Effects of -Dendrotoxin on K Currents and Action Potentials in Tetrodotoxin-Resistant Adult Rat Trigeminal Ganglion Neurons

To determine whether the -dendrotoxin ( -DTX)-sensitive current [D current, slow inactivating transient current (ID)] contributes to the modification of neuronal function in small-diameter adult rat trigeminal ganglion (TG) neurons insensitive to 1 M tetrodotoxin (TTX), we performed two different types of experiments. In the voltage-clamp mode, two distinct K current components, a fast inactivating transient current (IA) and a dominant sustained current (IK), were identified. -DTX (0.1 M), ranging from 0.001 to 1 M, maximally decreased IA by approximately 20% and IK by approximately 16.1% at a 50-mV step pulse, and 0.1 M -DTX application increased the number of action potentials without changing the resting membrane potential. Irrespective of the absence and presence of 0.1 M -DTX, applications of 4-aminopyridine (4-AP; 0.5 mM) and tetraethylammonium (TEA; 2 mM) inhibited approximately 50% inhibition of IA and IK, respectively. 4-AP (0.5 mM) depolarized the resting membrane potential and increased the number of action potentials in the absence or presence of 0.1 M -DTX. TEA prolonged the duration of action potentials in the absence or presence of 0.1 M -DTX. These results suggest that ID contributes to the modification of neuronal function in adult rat TTX-resistant TG neurons, but after the loss of ID due to 0.1 M -DTX application, 4-AP (0.5 mM) and TEA (2 mM) still regulate the intrinsic firing properties of action potential number and shape. The D current, slow inactivating transient current (ID) was first reported by Storm (1987) in hippocampal CA1 pyramidal neurons. The ID has been identified by its ability to delay the firing action potentials after a depolarizing current step and is sensitive to lower concentrations of -dendrotoxin ( -DTX) and 4-aminopyridine (4-AP) (Storm, 1987; Coetzee et al., 1999). Furthermore, there is a report demonstrating that a local modulation of ID exists in the form of an endogenous mast cell degranulating peptide-like molecule in the dorsal root ganglion (DRG) neurons (Stansfeld and Feltz, 1988). The trigeminal ganglion (TG) and DRG neurons express three distinct type of K currents in varying quantities: dominant sustained K current (IK), fast inactivating transient A current (IA), and ID (Puil et al., 1989; Gold et al., 1996; Everill et al., 1998; Everill and Kocsis, 1999; Seifert et al., 1999). In adult rat DRG neurons ranging 39 to 49 m in diameter, Everill et al. (1998) identified three different combinations of K currents (A, K, and D currents, A and K currents, and K and D currents) in the population of cells examined. Such a classification resembles a rapidly inactivating current, a slowly inactivating current, and a noninactivating current (IK), as reported by McFarlane and Cooper (1991) in neonatal rat sensory neurons. Recent evidence has demonstrated that the small-diameter TTX-resistant (TTX-R) neurons isolated from the neonatal TG expressed IK, IA, and ID and that activation of GABAB receptors inhibited the excitability due to the potentiation of IK and IA but not ID (Takeda et al., 2004). In comparison with properties of K currents in TG neurons of embryonic and juvenile rats, Seifert et al. (1999) found a higher 4-AP sensitivity of sustained K currents in the TG neuron of embryos, whereas the sensitivity of IA increased during development in juvenile animals. They also found that ID remained rather constant in TG neurons from a different age. However, there are no reports examining how ID functions in the adult rat TTX-R TG. The purposes of the present study were designed to examine relative contribution of ID to other K currents (IK and IA) Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.105.084988. ABBREVIATIONS: -DTX, -dendrotoxin; 4-AP, 4-aminopyridine; DRG, dorsal root ganglion; TG, trigeminal ganglion; TTX-R, tetrodotoxin resistance; TTX, tetrodotoxin; TEA, tetraethylammonium; 1–3T, 1 to 3 times threshold; NMDG, N-methyl D-glucamine; I-V, current-voltage; DDP, duration of depolarizing phase of action potentials; RMP, resting membrane potential. 0022-3565/05/3141-437–445$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 314, No. 1 Copyright © 2005 by The American Society for Pharmacology and Experimental Therapeutics 84988/3038441 JPET 314:437–445, 2005 Printed in U.S.A. 437 at A PE T Jornals on A ril 0, 2016 jpet.asjournals.org D ow nladed from and to assess their contribution to the firing properties of small-diameter adult rat TG neurons insensitive to TTX. Materials and Methods Cell Culture. For acute dissociation of the TG, adult Wistar rats (250–300 g) were deeply anesthetized with pentobarbital sodium (60 mg/kg i.p.) and were decapitated. A pair of the trigeminal ganglion were dissected and incubated in Hanks’ balanced salt solution (Invitrogen, Carlsbad, CA). They were incubated for 20 to 30 min at 35°C in Hanks’ balanced salt solution containing collagenase types XI (1 mg/ml; Sigma-Aldrich, St. Louis, MO) and I (1 mg/ml; SigmaAldrich). The cells were dissociated by trituration with a fire-polished Pasteur pipette and subsequently were plated onto poly-Llysine-pretreated 35-mm dishes. The plating medium contained Leibovitz’s L-15 solution (Invitrogen) supplemented with 10% newborn calf serum (0.09 v/v), penicillin-streptomycin (50 U/ml) (Invitrogen), 26 mM NaHCO3, and 30 mM glucose. The cells were maintained in a humidified atmosphere of 95% air and 5% CO2 at 37°C. The cells were used for recording between 2 and 10 h after plating. Neurons were accepted for study only if they showed a stable resting membrane potential 40 mV, an action potential overshoot 20 mV, and a whole-cell capacitance 30 pF throughout the experi-