX-R Na+ current in large cutaneous afferent neurons. Retrograde labeling permitted us to definitively identify cutaneous afferent neurons. Our results indicate that the slow TTX-R Na+ current in large cutaneous neurons is produced by alpha -SNS channels.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animal care

A breeding pair of SNS (+/-) mice (Akopian et al. 1999) was generously provided by Prof. John Wood and a colony of SNS (+/+) and SNS (-/-) animals were raised from this pair. Three month-old SNS (+/+) and SNS (-/-) mice (25-g weight) from the colony were used in this study. Animals were fed ad libitum and housed in a pathogen-free area at the Veterans Affairs Medical Center, West Haven. Animal care and surgical procedures followed a protocol approved by the Animal Care and Use Committee of Yale University.

Fluoro-Gold labeling of cutaneous neurons

DRG cell bodies giving rise to cutaneous afferent fibers were identified by retrograde labeling with Fluoro-Gold. Five to 10 µl of 4% solution of Fluoro-Gold dissolved in distilled water was injected sub-cutaneously in the lateral plantar region of both legs 1 wk before sacrifice for culture preparation (Honmou et al. 1994).

Culture of DRG neurons

The mice were exsanguinated under Ketamine/Xylazine anesthesia (38/5 mg/kg ip). Briefly, L4 and L5 lumbar DRG were freed from their connective sheaths in sterile calcium-free saline solution. The DRGs were then enzymatically digested for 15 min with collagenase A (1 mg/ml; Boehringer-Mannheim, Indianapolis, IN) in complete saline solution (CSS) containing 0.5 mM EDTA and 2 mg cysteine, and for 15 min with collagenase D (1 mg/ml; Boehringer-Mannheim, Indianapolis, IN) and papain (30 units/ml, Worthington Biochemical, Lakewood, NJ) in CSS containing 0.5 mM EDTA and 2 mg cysteine at 37°C. DRGs were carefully removed by pasteur pipette from the other cellular debri and placed in DRG culture medium (DMEM and F12 in a ratio of 1:1, 10% fetal calf serum, 100 U/ml penicillin and 0.1 mg/ml streptomycin) containing 1 mg/ml bovine serum albumin (Fraction V, Sigma Chemicals, St. Louis, MO) and 1 mg/ml trypsin inhibitor (Sigma Chemicals, St. Louis, MO). The DRGs were mechanically dispersed (5 strokes up and down) with a 1-ml pipette and plated on polyornithine and laminin-coated glass coverslips (100 µl suspension per coverslip). Neurons were placed in a 5% CO2-95% O2 incubator at 37°C and, 1 h after isolation, were fed with fresh culture medium. Na+ current properties in DRG neurons were investigated between 1 and 8 h after isolation. Short-term culture provided cells with truncated (<10 µm) axonal processes that can be voltage clamped readily and reliably, allowed the cells sufficient time to adhere to the glass coverslips, and were short enough to minimize changes in electrical properties that can occur in long-term cultures.

Electrophysiological recordings

Coverslips were mounted in a small flow-through chamber positioned on the stage of a Nikon Diaphot microscope (Nikon, Melville, NY) and were continuously perfused with the bath-external solution (see next paragraph) with a push-pull syringe pump (WPI, Saratoga, FL). Fluoro-Gold-labeled neurons were identified by fluorescence emission above 420 nm. A short arc mercury lamp was used as the excitation source, and it was defined by broadband (330-380 nm) pass filter (Nikon). Large (40-45 µm diam) Fluoro-Gold-labeled neurons were studied.

Cells were voltage clamped via the whole cell configuration of the patch-clamp with an Axopatch-200B amplifier (Axon Instruments, Foster City, CA) using standard techniques (Hamill et al. 1981). Micropipettes were pulled from borosilicate glasses (Boralex) with a Flaming Brown micropipette puller (P80, Sutter Instrument, Novato, CA) and polished by placing them close to a glass bead on a microforge (Narishige, Tokyo) to obtain electrode resistance ranging from 0.4 to 0.8 MOmega . To reduce the pipette capacitance, micropipettes were coated with a mixture prepared by adding approximately three parts of finely shredded parafilm to one part each of light and heavy mineral oil (Sigma, St. Louis, MO) and vigorously stirring over heat for 30-60 min. The pipette solution contained (in mM) 140 CsF, 1 EGTA, 10 NaCl, and 10 HEPES, pH 7.3, and was adjusted to 310 mosmol/l with glucose. The following bath solution was used (in mM): 140 NaCl, 3 KCl, 1 MgCl2, 1 CaCl2, 0.1 CdCl2, and 20 HEPES, pH 7.3; it was adjusted to 320 mosmol/l with glucose. CdCl2 was used to block Ca2+ currents. To eliminate the small residual sustained outward current that was seen at very depolarizing pulses, in some experiments we included 10 and 20 mM TEA in bath and pipette solutions, respectively, and CsF concentration was decreased to 120 mM in the pipette solution. Na+ current density did not appear to be different in these experiments. The pipette potential was zeroed before seal formation, and the voltages were not corrected for liquid junction potential. Capacity transients were canceled, and series resistance was compensated (>80%) as necessary. The leakage current was digitally subtracted on-line using hyperpolarizing control pulses, applied before the test pulse, of one-sixth test pulse amplitude (-P/6 procedure). Access resistance was monitored throughout the recording, and the cells were discarded if the resistance was >= 3 MOmega . Whole cell currents were filtered at 5 kHz and acquired at 50 kHz by a computer using Clampex 8.01 software (Axon Instruments). Digidata 1200B interface (Axon Instruments) was used for A-D conversion, and the data were stored on compact disk for analysis. For current density measurements, membrane currents were normalized to membrane capacitance. Membrane capacitance was calculated as the integral of the transient current in response to a brief hyperpolarizing pulse from -100 mV (holding potential) to -110 mV. The average of the membrane capacitance of the DRG neurons used for electrophysiological experiments was 54.56 ± 2.54 pF (mean ± SE, n = 21 from 5 different preparations) for SNS (+/+) mice and 58.92 ± 2.15 pF (n = 45; P > 0.05) for SNS (-/-) mice.

Calculation of fast and slow Na+ current density using prepulse inactivation

Prepulse inactivation takes advantage of the differences in the inactivation properties of the fast and slow Na+ currents (Cummins and Waxman 1997; McLean et al. 1988; Roy and Narahashi 1992). Na+ currents were elicited by 20-ms test pulses to -10 mV after 500 ms prepulses to potentials over the range of -130 to -10 mV. Fast Na+ currents were obtained by subtracting the current obtained at approximately -50 mV prepulse, which elicits only slow Na+ current, from the current obtained with more hyperpolarizing prepulses that elicit both fast and slow Na+ currents. Densities for fast TTX-S Na+ currents were calculated from cells that express fast Na+ currents and from cells expressing both fast and slow Na+ currents.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In addition to TTX-S fast Na+ currents, two distinct TTX-R Na+ currents in DRG neurons can be distinguished on the basis of their inactivation properties: slow TTX-R and persistent TTX-R Na+ currents (Cummins and Waxman 1997; Cummins et al. 1999). In this study we asked the following: 1) whether the slow Na+ currents are produced by SNS channels in large cutaneous DRG neurons and 2) whether a persistent Na+ current is present in these cells.

Identification of large cutaneous afferent DRG neurons by retrograde Fluoro-Gold labeling

DRG neurons isolated from the L4 and L5 ganglia of SNS (+/+) and SNS (-/-) mice consisted of small (<25 µm apparent diameter), medium (25-40 µm) and large DRG (>40 µm) cells. Of the overall population of DRG neurons, approximately 50% were small in size, 40% were medium in size, and 10% were large in size. The size and shape of these three types of neurons were similar in SNS (+/+) and SNS (-/-) mice. Figure 1A shows the optical image of large (>40 µm) and medium (<40 µm) size DRG neurons isolated from a SNS (-/-) mouse DRG. Figure 1B shows the retrograde labeling of one of these neurons following subcutaneous injection of Fluoro-Gold, indicating that it is a cutaneous afferent neuron. Only fluorescent neurons, which we could identify definitively as cutaneous afferents, were used in this study.



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Fig. 1. Bright-field and fluorescent images of dorsal root ganglion (DRG) neurons. A: bright-field image of large DRG neurons isolated from L4 and L5 ganglia of sensory neuron specific (SNS) (-/-) mouse is shown. B: fluorescence photomicrograph of the same neurons shows that the large DRG neuron is retrogradely labeled by subcutaneous injection of Fluoro-Gold and therefore can be identified as a cutaneous afferent neuron. Calibration bar is 100 µm.

Both fast and slow Na+ currents are present in SNS (+/+) large cutaneous afferent neurons

Sodium currents were elicited from large cutaneous afferent (40-45 µm diam) neurons with the whole cell patch-clamp technique by test pulses from -100 to +40 mV. Two overall patterns of Na+ current expression, one group of cells expressing only fast Na+ currents (Fig. 2A; 11 of 21 neurons tested) and the other group expressing fast and slow Na+ currents (Fig. 2B; 10 of 21 neurons tested), were seen in these cells. The Na+ current traces illustrated in Fig. 2A reach peak amplitude within 1 ms and inactivate back to the baseline within ~5 ms, and therefore these are fast Na+ currents. The Na+ current traces from another neuron, illustrated in Fig. 2B, also reach the peak amplitude within 1 ms but take more than 20 ms to inactivate back to the baseline, indicating the presence of slow Na+ currents in addition to fast Na+ currents. These results suggest that large cutaneous afferent neurons isolated from SNS (+/+) mouse can express either fast Na+ currents (Fig. 2A) or co-express fast and slow Na+ currents (Fig. 2B). The percentage of cells expressing only fast Na+ current is ~52%; and co-expressing fast and slow Na+ currents are ~48% (see below for the distribution of fast and slow Na+ currents). Large cutaneous afferent neurons expressing only slow Na+ currents were not observed in this study (n = 21; see Table 1).



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Fig. 2. Na+ currents in large cutaneous afferent neuron isolated from SNS (+/+) mouse. Families of Na+ current traces elicited in response to 80-ms depolarizing voltage steps ranging from -100 to +40 mV in 5-mV increments are shown. The holding membrane potential was -120 mV. Two overall patterns of Na+ current expression were seen in large cutaneous afferent neurons. A: the expression of only fast Na+ current in a large cutaneous afferent DRG neuron. B: the co-expression of fast and slow Na+ current in another large cutaneous afferent DRG neuron. The experimental conditions are given in detail in METHODS.


                              
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Table 1. Distribution of fast and slow Na+ currents in large (40-45 µm) cutaneous DRG neurons

The voltage dependence of steady-state inactivation of fast and slow Na+ currents are markedly different, and this has been used to separate fast and slow Na+ currents in DRG neurons (Cummins and Waxman 1997; McLean et al. 1988; Roy and Narahashi 1992). Recordings showing steady-state inactivation of Na+ currents expressing only fast Na+ current (11 neurons from 5 different preparations) or co-expressing fast and slow Na+ currents (10 neurons from 5 different preparations) are displayed in Fig. 3, A and D, respectively. Steady-state inactivation of Na+ currents was obtained using 40-ms test pulses to -10 mV after 500-ms prepulses to potentials from -130 to -10 mV. All of the current traces illustrated in Fig. 3A reach peak amplitude in <1 ms and inactivate completely to the baseline within a few milliseconds. The activation and inactivation of the fast Na+ currents of large cutaneous afferent neurons are similar to the fast Na+ currents present in small C-type DRG neurons (Cummins and Waxman 1997; Renganathan et al. 2000). Na+ current traces illustrated in Fig. 3D reach peak amplitude in <1 ms, but inactivation is incomplete and biphasic, indicating the presence of both fast and slow Na+ currents in this neuron.



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Fig. 3. Prepulse subtraction of fast and slow Na+ currents. A: records obtained using a steady-state inactivation protocol in a large cutaneous afferent neuron isolated form SNS (+/+) mouse expressing only fast Na+ currents are shown in A. Na+ currents were elicited by 40-ms test pulses to -10 mV, after 500-ms prepulses to potentials over the range of -130 to -10 mV, and these currents are termed as total Na+ current. B: prepulse subtraction shows the presence of only fast Na+ current. Fast Na+ currents were obtained by subtracting the response with -50-mV prepulse, which elicits only slow Na+ current, from the response with more hyperpolarizing prepulses that elicit both fast and slow Na+ currents. C: Na+ current traces from prepulse potentials of -50 and -45 mV demonstrates the absence of slow Na+ current in this neuron. D: records obtained using steady-state inactivation protocol in a large cutaneous afferent neuron isolated from SNS (+/+) mouse, co-expressing fast and slow Na+ currents, and these currents are termed as total Na+ current. E: the fast Na+ currents were obtained by subtracting the current obtained at -50-mV prepulse (slow Na+ current) from the current obtained with more hyperpolarizing prepulses (fast and slow Na+ currents). F: the slow Na+ currents obtained at prepulse potentials between -50 and -10 mV are shown.

Prepulse inactivation takes advantage of the differences in the inactivation properties of the fast and slow Na+ currents (Cummins and Waxman 1997; McLean et al. 1988; Roy and Narahashi 1992), i.e., fast Na+ currents inactivate completely at approximately -50 mV, whereas the slow Na+ currents do not inactivate at approximately -50 mV (see Cummins and Waxman 1997). In these studies, fast Na+ currents were obtained by subtracting the response with the prepulse that elicits only slow Na+ currents, from the response with more hyperpolarizing prepulses that elicit both fast and slow Na+ currents. The process of separating fast and slow Na+ currents by prepulse inactivation and subtraction will be hereafter termed as prepulse subtraction. Prepulse subtraction was used to separate the fast and slow Na+ currents in large cutaneous afferent DRG neurons: neurons that expressed only fast Na+ current in unsubtracted recordings showed fast current (Fig. 3B) and no slow Na+ current following prepulse subtraction (Fig. 3C; 11 neurons from 5 different preparations). In contrast, neurons that in unsubtracted records co-expressed both fast and slow Na+ current appeared to yield fast (Fig. 3E) and slow Na+ currents (Fig. 3F) after prepulse subtraction (10 neurons from 5 different preparations).

Consistent with earlier studies in C-type DRG neurons (Cummins and Waxman 1997; Elliott and Elliott 1993; Roy and Narahashi 1992), the fast Na+ currents in large cutaneous afferent neurons were sensitive to nanomolar concentrations of TTX, whereas the slow Na+ currents were resistant to 300 nM TTX (Fig. 4, n = 4). Prepulse inactivation and TTX subtraction give essentially the same results when used to separate fast and slow Na+ currents (Fig. 3F and Fig. 4), as reported previously in C-type DRG neurons (Cummins and Waxman 1997).



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Fig. 4. Slow Na+ currents of large cutaneous afferent neurons isolated from SNS (+/+) mouse are TTX-resistant. Families of Na+ current traces elicited in response to 80-ms depolarizing voltage steps ranging from -100 to +40 mV in 5-mV increments in the presence of 300 nM TTX are shown. The holding membrane potential was -120 mV.

Distribution of fast and slow Na+ currents in SNS (+/+) large cutaneous afferent neurons

In whole cell recordings of 1-8 h cultured cutaneous DRG neurons from SNS (+/+) mice, varying amounts of fast and slow Na+ currents were observed (see below in current densities). One hundred percent of large cutaneous neurons expressed fast Na+ currents, with 48% of the neurons co-expressing fast and slow Na+ currents. We did not observe any large cutaneous afferent neurons expressing only slow Na+ current (21 neurons from 5 different preparations).

Only fast Na+ currents are present in SNS (-/-) large cutaneous afferent neurons

Figure 5 illustrates a family of Na+ current traces elicited by depolarizing voltage test pulses from -100 to +40 mV from a typical large cutaneous afferent neuron isolated from SNS (-/-) mouse. The Na+ current traces inactivate back to the baseline completely within ~10 ms (see DISCUSSION for an explanation of the slower inactivation kinetics), suggesting that only fast Na+ current is present. Na+ current steady-state inactivation from a typical large cutaneous afferent neuron isolated from SNS (-/-) mouse indicates the presence of only fast Na+ current (Fig. 6A). When prepulse inactivation was used to separate the fast and slow Na+ currents, only fast Na+ current was observed (Fig. 6B), and no slow Na+ current was seen (Fig. 6C, 45 neurons from 5 different preparations).



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Fig. 5. Large cutaneous afferent DRG neurons isolated from SNS (-/-) mouse express only fast Na+ currents. Na+ current traces elicited in response to 80-ms depolarizing voltage steps ranging from -100 to +40 mV in 5-mV increments from a large cutaneous afferent neuron from SNS (-/-) mouse are shown. The holding membrane potential was -120 mV. The experimental conditions were similar to those described in Fig. 2.



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Fig. 6. Prepulse subtraction yields only fast Na+ currents in large cutaneous afferent neurons from SNS (-/-) mice. A: records obtained using a steady-state inactivation protocol in a large cutaneous afferent neuron isolated from SNS (-/-) mouse is shown. Na+ currents were elicited by 40-ms test pulses to -10 mV, after 500-ms prepulses to potentials over the range of -130 to -10 mV, and these currents are termed as total Na+ current. Note the absence of slow Na+ current at all prepulse potentials. B: as seen in large cutaneous afferent neurons from SNS (+/+) mice, fast Na+ currents inactivate completely at -55 mV; however, no slow Na+ currents were seen at this voltage. Subtraction of Na+ currents obtained from potentials ranging from -130 to -60 mV by -55 mV yields Na+ currents with similar amplitude to those shown in A, indicating that only fast Na+ currents are present. C: test pulses to -10 mV after 500-ms prepulse potentials ranging from -55 to -10 mV failed to elicit slow Na+ currents.

The mid-point potential and the slope of the steady-state activation curves for fast Na+ currents were -25.7 ± 4.5 mV and 6.8 ± 1.6 mV/e-fold in SNS (+/+) large cutaneous sensory neurons (n = 11), and -27.6 ± 3.8 mV and 5.9 ± 1.2 mV/e-fold, in SNS (-/-) large cutaneous sensory neurons (n = 20), respectively. The mid-point potential and the slope of the steady-state inactivation curves for fast Na+ currents were -79.62 ± 3.44 mV and -8.70 ± 2.39 mV/e-fold in SNS (+/+) large cutaneous sensory neurons (n = 11), and -72.84 ± 2.23 and -9.25 ± 1.20 mV/e-fold in SNS (-/-) large cutaneous sensory neurons (n = 45), respectively. These values are not significantly different between the large cutaneous sensory neurons of SNS (+/+) and SNS (-/-) mice (P > 0.05).

The fast Na+ currents in large cutaneous DRG neurons were sensitive to nanomolar concentrations of TTX, and no slow Na+ currents were seen in the presence of 300 nM TTX (Fig. 7, n = 10). A total of 45 large cutaneous afferent neurons isolated from 5 different SNS (-/-) mice were investigated for Na+ currents, and in all of them only fast TTX-S Na+ currents were seen. Slow Na+ currents could not be detected either using prepulse inactivation (n = 45) or in the presence of 300 nM TTX (n = 10), in large cutaneous afferent neurons isolated from SNS (-/-) mice. These results demonstrate that SNS produces the slow TTX-R Na+ currents in large cutaneous neurons.



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Fig. 7. Slow Na+ currents are absent in large cutaneous afferent neurons isolated from SNS (-/-) mouse in the presence of 300 nM TTX. Na+ current traces were elicited in response to 80-ms depolarizing voltage steps ranging from -100 to +40 mV in 5-mV increments in the presence of 300 nM TTX. The holding membrane potential was -120 mV. The experimental conditions were similar to the conditions described in Fig. 4.

Fast and slow Na+ current densities are both altered in SNS (-/-) mice

In these experiments the fast and slow Na+ current amplitudes were measured in 140 mM NaCl in bath solution, a concentration similar to that found in vivo and used routinely to quantify Na+ current density in DRG neurons (Cummins and Waxman 1997; Renganathan et al. 2000). The series resistance problem usually associated with large amounts of Na+ current and large DRG neurons were minimized by using a low-resistance pipette (0.4-0.8 MOmega ) and using 80-90% series resistance compensation. In a small number of large cutaneous afferent DRG neurons isolated from SNS (-/-) mice, the Na+ current amplitude exceeded 200 nA, reaching the limits of the amplifier, and these neurons were not included for the quantitation of Na+ currents. Prepulse subtraction (Cummins and Waxman 1997; McLean et al. 1988; Roy and Narahashi 1992) was used to separate and quantitate fast and slow Na+ currents expressed in each large cutaneous afferent neuron. The fast and slow Na+ current amplitudes in large cutaneous afferent neurons isolated from SNS (+/+) mice were 49.15 ± 5.14 nA and 18.82 ± 1.12 nA, respectively. The fast Na+ current amplitudes in large cutaneous afferent neurons from SNS (-/-) mice were 93.62 ± 8.64 nA (P < 0.01); slow Na+ currents, as noted above, could not be detected in cells from SNS (-/-) mice. To compensate for differences in cell size, currents were normalized to cell capacitance and expressed as current densities. The capacitance of the large cutaneous neurons isolated from SNS (+/+) and SNS (-/-) mice were 54.56 ± 2.54 pF and 58.92 ± 2.15 pF, respectively. The fast and slow Na+ current densities in large cutaneous neurons isolated from SNS (+/+) mice were 0.90 ± 0.12 and 0.39 ± 0.16 nA/pF, respectively. The fast Na+ current density in large cutaneous afferent DRG neurons isolated from SNS (-/-) mice, was 1.47 ± 0.14 nA/pF, approximately 60% higher than the current density observed in SNS (+/+) mice (P < 0.02).

Persistent Na+ currents are absent in large cutaneous afferent DRG neurons

Two TTX-R Na+ channels have been cloned from DRG neurons, SNS (Akopian et al. 1996; Sangameswaran et al. 1996) and NaN (Dib-Hajj et al. 1998; Tate et al. 1998). alpha -SNS transcripts produce a slow TTX-R Na+ current (Akopian et al. 1996), while NaN transcripts appear to produce a persistent TTX-R Na+ currents in small C-type DRG neurons (Cummins et al. 1999). Persistent TTX-R Na+ currents activate at low depolarizing voltages, i.e., -80 mV, whereas activation of fast and slow Na+ currents occurs at approximately -40 mV. Furthermore, at test potentials between -80 and -50 mV, the persistent Na+ currents do not inactivate or very slowly inactivate (Cummins et al. 1999; Dib-Hajj et al. 1999a; Renganathan et al. 2000). The pulse protocols employed in the experiments shown in Figs. 2, 4, 5, and 7 would have been expected to elicit persistent TTX-R Na+ currents if there were any present in large cutaneous afferent DRG neurons because the same protocols elicited both the persistent and slow TTX-R Na+ currents in small C-type DRG neuron (Fig. 8A, n = 4). However, we were unable to elicit persistent TTX-R Na+ currents in large cutaneous afferent neurons, i.e., at test potentials between -80 to -50 mV. Figure 8, C and D, juxtaposes Na+ current recordings from small (15 µm) and large cutaneous afferent neurons. As seen in this figure low depolarizing test pulses, i.e., from -80 to -50 mV, elicited a typical persistent TTX-R Na+ current in small C-type DRG neurons (Fig. 8C), but not in large cutaneous afferent DRG neurons (Fig. 8D, n = 45).



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Fig. 8. Persistent TTX-resistant Na+ currents are absent in large cutaneous afferent DRG neurons. A: families of Na+ current traces from a representative C-type DRG neuron in the presence of 300 nM TTX. Na+ current traces were recorded in response to 80-ms test pulses from a holding potential of -120 mV to test potentials ranging from -100 to +40 mV. B: pulse protocols, which distinguish the persistent TTX-R Na+ current from slow TTX-R Na+ current. Na+ current traces obtained from C-type DRG neuron isolated from SNS (+/+) mice (C) are compared with the traces obtained from a large cutaneous DRG neuron isolated from SNS (+/+) mice (D), at respective test pulses. The traces depicted in C were taken from A, and the traces depicted in D were taken from Fig. 4. Note the presence of a persistent Na+ current in C-type DRG neurons and their absence in large cutaneous afferent DRG neurons.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Large cutaneous afferent DRG neurons exhibit different functional properties compared with muscle afferent neurons and express a slow TTX-R Na+ current that is not present in muscle afferent neurons (Honmou et al. 1994). In this study we demonstrate that the slow TTX-R Na+ current is present in SNS (+/+) large cutaneous afferent neurons but absent in transgenic SNS (-/-) large cutaneous afferent neurons identified by retrograde Fluoro-Gold labeling. These results support the hypothesis that alpha -SNS transcript encodes the slow TTX-R Na+ current in large cutaneous DRG neurons. This conclusion is strengthened by our findings that the slow Na+ currents that are seen in SNS (+/+) large cutaneous afferent neurons are, as predicted by the SNS sequence, resistant to 300 nM TTX. Previous studies have demonstrated that, although SNS mRNA and protein are most highly expressed in small C-type DRG neurons, they are also present in large DRG neurons (Black et al. 1996; Dib-Hajj et al. 1996; Novakovic et al. 1998; Sangameswaran et al. 1996).

The size criteria used in the study are based on the assumption that DRG neurons <25 µm diam tend to give rise to C-fiber afferent axons, whereas neurons >40 µm diam tend to have low-threshold afferent fibers. This assumption is supported by the effects of capsaicin on DRG neurons from in vitro and in vivo mouse studies (Hiura and Sakamoto 1987; Urban and Dray 1993). Mouse DRG neurons with C-fiber characteristics (conduction velocity <0.6 m/s; broad action potential with shoulder on the descending slope) were depolarized and generated action potentials when 100-700 nM capsaicin was added to the bath solution for 30 s. However, capsaicin at these concentrations did not affect the membrane potential of DRG neurons with myelinated fibers (conduction velocity >2.0 m/s), indicating that neurons <25 µm diam have properties of nociceptors and neurons >40 µm tend to have low-threshold afferent fibers (Urban and Dray 1993). Capsaicin treatment of neonatal mice decreased the number of small DRG neurons (8-25 µm) by 51-77%, whereas the number of large-sized neurons (25-40 µm) decreased by 14-52%, suggesting that capsaicin affects small and certain types of large neurons (Hiura and Sakamoto 1987). Since a part of the 25-40 µm diam neuron population was affected by capsaicin treatment, we focused on neurons >40 µm diam with Fluoro-Gold labeling to unequivocally study large cutaneous afferent neurons in this investigation.

The total (and fast) Na+ currents in SNS (-/-) large sensory neurons (Figs. 5 and 6) appear to take longer to return to baseline than do the total and fast Na+ currents in SNS (+/+) large sensory neurons (Figs. 2 and 3). Analysis of the fast Na+ current traces obtained from SNS (-/-) or SNS (+/+) large cutaneous sensory neurons revealed that ~50% of these large cutaneous sensory neurons had current traces that inactivated back to baseline within 5 ms, whereas the other 50% of the large cutaneous sensory neurons had current traces that inactivated back to baseline within 10 ms. The inactivation of the fast Na+ currents (test potential, 0 mV) in approximately one-half the SNS (+/+) large cutaneous sensory neurons was best fitted with a single exponential function with the time constant of 0.47 ± 0.20 ms. In the other half of the large cutaneous sensory neurons, a second exponential function was needed to best fit the inactivation phase of the fast Na+ currents. The values for the time constant for inactivation from the fit were th1 = 0.69 ± 0.10 ms, amplitude = 0.8 ± 0.2; th2 = 6.70 ± 0.86 ms, amplitude = 0.2 ± 0.1. A similar pattern was observed in large cutaneous sensory neurons isolated from SNS (-/-) mice. In 40% of the neurons, the inactivation of fast Na+ currents could be fit with a single exponential function (th = 0.53 ± 0.20 ms), and in the other 60% of the neurons the inactivation of fast Na+ currents was best fitted with two exponential functions. The time constants for inactivation thus obtained were th1 = 0.65 ± 0.04 ms, amplitude = 0.83 ± 0.12; th2 = 4.94 ± 0.34 ms, amplitude = 0.17 ± 0.11. The inactivation time constants of the fast Na+ currents are not significantly different within the large cutaneous sensory neurons of SNS (+/+) and SNS (-/-) mice or between them (P > 0.05). The inactivation time constants obtained with a single exponential function or the smaller time constants obtained with two exponential functions from large cutaneous sensory neurons of SNS (+/+) and SNS (-/-) mice are similar to the values reported earlier (Cummins and Waxman 1997). We do not have a clear explanation for the observation that fast Na+ currents in some of the large cutaneous sensory neurons of SNS (+/+) or SNS (-/-) could be fit with two exponential functions. It is possible that fast Na+ currents in these neurons could originate from two types of TTX-S fast Na+ channels. The possibility that the fast Na+ currents in SNS (-/-) neurons contain a different subtype of Na+ channel that is up-regulated or expressed de novo, compared with fast Na+ currents in SNS (+/+) neurons, is under investigation.

Recently, a novel persistent TTX-R Na+ current has been described in small DRG neurons (Cummins et al. 1999). We found that neither SNS (+/+) nor SNS (-/-) large cutaneous afferent DRG neurons express persistent TTX-R Na+ currents. Previous studies (Dib-Hajj et al. 1998, 1999a; Fjell et al. 1999; Tate et al. 1998) indicate that NaN mRNA has a more restricted expression than SNS and is selectively expressed in small DRG neurons, and immunocytochemical studies with NaN-specific antibodies (Fjell et al. 2000) demonstrate NaN protein in unmyelinated and small-diameter myelinated axons that arise from small DRG neurons. Our results support the conclusion (Cummins et al. 1999) that NaN channels produce a persistent TTX-R Na+ current.

Differentiating large DRG neurons into cutaneous and muscle afferents by subcutaneous injection of Fluoro-Gold, Honmou et al. (1994) demonstrated the presence of slow TTX-R Na+ currents in large cutaneous afferent neurons. Consistent with these observations in rat DRG neurons (Honmou et al. 1994), the present results show that SNS (+/+) large cutaneous DRG neurons express two types of Na+ currents, a fast TTX-S Na+ current and slow TTX-R Na+ current. In the earlier study, fast Na+ current was observed together with slow Na+ current in 40%, and slow Na+ current alone was seen in ~60% of the large cutaneous DRG neurons (Honmou et al. 1994). However, we observed fast Na+ current in all large cutaneous afferent DRG neurons from mice (21 of 21 neurons) and slow Na+ current together with fast Na+ current in 48% of the neurons (10 of 21 neurons). These apparently contradictory observations may be reconciled by differences between the two studies: 1) there may be species difference between rat and mouse DRG neurons, 2) in the earlier study the rat neurons were cultured for 1-2 days prior to patch clamping, whereas, in this study, the neurons were studied between 1 and 8 h after plating.

The deletion of SNS gene leads to the loss of slow TTX-R Na+ current in large cutaneous DRG neurons (45 neurons of 45 neurons from 5 different preparations). The absence of slow TTX-R Na+ current in SNS (-/-) mice suggests that other types of sodium channels are unlikely to produce slow TTX-R Na+ current in large cutaneous afferent DRG neurons. Associated with the loss of SNS, we observed an increased expression of fast TTX-S Na+ currents in SNS (-/-) large cutaneous DRG neurons. This apparent up-regulation is similar to the increase observed in an earlier study in SNS (-/-) DRG neurons that focused on small cells (Akopian et al. 1999), where the fast TTX-S Na+ current density increased twofold. Akopian et al. (1999), using semi-quantitative polymerase chain reaction, could not detect changes in the less abundant TTX-S alpha -subunit transcripts; however, northern blots of the abundant PN1 channel transcript showed an increase in transcript expression of more than 50%. Patch-clamp studies on heterologously expressed PN1 channels have shown that they produce a fast TTX-sensitive current (Cummins et al. 1998). The increase of about 60% in fast Na+ current density and the similar increase in PN1 channel transcript in SNS (-/-) mice suggest that the increase in fast Na+ current density may be due to up-regulation of PN1 channel expression at the transcriptional level. Interestingly, the increase in PN1 expression does not appear to be part of a global or nonspecific up-regulation of sodium channel expression. Within the normal nervous system, PN1 is expressed in small and large diameter DRG neurons (Black et al. 1996; Toledo-Aral et al. 1997), whereas the expression of NaN is confined to small DRG neurons (Dib-Hajj et al. 1998, 1999a; Fjell et al. 1999, 2000; Tate et al. 1998). The lack of increased levels of NaN-type (persistent) Na+ currents in SNS (-/-) large cutaneous afferents, even though NaN and SNS are encoded by genes that are located within a cluster on a single chromosome (mouse chromosome 3) (Dib-Hajj et al. 1999b), suggests that compensatory up-regulation of the Na+ channel subtypes may be confined to channel isoforms that are normally synthesized in these cells.

Slow depolarizations, first described in sensory axons by Kocsis et al. (1983), have been associated with slow TTX-resistant Na+ channels (Honmou et al. 1994) and have been implicated in the generation of repetitive spike activity in large cutaneous afferents. Activity of these slow channels appears to contribute to abnormal bursting in injured sensory axons that can contribute to paresthesias (Kocsis et al. 1986). Here we have identified SNS as the channel that produces the slow TTX-resistant Na+ currents in large cutaneous afferents. Together with earlier studies that have correlated other Na+ channel isoforms in DRG neurons with specific physiological signatures (Cummins et al. 1998, 1999) and with future studies on channel isoforms whose physiological action in DRG are not yet understood, these results should ultimately contribute to an understanding of the molecular basis for electrogenesis in normal and injured spinal sensory neurons.


    ACKNOWLEDGMENTS

A breeding pair of SNS (+/-) mice was kindly provided by Prof. J. N. Wood, University College London. We thank Drs. S. D. Dib-Hajj and J. A. Black for help in genotyping and raising a colony of SNS (+/+) and SNS (-/-) mice from SNS (+/-) mice. We thank B. Tuftness for help with the figures.

This work was supported in part by grants from the Medical Research Service and Rehabilitation Research Service, Department of Veterans Affairs, and the National Multiple Sclerosis Society. We also thank the Eastern Paralyzed Veterans Association and the Paralyzed Veterans of America for support.


    FOOTNOTES

Address for reprint requests: S. G. Waxman, Dept. of Neurology, LCI 707, Yale School of Medicine, 333 Cedar St., New Haven, CT 06510 (E-mail: stephen.waxman{at}yale.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 2 March 2000; accepted in final form 26 April 2000.


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ABSTRACT
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