ATP Mediates Fast Synaptic Potentials in Enteric Neurons

Home , Hexamethonium, Mecamylamine, Nicotinic antagonist

The Journal of Neuroscience, December 1994, 74(12): 7563-7571

James J. Galligan and Paul P. Bertrand Department of Pharmacology and Toxicology, Michigan State University, East Lansing, Michigan 48824

Conventional intracellular electrophysiological methods were (Jahr and Jessell, 1983; Ueno et al., 1992; Shen and North, used to study fast synaptic transmission in the myenteric 1993) to causeresponses similar to thosecaused by acetylcholine plexus of guinea pig ileum in vitro. Fast excitatory postsyn- (ACh) acting at nicotinic receptors.ATP-activated ion channels aptic potentials (f EPSPs) were evoked in 98 neurons follow- are present in cell-free membrane patchesfrom neurons, indi- ing single stimuli applied to interganglionic connectives. The cating a close coupling between the ATP binding site and the nicotinic antagonist hexamethonium (100 AM) reduced fEPSPs cation channel (Krishtal et al., 1988; Bean et al., 1990; Silinsky by 83% in 37 neurons; these f EPSPs were considered to be and Gerzanich, 1993). Fast excitatory postsynaptic potentials cholinergic. In 61 neurons, hexamethonium reduced fEPSPs (fEPSPs), mediated by ATP acting at P, receptors, have been by 33%; fEPSPs recorded in the presence of hexamethon- recorded from guinea pig celiac ganglion neurons (Evans et al., ium were considered to be noncholinergic. Similar data were 1992; Silinsky et al., 1992) and from neurons in the medial obtained using the nicotinic antagonist mecamylamine (10 habenula of the rat (Edwards et al., 1992). @I) to block fEPSPs. Hexamethonium or mecamylamine The enteric nervous system (ENS) is the division of the au- completely blocked depolarizations caused by acetylcholine tonomic nervous system residing in the wall of the gastrointes- (ACh) applied by ionophoresis. The P, receptor antagonist tinal tract (Langley, 1921). The ENS is composedof many neu- suramin (l-300 PM) inhibited noncholinergic fEPSPs in 30 rochemically and functionally different neuronsthat contain a cells; the suramin IC, was 4 PM. Suramin (100 PM) did not variety of potential neurotransmitters and expressreceptors for block depolarizations caused by ACh or 5-HT, but suramin thesesubstances (Fumess and Costa, 1987; Wood, 1989). ACh, blocked depolarizations caused by ATP. Hexamethonium acting at nicotinic receptors, is the principal excitatory neuro- did not block ATP-induced depolarizations. The estimated transmitter in the gut and ACh is believed to be the solemediator reversal potential for suramin-sensitive f EPSPs and ATP- of enteric fEPSPs (Wood, 1989). However, there are other sub- induced depolarizations was - 25 and - 16 mV, respectively. stancescontained in the ENS that cause responsessimilar to ATP responses were reduced in low-sodium (26 mM) extra- nicotinic responses.5-Hydroxytryptamine (5-HT), acting at cellular solution, suggesting that ATP activates a cation 5-HT, receptors, causesshort-latency, short-duration depolar- channel. These data indicate that in myenteric nerves ATP, izations that are due to an increase in a cation conductance in addition to ACh, contributes to fast synaptic transmission. (Mawe et al., 1986; Surprenant and Crist, 1988) and 5-HT- [Key words: enferic nerves, synaptic transmission, purine activated channels are present in cell-free patches of enteric receptors, ligand-gated ion channels, autonomic nervous neurons (Derkach et al., 1989). In the ENS, ATP also causes system, gastrointestinal tract] fast inward currents and activates cation channels in cell-free patchesof enteric nerves(Bertrand and Galligan, 1992; Barajas- ATP is a cotransmitter releasedwith norepinephrine from sym- Lopez et al., 1993, 1994). Although receptors mediating re- pathetic nervesthat mediate contractions of somesmooth mus- sponsessimilar to nicotinic responsesare present in the ENS, cle (Burnstock and Kennedy, 1986; von Kugelgen and Starke, enteric noncholinergic fEPSPs have not been identified (Evans 1991). ATP is an agonistat P, purine receptors(Bumstock and and Surprenant, 1992). Kennedy, 1985) and in smooth muscle cells, ATP acts on P, In the present study, conventional electrophysiologicalmeth- receptors to cause depolarization and contraction (Benham et ods were used to record fEPSPs from myenteric neurons of al., 1987; Friel, 1988). Some P, receptorsare likely to be ligand- guinea pig ileum in vitro. Acutely isolatedpreparations of myen- gated cation channels, as they are permeableto sodium, potas- teric plexus were used,as most synaptic connections present in sium, and calcium and mediate short-latency, short-duration the intact intestine are preserved. We have shown that ATP responses(Benham et al., 1987; Friel, 1988). ATP also acts at mimics fEPSPs and that the P, receptor antagonist suramin P, receptors on sensory neurons (Krishtal et al., 1988; Bean, (Dunn and Blakely, 1988; Hoyle et al., 1990)blocks somefEPSPs. 1990), autonomic neurons (Allen and Bumstock, 1990; Fieber We conclude that ATP is a mediator of noncholinergic fEPSPs and Adams, 1991 b; Nakazawa, 1994), and neuronsin the CNS in the ENS. Materials and Methods Received Mar. 25, 1994; revised May 23, 1994; accepted June 1, 1994. This work was supported by Grants DK40210 and NS01738 from NIH. We Tissuepreparation. Male guineapigs weighing 250-350 gm wereanes- thank John Beecher, Drug Services, Center for Disease Control, Atlanta, GA, for thetizedvia halothaneinhalation, stunned, and bled throughthe neck. the sift of suramin. A segmentof ileum wasexcised 10-20 cm proximal to the ileocecal C&respondence should be addressed to Dr. James J. Galligan, Department of junction and placedin oxygenated(95% O/5% CO,) Krebssolution of Pharmacology and Toxicology, Life Sciences Building B440, Michigan State Uni- the followingcomposition (millimolar): NaCl, 117;KCl, 4.7; CaCl,,2.5; versity, East Lansing, MI 48824. MgC&, 1.2; NaHCO,, 25; NaH,PO,, 1.2; glucose,11. The Krebsso- Copyright 0 1994 Society for Neuroscience 0270-6474/94/147563-09$05.00/O lution containednifedipine (1 PM) and scopolamine(1 PM) to block 7564 Galligan and Bertrand * ATP-mediated Fast Synaptic Potentials longitudinal muscle contractions and muscarinic receptors on myenteric was 3 PM at a membrane potential of -88 * 5 mV (n = 9). nerves during intracellular recordings. A 1.5 cm segment of ileum was Similar experiments were done using the nicotinic antagonist cut open along the mesenteric border and pinned out flat (mucosal surface up) in a petri dish lined with Silastic elastomer. A longitudinal mecamylamine, which also inhibited ACh-induced depolari- muscle-myenteric plexus preparation was made by stripping away the zations in a concentration-dependent manner (Fig. 1B). Mec- mucosal, submucosal, and circular muscle layers using fine forceps and amylamine (10 KM) completely blocked the ACh responseand scissors. A 5 mm* piece of longitudinal muscle myenteric plexus was the mecamylamine IC,, was 0.12 PM at a membrane potential transferred to a small recording chamber (2 ml volume) lined with of -88 f 6 mV (n = 7). These data indicate that hexamethon- Silastic elastomer. The preparation was stretched lightly and pinned to the chamber bottom using small stainless steel pins. The preparation ium and mecamylamine can completely block nicotinic recep- was superfused with 36°C oxygenated Krebs solution at a flow rate of tors activated by ACh when it is applied in a manner that mimics 7 ml/min. the amplitude and duration of fEPSPs. Intracellular recording. Individual myenteric ganglia were visualized ACh was applied by ionophoresisin the absenceand presence at 200 x magnification using an inverted microscope (Nikon Diaphot or Olympus CK-2) with differential interference contrast optics (Hoff- of the P, receptor antagonist suramin (Fig. 2A). Suramin did man Modulation). Intracellular recordings were obtained from single not changethe amplitude of the ACh responsein any neuron neurons using glass microelectrodes filled with 2 M KC1 (tip resistance, tested (Fig. 2B). Furthermore, suramin (100 PM) did not block 80-l 20 MQ). An amplifier with an active bridge circuit (Axoclamp 2A, depolarizations causedby 5-HT (control, 23 ? 1 mV; suramin, Axon Instruments, Foster City, CA) was used for membrane potential 23 + 5 mV, II = 2). The 5-HT depolarizations were blocked by recordings and for injection of current into neurons via the recording microelectrode. In most experiments, the membrane potential was ad- the 5-HT, receptor antagonist ondansetron (1 PM) (control, 26 justed to a potential negative to -70 mV using constant DC current to + 4 mV, ondansetron, 6 +- 4 mV, n = 3, p < 0.01). avoid action potentials when evoking fEPSPs. Signals were filtered at 1 kHz using a 4-pole Bessel filter (Warner Instruments, New Haven, Noncholinergic fEPSPs in myenteric neurons CT), digitized at 1 kHz using a Labmaster TL-1 or Digitdata 1200 analog/digital converter (both from Axon Instruments) and acquired Single electrical stimuli applied to interganglionic nerve strands and stored using AXOTAPE 2.0 (Axon Instruments) and a personal com- evoked an fEPSP in 98 neurons; the average amplitude of the puter. fEPSP was 23 + 4 mV. None of theseneurons exhibited a spike Synaptic potentials were evoked using a broken-back glass pipette afterhyperpolarization that lasted longer than 1 set, and these (tip diameter 40-60 PM) filled with Krebs solution as a focal stimulating cells would be classified as “S” neurons according to the criteria electrode. The stimulating electrode was positioned on an interganglionic nerve strand. To evoke an fEPSP, nerve fibers were stimulated using of Hirst et al. (1974). Hexamethonium (100 PM) reduced the single stimuli of 0.3 msec duration applied at a rate of 0.2 Hz. When fEPSP by 83 ? 1% in 37 neurons;in the presenceof hexame- studying fEPSPs, 6-12 individual responses were averaged. In some thonium the fEPSP was 4 f 0.4 mV at a membrane potential experiments, slow excitatory postsynaptic potentials (sEPSPs) were of -90 f 4 mV (Fig. 3A, left). These fEPSPs were considered evoked using trains of stimuli (20 Hz for 500 msec). The amplitude of single sEPSl% before and after hrug treatment was ‘measured: to be cholinergic. In 6 1 neurons, hexamethonium (100 KM) re- Drug application. Antagonists were applied by superfusion in a known duced the fEPSP by 34 f 3%; in thesecells, fEPSP amplitude concentration by addition to the superfusing Krebs solution. Antago- was 15 f 1 mV in the presenceof the antagonist at a membrane nists were applied for 4-12 min prior to measuring the amplitude of potential of -97 f 3 mV. Hexamethonium-resistant fEPSPs the evoked response. When constructing antagonist concentration-re- were completely blocked by tetrodotoxin (0.3 KM, n = 9) (Fig. sponse curves, successive antagonist concentrations were applied cu- mulatively and each concentration was present for a minimum of 4 min 3A, right). The control distribution of fEPSP amplitude was before measuring the amplitude of the evoked response. ACh was ap- dispersed over a range of amplitudes between 10 and 40 mV plied by ionophoresis from an electrode (tip resistance < 20 MS2) placed (Fig. 3B). The distribution of fEPSP amplitudesin the presence directly above the impaled neuron. The concentration of ACh in the of hexamethonium was skewed towards smaller amplitudes with electrode was 1 M. A retaining current of - 6 nA was used and cathodal current pulses of l-20 msec duration and 100-199 nA intensity were a peak at lessthan 10 mV (Fig. 3C). There was a large overlap used. An average of 6-12 individual ACh responses was obtained for of the two distributions indicating that most fEPSPs in the each treatment. ATP was applied by pressure ejection (10 psi) from a myenteric plexus are a mixture of hexamethonium-sensitive and fine-tipped (< 10 pm) pipette positioned within 200 pm of the impaled -insensitive components. neuron. The concentration of ATP in the pipette was 100 or 300 PM. Although it was shown above that 100 FM hexamethonium Statistics. All data are expressed as the mean + SEM. Student’s t test for paired data or analysis of variance were used to establish significant completely blocked responsesto ACh, it is possible that ion- differences between control and treatment groups. Antagonist inhibition tophoretically applied ACh and neurally releasedACh act at of EPSPs, ACh, or ATP responses is expressed as percentage change different sitesand that higher concentrations of hexamethonium from control amplitude. Concentration-response curves were plotted may required to block nicotinic fEPSPs. This differential sen- and concentrations of antagonist causing half-maximal inhibition (IC,,) were determined from average concentration-response curves. sitivity of receptors could account for the occurrence of hexa- Drugs. Mecamylamine was obtained from Research Biochemicals methonium- resistant fEPSPs. This possibility was tested in two Inc. (Natick, MA). Suramin was a gift from the Center for Disease ways. First, in 12 neurons in which the fFPSP was partly reduced Control (Atlanta, GA). All other drugs and chemicals were obtained by 100 FM hexamethonium, it was found that a higher concen- from Sigma Chemical Co. (St. Louis, MO). tration of hexamethonium (300 PM) did not further reduce the fEPSP (Fig. 4A,B). Second, mecamylamine was also used to Results determine if some fEPSPs recorded from myenteric neurons Nicotinic receptor antagonists, but not suramin, block were noncholinergic. In 4 of 11 neurons, mecamylamine (10 ACh-induced depolarizations PM) reduced the fEPSP by 77 +- 2% (Fig. 4C), while in 7 of 11 ACh was applied by ionophoresis to cause a depolarization that neurons, mecamylamine reduced the fEPSP by only 32 f 9% was similar in amplitude and time course to fEPSPs recorded (Fig. 40). Taken together, thesedata indicate that the failure of from myenteric neurons (Fig. 1A). Hexamethonium inhibited 100 PM hexamethonium to block completely the fEPSP in some ACh-induced depolarizations in a concentration-dependent neurons cannot be attributed to the use of an insufficient con- manner and the ACh response was completely blocked by 100 centration of antagonist or to a population of hexamethonium- PM hexamethonium (Fig. lA,B). The IC,, for hexamethonium resistant nicotinic receptors. Instead, the fEPSP in most myen- The Journal of Neuroscience, December 1994, 14(12) 7585

ACh ionophoresis ACh response A Control Control Suramin 500 MM A \ /

Haxamethonium (PM) 10 mV I ‘\ 10 ms 20 ms B Mecamylamine

80 60 40 20 0 I ...... a ...... I ...... I ...... I ....ul 10 -* 10 -’ 10 -* 10 -5 10 -’ 10 -) 0 300 [Suramin](flM) [Antagonist](M) Figure 2. Suramin does not alter ACh-induced depolarizations. A, ACh applied by ionophoresis (5 msec, 199 nA) causes a denolarization Figure 1. Nicotinic receptor antagonists block ACh-induced depolar- that is not reduced in amplitude by suramin. B; Data from experiments izations of myenteric neurons. A, ACh applied by ionophoresis (2 msec, 100 nA) to a myenteric neuron causes a depolarization that is inhibited similar to A demonstrating that suramin (300 MM) does not inhibit ACh by 10 and 100 PM hexamethonium. B, Concentration-response curves responses in myenteric neurons (n = 6). for inhibition of ACh-induced depolarizations by hexamethonium and mecamylamine. Data obtained from experiments similar to that shown cells, suramin reduced the noncholinergic fEPSP by less than in A and are the mean f SEM of three to nine cells per point. 20%. In 30 cells, suramin (100 FM) reduced the noncholinergic fEPSP by 7 1 + 4% (Fig. 54). The control noncholinergic fEPSP teric neurons is due to the action of a neurotransmitter or was 17 + 1 mV, and in the presence of suramin the fEPSP was neurotransmitters in addition to ACh and the hexamethonium- 5 + 1 mV (p < 0.01). Inhibition of the noncholinergic fEPSP resistant fEPSP is a noncholinergic synaptic event. The data by suramin was concentration dependent; the suramin IC,, was presented in Table 1 show that the latency, time to peak, and 4 PM (Fig. 5B). Although it was shown above that suramin did time to half-decay for noncholinergic f’EPSPs were similar to not block responses to ACh or 5-HT, it is possible that suramin the time course of nicotinic fEPSPs. could act nonspecifically to disrupt synaptic transmission. This possibility is unlikely because there were some noncholinergic Noncholinergic fEPSPs blocked by the P2 receptor antagonist fEPSPs that were unaffected by suramin and it was also found suramin that suramin (100 PM) did not change the amplitude of sEPSPs Suramin (100 PM) was applied to 35 of 6 1 neurons in which the elicited by brief trains of stimuli (control sEPSP, 22 + 4 mV, fEPSP was partly reduced by hexamethonium (100 PM). In five sEPSP in the presence of suramin, 17 + 3 mV; n = 5, p > 0.05).

Table 1. Time coarse of cholinergic and noncholinergic fEP!SPs in myenteric neurons

Time to peak Half-decay Amplitude Latency(msec) (msec) (msec) @VI CholinergicIEPSP (n = 11) 3.1 -+ 0.4 4.0 + 0.6 13.1 * 2.5 20.7 * 2.5 NoncholinergictEPSP (n = 10) 3.5 t 0.2 4.1 z!z0.6 12.3 iz 1.2 20.6 f 1.2 Data are mean 2~ SEM. There were no differences between choline& and noncholinergic fEPSPs @ > 0.05, Student’s t test). 7566 Galligan and Bertrand l ATP-mediated Fast Synaptic Potentials

fEPSP A .

1 Hexamethonium 100 p M -J 10 mV 15 10 ms B 12 I Control 9

6 3 0 0 10 20 30 40 50 Amplitude (mV)

12 Hexamethonium Figure 3. Cholinergic and noncholinergic fEPSPs. A: L@, fEPSP that was 9 completely blocked by hexamethonium. Right, Recording from a different neuron with an fEPSP that was partly 6 reduced in amplitude by hexamethonium. The noncholinergic fEPSP was completely blocked by tetrodotoxin 3 (TTX). B, Amplitude-frequency distribution for fEPSPs recorded in the ab- 0 sence of hexamethonium. C, Ampli- tude-frequency distribution for fEPSPs 0 10 20 30 40 50 recorded in the presence of hexamethonium (100 PM). Amplitude (mV)

The reversal potential for the noncholinergic fEPSP was de- associatedwith a decreasein membrane input resistance(Fig. termined in experiments similar to that shown in Figure 6, A 7A). In 8 of 10 neuronsin which the fEPSP was suramin sen- and B. The amplitude of the suramin-sensitivefEPSP was mea- sitive, ATP caused a fast depolarization. ATP caused a fast sured at different membrane potentials in the presenceof hex- depolarization in only one of eight neuronsin which the fEPSP amethonium (100 PM). In this cell, the estimated reversal po- was completely blocked by hexamethonium. The fast depolar- tential was - 15 mV (Fig. 6C). This experiment was repeated ization causedby ATP was blocked by suramin (100 PM) (Fig. in six additional neurons and the average estimated reversal 7B). Hexamethonium (100 PM) did not block ATP-induced de- potential was -25 + 7 mV. This value was similar to the es- polarizations (control, 22 + 4 mV, hexamethonium, 26 + 3 timated reversal potential for fEPSPs measuredin the absence mV; n = 2). In somecells, ATP alsocaused a slow depolarization of hexamethonium (-24 + 6 mV, n = 8). (Fig. 7A), but this responsewas not studied. The reversal potential for the ATP responsewas estimated ATP mimics noncholinergic fEPSPs in experiments similar to that shown in Figure 8 where the ATP was applied by pressureejection from a micropipette onto amplitude of the ATP responsewas measuredat different mem- neurons exhibiting noncholinergic fEPSPs. ATP caused a de- brane potentials. In the experiment shown in Figure 8, the es- polarization of l-2 set duration and the depolarization was timated reversal potential was -2 mV. Similar experiments The Journal of Neuroscience, December 1994, 74(12) 7567 fEPSP

Hexame’thonium (IIM) Control 100 300 10 mV @ [Hexamethonium]( VM) I 10 ms

C D

, Control

I b--tiecamylamine 10 p M Mslcamylamine 10 ~ M Figure 4. Effectsof hexamethoniumand meeamylamineon fEPSPs.A, fEpSP reducedin amplitudeby 100 PM hexamethoniumand 300 LLM hexamethoniumdoes not further reducefEPSP amplitude. B,. Data from experimentssimilar to that shownin A (n = 12).Heliamethonium at 100 and 300 PM significantlyreduced the amplitudeof the fEPSP (p < 0.05) but the effectsof 100PM are not differentfrom the effectsof 300 PM (p > 0.05).Data wereanalyzed by ANOVA and the leastsignificant difference test. were repeated in three additional neurons, and the averagees- The failure of hexamethonium to block the fEPSP was not timated reversal potential was - 16 + 5 mV. The decreasein due to the use of insullicient concentrations of antagonist. ACh input resistanceduring the ATP responseand the reversal po- was applied by ionophoresis onto neurons, and the ACh re- tential near 0 mV suggeststhat the ATP responsein myenteric sponsewas similar in time courseand amplitude to the fEPSP. neurons may be due to an increasein a nonspecificcation con- This observation indicates that ionophoretically applied ACh ductance. Consistent with this suggestion,it was found that and neurally releasedtransmitter were acting at similar sitesto reducing the extracellular sodium concentration from 145 mM cause a depolarization. The concentration of hexamethonium to 26 mM (choline chloride substitution for sodium chloride) (100 PM) usedto study fEPSPs was 30-fold higher than its IC,, shifted the reversal potential to -31 f 1 mV (n = 3) (Fig. 8B). value (3 PM) determined in studiesof the ACh response,and at a concentration of 100 PM hexamethonium completely blocked Discussion ACh responses.In addition, in neurons in which 100 NM hex- Noncholinergic fast synaptic transmissionin the myenteric amethonium did not completely block the fEPSP, a higher con- plexus centration of hexamethonium (300 PM) did not further reduce There are many potential neurotransmitters contained in the the amplitude of the fEPSP. ENS (Fumessand Costa, 1987). In addition to ACh, thesepo- The fEPSPs that were not blocked by hexamethonium were tential neurotransmitters include 5-HT, GABA, nitric oxide, not due to an action of neurally releasedACh at a population and many neuropeptides. Studies of the contribution of non- of hexamethonium-insensitive nicotinic receptors. Mecamyla- choline& neurotransmitters to synaptic transmission in the mine, another antagonist of ganglionic nicotinic receptors(Fie- ENS have focused on slow synaptic events and it has been ber and Adams, 1991 a), wasused to determine if somenicotinic assumedthat ACh is the sole mediator of fast synaptic trans- receptors in the ENS were resistant to hexamethonium. Mec- mission between enteric nerves (Wood, 1989). However, in the amylamine was an effective antagonist of ACh responses,and present study, we have shown that in most myenteric neurons the concentrationof mecamylamine( 10FM) usedto study lEPSPs of guinea pig small intestine fEPSPs are mediated by neuro- was almost loo-fold higher than the IC,, value (0.12 pM) de- transmitters in addition to ACh acting at nicotinic receptors. termined for block of the ACh response.At a concentration 10 This conclusion is basedon the observation that most fEPSPs PM, mecamylamine did not block all fEPSPs recorded from were not completely blocked by the nicotinic antagonist hexa- myenteric neurons. In addition, mecamylamine and hexame- methonium. thonium are open channel blockers of the nicotinic receptor, 7566 Galligan and Bertrand l ATP-mediated Fast Synaptic Potentials

mylamine, the remainingfEPSPs are noncholinergic.The marked Noncholinergic fEPSP overlap of the frequency distributions of fEPSP amplitudes in A the absenceand presenceof hexamethonium indicates that most fEPSPs elicited by electrical stimulation in the ENS are due to the action of a transmitter or transmitters in addition to ACh. ATP mediates noncholinergic fEPSPs ATP acts at P, receptors on smooth muscle cells (Benham et al., 1987; Friel, 1988), sensoryneurons (Krishtal et al., 1988; Bean, 1990), autonomic neurons (Fieber and Adams, 199 lb; Nakazawa, 1994), and neurons in the CNS (Ueno et al., 1992; Shen and North, 1993) to cause a depolarization or inward current. The short latency, time course, and reversal potential Suramin (Ir Ml 10 mV (near 0 mV) of theseresponses have led to the conclusion that the purine receptor at which ATP acts is a ligand-gated cation I 1 channel with properties similar to the nicotinic receptor (Bean, 5 ms 1992). The P, receptor antagonist suramin (Dunn and Blakely 1988; Hoyle et al., 1990) blocks ATP-induced responsesin peripheral neurons(Nakazawa, 1994) and PC12 cells (Nakazawa B et al., 1991 a). Suramin has also been usedto show that synaptic 80 - responsesrecorded from medial habenulaneurons (Edwardset al., 1992) and neuronsfrom guinea pig celiac ganglion in tissue 60 - culture are mediated by ATP (Evans et al., 1992; Silinsky and Gerzanich, 1993). In the present study, suramin reduced non- 40 - cholinergic fEPSPs in 30 of 35 neurons tested. The inhibition of noncholinergic fEPSPs by suramin was concentration dependent and, on average, suramin reduced the noncholinergic 20 - component of the fEPSP by a maximum of 70%. In neuronsexhibiting a suramin-sensitiveWPSP, ATP caused 0 ‘..‘...... ““‘n . .‘..‘.‘I . ‘“‘- a fast depolarization (~2 set duration), and the fast depolar- lo-* 1o-6 10” 10’” ization was associatedwith a decreasein input resistance.ATP generally did not causea fast depolarization in neuronsin which the fEPSP was blocked by hexamethonium. The depolarization [Suramin]M causedby ATP was blocked by suramin but suramin did not block ACh responsesor depolarizations causedby 5-HT acting Figure 5. Suramin inhibits noncholinergic fEPSPs. A, In the presence at 5-HT, receptors.Depolarizations causedby 5-HT were blocked of hexamethonium (100 PM) suramin at 10 and 100 PM reversibly in- by the 5-HT, receptor antagonist ondansetron (Derkach et al., hibits the noncholinergic WPSP. Wash indicates recovery from sura- 1989; Vanner and Surprenant, 1990). Suramin did not block min; hexamethoniumwas presentthroughout. B, Concentration-re- noncholinergic fEPSPs via a nonspecific action on synaptic sponses curve for inhibition of noncholinergic fEPSP by suramin. Data transmission,as some noncholinergic fEPSPs were not blocked are the mean& SEM of the indicatednumber of cellsper data point. by suramin and slow synaptic potentials evoked by trains of stimuli were not affected by suramin. and the block caused by these drugs is enhanced at hyperpo- In other cells, ATP activates a nonspecific cation channel as larized membrane potentials (Ascher et al., 1979; Fieber and ATP-induced currents reverse polarity near 0 mV and reducing Adams, 1991 a). We studied the action of hexamethonium and extracellular sodium shifts the reversal potential in a negative mecamylamine on fEPSPs at potentials near -90 mV, which direction (Benhamet al., 1987; Friel, 1988; Krishtal et al., 1988; would increasethe block of the nicotinic receptor produced by Fieber and Adams, 1991 b; Nakazawa et al., 1991 b). As the ATP these drugs when compared to their effects at potentials near responserecorded from myenteric neuronswas associatedwith rest (- 60 mV). Finally, there was a subpopulation of neurons a decreasedinput resistance,the ATP responseand noncholi- in which hexamethonium or mecamylaminereduced the fEPSP nergic fEPSP could be due to activation of a cation conductance. by more 80%, indicating that some fEPSPs were largely me- The estimated reversal potential for the noncholinergic fEPSP diated by neurally released ACh acting at nicotinic receptors (-25 mV) and the ATP response(- 16 mV) were similar and and that the nicotinic antagonistscan block thesereceptors. were not different from that measuredfor the nicotinic fEPSP The noncholinergic responsewas completely blocked by te- (Wood, 1989).These data are consistentwith the noncholinergic trodotoxin, indicating that it was dependent on presynaptic ac- fEPSP and ATP responsesbeing due to the activation of a tion potentials. The latency, rise time, and decay time of the nonspecific cation channel similar to the nicotinic receptor. noncholinergic responsewere similar to the observed times for However, our estimatesof the ATP reversal potential and the fEPSPs that were blocked by the nicotinic receptor antagonists. reversal potential of the noncholinergic fEPSP are morenegative Therefore, the noncholinergic responseis likely to be due to the than those obtained by others for ATP responsesand suramin- action of a neurotransmitter at a ligand-gated ion channel sim- sensitive synaptic responses(Benham et al., 1987; Friel, 1988; ilar to the nicotinic receptor. These data indicate that when Bean et al., 1990; Fieber and Adams, 1991b; Edwards et al., nicotinic receptors are blocked by hexamethonium or meca- 1992; Evans et al., 1992; Nakazawa, 1994; Silinsky and Ger- The Journal of Neuroscience, December 1994, f4(12) 7569

Noncholinergic fEPSP

B mV

-46

Figure 6. Reversal potential for sur- -60 amin-sensitive fEPSP. A, Suramin re- duces the amplitude of the noncholinergic fEPSP. B, Amplitude of the 0’ \ suramin-sensitive fEPSP measured at the indicated membrane potentials. C, -90 -60 -30 0 Plot of data shown in B; the estimated reversal potential for the fEPSP in this Potential (mV) cell was - 15 mV.

zanich, 1993). This discrepancy may be due to the inward rec- ATP response tification exhibited by ATP-induced currents and suramin-sensitive fEPSPs. We used high-resistance intracellular microelectrodes(80-l 20 MO). Therefore, the amplitude of noncholinergic fEPSPs and ATP responsescould be measuredat k potentials only as positive as -20 mV, and the reversal potential was estimatedby extrapolation. As we did not measurethe ATP responseor the noncholinergic fEPSP in the range of potentials where ATP currents rectify, it is likely that our estimate of the reversal potential is more negative than the actual reversal potential. Alternatively, the properties of the ATP-activated channel in myenteric neuronsmay be different from the samechan- A 10 mV nels in other cell types. ATP --l The ATP responsewas reduced when it was studied in low 2 s extracellular sodium solutions (26 mM). In low-sodium solutions, the ATP reversal potential shifted to -30 mV; this shift is lessthan that would be predicted when reducing the extracellular sodium concentration by 80%. Under theseconditions, the ATP reversal potential should have shifted in the negative direction by 50 mV. The smaller shift measuredhere could be attributed to the rectifying properties of the ATP-activated channel or the channel in myenteric neurons may be somewhat permeable to choline and other cations including calcium, as shown in other cell types (Benham et al., 1987; Friel, 1988; Fieber and Adams, 1991 b; Nakazawa et al., 1991 b; Ueno et al., 1992). In some neurons, the fast depolarization causedby ATP was followed by a slow depolarization. The slow depolarization causedby ATP in enteric neuronshas been shown by others to be due to an inhibition of restingpotassium channels (Katayama 0 100 and Morita, 1989). It remainsto be determined if there are slow [Suramin](vM) synaptic potentials in the ENS that are also mediated by ATP. Figure 7. ATP mimics the noncholinergic fEPSP. A, Depolarization t caused by ATP (triangle) applied by pressure ejection from a micropipette. Downward deflections are voltage responses to 200 pA, lOO- sponses indicates a decrease in input resistance during the ATP response. msec-duration hyperpolarizing current pulses passed through the B, ATP responses are blocked by suramin. Asterisk indicates signifi- recording microelectrode. Decrease in amplitude of these voltage re- cantly different from control (p < 0.05, n = 6). 7570 Galligan and Bertrand * ATP-mediated Fast Synaptic Potentials

ATP Response A B

60 1 Normal Krebs 50 Low sodium Krebs -58 --bh--- A 40

4()---b?-- A 01 -100 -80 -60 -40 -20 0

-g5-JhL--- I Membrane Potential (mV) ATIP I 20 mL I 1 s

Figure 8. Reversal potential for ATP response. A, Amplitude of ATP response at the indicated membrane potentials. B, Plot of data shown in A. In this cell, the estimated reversal potential was -2 mV. In the same cell, the reversal potential for the ATP response shifted to -30 mV when the sodium concentration was reduced to 26 mM (choline chloride substitution for sodium chloride).

guinea pig small intestine release ATP in a calcium-dependent Significance of noncholinergic fEPSPs manner (White and Leslie, 1982). These histochemical and neu- ATP induces a depolarization in many neurons in situ and in rochemical data are consistent with the electrophysiological data culture, but the functional significance of the ATP-induced re- presented here indicating that ATP is a fast synaptic transmitter sponse in these neurons is not clear (Bean, 1992). Recently, it in the ENS. has been shown that when glutamate and GABA receptors are Reflex peristaltic contractions can be elicited in vitro in iso- blocked, fast synaptic currents recorded from medial habenula lated segments of intestine when the lumen of the segment is neurons are mediated by ATP (Edwards et al., 1992). As these slowly filled with fluid (Trendelenburg, 1917). The peristaltic studies were done in acutely isolated brain slices, the synaptic reflex involves a filling phase in which the intestinal segment responses recorded in these studies are likely to be present in accommodates to the volume of fluid and an emptying phase vivo. Celiac ganglion neurons maintained in tissue culture for 1 that occurs when intraluminal pressure has reached a critical week or more form autapses which use ATP as the excitatory level. The emptying phase of the peristaltic reflex is generally neurotransmitter (Evans et al., 1992; Silinsky and Gerzanich, blocked by nicotinic antagonists (Furness and Costa, 1987). 1993). Although occasional ATP-mediated synaptic responses However, distention-evoked peristalsis in guinea pig ileum can occur in the intact celiac ganglion (Silinsky and Gerzanich, 1993), be elicited in the presence of hexamethonium when the tissues most fEPSPs in this tissue are blocked by nicotinic antagonists, are also incubated with the opipid receptor antagonist naloxone and the functional significance of ATP-mediated synaptic po- (Barth0 et al., 1987). Recently, it has been shown that at low tentials recorded in tissue culture are unknown (McLachlan and rates of distention, the nerve-mediated accommodation that Meckler, 1989; McLachlan and Keast, 1993). We used acutely occurs during the filling phase of the peristaltic reflex is not isolated preparations of myenteric plexus, and most synaptic blocked by hexamethonium (Waterman et al., 1994). Both groups connections present in the intact intestine would be preserved concluded that non-nicotinic ganglionic transmission was re- in this preparation. Therefore, the ATP-mediated fEPSPs re- sponsible for the hexamethonium-resistant reflexes they had corded here are likely to contribute to ganglionic neurotrans- described. Although previous work dealing with noncholinergic mission in intact tissues. Histochemical methods using quina- ganglionic transmission in the ENS has focused on slow synaptic crine to reveal neuronal stores of ATP have demonstrated that transmission (Wood, 1989), it is possible that ATP acting as a quinacrine-induced fluorescent nerve fibers and nerve cell bod- fast synaptic transmitter mediates these hexamethonium-resis- ies are found throughout the myenteric plexus of the small in- tant reflexes. testine of mouse, guinea pig, and rat (Olson et al., 1976). In In conclusion, ATP-mediated fEPSPs contribute to excitatory addition, synaptosomes prepared from the myenteric plexus of ganglionic transmission in the myenteric plexus of guinea pig The Journal of Neuroscience, December 1994, 14(12) 7571

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