1. Rat dorsal root ganglion (DRG) cell bodies were screened according to action potential (AP) duration, capsaicin sensitivity, expression of IH, IA, and N-, L-, and T-type Ca2+ channel currents. AP duration was measured at half of total amplitude at a membrane potential of -60 mV. Sensitivity to capsaicin was defined as production of an inward current at a holding potential (HP) of -60 mV by 1 microM capsaicin. IH was evoked by a 787-ms hyperpolarization to -110 mV from an HP of -60 mV. IA was evoked by repolarization to -60 mV after a 787-ms hyperpolarization to -110 mV. High-threshold Ca2+ channel current was evoked by a depolarization to -10 or 0 mV from an HP of -60 mV, and L- and N-type Ca2+ channel current was fractionated using selective Ca2+ channel blockers (nimodipine and omega-conotoxin GVIA). T-type Ca2+ channel current was evoked by a depolarization to -40 mV from an HP of -90 mV. Ninety-seven of the 116 DRG cells studied fit closely into one of four categories based on expression of the above characteristics. These four categories, referred to as types 1-4, are described below. 2. Type 1 DRG cells (soma diameter 24.6 +/- 0.5 microns, mean +/- SE; n = 34) had long-duration APs (average = 9.8 ms) with a prominent shoulder on the falling limb and were capsaicin sensitive. Significant IH or IA was not expressed. High-threshold Ca2+ channel current was on average 28% omega-conotoxin GVIA sensitive (N-type) and 46% nimodipine sensitive (L-type); 26% was resistant to both blockers (resistant). T-type Ca2+ channel currents averaged 245 pA. 3. Type 2 DRG cells (soma diameter 25.2 +/- 0.9 microns, n = 19) had short-duration APs (average = 2.9 ms) with a small shoulder on the falling limb and were capsaicin sensitive. IH was negligible but IA averaged 184 pA. High-threshold Ca2+ channel current averaged 42% N-type, 23% L-type, and 35% resistant. T-type Ca2+ channel currents averaged 47 pA. 4. Type 3 DRG cells (soma diameter 18.6 +/- 0.8 microns, n = 21) had short-duration APs (average = 1.8 ms) and were insensitive to capsaicin. IA was not expressed but IH averaged 147 pA. High-threshold Ca2+ channel current averaged 27% N-type, 44% L-type, and 29% resistant. T-type Ca2+ channel currents averaged 306 pA. 5. Type 4 DRG cells (soma diameter 33.9 +/- 0.4 microns, n = 23) had short-duration APs (average = 1.1 ms) and were capsaicin insensitive. IA was not expressed but IH averaged 810 pA. High-threshold Ca2+ channel current was 16% N-type, 4% L-type, and 80% resistant. T-type Ca2+ channel currents averaged 4,031 pA. 6. There was a large variation in the inhibition of high-threshold Ca2+ channel currents by serotonin (5-HT) and (+)8-OH-DPAT in type 1 DRG cells versus types 2-4. On average, 5-HT (10 microM) inhibited high-threshold Ca2+ channel current by an average of 42% in type 1 DRG cells, compared with 15%, 18%, and 7% inhibition in types 2-4, respectively. Similarly, (+)8-OH-DPAT (1 microM) inhibited high-threshold Ca2+ channel current by an average of 35% in type 1 DRG cells, compared with 5%, 8%, and 3% inh...

The distribution of tetrodotoxin (TTX)-sensitive and -insensitive Na ϩ currents and their modulation by serotonin (5HT) and prostaglandin E 2 (PGE 2 ) was studied in four different types of dorsal root ganglion (DRG) cell bodies (types 1, 2, 3, and 4), which were previously identified on the basis of differences in membrane properties (Cardenas et al., 1995). Types 1 and 2 DRG cells expressed TTX-insensitive Na ϩ currents, whereas types 3 and 4 DRG cells exclusively expressed TTX-sensitive Na ϩ currents. Application of 5HT (1-10 M) increased TTX-insensitive Na ϩ currents in type 2 DRG cells but did not affect Na ϩ currents in type 1, 3, or 4 DRG cells. The 5HT receptor involved resembled the 5HT 4 subtype. It was activated by 5-methoxy-N,Ndimethyltryptamine (10 M) but not by 5-carboxyamidotryptamine (1 M), (ϩ)-8-hydroxydipropylaminotetralin (10 M), or 2-methyl-5HT (10 M), and was blocked by ICS 205-930 with an EC 50 of ϳ2 M but not by ketanserin (1 M). PGE 2 (4 or 10 M) also increased Na ϩ currents in varying portions of cells in all four groups.The effect of 5HT and PGE 2 on Na ϩ currents was delayed for 20-30 sec after exposure to 5HT, suggesting the involvement of a cytosolic diffusible component in the signaling pathway. The agonist-mediated increase in Na ϩ current, however, was not mimicked by 8-chlorophenylthio-cAMP (200 M), suggesting the possibility that cAMP was not involved.The data suggest that the 5HT-and PGE 2 -mediated increase in Na ϩ current may be involved in hyperesthesia in different but overlapping subpopulations of nociceptors. Key words: serotonin; PGE 2 ; capsaicin; nociceptor; tetrodotoxin; cAMP; dorsal root ganglion; 5HT 4 receptorPrimary hyperesthesia is thought to be a consequence of the release of proinflammatory mediators in the vicinity of nociceptor endings. Proinflammatory agents such as serotonin (5HT), prostaglandins, and adenosine are derived from a number of sources (Cooper and Sessle, 1992). Interactions between such agents and endings of thinly myelinated and unmyelinated nociceptive afferents induce activity, decrease threshold, and increase suprathreshold mechanothermal reactivity (Handwerker, 1976;Kumazawa and Mizumura, 1980;Mense, 1981;Martin et al., 1987;Schaible and Schmidt, 1988;Lang et al., 1990;Grubb et al., 1991;Herbert and Schmidt, 1992).Contributions to hyperesthesia by proinflammatory agents are possibly achieved by modulation of membrane currents involved in the initiation and repolarization of action potentials in nociceptive endings, as well as induction of edema in the surrounding matrix (Cooper, 1993). The study of acutely isolated dorsal root ganglion (DRG) cell bodies may be usef ul in determining the roles of various ion channels in the actions of proinflammatory agents on nociceptor f unction. A number of studies have shown that afferent cell bodies exhibit properties of nociceptor endings in vitro. These include expression of currents sensitive to proinflammatory mediators as well as features of peripheral transduction mechanisms (Baccaglini and Hogan...

The hyperpolarization-activated cation current (IH) is expressed differentially in various subpopulations of rat sensory neurons. For example, IH is infrequently expressed by C-and Aä-type dorsal root ganglion (DRG) neurons but is prominent in most Aá-and Aâ-type DRG neurons (Harper & Lawson, 1985a,b;Scroggs et al. 1994; Valiere & McLachlan, 1996). Since C-and Aä-type DRG neurons are typically involved in the transmission of different types of information than Aâ-and Aá-type DRG neurons, it is likely that agents which modulate IH may selectively affect certain types of sensory information. Serotonin (5_HT) has previously been shown to increase IH in rat nucleus prepositus hypoglossi neurons and in guinea-pig thalamic neurons through an increase in cAMP. However, the possibility that 5_HT may modulate IH in DRG neurons, as well as the potential mechanisms involved, have not previously been explored.In an earlier study we developed a set of criteria which appeared useful in categorizing small-and medium-diameter DRG cell bodies into four groups, types 1, 2, 3 and 4, based on capsaicin sensitivity, expression of IH, IA (a transient, 4_aminopyridine-sensitive outward K¤ current) and T_type Ca¥ current amplitude (Cardenas et al. 1995). The likelihood that these categories have some functional significance is supported by subsequent findings that 5_HT1A receptor activation selectively inhibited high-threshold Ca¥ currents in type 1 DRG cells and 5_HTÚ receptor activation selectively increased TTX-insensitive Na¤ currents in type 2 DRG cells (Cardenas et al. 1995(Cardenas et al. , 1997a. In the present study we observed that 5_HT increased IH in type 4 DRG cells and a subpopulation of large-diameter DRG cells, but not in type 1, 2, or 3 DRG cells. The 5_HT receptor involved responded to several selective 5_HT Journal of Physiology (1999) 1. The effect of serotonin (5_HT) on the hyperpolarization-activated cation current (IH) was studied in small-, medium-and large-diameter acutely isolated rat dorsal root ganglion (DRG) cells, including cells categorized as type 1, 2, 3 and 4 based on membrane properties. 5_HT increased IH in 91% of medium-diameter DRG cells (including type 4) and in 67% of large-diameter DRG cells, but not other DRG cell types. 2. The increase of IH by 5_HT was antagonized by spiperone but not cyanopindolol, and was mimicked by 5-carboxyamidotryptamine, but not (+)_8-hydroxydipropylaminotetralin (8_OH_DPAT) or cyanopindolol. These data suggested the involvement of 5_HTÝ receptors, which were shown to be expressed by medium-diameter DRG cells using RT-PCR analysis. 3. 5_HT shifted the conductance-voltage relationship of IH by +6 mV without changing peak conductance. The effects of 5_HT on IH were mimicked and occluded by forskolin, but not by inactive 1,9-dideoxy forskolin. 4. At holding potentials negative to −50 mV, 5_HT increased steady-state inward current and instantaneous membrane conductance (fast current). The 5_HT-induced inward current and fast current were blocked by Cs¤ but not Ba¥ and re...

The physiological effects of 5HT receptor coupling to TTX-resistant Na(+) current, and the signaling pathway involved, was studied in a nociceptor-like subpopulation of rat dorsal root ganglion (DRG) cells (type 2), which can be identified by expression of a low-threshold, slowly inactivating A-current. The 5HT-mediated increase in TTX-resistant Na(+) current in type 2 DRG cells was mimicked and occluded by 10 microM forskolin. Superfusion of type 2 DRG cells on the outside with 1 mM 8-bromo-cAMP or chlorophenylthio-cAMP (CPT-cAMP) increased the Na(+) current, but less than 5HT itself. However, perfusion of the cells inside with 2 mM CPT-cAMP strongly increased the amplitude of control Na(+) currents and completely occluded the effect of 5HT. Thus it appears that the signaling pathway includes cAMP. The phosphodiesterase inhibitor 3-isobutyl-L-methylxanthine (200 microM) also mimicked the effect of 5HT on Na(+) current, suggesting tonic adenylyl cyclase activity. 5HT reduced the amount of current required to evoke action potentials in type 2 DRG cells, suggesting that 5HT may lower the threshold for activation of nociceptor peripheral receptors. The above data suggest that serotonergic modulation of TTX-resistant Na(+) channels through a cAMP-dependent signaling pathway in nociceptors may participate in the generation of hyperalgesia.

. Ca 2ϩ channel subtypes expressed by dopaminergic (DA) neurons in the substantia nigra pars compacta (SNc) were studied using whole cell patch-clamp recordings and blockers selective for different channel types (L, N, and P/Q). Nimodipine (Nim, 2 M), -conotoxin GVIA (Ctx, 1 M), or -agatoxin IVA (Atx, 50 nM) blocked 27, 36, and 37% of peak whole cell Ca 2ϩ channel current, respectively, indicating the presence of L-, N-, and P-type channels. Nim blocked approximately twice as much Ca 2ϩ channel current near activation threshold compared with Ctx or Atx, suggesting that small depolarizations preferentially opened L-type versus N-or P-type Ca 2ϩ channels. N-and L-channels in DA neurons opened over a significantly more negative voltage range than those in rat dorsal root ganglion cells, recorded from using identical conditions. These data provide an explanation as to why Ca 2ϩ -dependent spontaneous oscillatory potentials and rhythmic firing in DA neurons are blocked by L-channel but not N-channel antagonists and suggest that pharmacologically similar Ca 2ϩ channels may exhibit different thresholds for activation in different types of neurons. I N T R O D U C T I O NNumerous studies have indicated that Ca 2ϩ entry is important in regulating the firing patterns of dopaminergic (DA) neurons in the substantia nigra. The pacemaker-like slow depolarizations (PLSD), spontaneous oscillatory potentials (SOPs), and slow afterhyperpolarizations involved in regulation of DA neuron firing activity have all been shown to be Ca 2ϩ dependent (Fujimura and Matsuda 1989; Grace and Onn 1989; Harris et al. 1989; Kang and Kitai 1993a,b; Mercuri et al. 1994; Nedergaard et al. 1993; Ping and Shepard 1996; Yung et al. 1991). A previous study suggests that DA neurons express L-, N-, P/Q-, and R-type Ca 2ϩ channels (Cardozo and Bean 1995). However, the Ca 2ϩ channel subtype(s) involved in regulation of DA neuron firing behavior have not been conclusively determined. In two studies, DA neuron spontaneous firing and SOPs were abolished by dihydropyridines but not -conotoxin GVIA (Ctx), suggesting a prominent role for L-, but not N-type Ca 2ϩ channels in these phenomena (Mercuri et al. 1994; Nedergaard et al. 1993). However, Kang and Kitai (1993b) provided evidence that DA neuron pacemaker activity was underlain by N-but not L-type Ca 2ϩ channels. Finally, a study by Fujimura and Matsuda (1989) suggested that neither N-nor L-channels were involved in DA neuron pacemaker activity.The present study investigated the relative contribution of L-, N-, and P-type Ca 2ϩ channels to whole cell Ca 2ϩ channel current at different membrane potentials, to see which of the three channels opened at membrane potentials that would be commensurate with a role in the PLSD. We also compared the voltage dependence of DA neuron Ca 2ϩ channel activation to that of dorsal root ganglion (DRG) neurons. Our data indicate that a larger proportion of the whole cell Ca 2ϩ current observed near activation threshold is conducted through L-channels versus N-or P-chann...

The coupling of serotonin receptors to Ca2+ channels was studied in a subpopulation of acutely isolated rat dorsal root ganglion (DRG) cell bodies (type 1 DRG cells), which have membrane properties similar to C-type nociceptive sensory neurons. In these cells, serotonin (5HT) inhibited high-threshold Ca2+ channel current and decreased action potential duration. The inhibitory effects of 5HT and the 5HT1A agonist 8-OH-DPAT were shown to be antagonized by the 5HT1A antagonists spiperone and pindolol, respectively, indicating involvement of a 5HT1A receptor. Several observations suggest that 5HT1A receptors couple to N- and L-type Ca2+ channels by two different signaling pathways in type 1 DRG cells. The inhibition of Ca2+ channel currents produced by 10 microM 5HT occurred in two phases, an initial slowing of current activation rate (kinetic slowing), which was complete within 10 s, and a simultaneous reduction in steady state current amplitude (steady state inhibition), which peaked in approximately 1 min. The kinetic slowing, but not steady state inhibition, was reversed by a positive prepulse to +70 mV (prepulse). Blockade of N-type Ca2+ channels selectively reduced the kinetic slowing and its reversal by prepulses. Chelation of intracellular Ca2+ or blockade of L-type Ca2+ channels selectively reduced the steady state inhibition. Recordings using the cell-attached patch configuration suggest that steady state inhibition required a component that was diffusible in the cytosol, while kinetic slowing occurred via a membrane delimited pathway. The application of 5HT to the cell body outside the patch pipette reduced macroscopic Ca2+ channel currents in 33% of small-diameter DRG cells tested, indicating the participation of a cytosolic diffusible component. Application of 5HT (a membrane impermeant compound) outside the patch pipette produced steady state inhibition only, whereas similar application of membrane permeant 5HT1A agonists, 8-OH-DPAT or 5-methoxy-N,N-dimethyl-tryptamine, produced kinetic slowing and steady state inhibition. Together these data suggest that 5HT1A receptors couple negatively to Ca2+ channels via two pathways: a membrane-delimited pathway that couples to N-channels and actuates voltage-sensitive kinetic slowing and a pathway dependent on a cytosolic diffusible component and free intracellular Ca2+, which couples to L channels and actuates steady state inhibition.

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