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Current, TTX-Resistant Na؉ Current, and Ca2؉ Current in the Action Potentials of Nociceptive Sensory Neurons

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Current, TTX-Resistant Na؉ Current, and Ca2؉ Current in the Action Potentials of Nociceptive Sensory Neurons The Journal of Neuroscience, December 1, 2002, 22(23):10277–10290 Roles of Tetrodotoxin (TTX)-Sensitive Na؉ Current, TTX-Resistant Na؉ Current, and Ca2؉ Current in the Action Potentials of Nociceptive Sensory Neurons Nathaniel T. Blair and Bruce P. Bean Department of Neurobiology, Harvard Medical School, Boston, Massachusetts 20114 Nociceptive sensory neurons are unusual in expressing carry the largest inward charge during the upstroke of the voltage-gated inward currents carried by sodium channels re- nociceptor action potential (ϳ58%), with TTX-sensitive sodium sistant to block by tetrodotoxin (TTX) as well as currents carried channels also contributing significantly (ϳ40%), especially near by conventional TTX-sensitive sodium channels and voltage- threshold, and high voltage-activated calcium currents much dependent calcium channels. To examine how currents carried less (ϳ2%). Action potentials had a prominent shoulder during by each of these helps to shape the action potential in small- the falling phase, characteristic of nociceptive neurons. TTX- diameter dorsal root ganglion cell bodies, we voltage clamped resistant sodium channels did not inactivate completely during cells by using the action potential recorded from each cell as the action potential and carried the majority (58%) of inward the command voltage. Using intracellular solutions of physio- current flowing during the shoulder, with high voltage-activated logical ionic composition, we isolated individual components of calcium current also contributing significantly (39%). Unlike current flowing during the action potential with the use of calcium current, TTX-resistant sodium current is not accompa- channel blockers (TTX for TTX-sensitive sodium currents and a nied by opposing calcium-activated potassium current and mixture of calcium channel blockers for calcium currents) and may provide an effective mechanism by which the duration of ionic substitution (TTX-resistant current measured by the re- action potentials (and consequently calcium entry) can be placement of extracellular sodium by N-methyl-D-glucamine in regulated. the presence of TTX, with correction for altered driving force). Key words: action potential; excitability; dorsal root ganglion; TTX-resistant sodium channels activated quickly enough to nociceptor; sodium channel; tetrodotoxin Unmyelinated C-fibers originate from small primary sensory neu- different time course during the action potential. The action rons and transmit nociceptive information into the CNS. Noci- potentials of nociceptors associated with C-fibers have unusually ceptive neurons are unusual in expressing voltage-gated sodium wide action potentials with a characteristic hump or shoulder current resistant to tetrodotoxin (TTX; Kostyuk et al., 1981) (for on the falling phase (Gallego, 1983; Ritter and Mendell, 1992; review, see McCleskey and Gold, 1999; Waxman et al., 1999), and Djouhri et al., 1998; Lopez de Armentia et al., 2000). The genes encoding two TTX-resistant (TTX-R) sodium channels, shoulder generally has been attributed to calcium current (Dich- NaV1.8 and NaV1.9, are expressed exclusively in peripheral sen- ter and Fischbach, 1977; Ransom and Holz, 1977; Yoshida et al., sory neurons (Akopian et al., 1996; Dib-Hajj et al., 1998; Amaya 1978; Gallego, 1983; Renganathan et al., 2001). Consistent with et al., 2000). The expression of NaV1.8 has been implicated in this, the shoulder is still present in neurons from NaV1.8-null mediating inflammatory pain (Akopian et al., 1999; Porreca et al., mice (Renganathan et al., 2001). On the other hand, computer 1999). modeling has raised the possibility of substantial current from Nociceptive sensory neurons also express TTX-sensitive TTX-R channels during the shoulder (Schild and Kunze, 1997). (TTX-S) sodium channels (Kostyuk et al., 1981; Caffrey et al., Such modeling depends critically on extrapolated inactivation 1992; Roy and Narahashi, 1992; Elliott and Elliott, 1993) and kinetics at voltages near the peak of the action potential, where multiple types of voltage-dependent calcium channels (Scroggs kinetics of the current cannot be measured easily, so experimental and Fox, 1992a). It is unknown exactly how each of these three tests of the predictions of the model would be useful. depolarization-activated inward cation currents helps to shape Currents flowing during an action potential can be measured the action potential and contributes to the excitability of the directly by using the action potential clamp method (Llina´s et al., neurons. TTX-R sodium current has much slower activation and 1982; McCobb and Beam, 1991; Scroggs and Fox, 1992b) in which inactivation kinetics than the TTX-S sodium current (Kostyuk et an action potential waveform is used as the command voltage in al., 1981; Elliott and Elliott, 1993) and is likely to follow a voltage-clamp experiments. This offers a more direct approach than either computer modeling or experiments testing the effect of blockers in current clamp in which the blocking of one current Received June 29, 2002; revised Sept. 17, 2002; accepted Sept. 24, 2002. changes the voltage trajectory, producing indirect effects on all This work was supported by National Institutes of Health Grants HL35034, NS36855, and NS38312. N.T.B. was supported by the Stuart H. Q. and Victoria Quan other voltage-gated conductances. Ideally, the action potential Fellowship. clamp should be performed with action potentials recorded in the Correspondence should be addressed to Nathaniel Blair, Department of Neuro- biology, Harvard Medical School, 220 Longwood Avenue, Boston, MA 02115. same cell (de Haas and Vogel, 1989; Doerr et al., 1989; Taddese E-mail: [email protected]. and Bean, 2002) rather than generic action potentials, because Copyright © 2002 Society for Neuroscience 0270-6474/02/2210277-14$15.00/0 there is substantial cell-to-cell variability in current densities and 10278 J. Neurosci., December 1, 2002, 22(23):10277–10290 Blair and Bean • Inward Currents during Nociceptor Action Potentials action potential shapes. Using small dorsal root ganglion (DRG) for half-block (Roy and Narahashi, 1992; Elliott and Elliott, 1993; Ogata neurons with electrophysiological properties matching those ex- and Tatebayashi, 1993). There was often considerable sweep-to-sweep decline in potassium currents, especially when cells were stimulated at pected of nociceptors, we used the action potential clamp method high frequencies. Because of this, action potential commands were ap- with individual neurons to examine the contribution of TTX- plied at low rates (generally 0.1 Hz), and only two to three sweeps in each sensitive, TTX-resistant, and calcium currents at various times solution were collected and averaged. Even so, in some cases the TTX- during the action potential. sensitive currents defined by TTX subtraction were sometimes outward, almost certainly because the error resulting from nonstationary potas- MATERIALS AND METHODS sium currents was larger than any TTX-sensitive sodium current. When the value for TTX-sensitive charge transfer was calculated for the data Cell preparation. DRG neurons were prepared from Long–Evans rats, summarized in Figure 11, a positive (outward) value was considered as postnatal day 14–18. Animals were anesthetized with isoflurane and zero. decapitated, and ganglia were removed and chopped in half. Pieces of The TTX-R sodium current was isolated by using complete replace- ganglia were treated at 37°C for 20 min in 100 U/ml papain (Worthington 2ϩ 2ϩ ment of Na by N-methyl-D-glucamine (NMDG), both solutions contain- Biochemical, Lakewood, NJ) with 5 mM cysteine in Ca ,Mg -free ing 300 nM TTX to block TTX-S current, as well as a mixture of calcium Hank’s solution containing (in mM): 136.9 NaCl, 5.4 KCl, 0.34 Na HPO , 2 4 channel blockers (10 ␮M nimodipine, 1 ␮M ␻-conotoxin-GVIA, and 250 0.44 KH PO , 5.55 glucose, 5 HEPES, and 0.005% phenol red, pH 7.4. 2 4 ϩ ϩ nM ␻-agatoxin (Aga)-IVA) and also 5 mM TEA to reduce potassium After this, the ganglia were transferred to Ca 2 ,Mg2 -free Hank’s currents. Eliminating calcium currents was necessary when measuring solution containing 3 mg/ml collagenase (type I; Sigma-Aldrich, St. TTX-R current by NMDG replacement for Na, because calcium currents Louis, MO) and 4 mg/ml Dispase II (Boehringer Mannheim, Indianap- themselves are reduced slightly by the substitution of NMDG for Na olis, IN) and were incubated for 20 min at 37°C. Ganglia were placed in (Zhou and Jones, 1995); reducing potassium currents was necessary Leibovitz’s L-15 medium (Invitrogen, San Diego, CA) supplemented because potassium currents were found to be reduced by 10–15% when with 10% fetal calf serum, 5 mM HEPES, and 100 ng/ml NGF (Invitro- NMDG replaced Na. gen); individual cells were dispersed by trituration with a fire-polished The high voltage-activated (HVA) calcium current was obtained as the Pasteur pipette and plated on glass coverslips treated with 100 ␮g/ml current blocked by a mixture containing 10 ␮M nimodipine, 1 ␮M poly-D-lysine. Cells were incubated in the supplemented L-15 solution ␻ ␻ (in room air) at 33°C for 2–4 hr, after which they were stored at 4°C. -conotoxin-GVIA, and 250 nM -Aga-IVA. The calcium channel When used within 48 hr, these cells retained a healthy appearance and blocker mixture was applied in external solutions designed to reduce had negative resting potentials and overshooting action potentials. There calcium-activated and purely voltage-activated potassium currents, either was no obvious systematic difference in action potential parameters Tyrode’s solution with 5 mM TEA-Cl added or an external solution with between cells used the day of preparation and those kept at 4°C for up to Na and K completely replaced with TEA-Cl. In early experiments the HVA calcium current was obtained as the current that was sensitive to 48 hr. ␮ General electrophysiology. Whole-cell recordings from DRG neurons block by 30 M CdCl2. This gave similar results as the calcium channel blocker mixture used in the succeeding experiments, but the blocker were made with an Axopatch 200B amplifier (Axon Instruments, Union 2ϩ City, CA).
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