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Proc. Natl. Acad. Sci. USA Vol. 96, pp. 7650-7657, July 1999 Colloquium Paper This paper was presented at the National Academy of Sciences colloquium "The Neurobiology of Pain, " held December 11-13, 1998, at the Arnold and Mabel Beckman Center in Irvine, CA. Calcium regulation of a slow post-spike hyperpolarization in vagal afferen~r neurons (spike frequency adaptation/ryanodine receptor/autacoids/allergic inflammation/mast cell) RUTH CORDOBA-RODRIGUEZ*, KIMBERLY A.MOORE*,JOSEPHP.Y.KAO!,ANDDANTEEWEINRElCH*t *Department of Pharmacology and Experimental Therapeutics and iMedica1 Ric~technolo~v (~.nter ~nr1 nen~rtm~nt of Phv~i~l~v r Inivercitv of M~r~rl~nr1 School of Medicine, Baltimore, MD 21201-1559 ABSTRACT Activation of distinct classes of potassium channels can dramatically affect the frequency and the pat- tern of neuronal firing. In a subpopulation of vagal afferent neurons (nodose ganglion neurons), the pattern of impulse activity is effectively modulated by a Ca2+-dependent K+ current. This current produces a post-spike hyperpolarization (AHPslow3 that plays a critical role in the regulation of membrane excitability and is responsible for spike-frequency accommodation in these neurons. Inhibition of the AHPs~ow by a number of endogenous autacoids (e.g., histamine, serotonin, prostanoids, and bradykinin) results in an increase in the firing frequency of vagal afferent neurons from 10 Hz. After a single action potential, the AHPs,ow in nodose neurons displays a slow rise time to peak (0.3-0.5 s) and a long duration (3-15 s). The slow kinetics of the AHPs~ow are due, in part, to Ca2+ discharge from an intracellular Ca2+-induced Ca2+ release (CICR) pool. Action potential-evoked Ca2+ in- flux via either L or N type Ca2+ channels triggers CICR. Surprisingly, although L type channels generate 60% of action potential-induced CICR, only Ca2+ influx through N type Ca2+ channels can trigger the CICR-dependent AHPs~ow. These observations suggest that a close physical proximity exists between endoplasmic reticulum ryanodine receptors and plasma membrane N type Ca2+ channels and AHPs~ow potassium channels. Such an anatomical relation might be particularly beneficial for modulation of spike-frequency ad- aptation in vagal afferent neurons. Activation and sensitization of primary afferent nerve fibers during allergic inflammation are orchestrated by inflamma- tory mediators released from various cells, including tissue mast cells. Inf lammatory mediators provoke excitability changes in sensory nerves through diverse mechanisms, in- cluding (i) modification of the density and coupling efficacy of ligand-gated ionic channels; (ii) alteration in voltage-gated sodium, potassium, and calcium channels; and (iii) manipula- tion of cellular mechanisms that control spike-frequency ad- aptation. After immunologic activation of mast cells in airway in vivo or in sensory ganglia in vitro, a wide range of electrophysio- logical changes can be detected in peripheral sensory nerve terminals of the vagus (1) and in vagal primary afferent somata (located in the nodose and jugular ganglia) (24. These changes range from transient (minutes) membrane depolarizations that sometimes reach action potential (AP) threshold (3) to a sustained (days) unmasking of functional NK-2 tachykinin receptors (4, 5~. One electrical membrane property that is particularly sensitive to inflammatory mediators is a slow post-spike afterhyperpolarization (AHPs~ow; see Fig. 1) (34. PNAS is available online at =, ~ i =, . . , . This slow afterpotential influences neuronal excitability and determines the frequency and pattern of neuronal discharge. We have found that the amplitude and duration of the AHPs~ow are exquisitely sensitive to known inflammatory mediators such as prostanoids, amines, and kinins applied exogenously (Table 1) or released endogenously (i.e., after immunologic activation of mast cells) (3, 6~. Inhibition of the AHPs~ow is accompanied by a loss of spike-frequency adaptation. Thus, modulation of the AHPs~ow amplitude and duration provides a mechanism for neuronal sensitization. We are interested in identifying the ionic channels and second-messenger transduction pathways that participate in the initiation and maintenance of the AHPs~ow in vagal primary afferent neurons. In this report, we describe the general properties of this slow afterpotential and our progress in its characterization. Our working hypothesis is that a close func- tional proximity between three separate channels tN type voltage-sensitive calcium channels, ryanodine (RY)-sensitive Ca2+-induced Ca2+ release (CICR) calcium channels, and AHPs~ow K+ (SK) channels that underlie the AHPs~ow] is essential for the initiation of the AHPs~ow. RESULTS General Properties of Vagal Afferent AHPslow. The AHPslow is observed in a wide variety of peripheral and central neurons (for review, see ref. 7~. In nodose neurons, AHPslow is always preceded by a fast post-spike afterhyperpolarization (AHPfaSt, 10-50 ms) that occurs at the end of the AP repolarization. In some neurons, the AHPfaS~ is followed by a second afterpo- tential that lasts 50-300 ms (AHPmeuiUm). The AHPme~jum is voltage- and Ca2+- dependent and blocked by 10 mM tetra- ethylammonium in ~50% of neurons, suggesting that it is mediated by large-conductance Ca2+-activated K+ channels (BK channels) (8~. In vagal afferent somata, the AHPs~ow is particularly robust. After a single AP, the AHPs~oW displays a delayed onset (100-500 ms), a slow rise time to peak (0.3-5 s), and a long duration (2-15 s; see Fig. 1~. The proportion of AHPs~Ow neurons within nodose ganglia varies among species: ~20% in the guinea pig, ~35% in rabbit, and ~85% in ferret. Only nodose neurons classified as C fibers (conduction velocity <1 m/s) possess AHPs~ow. To date, there have been few species differences in the pharmacological or physiological properties Abbreviations: AP, action potential; BK, large-conductance Ca2+- activated K+ channels; SK, small-conductance Ca2+ -activated K+ channels; CICR, Ca2+-induced Ca2+ release stores; RY, ryanodine; VDCC, voltage-dependent Ca2+ channels; L, N, R, L type, N type, and R-type VDCC; AHP, afterhyperpolarization; DBHQ, 2,5,-di~t- butyl~hydroquinone. lTo whom reprint requests should be addressed. e-mail: dweinrei@ 7650

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Colloquium Paper: Cordoba-Rodriguez et al. . -54 mV V / 10 mV 1 50 msec -58 mV -53 mV 10 mV ~ 2 see FIG. 1. A single AP can evoke three types of AHP in nodose neurons. (Top) A neuron with a single-component afterpotential lasting ~30 ms. This AHP is designated AHPfas~. All neurons have this short duration afterpotential. (Middle) Example of a neuron with two afterpotentials, an AHPfasr followed by a longer lasting afterpotential (~300 ms), the AHPmeciium. In approximately half of the neurons, the AHPme~ium is Ca2+-dependent. ( OCR for page 7650
7652 Colloquium Paper: Cordoba-Rodriguez et al. A. 2.0 mM Ca2' 1.5 mM Ca2+ 1.0 mM Ca2' A. - .. AHP slow Proc. Natl. Acad. Sci. USA 96 (1999J '''';,; ~ ~ see I 0.5 mM Ca2+ 0.0 mM Ca2+2.0 mM Ca2+ B. , ~at ~cat AHP slow AHP medium AHP fast B. 100 ~/6 C. x 80 60 - 40 at: 20 66/ /1 an' 0.0 0.5 1.0 1.5 2.0 [Ca2+10 (mM) FIG. 3. Effects of varying [Ca2+30 on the amplitude of the AHPs~ow recorded in isolated nodose neurons. (A) Sample traces of AHPs~ow evoked by a train of four APs in the presence of different [Ca2+]O. APs are evoked by transmembrane depolarizing current pulses (2 nA, 3 ms, 10 Hz) and are truncated. tCa2+]O was varied from 2.0 to 0.0 mM in 0.5 mM decrements. The AHPs~ow is completely blocked when (Ca2+30 is reduced to nominally zero. On returning to 2.0 mM [Ca2+iO, the AHPs~ow recovers to its original amplitude. (B) Relation between [Ca2+]O and AHPs~ow amplitude recorded in several neurons. Values are means + SEM of the number of observations indicated near each data point. Data are normalized to the maximum response recorded . . In a given neuron. Linear regression analysis yields the solid line (r = 0.993). Fig. 5 shows an overlay of the outward current responses evoked by Ca2+ injection in a single nodose C type neuron at holding potentials of -20 mV and -50 mV. The kinetic differences between IK medium and IK SIOW after Ca2+ injection are dramatic. In contrast to the rapid activation of IK medium, the onset of IK-SIOW is delayed, and the decay of IKmeuiUm is nearly complete before the peak amplitude of the IK SIOW is reached. These two outward currents mirror the temporal and pharmacological differences between AHPme~iUm and AHPSIOW. IKSIOW, like the AHPs~ow, was blocked by 100 nM prostaglandin D2. The data shown in Table 2 summarize quantitative differences between these two Ca2+-induced out- ward currents. It is possible that the delayed onset of IK SIOW compared with IK medium results from unequal Ca2+ diffusion distances from the injection site to the two types of K+ channels. This cause seems unlikely because the orientation of impalement was random, and the plasma membranes of dissociated nodose neurons appear devoid of processes that would provide semi- isolated regions where IK SIOW might be generated. An alterna- tive possibility is that additional intermediate steps, such as the synthesis or release of a second messenger, are required to activate IK SIOW. The large Qua (>3.0; ref. 14) supports the latter alternative. One candidate is mobilization of intracellularly stored Ca2+. Ca2+ Released by the CICR Pool Is Essential for the Generation of the AHPs,ow. Single APs produce transient increases in tCa2+]i (micas) as measured by the fluorescent r 4 . ~ FIG. 4. Effects of BAPTA on the AHPs~ow and on the excitability of an acutely dissociated rabbit nodose neuron. (A) Bath-applied BAPTA/acetomethylester (10 AM) blocks the AHPs~ow within 5 min without changing the resting membrane potential or membrane input resistance. APs were evoked by transmembrane depolarizing current pulses (4 nA, 1.5 ms, 10 Hz) and are truncated. (B) Responses recorded at a faster sweep speed to illustrate the kinetics of the AHPfast and AHPme~ium, which precede the AHPs~ow. The AHPfasr iS unaffected by 10 EM BAPTA/acetomethylester (compare a with b). The Ca2+ dependence of the AHPme~ium is illustrated in c, where the neuron is superfused with 100 ,uM CdCl2 for 30 s, which blocks most of the AHPme~ium. The residual component of the AHP recorded in CdCl2 is the AiIPfas~' which is mediated by delayed rectifier K+ channels. (C) Depression of the AHPs~ow markedly increases neuronal excitability. The average AP firing frequency induced by a current ramp protocol (1 nA, 2 s) increased from 1 to 5.5 Hz when the AHPs~ow was blocked. Similar loss of spike-frequency adaptation was observed with brady- kinin, prostaglandin D2, histamine, and other inflammatory autacoids (see Table 2~. The scale bar represents 3 mV, 2 s inA; 15 mV, 0.25 s in B.; and 15 mV, 0.5 s in C. The dashed line represents the resting membrane potential (-60 mV). Resting membrane input resistance was 70 ME. Data is from ref. 19 with permission from the American Physiological Society. indicator fura-2. The magnitude of the l\Ca~ depends on both [Ca2+]O and the number of APs. Over the range of one to eight APs, there is an approximately linear relation between the magnitude of the i\Cat and the number of APs (Fig. 6). In the presence of drugs that block CICR but do not significantly affect AP-induced Ca2+ influx [(RY, 10 ,uM), 2,5,-di(t- butyl)hydroquinone (DBHQ, 10 ,uM), or thapsigargin (TG, 100 nM)], we found that at least eight APs were required to evoke a detectable i\Cat (Fig. 6). In the presence of RY, DBHQ, and TG, the l\Cat-AP relation exhibits slopes of 0.5, 1.1, and 0.8 nM per AP, respectively. When compared with the slope of 9.6 nM per AP in control neurons, Ca2+ influx produced by a single nodose AP is amplified by 5- to 10-fold by CICR (16). Nodose neurons demonstrate a relatively low stimulus threshold for eliciting CICR. For instance, a robust CICR response can be observed after a single AP stimulus in nodose neurons, whereas many tens of APs are required in dorsal root ganglion neurons (17). The greater CICR response in nodose neurons is not due to greater Ca2+ influx through voltage-dependent Ca2+ channels (VDCCs); a single AP produces comparable Ca2+ influx in nodose and dorsal root ganglion neurons (39 vs. 49 pC, respectively; refs. 16 and 18). Rather, the more responsive CICR pool in nodose neurons

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Colloquium Paper: Cordoba-Rodriguez et al. 1 1 t tA 5sec 1 A Control ~ 0.2 nA Ryanodine 2 FIG. 5. Comparison of two outward K+ currents evoked by intra- cellular Ca2+ injection. Recordings were made in a single acutely isolated adult rabbit nodose neuron. A slow outward current (IK-SIOW) was activated by a 5-nA, 1-s iontophoretic Ca2+ injection at a holding potential of -50 mV. A second outward current (IKme~iUm) was activated at -20 mV (5 nA, 0.5 see). IK medium activates and decays completely before IK-SIOW reaches peak amplitude. IK medium was blocked by 10 mM tetraethylammonium; IK-SIOW was blocked by 100 nM prostaglandin D2. The iontophoretic pipette was filled with a 0.2 M CaCl2 solution. Voltage-clamp currents were recorded with a second intracellular pipette. The discontinuous (switched) current injec- tion mode of an Axoclamp II amplifier was used for both current- and voltage-clamp applications. The larger calibration value is for IK medium. Population data is shown in Table 2. may reflect either a closer proximity between plasma mem- brane Ca2+ influx channels and endoplasmic reticulum RY receptors or a more sensitive RY receptor. By using physiological stimuli (APB) in conjunction with pharmacological manipulations of CICR, we have demon- strated that CICR is essential for the development of the AHPs~oW. Over the range of 1-16 APs, the magnitudes of the AP-induced AHPs~ow and the 1\ Cat (a monitor of CICR in these neurons) were highly correlated (r = 0.985~. Simultaneous recordings of l\Cae and AHPs~oW before and during bath application of CICR inhibitors (RY, TG, DBHQ, or 10 ,uM cyclopiazonic acid) revealed that both responses were blocked in a parallel fashion (Fig. 7; see also Table 1 in ref. 19~. These data indicate that a CICR pool is essential for the generation of the AHPs~ow. They also provide a potential explanation for the slow kinetics of the AHPs~ow, namely Ca2+ mobilization from CICR. Effects of Changing [Ca2+ l 0 on the AHPs,ow, Scat, and Ca2+ influx. If the AHPs~oW depends on Ca2+ released from the CICR pool triggered by AP-induced Ca2+ influx, it would follow that changes in [Ca2+]O should produce corresponding effects on both the AHPs~oW and the I\Ca~. The data shown in Fig. 3A illustrate the effects of progressively lowering tCa2+]O from 2.0 mM to nominally zero on the amplitude of the AHPs~oW recorded in a single nodose neuron. As tCa2+30 was decreased, the amplitude of the AHPs~ow was reduced propor- tionally. When the results from this and five additional neurons were plotted (Fig. 3B), the relation between tCa2+]O and the amplitude of the AHPs~ow was linear (r = 0.993; n = 6, pooled data from three current-clamp and three hybrid voltage-clamp experiments). Table 2. Comparison of IK-SIOW and IK-me~ium Peak Holding potential, mV conductance, Current nS n IK-SIOW 27.9 + 6.5 14 -55.4 + 2.7 IK medium 53.2 + 16.5 6 -20 + 3.7 IK-S1OW and IK-medium are outward currents elicited by iontophoretic injection Ca2+ into acutely isolated nodose neurons of the rabbit. The peak conductance is the largest conductance elicited, independent of membrane potential. The holding potential is the potential at which the peak conductance was measured. The decay time constant was measured by fitting a line, by eye, to the log transform of the decay of the current. The duration was calculated from the onset of Ca2+ injection to the time at which the current had decayed to 20% of its peak value. Data are summarized as the mean + SEM. Proc. Natl. Acad. Sci. USA 96 (1999J 7653 4 l 1\` 8 -~,~% :30 nM 3 sec ~^ ~. 8 90 a . 50- / '_ 40 E 30 ~ , , 0 4 8 12 16 20 24 28 32 Number of action potentials FIG. 6. (Upper) Effect of RY on AP-induced Ca2+ transients. Traces are Ca2+ transients evoked by varying numbers of APs, as indicated below each trace. In control neurons, distinct Ca2+ transients can be elicited by very few APs. In contrast, in the presence of 10 ,uM RY, a CICR inhibitor, at least eight APs are required to generate a discernible change in [Ca2+]i. Suppression of the Ca2+ transient by RY is due to its effect on CICR and not the result of nonspecific effects on Ca2+ channels; the kinetics and amplitude of ICa elicited by APs are completely unaffected by RY. (Lower) Effect of RY on the relation between the amplitude of Ca2+ transients and number of APs. o and are mean amplitudes of Ca2+ transients evoked by varying numbers of action potentials for control (n = 10) and for RY-treated nodose neurons (n = 3), respectively. Linear regression of data from control (c4 action potentials) and RY-treated cells yielded slopes of 9.6 + 0.01 and 0.5 + 0.23 nM per AP, respectively. Comparison of the slopes illustrates that CICR is capable of amplifying the "trigger" Ca2+ resulting from AP-induced Ca2+ influx by 20-fold. Data is modified from ref. 16 with permission from Journal of Physiology (London). Next, we examined the relation between tCa2+]O and the magnitude of the AP-induced ACa~. Fig. SA illustrates l`Ca~s elicited by varying numbers of APs recorded from a single neuron in Locke solution containing 2.2 or 1.1 mM Ca2+. The population results relating the normalized amplitude of the ACa~s recorded in four neurons to the number of APs is shown in Fig. 8B. In 1.1 mM tCa2+]O, the first few APs did not elicit a measurable l\Ca~. For the neuron shown in Fig. 8A, at least eight APs were necessary to evoke a detectable /\Ca~. In three additional neurons, the minimum number of APs necessary to Decay Time-to-peak, time constant, Duration, n ms n ms n s n 14 6,570 + 1085 12 6,735 + 789 5 23 + 3.4 14 6 958 + 56 6 818+ 97 6 2.5 +0.16 6

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7654 Colloquium Paper: Cordoba-Rodriguez et al. Proc. Natl. Acad. Sci. USA 96 (1999 A it. 2.2 mM 1Ca2 lo ~ ~1\~ ^6 h 150nM h / ~3sec ~ \. 110nM B. I 3 mV 2 sect .. ~ .~' FIG. 7. Effect of DBHQ, a functional CICR inhibitor, on the AP- induced Ca2+ transient and on the AHPs~ow recorded simultaneously in an acutely isolated rabbit nodose neuron. Upper traces represent superimposed Ca2+ transients evoked by a train of four APs (10 Hz) recorded in control Locke solution and 7 min after switching to Locke solution containing 10 ,uM DBHQ. The lower pair of traces shows AHPs~ow DBHQ treatment completely blocked both the Ca2+ tran- sient and the AHPs~ow. Resting [Ca2+]i was 91 nM. Fluorescence data were acquired at 10 Hz. Resting membrane potential was -67 mV. AP amplitudes are truncated. Data are from ref. 19 with permission from the American Physiological Society. elicit a detectable ACa~ ranged from 4 to 32. The 1\Ca~-AP relation recorded in 1.1 mM tCa2+]O, as in Locke solution containing normal tCa2+]O, followed a hyperbolic relation (x2 = 6.75 and 0.31; r = 0.988 and 0.999 for 2.2 and 1.1 mM Ca2+,respectively; Fig. 8B and see also Fig. 1 in ref. 16~. Given the hyperbolic nature of the i\Ca~-AP relation, deducing the effects of altered tCa2+~0 on the magnitude of the i\Ca~ clearly depends on where along this relation the comparison is made. At one extreme, there is a ~2-fold change when comparing the plateau phases of the curves in normal and one-half normal tCa2+]O. It is also possible to calculate the limiting initial slopes for the rising phase of the curves (dashed lines in Fig. 8B). The limiting slopes, which represent the full Ca2+ release potential of the CICR pool before any release has actually occurred, were 15 + 3.8 and 2 + 0.7 nM per AP in 2.2 and 1.1 mM tCa2+]O, respectively. Thus, reducing [Ca2+]O by a factor of 2 results in a reduction of the l\Ca~ by a factor of 7 + 2.8 when the rising phases of the two curves are compared. The ~7-fold reduction of the i\Ca~ associated with halving [Ca2+~0 is much larger than the 2-fold reduction in the AHPs~ow amplitude (Fig. 3), suggesting that some, but not all, of the Ca2+ released from the CICR pool is required for the generation of the AHPs~ow. The disproportionate effect of reduced tCa2+~0 on the i\Ca~ versus the AHPs~ow could arise from a nonlinear reduction of Ca2+ influx per AP and/or from a decreased Ca2+ release from CICR pool per unit Ca2+ influx. To study these possi- bilities, we examined the effect of lowering tCa2+]O on AP- induced Ca2+ influx. The amount of Ca2+ entering a neuron with each AP in normal and in reduced tCa2+]O was deter- mined by using a prerecorded AP as whole-cell voltage-clamp command under experimental conditions where the major inward charge carrier is Ca2+ (for details, see Fig. 2 in ref. 16~. When [Ca2+]O was decrementally reduced from 2 mM to nominally zero, the magnitude of the ICa decreased propor- tionally. The charge movement caused by Ca2+ influx, nor- malized to cell membrane capacitance (pC/pF), was plotted against varying (Ca2+]O for 12 neurons. Over the range of 0-2.0 mM [Ca2+iO, Ca2+ influx varied linearly with tCa2+]O (r = 0.974~. These results indicate that changes in Ca2+ influx alone x ~oo - o O~ ._ .. 3 ~_ ~n 20 0 6~ /~ ~ ,~ {W. . . . . . . . . . . . 0 8 1 6 24 32 40 48 56 64 Number of action potentials FIG. 8. Effect of varying [Ca2+]O on the amplitude of AP -induced Ca2+ transients. (A) Representative traces of Ca2+ transients evoked by varying numbers of APs in normal (2.2 mM) and reduced (1.1 mM j tCa2+iO. APs were elicited by transmembrane depolarizing current pulses (2 nA, 1.5 ms, 10 Hz). The number of APs is indicated below each trace. (B) The normalized (mean + SEM) amplitude of Ca2+ transients recorded in four neurons is plotted against varying numbers of APs. Data are normalized to the maximal response recorded in a given neuron. 0 represents Ca2+ transients recorded in 2.2 mM [Ca2+iO; ~ represents Ca2+ transients recorded in the same neurons in 1.1 mM [Ca2+]O. Continuous curves are rectangular hyperbolas fit to the data (x2 = 6.75 and 0.31, r = 0.988 and 0.999 for 2.2 and 1.1 mM tCa2+iO, respectively). The dashed lines represent the limiting initial slopes (15 + 3.8 and 2 + 0.7 nM per AP for 2.2 and 1.1 mM [Ca2+]O, respectively). cannot account for the disproportionate reduction in the l\Ca~ relative to the AHPs~ow that is observed when tCa2+]O is reduced. The disproportionate effect of reduced [Ca2+]O on the i\Ca~-AHPs~Ow relation could arise from a diminution in the amount of Ca2+ released from the CICR pool. Caffeine, a known agonist of CICR, is traditionally used to assess the releasable content of the CICR pool. In 8 of the 13 neurons studied, halving tCa2+~0 reduced the caffeine-induced l\Ca~ by 20-79% (100% vs. 47 + 7.2% in 2.2 and 1.1 mM tCa2+iO, respectively; P = 0.0002). In other words, decreasing [Ca2+~0 by a factor of 2 caused a 1.25- to 5-fold reduction in the caffeine response. On returning to normal Locke solution, the caffeine response was restored to near control values. In the remaining five neurons, the caffeine-induced ACa~ was unaffected by reducing tCa2+iO (100% vs. 112 + 8.4% in 2.2 and 1.1 mM tCa2+iO, respectively; P = 0.690~. There was no significant difference in resting levels of [Ca2+]i between these two groups of neurons (93 + 29.5 nM vs. 111 + 29.7 nM; P = 0.530~. Unfortunately, the wide variability in the effects of reduced [Ca2+]O on the caffeine responses prevents a meaningful interpretation of the effect of [Ca2+]O on the releasable content of the CICR pool. Ca2+ Influx Through N Type Calcium Channels Selectively Elicits AHPs,ow. Six types of VDCCs have been described in neurons: L, N, P, Q, R, and T (20~. Nodose neurons express several types of VDCCs. By using a panel of pharmacologic reagents that are selective for different types of VDCCs, we tested the contribution of each to the total AP-induced Ca2+ current. Our results, summarized in Table 3, reveal that ~85%

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Colloquium Paper: Cordoba-Rodriguez et al. Table 3. Effects of Ca2+ channel blockers on action potential-induced inward Ca2+ currents Proc. Natl. Acad. Sci. USA 96 (1999J 7655 Table 4. Actions of specific Ca2+ channel blockers on the action potential-induced Ca2+ transient and the AHPs~ow Concentration Reduction, % Channel type Channel blocker ,uM Reduction n Channel ca2+ AHPs~ow T Amiloride 500 0 + 0 18 type Channel blocker transient namplitude n L Nifedipine 10 44 + 5.6 9 L Nifedipine 57 + 7.7 210 + 0 5 P/Q co-AGA IVA 0.2 0 + 0 2 N cl)-CTX GVIA 39 + 6.2 4100 + 0 6 Q w-CTX MVIIC 0.25 0 + 0 6 T. R Nickel nd 0 + 0 5 N co-CTX GVIA 1 40 + 4.0 15 All Cadmium 100 + 0 2100 + O The blocking effect of amiloride, nifedipine, co-agatoxin (AGA) IVA, m-conotoxin (CTX) MVIIC, and co-conotoxin (CTX) GVIA is expressed as percent reduction in the peak amplitude of the total calcium current + SEM. n corresponds to the number of cells for each condition. of the AP-induced inward Ca2+ current is shared by L and N type Ca2+ channels (Fig. 9~. P. Q. and T type Ca2+ channel antagonists were ineffective, suggesting that the remaining Ca2+ current is associated with Ca2+ influx through R type channels. Nifedipine (10 ,uM), an L type Ca2+ channel blocker, produced no measurable effect on either the AHPfaS~, the AHPmeuiUm, or the AHPs~oW. By contrast, c')-conotoxin-GVIA (0.5 ,uM), a selective N type Ca2+ channel blocker, always A B V The following concentrations of antagonists were used: nifedipine (10 ~M), co-conotoxin GVIA (0.5 ~M or 1 ,uM), nickel (50-500 ,(1M), and cadmium (100 ,uM). nd, not determined. Obliterated the AHPs~ow, and in ~50% of the neurons abol- ished the AHPme~iUm (about half of the AHPm~jUm are Ca2+- sensitive, see above), while leaving the AHPfaS~ unaffected (Fig. 9 and Table 4.~. These results indicate that the SK and BK type K+ channels are both regulated by Ca2+ influx through N type channels. BK channels are gated by influx Ca2+ directly (8), whereas SK channels are affected by influx Ca2+ indirectly (i.e., Ca2+ entering through N type VDCC triggers RY recep- tors to release Ca2+ from CICR pools). Such a sequence implies a functional coupling between N type Ca2+ channels Contro! Nifedipine 6~-CTX GVIA Cadmium Control CdCI2 Wash m-C1X GVIA Wash ~. , 1~ ~ C c~ o I Control Nifedipine Control 2ms L m-CTX GVIA 5 s . _ _ _ ~1 ~1 o I ~ 1 4s 4s FIG. 9. Effects of VDCC antagonists on AP-induced calcium currents, AHPs~ow and AP-induced Ca2+ transients. (A) Inward calcium currents recorded in isolated nodose neurons evoked by a prerecorded AP waveform from a holding potential of - 60 mV. From Left to Right, control inward current in the presence of 2 mM tCa2+30 and in the presence of 10 ,uM nifedipine. After reestablishing control conditions, the neuron was exposed to 1 ,uM co-conotoxin-GVIA. The effects of 500 ,uM cadmium were recorded in another neuron; the control current for this cell was similar to the first trace. (B) AHPs~ow evoked by a train of four APs (10 Hz) recorded in another nodose neuron. From Left to Right, AHPs~ow evoked in control conditions, in the presence of 100 ,uM CdC12, after washout, in the presence of 500 nM co-conotoxin-GVIA, and after washout. (C) AP-induced Ca2+ transients recorded in two nodose neurons. From Left to Right, Ca2+ transients evoked by a train of eight APs in normal Locke solution, and in Locke solution containing 10 ,uM nifedipine. In another neuron, 1 ,uM co-conotoxin-GVIA reduced the Ca2+ transient ~50% (see Table 4~. APs were evoked by 2.5-ms, 10-Hz depolarizing current pulses.

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7656 Colloquium Paper: Cordoba-Rodriguez et al. and RY channels in the endoplasmic reticulum. We tested this proposition by examining the effects of VDCC antagonists on the magnitude of AP-induced Decal. Ca2+ influx through both L and N type Ca2+ channels triggers CICR. The magnitude of the focal is a sensitive indicator of Ca2+ release from the CICR pool. To determine the relative influence of Ca2+ influx through L and N type channels on release from the CICR pool, we applied selective VDCC antagonists and monitored the amplitude of /\Ca~. Nifedipine (10 EM) and co-conotoxin-GVIA (0.5-1.0 EM) diminished the amplitude of the ACa~ by 57% and 39%, respectively (Fig. 9 and Table 4~. These results reveal that Ca2+ entering through either L or N type Ca2+ channels provides "trigger" Ca2+ to stimulate CICR. Given that the amount of Ca2+ influx through L and N type Ca2+ channels is comparable (44% and 40%, respectively, of total AP-induced Ca2+ influx; see Table 3), there must be a remarkable spatial arrangement between plasma membrane N type Ca2+ channels, endoplas- mic reticulum RY receptors, and plasma membrane SK chan- nels. Our working hypothesis concerning the regulation of the AHPs~ow by Ca2+ is illustrated schematically in Fig. 10. DISCUSSION Whether recorded in intact vagal sensory ganglia or in acutely isolated vagal afferent somata (nodose neurons), single APs can elicit an AHPs~ow that exhibits a delayed onset (50-300 ms), a slow time to peak amplitude (0.3-0.5 s), and a particularly long duration (2-15 s) (14, 21~. Inhibition of the AHPs~ow by numerous inflammatory mediators (e.g., bradykinin, prosta- noids, histamine, serotonin, leukotriene C4; see Table 1) results in an increased neuronal excitability and a loss of spike-frequency adaptation. Thus, modulation of the AHPs~ow by these mediators provides a mechanism for peripheral nociceptor sensitization that may underlie allergic inflamma- tion-induced hyperalgesia. One unresolved but important mechanistic question re- volves around the delayed onset and protracted duration of the AHPs~ow. Many of our studies of nodose AHPs~ow were per- formed with acutely dissociated adult neurons, which are essentially spherical structures lacking dendritic and axonal processes. Thus, the delayed onset of the AHPs~ow cannot be due to slow diffusion of Ca2+ from distal sites of influx to somal SK channels. The high temperature coefficient (Qua > 3.0) for the rising phase and the decay time constant of the nodose AHPs~ow (14) also argues against simple Ca2+ diffusion as an explanation for the slow kinetics of the AHPs~ow. The time Ca2+ / ~ i_ RYR I_ _~_ I Cacti_ Ca2+ ~CICR BE SKI FIG. 10. Schematic diagram of the relation between plasma mem- brane Ca2+ channels, BK, and SK potassium channels and endoplas- mic reticulum RY receptors in primary vagal afferent neurons. Single APs evoke Ca2+ influx through L and N type VDCCs. Ca2+ influx through either of these channels can trigger release of Ca2+ from the endoplasmic reticulum via RY receptors. Whereas BK channels are activated directly by Ca2+ entering the neuron via N type VDCC, SK channels are activated indirectly. SK channels require Ca2+ from CICR pools released after Ca2+ influx through N type channels. Proc. Natl. Acad. Sci. USA 96 (1999J course of the AHPs~ow could arise from unusual channel kinetics of the SK channels. This also appears unlikely if SK channels in nodose neurons have activation kinetics similar to those cloned from rat brain (224. Recombinant SK channels from rat brain have activation time constants that are orders of magnitude shorter than the rise time of the AHPs~ow. It is more likely that the time course of the AHPs~ow is a conse- quence of the fecal because of CICR. If the AHPs~ow is directly dependent on Ca2+ released from the CICR pool, the AHPs~ow and the AP-induced rise in [Ca2+]i should display similar kinetics. Quantitative kinetic compari- sons between these two variables are subject to some uncer- tainty, because the time course of the /\Ca~ reflects global changes in [Ca2+ iT, whereas the kinetics of the AHPs~ow are determined by events at the plasma membrane. Nonetheless, we determined the time-to-peak and 10-to-90% decay time for both the AHPs~oW and the i\Ca~ elicited by one to eight APs (19~. The time-to-peak for AHPs~ow was significantly slower than the ACa~ by nearly a factor of a two (1.0 s vs. 1.9 s); the i\Ca~ also decayed more rapidly than the AHPs,oW (3 s vs. 7 s). Analogous temporal discrepancies have been reported be- tween the fecal and AHPs,oW in vagal motoneurons (23~. Such temporal differences suggest that Ca2+ released from CICR pools does not act alone to gate AHPs~ow K+ channels. Cloned SK channels contain many potential phosphorylation sites (15~; Ca2+-dependent phosphorylation and/or dephosphory- lation may thus be additional processes in the signal- transduction pathway of AP-evoked AHPs~ow. Unambiguous data now exist showing that Ca2+ can directly activate SK channels in hippocampal neurons (24) and in Xenopus oocytes (224. In nodose neurons, it is less clear whether Ca2+ alone is sufficient to activate and sustain the AHPs~ow after an AP. In hippocampal neurons, flash photolysis of a "caged" Ca2+ chelator immediately truncates AP-induced AHPs~ow' suggesting that elevated intracellular Ca2+ is re- quired to maintain the AHPs~ow (25~. These results do not, however, distinguish between continuous Ca2+ gating of SK channel and the involvement of other Ca2+-dependent factors sustaining the longevity of the AHPsjow. It is also possible that Ca2+-dependent factors act synergistically with Ca2+ to control SK channels (234. The nearly spherical morphology and large size of acutely isolated adult nodose neurons provide a favor- able preparation to determine the nature of second messen- gers required to activate and sustain the AHPs~ow. In conclusion, a subset of vagal primary afferent neurons possess a slowly developing and long-lasting spike afterhyper- polarization, the AHPs~ow' that can profoundly affect the discharge frequency of these visceral afferent neurons. Al- though AP-evoked Ca2+ influx via both L and N type Ca2+ channels triggers CICR, only Ca2+ flux through N type chan- nels activates the CICR-dependent AHPs~ow. This type of specificity suggests that spatial coupling between N type Ca2+ channels and SK channels may be critical for the generation of the AHPs~ow in nodose neurons. The exact mechanism coupling ACa~ to the AHPs~ow current remains to be determined. We thank our coworkers who participated in many of the experi- ments described in this manuscript: Drs. Akiva Cohen, Samir Jafri, and Bill Wonderlin, and Mr. Glen Taylor. The authors also thank Dr. Liz Katz and Mr. Eric Lancaster for their constructive suggestions on an earlier draft of this manuscript. This work was supported by National Institutes of Health Grants GM-46956 to J.P.Y.K., NS-22069 to D.W. and Training Grant NS-07375 to K.A.M. 1. Undem, B. J. & Riccio, M. M. (1997) inAsthma, eds. Barnes, P. J., Grunstein, M. M., Leff, A. & Woolcock, A. J. (Lippincott, Philadelphia), pp. 1009-1026. 2. Weinreich, D. (1995) Pulm. Pharmacol. 8, 173-179. 3. Undem, B. J., Hubbard, W. & Weinreich, D. (1993) J. Auton. Nerv. Syst. 44, 35-44.

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Colloquium Paper: Cordoba-Rodriguez et al. 4. Weinreich, D., Moore, K. A. & Taylor, G. E. (1997) J. Neurosci. 17, 7683-7693. 5. Moore, K. A., Taylor, G. E. & Weinreich, D. (1999) J. Physiol. (London) 514.1, 111-124. 6. Greene, R., Fowler, J. C., MacGlashlan, D., Jr. & Weinreich, D. (1988) J. Appl. Physiol. 64, 2249-2253. 7. Sah, P. (1996) Trends Neurosci. 19, 150-154. 8. Blatz, A. L. & Magleby, K. L. (1987) Trends Neurosci. 10, 463-467. 9. Gold, M. S., Shuster, M. J. & Levine, J. D. (1996) Neurosci. Lett. 205, 161-164. 10. Villiere, V. & McLachlan, E. M. (1996) J. Physiol. (London) 76, 1924-1941. 11. Coleridge, J. C. G. & Coleridge, H. M. (1984) Rev. Physiol. Biochem. Pharmacol. 99,1-110. 12. Weinreich, D. & Wonderlin, W. F. (1987) J. Physiol. (London) 394, 415-427. 13. Higashi, H., Morita, K. & North, R. A. (1984) J. Physiol. (London) 355, 479-492. 14. Fowler, J. C., Greene, R. & Weinreich, D. (1985) J. Physiol. (London) 365, 59-75. Proc. Natl. Acad. Sci. USA 96 (1999) 7657 17. Shmigol, A., Verkhratsky, A. & Isenberg, G. (1995) J. Physiol. (London) 489, 627-636. 18. Scroggs, R. S. & Fox, A. P. (1992) J. Neurosci. 12, 1789-1801. 19. Moore, K. A., Cohen, A. S., Kao, J. P. Y. & Weinreich, D. (1998) J. Neurophysiol. 79, 688-694. 20. Dunlap, K., Luebke, J. I. & Turner, T. J. (1995) Trends Neurosci. 18, 89-98. 21. Leal-Cardosa, H., Koschorke, G. M., Taylor, G. & Weinreich, D. (1993) J. Auton. Nerv. Syst. 45, 29-39. 22. Hirschberg, B., Maylie, J., Adelman, J. P. & Marrion, N. V. (1998) J. Gen. Physiol. 111, 565-581. 23. Lasser-Ross, B., Ross, W. N. & Yarom, Y. (1997) J. Neurophysiol. 78, 825-834. 24. Marrion, N. V. & Tavalin, S. J. (1998) Nature (London) 395, 900-905. 25. Lancaster, B. & Zucker, R. S. (1994) J. Physiol. (London) 475, 229-239. 26. Weinreich, D., Koschorke, G. M., Undem, B. J. & Taylor, G. E. (1995) J. Physiol. (London) 483.3, 735-746. 27. Jafri, M. S., Moore, K. A., Taylor, G. E. & Weinreich, D. (1997) 15. Kohler, M., Hirschberg, B., Bond, C. T., Kinzie, J. M., Marrion, N. V. & Adelman, J. P. (1996) Nature (London) 273, 1709-1714. J. Physiol. (London) 503.3, 533-546. 16. Cohen, A. S., Moore, K. A., Bangalore, R., Jafri, M. S., Wein- 28. Christian, E. P., taylor, G. E. & Weinreich, D. (1989) J. Appl. retch, D. & Kao, J. P. Y. (1997) J. Physiol. (London) 499, 315-328. Physiol. 67, 584-591.