The Journal of Neurophysiology Vol. 78 No. 5 November 1997, pp. 2396-2401
Copyright ©1997 by the American Physiological Society

Inhibitory Effects of Arachidonic Acid on Nicotinic Transmission in Bullfrog Sympathetic Neurons

Shoichi Minota and Sadahiro Watanabe

Division of Basic Medical Science, Kobe City College of Nursing, Nishi-ku, Kobe 651-21 Japan

	ABSTRACT

Abstract Introduction Methods Results Discussion References

Minota, Shoichi and Sadahiro Watanabe. Inhibitory effects of arachidonic acid on nicotinic transmission in bullfrog sympathetic neurons. J. Neurophysiol. 78: 2396-2401, 1997. Arachidonic acid (AA, 0.2-40 µM) reversibly reduced the amplitude of the fast excitatory postsynaptic potentials and the underlying currents (fast EPSCs) of bullfrog sympathetic neurons evoked by preganglionic nerve stimulation in a Ca²⁺-deficient solution. AA reduced the acetylcholine (ACh)-induced nicotinic currents (nI_ACh) evoked by brief applications of ACh to the ganglion cells in a dose-related manner. AA reduced the maximum amplitude of nI_ACh estimated from the dose-response relationship without causing an appreciable change in the apparent dissociation constant. Indomethacin (2 µM) and nordihydroguaiaretic acid (20 µM), blockers of cyclooxygenase and lipoxygenase pathways, respectively, had no effect on the inhibition of fast EPSC by AA. AA did not obviously affect the preganglionic nerve terminal spike configuration, synaptic delay, facilitation, quantal content of transmitter release, or the presynaptic long-term potentiation elicited by the repetitive stimulation applied to the preganglionic nerve fibers. These results suggest that AA acts on an allosteric site of the nicotinic receptor-channel complex either directly or indirectly and in turn inhibits ion permeation through these channels without affecting the release of ACh from preganglionic nerve terminals.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

Arachidonic acid (AA), a cis-unsaturated fatty acid, can be released from the plasma membrane by activation of either phospholipase A₂ (PLA₂) or a combination of phospholipase C and diacylglycerol lipase (Axelrod 1990; Axelrod et al. 1988). The released AA can be metabolized via both cyclooxygenase and lipoxygenase pathways, resulting in the formation of prostaglandins and leukotrienes (Axelrod et al. 1988; Piomelli and Greengard 1990; Shimizu and Wolfe 1990). AA enhances the activity of protein kinase C in human neutrophils (McPhail et al. 1984) and in neuroblastoma cells (Linden and Routtenberg 1989). It also releases Ca²⁺ from the internal storage site, possibly in cooperation with inositol 1,4,5-triphosphate in islet cells of the pancreas (Maruyama 1993). In the CNS, AA released from postsynaptic cells is thought to be a retrograde messenger where its function is to maintain long-term potentiation (LTP) in hippocampal neurons (Williams et al. 1989). AA also modulates, directly or indirectly, the activity of various types of ion channels (Ordway et al. 1991). AA reduces activity of the voltage-dependent Ca²⁺ channels of rabbit intestinal smooth muscle cells (Shimada and Somlyo 1992), Na⁺ channels of cultured striatal neurons (Fraser et al. 1993), and -aminobutyric acid-gated Cl channels in cerebral cortical synaptoneurosomes (Schwartz and Yu 1992). On the other hand, AA enhances the activity of M channels in hippocampal neurons (Schweitzer et al. 1990) and in bullfrog sympathetic neurons (Yu 1995), outwardly rectifying K⁺-selective channels in rat atrial cells (Kim and Clapham 1989) and N-methyl-D-aspartate (NMDA) receptor-mediated currents in cerebellar granule cells (Miller et al. 1992). An inhibitory action of AA on acetylcholine (ACh) receptor function was shown by means of ²²Na⁺ efflux from membrane vesicles prepared from Torpedo electroplax (Andreasen and McNamee 1980) and by catecholamine release from bovine adrenal medulla (Ehrengruber and Zahler 1991). Vijayaraghavan et al. (1995) reported that 20 µM AA reversibly inhibited the nicotine-induced responses in cultured chick ciliary ganglion neurons dissociated from embryos. They further demonstrated that AA at low concentrations also reversibly inhibited 7-containing receptors expressed in Xenopus oocytes after injection of 7 cRNA. However, the effects of AA on nicotinic receptor-mediated responses in sympathetic ganglion neurons have not been studied in detail.

The present study was undertaken to study the action of AA on nicotinic receptor-mediated synaptic transmission in bullfrog sympathetic neurons.

Some of these data have appeared in an abstract (Minota 1992, 1993, 1996).

	METHODS

Abstract Introduction Methods Results Discussion References

Bullfrogs (Rana catesbeiana) were used. After decapitation and pithing, the sympathetic chains, including the paravertebral sympathetic ganglia, were isolated rapidly. The ninth or tenth sympathetic ganglia with their preganglionic nerve trunks were mounted in a small chamber and connective tissues were removed by fine forceps without the use of collagenase and/or trypsin. Single-electrode current- or voltage-clamp recordings were made from B-type neurons, which were impaled with conventional microelectrodes filled with 3 M KCl (tip resistance 30-60 M). Neurons that showed a resting membrane potential more negative than 55 mV were used in the experiments.

A Nihon Kohden CEZ-3100 amplifier was used for both current- and voltage-clamp experiments. The discontinuous single-electrode voltage clamp was employed at a sampling frequency of 5-6 kHz. The changes of both voltage and current were recorded continuously by the use of pen-writing recorder (Nihon Kohden RJG-4024, flat response 100 Hz) and stored on FM tape (Sony NFR3515W, 1 kHz) for later analysis.

The composition of the Ringer solution was (mM) 115.5 NaCl, 2 KCl, 1.8 CaCl₂, and 2.4 NaHCO₃, pH 7.4. A Ca²⁺-deficient solution was prepared by a mixture of the Ringer solution and a Ca²⁺-free and high-Mg²⁺ solution. In preparing a Ca²⁺-free and high-Mg²⁺ solution, CaCl₂ was omitted from the Ringer and 12 mM MgCl₂ was added; osmolarity was adjusted by changing NaCl concentration. Atropine (0.1 µM) sometimes was added to the Ringer solution to block muscarinic receptors.

The iontophoretic electrode filled with 2 M ACh (tip resistance 35-50 M) was positioned within 50 µm of the recording cell. To minimize the leakage of ACh, a negative retaining DC current (10-20 nA) was applied continuously to the ACh electrode. The ACh was applied iontophoretically by a positive current pulse with a duration of 500 ms, and the amplitude of the pulse was varied, often 250 nA. The fast excitatory postsynaptic potentials (EPSPs) were elicited by stimulating the preganglionic chain above the seventh ganglion through a glass suction electrode in a Ca²⁺-deficient solution.

When repetitive stimuli with brief interval were applied to the presynaptic nerve, the synaptic potentials at first grow in amplitude. This growth of synaptic potential is called "facilitation" (Katz 1966) and is caused by an increase in transmitter release during repetitive nerve excitation. To examine the effects of AA on facilitation of the fast excitatory postsynaptic current (EPSC), facilitation of the fast EPSC was induced by paired pulses applied at an interval of 50 ms every 3 s in a Ca²⁺-deficient solution. Because the fast EPSC amplitude fluctuated in a Ca²⁺-deficient solution, the magnitude of facilitation was calculated from averaged values of 32 consecutive paired responses. The magnitude of facilitation was expressed as the ratio of the amplitude of the second fast EPSC (E₂) over the first one (E₁), i.e., (E₂/E₁  1) × 100.

The effects of AA on the transmitter release were examined by means of the quantal analysis. The quantal content of the fast EPSPs induced at 0.2 Hz was calculated by variance and/or failure methods in applying Poisson's law using a computer (NEC 9801) after digitization of the records. Data were analyzed for each sample of 60 consecutive responses (in a 5-min period) obtained in a Ca²⁺-deficient solution. If the failure of a response was <1% or the recording was accompanied by a high basal noise, the data were analyzed only by the variance method (Koyano et al. 1985 for detailed methods). The apparent quantal size was computed by dividing mean amplitude of the fast EPSP by quantal content.

An increase in synaptic efficacy lasting more than hours as a result of tetanic synaptic activities was first found in the hippocampus and termed LTP (see review by Bliss and Lynch 1988). In bullfrog sympathetic ganglia, a long-lasting increase in amplitude of the fast EPSP also occurred after repetitive stimulations applied to the preganglionic nerve fibers. This increase in synaptic efficacy resulted from an increase in the release of transmitter and termed the presynaptic LTP (pre-LTP) (Koyano et al. 1985). In the present experiment, the pre-LTP was induced by repetitive stimulation applied to the preganglionic nerve fibers (33 Hz for 5 s) in a Ca²⁺-deficient solution.

The drugs used were AA (Sigma), ACh chloride (Wako, Japan), indomethacin (Sigma), nordihydroguaiaretic acid (NDGA; Sigma), and atropine sulfate (Tokyo Kasei, Japan). Stock solution of AA was prepared in dimethyl sulfoxide (DMSO) and stored under N₂ atmosphere in the dark at 20°C. When required, this stock was thawed and added to the superfusion solution, which was sonicated for 1 min immediately before applying it to the cells. Stock solutions of indomethacin and NDGA also were kept dark at 20°C. The drugs were added to the superfusion solution from stock solutions, and the final DMSO concentration in the bath perfusion solution was 0.1%; this concentration did not affect the transmitter release and the membrane excitability (Minota et al. 1991). Tetrodotoxin (0.1 µM; Sankyo, Japan), if necessary, was added to the Ringer solution to block voltage-dependent sodium currents.

	RESULTS

Abstract Introduction Methods Results Discussion References

AA inhibits nicotinic synaptic transmission

Figure 1 shows the decrease in amplitude of the fast EPSPs (Fig. 1A) and the fast EPSCs (Fig. 1B) in the presence of 40 µM AA. Fast EPSPs or EPSCs were elicited continuously by single-electrical stimulation applied to the preganglionic nerve fibers every 5 s in a Ca²⁺-deficient solution. Because of the amplitude of synaptic responses induced by preganglionic nerve stimulations fluctuated in a Ca²⁺-deficient solution, 16-32 consecutive synaptic responses were averaged and shown in Fig. 1. AA reversibly reduced the amplitude of both fast EPSPs and fast EPSCs. Themean reduction of fast EPSPs and fast EPSCs was 43 ± 6%(mean ± SD; n = 8) and 42 ± 6% (n = 15), respectively, 15 min after start of perfusion of 20 µM AA.

View larger version (9K): [in this window] [in a new window]   FIG. 1. Inhibition of fast excitatory postsynaptic potentials (EPSPs) and fast excitatory postsynaptic currents (EPSCs) by 40 µM arachidonic acid (AA) in sympathetic neuron. A: 32 consecutive fast EPSPs were averaged in control and wash, whereas 16 consecutive fast EPSPs recorded 8 min after application of 40 µM AA were averaged. Wash was obtained 20 min after removal of AA. Resting membrane potential was 58 mV. B: 32 consecutive fast EPSCs recorded before, 12 min after application of 40 µM AA, and 16 min after removal of AA were averaged. Same neuron in A was held at a holding potential of 58 mV. Preganglionic nerve fibers were stimulated every 5 s in a Ca2+-deficient solution.

In addition to reducing the amplitude of fast EPSCs, AA elicited an outward current in 13 of 21 neurons. The amplitude of outward current measured 15 min after start of perfusion of 20 µM AA was 0.09 ± 0.07 nA (SD; n = 13) at a holding potential of 60 mV. AA did not affect the holding current in the remaining neurons (8 of 21) examined, although it reduced the amplitude of fast EPSCs. Under the current-clamp condition, AA (20 µM) hyperpolarized the membrane in 7 of 17 neurons examined with a mean of 3.1 ± 1.1 (SE) mV and caused no detectable change in the remaining 10 neurons.

A noncompetitive inhibitor

The effect of AA on the acetylcholine (ACh)-induced nicotinic currents (nI_ACh) induced by brief iontophoretic application of ACh to the cells was examined in the presence of the muscarinic receptor blocker atropine (0.1 µM). AA (0.2-20 µM) reduced the amplitude of nI_ACh in a concentration-dependent manner as shown in Fig. 2. Twenty micromolar AA reduced the nI_ACh to 16% of the control. The mean reduction was 88 ± 14% (n = 9) at a holding potentialof 60 mV, which was about two times larger than that of the inhibition of fast EPSC by AA. Kuffler and Yoshikami (1975) demonstrated a linear relationship between the charge applied to the iontophoretic pipette and the dose of ACh extruded from the pipette. We assumed that there is a linear relationship between the charge applied to the pipette and the dose of ACh in the present experiment and constructed the dose-response curves from the data obtained in the presence and the absence of AA. As seen in Fig. 3A, the amount of ACh released from the ACh pipette by brief current pulse was not enough to produce a maximum response due to a limitation of the iontophoretic methods used here. Therefore it is difficult to directly determine whether AA acts on the maximum response or on the affinity of ACh for the receptors. However, it may be possible to estimate the effects of AA on these factors if the Lineweaver-Birk plot is constructed. When the Lineweaver-Birk plot was constructed with the assumption of a Hill number of 2.0, there was a linear relationship between the reciprocal value of the amplitude of nI_ACh and the square of current intensity used for ACh application (Fig. 3B). The points of intersection of each straight line with vertical and horizontal axis represent the reciprocal values of the maximum amplitude of the nI_ACh and the apparent dissociation constant (appKm) for ACh, respectively. It was clear that AA reduced the maximum nI_ACh without affecting the appKm. The exact value of the appKm could not be determined because the concentration of ACh around neurons was not known under the present experimental conditions.

View larger version (14K): [in this window] [in a new window]   FIG. 2. Effects of 0.2, 2 and 20 µM AA on acetylcholine (ACh)-induced nicotinic currents (nIACh). Bars in each record indicate the period of ACh application (duration of 500 ms). Magnitude of current strength for iontophoretic application of ACh were shown (left). nIAChs were induced every 10 s in the Ringer solution. Holding potential was 57 mV.

View larger version (16K): [in this window] [in a new window]   FIG. 3. Concentration-response data for the inhibition of nIACh by AA. A: peak responses of nIACh in the absence and presence of AA shown in Fig. 2 were measured and plotted against current strength (nano-coulomb; nC) of ACh applications. B: Lineweaver-Birk plot was constructed from the data shown in A. Linear lines could be drawn in all cases when Hill number was assumed to be 2.

AA-pathway inhibitors

When the neurons were incubated with 20 µM NDGA, a blocker of lipoxygenase pathway, the amplitude of fast EPSCs was increased slightly. However, NDGA did not block the inhibition of the fast EPSCs induced by AA (Fig. 4A). The mean reduction of fast EPSCs induced by AA in the presence of 20 µM NDGA was 41 ± 5% (n = 4) 10 min after start of perfusion of 40 µM AA. The inhibitory effect was reversible within 20 min after removal of AA from a Ca²⁺-deficient solution containing NDGA. Indomethacin (2 µM), a blocker of cyclooxygenase pathways, also did not prevent the blocking action of AA on the fast EPSCs (Fig. 4B). The mean reduction of fast EPSCs induced by AA in the presence of 2 µM indomethacin was38 ± 12% (n = 4) 10 min after start of perfusion of 40 µM AA. The recovery of fast EPSCs could not be observed 20 min after removal of AA from a Ca²⁺-deficient solution containing indomethacin.

View larger version (10K): [in this window] [in a new window]   FIG. 4. Effects of AA-pathway inhibitors on the inhibition of fast EPSCs by AA. Twenty micromolar nordihydroguaiaretic acid (NDGA; A) and 10 µM indomethacin (B) did not significantly block the effect of AA. Forty micromolar AA reduced the fast EPSCs in the presence of NDGA (A) and indomethacin (B). Recovery of fast EPSCs in B could not be observed 20 min after removal of AA in the presence of indomethacin (not shown). Sixteen consecutive fast EPSCs were averaged. Fast EPSCs in A and B were elicited by stimulation of preganglionic nerve fibers every 3 and 4 s, respectively, in a Ca2+-deficient solution.

Excitability of preganglionic nerve fibers

The effect of AA on the spike potential originating in nerve fibers was examined. When the preganglionic nerve fibers were stimulated, the small biphasic electrical response could be recorded immediately before generation of the fast EPSC in some neurons (Fig. 5, ). This small spike response reflects the impulse arriving at preganglionic nerve terminals that contact the neuron impaled by the recording electrode (Katz and Miledi 1965). Each record in Fig. 5 shows the average recording of 32 consecutive recordings. Records on the right were obtained at higher amplification than those on the left. The time interval between the artifact of electrical stimulus and small spike response was not changed by 20 µM AA. Thus AA had no effect on the conduction of action potential along the preganglionic nerve fibers. The conduction velocity shown in Fig. 5 was 4.8m/s. The mean conduction velocity was 6.3 ± 1.8 m/s(n = 5). Similarly, AA did not change the synaptic delay (1.3 ms) estimated from the time interval between the generation of small spike response and the initiation of the fast EPSC (Fig. 5). The mean synaptic delay was 1.3 ± 0.2 ms(n = 5). Furthermore, AA did not affect either the amplitude or configuration of small spike response.

View larger version (7K): [in this window] [in a new window]   FIG. 5. Effects of 20 µM AA on the spike response of preganglionic nerve terminals obtained 15 min after application of AA. Initial part of the records (left) was shown on expanded current scale (right). , spike responses originating in the preganglionic nerve terminals. Thirty-two consecutive fast EPSCs were averaged.

ACh release

The action of AA on paired pulse-facilitation of transmitter release was examined. The facilitation was induced by paired pulses applied at an interval of 50 ms. As seen in Fig. 6, the amplitude of the fast EPSC induced by the second pulse of paired pulses was larger than that of the first one, indicating the existence of facilitation. The magnitude of facilitation, which was calculated from averaged values of 32 consecutive paired responses, fluctuated in the range between 90 and 120% in the control condition. AA (20 µM) did not significantly affect the magnitude of facilitation, although it reduced the amplitude of fast EPSC (Fig. 6).

View larger version (6K): [in this window] [in a new window]   View larger version (9K): [in this window] [in a new window]   FIG. 6. Effects of AA on facilitation of fast EPSCs. Top: currents obtained before and during application of 20 µM AA at 56 mV, respectively. Paired pulses having an interval of 50 ms were applied continuously to the preganglionic nerve fibers every 3 s in a Ca2+-deficient solution. Thirty-two consecutive fast EPSCs were averaged in each record. Bottom: changes in the magnitude of facilitation before, during, and after application of 20 µM AA.

The quantal analysis of fast EPSPs was carried out. As shown in Fig. 7, the amplitude of fast EPSPs () decreased in the presence of 10 µM AA, whereas the quantal content of transmitter release () was not significantly changed. However, the apparent quantal size () was decreased to the same extent as the fast EPSPs. The time course of the reduction of quantal size seemed to parallel that of the amplitude of the fast EPSPs.

View larger version (16K): [in this window] [in a new window]   FIG. 7. Effects of AA on the pre-long-term potentiation (pre-LTP) in sympathetic neuron. The changes in the amplitude (), quantal content (), and quantal size () of fast EPSPs before and during application of 10 µM AA are shown as a value relative to the mean before application of AA. Preganglionic nerve fibers were continuously stimulated every 5 s in a Ca2+-deficient solution. Presynaptic tetanus (33 Hz, 5 s) was applied 51 min after start of AA-perfusion.

The large increase in the transmitter release that occurs after repetitive presynaptic activity in bullfrog sympathetic ganglia is termed the pre-LTP (see METHODS) (Koyano et al. 1985). AA in the concentration of 10 µM not only didn't affect the generation of pre-LTP, but also didn't affect the maintenance of it (Fig. 7). Furthermore, the magnitude of pre-LTP measured 20 min after tetanus (33 Hz, 5 s in duration) in the presence of AA was 35%, which was within the range of the magnitude obtained in the absence of AA (see Koyano et al. 1985).

	DISCUSSION

Abstract Introduction Methods Results Discussion References

The present experiment demonstrated the inhibitory action of AA on nicotinic synaptic transmission in bullfrog sympathetic neurons.

Micromolar levels of AA reduced the amplitude of fast EPSPs and associated current to ~55% of the control in a Ca²⁺-deficient solution (Fig. 1). AA was more effective in reducing the amplitude of nI_ACh to ~15% of the control in the Ringer solution (Figs. 2 and 3). The observation that AA suppresses the nI_ACh indicates that AA acts on the nicotinic receptor-channel complex rather than the presynaptic nerve terminals in sympathetic ganglia. According to the results obtained from the Lineweaver-Birk plot, AA reduced the maximum response of nI_ACh without affecting the appKm for ACh (Fig. 3B). These results suggest that AA does not alter the affinity of the nicotinic receptor for ACh. In the present study, the dose-response curves were constructed by assuming a linear relationship between the charge applied to the pipette and the dose of ACh extruded from the pipette. If there is not direct proportionality between the charge and the dose of ACh, the Lineweaver-Birk curve may not be constructed by simple manner done in this study. In this case, a possibility is not necessary ruled out that AA may inhibit the affinity of the nicotinic receptor for ACh.

The mechanism by which AA inhibits the nicotinic transmission is unknown. One possibility is that AA binds to allosteric sites of the nicotinic receptor-channel complex, such as a channel pore, and in turn inhibits synaptic transmission without affecting the binding of ACh to the nicotinic receptors. Another possibility is that AA perturbs the local environment of the receptors by dissolving into the membrane and in turn indirectly inhibits the receptor functions. We do not have direct evidence at present to support the contention either directly or indirectly. Ehrengruber and Zahler (1991) reported that 10 µM AA blocked receptor-mediated Ca²⁺ influx and the secretion of catecholamine measured fluorimetrically in bovine chromaffin cells, probably by directly blocking the nicotinic channels. Vijayaraghavan et al. (1995) recently reported that the nicotine-induced response in chick ciliary neurons dissociated from embryos was inhibited reversibly in the presence of AA. These results are consistent with the present result obtained electrophysiologically by intracellular recording from single sympathetic neurons.

The magnitude of inhibition of nI_ACh by AA was larger than that of fast EPSCs. This may suggest that subtypes of ACh receptors (AChRs) are localized to synaptic versus nonsynaptic regions of postsynaptic neurons and that they differ in their sensitivity to AA. The fast EPSC would result from activation of AChRs located in synaptic regions. On the other hand, if there are abundant AChRs located in nonsynaptic regions on the sympathetic neurons, nI_ACh would be mediated primarily through activation of these receptors. The ciliary neurons have two major classes of nicotinic AChRs. One class is primarily synaptic in location and binds to the monoclonal antibody (mAb) 35, and contains the 3, 4, and 5 AChR gene products (Halvorsen and Berg 1987; Loring and Zigmond 1987; Sargent 1993; Vernallis et al. 1993). The second class of receptors, termed Bgt-AChRs, is 5- to 10-fold more abundant, binds -bungarotoxin (Bgt), and is located primarily in nonsynaptic regions on the neurons (Dun and Karczmar 1980; Jacob and Berg 1983; Loring et al. 1985). Bgt-AChRs contain only 7 of known neuronal AChR gene products (Vernallis et al. 1993; Zhang et al. 1994, 1996). Micromolar concentrations of AA reversibly block both Bgt-AChRs and mAb 35-AChRs, but the effect of AA was most pronounced on Bgt-AChRs (Vijayaraghavan et al. 1995). Viewed in this context, AA may inhibit the nI_ACh more effectively than the fast EPSC.

Zhang et al. (1994, 1996) reported that 7 AChRs on ciliary ganglion cells cannot be activated readily with application of ACh from a puffer pipette or from a distance and have shown that they might be activated either by very rapid application of ACh with a sewer pipe-solenoid system or by nerve released ACh. If the same 7 AChRs exist on the bullfrog sympathetic ganglion neurons, they might be activated by nerve released ACh but could not be activated when the ACh is applied by iontophoresis through the ACh pipette used in this experiment. In this case, AA may inhibit the fast EPSC more effectively than the nI_ACh. This is the inverse result compared with the present observation. Therefore, it seems that the nicotinic AChRs located in nonsynaptic regions on bullfrog sympathetic neurons may possess different properties from that of the AChRs on ciliary neurons and might be activated by iontophoretic application of ACh.

The inhibition of the nicotinic responses by AA had a relatively slow time course as compared with that reported by Vijayaraghavan et al. (1995). The reason is not known at present. The ganglion preparations dissociated from matured bullfrog were not treated with collagenase in the present experiment. Therefore the connective tissues including satellite cells still remain and cover tightly the sympathetic ganglion neurons. These connective tissues may interfere with rapid movement of AA to the surface of target neurons. Another speculation is that the sensitivity of the nicotinic AChRs on sympathetic neurons to AA may differ from that on ciliary neurons.

Indomethacin and NDGA, blockers of cyclooxygenase and lipoxygenase pathways, respectively, did not prevent the blocking action of AA on the nicotinic responses. These results suggest that AA directly regulates the nicotinic currents and that its metabolites do not play a major part in the AA-induced blockade of the nicotinic response in the bullfrog sympathetic ganglia. In this respect, the question whether other fatty acids, especially unsaturated fatty acids, may inhibit the nicotinic-receptor channels of bullfrog sympathetic neurons is of interest. In ciliary neurons, fatty acids having either no double bonds (stearic acid, arachidic acid) or only one double bond (oleic acid, elaidic acid) had little or no effect on the ACh response, and fatty acids having two (linoleic acid) or three (linolenic acid) double bonds produced partial inhibition but were not as effective as AA (Vijayaraghavan et al. 1995). Furthermore, the secretion of catecholamine by ACh in chromaffin cells has been shown not to be mediated by the metabolite of lipoxygenase pathway (Ehrengruber and Zahler 1991). These results are consistent with the present conclusion that AA may inhibit directly the nI_ACh without via breakdown products.

The observation that AA had no effect on the conduction of action potential along the preganglionic nerve fibers, the configuration of preganglionic nerve terminal spike response, or the synaptic delay suggests that AA may not affect the excitability of preganglionic nerve fibers and preganglionic nerve terminals in sympathetic ganglia. AA does not affect the magnitude of facilitation of fast EPSCs, although it inhibits the fast EPSC by ~50% (Fig. 6). The facilitation is thought to be caused by the residual Ca²⁺ that remains in the nerve terminal after a preceding impulse and enhances transmitter release by acting cooperatively with Ca²⁺ entering during a second impulse (Katz and Miledi 1968; Rahamimoff 1968). The lack of significant effect of AA on the facilitation suggests that AA does not modify the mobilization of Ca²⁺ in sympathetic preganglionic nerve terminals. The quantal analysis of the fast EPSPs showed that the quantal size but not quantal content of transmitter release was significantly reduced by AA. The decrease in the quantal size in the presence of AA seems to reflect the inhibition of the activity of nicotinic receptor-channel complex by AA rather than a decrease in the ACh concentration in individual synaptic vesicles. These results suggest that AA does not act on the release process of ACh in sympathetic preganglionic nerve terminals.

The binding of a neurotransmitter, glutamate, to the NMDA receptors raises intracellular Ca²⁺ concentration (Ascher and Nowak 1988; MacDermott et al. 1986) and leads to the activation of PLA₂, which can release AA from plasma membrane (Axelrod 1990; Axelrod et al. 1988). Released AA is thought to be a retrograde transmitter for the initiation of LTP in hippocampus (Williams et al. 1989). However, in the case of bullfrog sympathetic ganglia, AA did not significantly affect the pre-LTP evoked by repetitive stimulation applied to the preganglionic nerve fibers (Fig. 7). Furthermore, 4-bromophenacylbromide, an inhibitor of PLA₂, does not block the pre-LTP (not shown). These results suggest that AA may not have a major role in the generation and maintenance of pre-LTP in sympathetic neurons.

	ACKNOWLEDGEMENTS

  The authors express gratitude to Dr. N. J. Dun for reading the manuscript and for helpful comments.

	FOOTNOTES

  Address reprint requests to S. Minota.

  Received 12 March 1997; accepted in final form 7 July 1997.

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