Journal of Physiology (1990), 426, pp. 453-471 With 8 figures Printed in Great Britain

453

SUBSTANCE P MEDIATES SYNAPTIC TRANSMISSION BETWEEN RAT MYENTERIC NEURONES IN CELL CULTURE

BY ALAN L. WILLARD From the Department of Physiology, University of North Carolina, Chapel Hill, NC 27599-7545, USA

(Received 29 November 1989) SUMMARY

1. Whole-cell patch-clamp recordings were made from pairs of neurones in cell cultures of rat myenteric neurones. In some pairs, action potentials evoked in the first neurone evoked a slow excitatory postsynaptic potential (EPSP) in the second neurone. 2. Action potentials at a frequency of at least 5 Hz were required to evoke slow EPSPs. In one group of cells, the slow EPSP followed a series of nicotinic fast EPSPs; in another group, fast EPSPs did not precede the slow EPSP. 3. The slow EPSPs were 2-16 mV in amplitude and were accompanied by decreased resting potassium conductance. 4. Most (17/28) neurones in which action potentials evoked only slow EPSPs in a follower cell contained substance P (SP)-like immunoreactivity; they were not immunoreactive for 5-hydroxytryptamine (0/15) or vasoactive intestinal peptide (0/22). 5. Postsynaptic responses to SP, neurokinin A and a synthetic tachykinin ([pGlu6, Pro9]SP6 l1) mimicked the slow EPSPs. The non-tachykinin peptide vasoactive intestinal polypeptide (VIP), which was not found in neurones that evoked only slow EPSPs, also mimicked the slow EPSPs. Responsiveness to SP decreased significantly during slow EPSPs. 6. Desensitization to either SP or VIP reduced or prevented the slow EPSPs and also responses to each other. Two proposed antagonists of SP receptors, [D-Arg1,DPro2,D-Trp7 9,Leu"1]substance P and [D-Arg',D-Trp7' 9,Leu11]substance P, did not affect the slow EPSPs significantly. 7. Antisera against SP reversibly blocked or reduced slow EPSPs evoked by eight of thirteen presynaptic neurones that evoked slow EPSPs without evoking fast EPSPs. All eight of the presynaptic neurones that evoked anti-SP-sensitive slow EPSPs contained SP-like immunoreactivity. None of the presynaptic neurones that evoked anti-SP-insensitive slow EPSPs contained detectable SP-like immunoreactivity. Normal sera and anti-VIP antisera did not alter the slow EPSPs detectably. 8. It is concluded that subsets of myenteric neurones release an SP-like transmitter to evoke slow EPSPs. These neurones appear to lack a 'classical' neurotransmitter that evokes fast EPSPs. MS 8107

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A. L. WILLARD INTRODUCTION

Much indirect evidence suggests that substance P (SP) is a neurotransmitter in the enteric nervous system. Enteric neurones contain SP (reviewed by Pernow, 1983; Furness & Costa, 1987; Furness, Llewellyn-Smith, Bornstein & Costa, 1988), electrical stimulation causes its Ca2+-dependent release (Baron, Jaffe & Gintzler, 1983; Holzer, 1984), and SP mimics non-cholinergic slow EPSPs evoked in enteric neurones by stimulation of axon bundles between the enteric ganglia (Katayama & North, 1978; Katayama, North & Williams, 1979; Johnson, Katayama, Morita & North, 1981; Surprenant, 1984; Mihara, Katayama & Nishi, 1985). Further indirect support comes from the observation that slow EPSPs can be evoked in myenteric ganglia following surgical procedures that spare SP-positive axons while eliminating many classes of axons containing other transmitter candidates that mimic the slow EPSPs (Bornstein, North, Costa & Furness, 1984). Thus, its presence and pharmacological actions make SP a strong candidate to be at least one of the transmitters mediating slow non-cholinergic EPSPs in enteric ganglia. However, all the evidence cited above is indirect because the presynaptic neurones that elicit the slow EPSPs were not identified directly. There has been no direct demonstration that stimulation of SP-containing neurones is both necessary and sufficient to evoke slow EPSPs. In the experiments reported here, cell cultures were used to investigate slow synaptic transmission between myenteric neurones. Previous studies have shown that individual neurones in cell cultures prepared from rat myenteric plexus can evoke slow EPSPs in other neurones (Willard & Nishi, 1985 a, 1987; Willard, 1990). In this study, immunocytochemical staining has been used to demonstrate that subsets of myenteric neurones that evoke EPSPs contain immunoreactive SP. This paper describes the synaptic potentials evoked by SP-containing myenteric neurones and presents pharmacological data to support the argument that SP is a transmitter that evokes slow EPSPs. Some of these results have appeared in an abstract (Willard, 1989). METHODS

Cell cultures Cultures of myenteric neurones were prepared as described by Nishi & Willard (1985) and Willard (1990) from tissue strips removed from the small intestines of 1- to 3-day-old rat pups (CD strain; Charles River) that had been killed by decapitation.

Electrophysiology Experiments were done at room temperature. Cultures were viewed through an inverted microscope (Nikon Diaphot) with epifluorescence and Hoffman modulation contrast optics. Recording solution (see below) replaced culture medium. Whole-cell recordings (Hamill, Marty, Neher, Sakmann & Sigworth, 1981) were made with patch pipettes pulled from haematocrit glass (Fisher Blue Tip). Pipettes filled with internal solution had DC resistance of 4-8 MO. Either a Dagan 8900 patch-clamp amplifier (100 MO feedback resistor in the headstage) or a WPI M-707A electrometer were used for current-clamp recordings. Voltage-current (V-I) relations were determined by measuring the voltage changes evoked by families of current pulses delivered at rest or during slow EPSPs and responses to exogenously applied compounds. Reversal potentials were estimated by two methods. The first was to evoke

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slow EPSPs while the postsynaptic membrane potential had been altered by injection of steady currents. The second was to determine the point of intersection of V-I curves obtained before, during and after evoking slow EPSPs (for theory, see Ginsborg, 1973). This latter method had the advantage of requiring that fewer slow EPSPs be evoked. Synaptically connected pairs of neurones were identified either by making recordings from randomly selected pairs of neurones and testing each for the ability to elicit synaptic potentials in the other or by the dye injection procedure described by Willard (1990). Briefly, patch pipettes were used to inject Lucifer Yellow into neurones. After dye had diffused into processes and terminals of injected cells for about 15 min, the field of view was examined briefly to locate neuronal cell bodies that were contacted by varicosities of the injected cell. Injected neurones were then tested for their ability to evoke synaptic responses in contacted neurones. Solutions were added to the entire culture dish via a gravity-driven perfusion system. When it was necessary to minimize the volumes of test solutions used, such as when using antisera, test solutions were added to only a restricted area in the centre of the culture dish. This was accomplished by removing all fluid from the culture, except for about 30 Itl that remained in a paraffin ring surrounding cells in the centre of the culture dish. This ring, which could hold up to 300 /u1, was then superfused via a microperfusion system described by Willard (1990). Solution Recording solution. This contained 137 mM-NaCl, 5'4 mm-KCl, 0-44 mM-KH2PO4, 0-34 mMNa2HPO4, 10 mM-glucose, 2-5 mM-CaCl2, 5 mM-HEPES-NaHEPES (pH 7-35) and 28 /uM-Phenol Red. Test solutions of peptides also contained 1-2'5 mg/ml bovine serum albumin. Internal solution. This contained 150 mM-KCl, 1 mM-EGTA-KOH, 2 mM-MgCl2, 2 mM-Na2ATP, 5 mM-HEPES (pH 7 35) and 28 #uM-Phenol Red.

Immunocytochemistry Methods for immunocytochemical staining and for relocation of electrophysiologically characterized cells have been described previously (Nishi & Willard, 1985; Willard & Nishi, 1987; Willard, 1990). Specific staining was defined as that which could be blocked by pre-adsorbing the primary antiserum with the antigen against which it had been raised and not by pre-adsorption with related molecules. Pre-incubation with synthetic SP (1 ug/ml) abolished immunocytochemical staining with both anti-SP antisera used. Pre-incubation with 1 Itg/ml of neurokinin A (substance K) did not detectably affect staining. Other controls included omitting the primary antiserum in order to test for non-specific binding of the secondary antiserum. Materials Lyophilized peptides were purchased from Peninsula Laboratories and from Sigma. Stock solutions (1 mg/ml in 10 mM-acetic acid) were prepared and then stored frozen in 10 ,1 aliquots until immediately before use. Two anti-SP antisera were used. They were purchased from IncStar(17H2T) and Peninsula (RAS 7451). Normal rabbit serum was purchased from Dako and Vector. All other chemicals were purchased from Sigma.

Statistics All means are presented + S.D. x2 tests were used to determine the significance of the proportions of identified presynaptic neurones that contained particular neurotransmitter candidates. RESULTS

General observations These results came from myenteric neurones grown in culture for 3-7 weeks. All presynaptic neurones described in this study caused slow non-cholinergic EPSPs in one or more postsynaptic neurones without directly causing detectable fast EPSPs. These neurones, which will be referred to as 'drivers' of slow EPSPs, comprise only 10-20 % of all neurones in these cultures. Approximately 40 % of neurones in these

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cultures evoke only fast nicotinic EPSPs (Willard & Nishi, 1985a, 1987) and about 5 % evoke both fast nicotinic EPSPs and slow non-cholinergic EPSPs mediated by a VIP-like co-transmitter (Willard, 1990). Spontaneous nicotinic EPSPs occurred in about half of all neurones that received slow EPSPs. Stimulation of drivers of slow 1 Hz

2 Hz

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Fig. 1. Repetitive stimulation evokes slow EPSPs. Lower and upper traces are chart records of intracellular recordings from pre- and postsynaptic myenteric neurones, respectively. Fifty action potentials were evoked in the presynaptic neurone at the indicated frequencies. Stimulation at 7 Hz and above evoked a slow EPSP in the postsynaptic neurone. A photomicrograph of these neurones appears in Fig. 3A. Calibration: 30 s, 20 mV.

EPSPs occasionally caused increases in the frequency of fast EPSPs recorded in postsynaptic neurones, presumably due to activation of cholinergic neurones contacting the postsynaptic neurones. Frequency dependence of slow EPSPs Single presynaptic action potentials never evoked slow EPSPs. In the vast majority of cases, detectable (a 2 mV) slow EPSPs occurred only after repetitive stimulation at 5-20 Hz. Of sixty-seven neurones tested, two evoked slow EPSPs at 3 Hz stimulation, nineteen did so when stimulated at 5 Hz, thirty-three required 10 Hz stimulation, and thirteen did not evoke slow EPSPs until stimulated at 20 Hz. Figure 1 shows an example of a neurone that evoked slow EPSPs when stimulated at frequencies > 7 Hz. For any particular neurone, the minimum number of action potentials necessary to evoke a detectable slow EPSP decreased as the frequency of stimulation increased. For seventeen neurones tested at both 10 and 20 Hz, the mean minimum number of action potentials required to evoke a slow EPSP was 27 + 37 (range 4-200) at 20 Hz and 64 + 53 (range 10-300) at 10 Hz. Neurones failing to evoke detectable slow EPSPs in other neurones after 30 s of 20 Hz stimulation were categorized as not making functional connections with those neurones. It is possible that different patterns of stimulation, such as trains of brief bursts (e.g. Gardner & Potter, 1988), might have revealed connections not evoked by single continuous trains. However, detailed studies of optimal patterns of stimulation were not

performed. The amplitude and duration of slow EPSPs, which depended on both frequency

457 SUBSTANCE P-MEDIATED SLOW EPSPs and duration of presynaptic stimulation, varied considerably. Slow EPSPs evoked by 10 Hz stimulation usually reached maximal amplitudes within 10 s. Longer trains tended to prolong the slow EPSPs without increasing their amplitudes. Twelve neurones stimulated at 10 Hz for 5 s evoked slow EPSPs with a mean amplitude of

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Fig. 2. 'Run-down' of slow EPSPs. In this and subsequent figures, only recordings from the postsynaptic neurones are shown. Arrow-heads indicate when presynaptic stimulation began. A, stimulation of a presynaptic neurone at 20 Hz for 4 s evoked a slow EPSP. The slow EPSPs in panels B, C and D were evoked 7, 9 and 19 min later. Slow EPSPs in panels, A, B and D caused spiking of the postsynaptic neurone. In contrast, the slow EPSP in C, evoked only 1 min after the end of the slow EPSP in panel B, was significantly smaller. Calibration: 30 s, 20 mV.

8 + 5 mV (range 2-16 mV) and with durations ranging from 15 to 190 s. Amplitudes of consecutively evoked slow EPSPs declined markedly when fewer than 5 min had passed since the end of the preceding slow EPSP (Fig. 2). This 'run-down' was especially pronounced in the case of slow EPSPs of maximal amplitude and duration. Accordingly, during experiments that required eliciting several slow EPSPs, it was often convenient to evoke submaximal (but still easily detected) slow EPSPs.

Immunocytochemical properties of neuronea that evoked slow EPSPs Subsets of cultured myenteric neurones respond to application of several enteric neurotransmitter candidates, including ACh, 5-HT, substance P and vasoactive intestinal polypeptide (VIP), with slow depolarizations similar to the slow EPSPs (Willard & Nishi, 1985b). Willard & Nishi (1985a) showed that slow EPSPs evoked by neurones that do not evoke fast EPSPs are non-cholinergic. That finding was confirmed in this study; the muscarinic antagonist atropine did not alter the slow EPSPs, even when applied at concentrations as high as 10 /M. Furthermore, although application of 100 ,sM-ACh elicited nicotinic responses in eighteen of eighteen neurones that received slow EPSPs, it elicited muscarinic responses in only two of those eighteen neurones. Thus, it is unlikely that a cholinergic neurone would have evoked muscarinic slow EPSPs without also evoking nicotinic fast EPSPs. To test whether they contained any of the non-cholinergic transmitter candidates whose actions mimicked slow EPSPs, sixty-five physiologically identified drivers of slow EPSPs were tested for their content of SP-like, 5-HT-like or VIP-like immunoreactivity (LIR). Seventeen of twenty-eight (61 %) contained SP-LIR, none of fifteen contained 5-HT-LIR, and none of twenty-two contained VIP-LIR. Figure 3 shows examples of SP-positive and SP-negative presynaptic neurones. Because the overall proportion of SP-positive neurones in these cultures is only about 15 % (Nishi

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& Willard, 1985), the observation that more than 60 % of drivers of slow EPSPs were SP-positive is highly significant (P < 0-001). Similarly the absence of VIP-LIR, which is found in about 30% of neurones in these cultures, is highly significant

(P < 0-001). Comparison of slow EPSPs and responses to substance P and related peptides Because a significant fraction of neurones that evoked slow EPSPs contained immunoreactive SP, the ionic dependence and reversal potential of responses to SP were analysed and compared to those of slow EPSPs. In addition to SP, three related peptides that have been proposed to be relatively selective agonists for three different classes of neurokinin receptors in several tissues were tested for their ability to elicit responses that mimicked slow EPSPs. Those three peptides were septide ([pGlu6,Pro9]SP6 l), neurokinin A (NKA; formerly called substance K), and senktide (succinyl-[Asp6,MePhe8]SP6,1), which have been proposed to prefer NK-1, NK-2 and NK-3 receptors respectively (Wormser, Laufer, Hart, Chorev, Gilon & Selinger, 1986; Laufer, Gilon, Chorev & Selinger, 1988). Substance P, NKA and septide all evoked depolarizations similar to slow EPSPs. Examples of responses to SP and septide are shown in Fig. 4A. Because these responses were not significantly affected by extracellular solutions containing reduced calcium and elevated magnesium (Fig. 4A), they probably resulted from direct postsynaptic actions of the peptides rather than from indirect actions caused by excitation of nearby terminals. Detailed dose-response studies were not done, but for most neurones the relative order of potency was SP > septide Z NKA > senktide. When tested at 5/M on eight neurones, SP, NKA and septide evoked mean depolarizations of 9 + 5, 5 + 4 and 4 + 4 mV respectively. Senktide (5 FM) elicited no response on seven neurones and a 1-2 mV depolarization in one neurone. Cross-desensitization During responses to SP (and for about 1-5 min following responses to SP), presynaptic stimulation elicited either no or significantly smaller slow EPSPs (Fig. 4B). Similarly, responses to SP were reduced or absent during and for about 1-5 min following slow EPSPs (Fig. 4 C). The most likely explanation of these results is either desensitization of synaptic receptors or occlusion of second messengers or channels that participate in the generation of slow EPSPs and of responses to SP.

Conductance changes Apparent input resistance, determined by measuring voltage changes caused by injected current pulses, increased during slow EPSPs and during responses to SP (Fig. 5A). This result suggests that a decreased conductance causes the slow EPSPs. Fig. 3. Photomicrographs of neurones that caused slow EPSPs. Cultures were fixed after recording and then stained with immunoperoxidase techniques to reveal SP-LIR. In each panel, double arrow-heads indicate physiologically identified presynaptic neurones and asterisks mark identified postsynaptic neurones. Recordings from neurones in panels A, B, C and D are shown in Figs 1, 8A, 8B and 8C, respectively. The presynaptic neurones in A, B and C were SP-positive. The presynaptic neurone in D is contacted by SP-positive boutons but does not contain detectable levels of SP-LIR itself. Calibration: 40 ,gm.

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Comparison of voltage-current (V-I) curves obtained before and during slow EPSPs and responses to SP revealed that the conductance was not detectably voltagesensitive at membrane potentials between -50 and -90 mV, the normal range within which slow EPSPs occur (Fig. 5B). A ANS

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Fig. 4. Tachykinins evoke responses that mimic and occlude slow EPSPs. A, B and C were obtained from neurones in three different cultures. A: left, stimulation of a presynaptic neurone for 2 s at 12 Hz (indicated by the arrow-head labelled NS for neural stimulation) evoked a slow EPSP. Middle and right, pressure ejection of 2 /zM-SP and 5 /LM-septide (Sep) elicited similar responses. During the test applications of SP and septide, the culture was superfused with recording solution containing reduced calcium (0-36 mM) and elevated magnesium (10 mM) to block synaptic activity. B, desensitization to SP prevents slow EPSPs. Left, slow EPSP evoked by stimulation of a presynaptic neurone for 3 s at 10 Hz. Middle, presynaptic stimulation during a depolarization evoked by superfusion with 10 ,/M-SP failed to elicit a detectable slow EPSP. Right, 20 min after washing out the SP, presynaptic stimulation again evoked a slow EPSP. Prior to stimulation of the presynaptic neurone, current was injected into the postsynaptic neurone to depolarize it to a membrane potential similar to that caused by superfusion with SP. This shows that the failure of presynaptic stimulation to evoke a slow EPSP was not due to the postsynaptic depolarization caused by the SP. C, 'run-down' of responsiveness to SP and decreased responsiveness to SP during a slow EPSP. Arrow-heads labelled SP indicate time of pressure ejection of 5 4aM-SP onto a postsynaptic neurone. Left, SP evoked a much smaller response when applied immediately after the decline of a previous response to SP. Middle, 7 min later, stimulation of a presynaptic neurone at 15 Hz for 2-5 s evoked a slow EPSP and spiking of the postsynaptic neurone. Application of SP during the falling phase of the slow EPSP failed to evoke a detectable response. Right, 10 min later, SP evoked a response similar to that evoked in the left panel. Calibration: 30 s, 15 mV.

Reversal potentials and ionic dependence Reversal potentials of slow EPSPs and of responses to SP were estimated by two methods, each of which gave similar results. They were estimated from the intersection of V-I curves obtained at rest and during slow EPSPs (e.g. Fig. 5B) and also by evoking slow EPSPs after altering postsynaptic membrane potential by injection of current. With control extracellular and internal solutions, the mean estimates of reversal potential were close to the estimated potassium equilibrium potential of -84 mV. Changing the concentration of extracellular potassium caused the reversal potentials of the slow EPSPs and of responses to SP to shift by 53 + 6 and 59+7 mV per 10-fold change, respectively (Fig. 6). These values were not significantly different from the 58 mV shifts predicted by the Nernst equation.

461 SUBSTANCE P-MEDIATED SLOW EPSPs Alterations of extracellular concentrations of sodium and chloride did not cause significant changes in reversal potential, suggesting that channels selective for potassium ions mediate both the slow EPSPs and the slow depolarizing responses to SP. SP

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Fig. 5. Input resistance increases during slow EPSPs. A, hyperpolarizing current pulses of constant amplitude evoked larger voltage changes during a slow EPSP (1), during a response to 3 /sM-SP (2) and during a manually clamped slow EPSP (3). Calibration: 30 s, 15 mV. B, voltage-current curves for a postsynaptic neurone at rest (0), during the peak of a slow EPSP (@) and during the peak of a depolarization caused by superfusion with 3 1srm-SP (A). All three curves intersect at about -85 mV, indicating that the slow EPSP and the response to SP reverse at that potential, which is close to the estimated potassium equilibrium potential of -84 mV. Data in A and B were obtained from neurones in two different cultures.

Similarity of responses to substance P and vasoactive intestinal polypeptide The responses elicited by SP were very similar to responses evoked by VIP (Willard, 1990). Figure 7A compares the responses of a single postsynaptic neurone to both peptides. Figure 7B shows that during responses to each of these two peptides, neurones were unresponsive to the other. Furthermore, in four neurones, presynaptic stimulation failed to evoke a slow EPSP during a postsynaptic response evoked by VIP (data not shown). Figure 7C shows that responses to SP and VIP reversed at the same membrane potential as did the slow EPSPs. The ability of VIP and SP to evoke similar if not identical responses is important because it means that very different transmitter candidates, including one not found in identified drivers of

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slow EPSPs, can evoke postsynaptic responses that mimic slow EPSPs. Thus it is possible that the slow EPSPs are not caused by SP but rather by a different colocalized substances) with a similar postsynaptic mechanism of action.

Effects of synthetic substance P antagonists and of anti-substance P Synthetic substance P antagonists [D-Arg',D-Pro2,D-Trp7' 9,Leu'1]substance P and spantide ([D-Arg1,D-Trp7 9,Leu'1]substance P) are compounds that antagonize tachykinin responses in several -60

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Fig. 6. Effects of altered extracellular potassium on the reversal potential of slow EPSPs (upper panel) and 5 /SM-SP (lower panel). Reversal potentials were estimated from the intersections of V-I curves obtained at rest and during slow EPSPs. The slopes of the regression lines were 53 and 59 mV per 10-fold change in potassium concentration. Error bars are S.D. Numbers in parentheses are the number of postsynaptic neurones tested for each point.

neuronal systems (Rosell, Bjorkroth, Xu & Folkers, 1983; Konishi & Otsuka, 1985; Featherstone, Fosbraey & Morton, 1986; Randic, Jeftinija, Urban, Raspantini & Folkers, 1988) but not in neurones of the guinea-pig enteric nervous system (Galligan, Tokimasa & North, 1987; Surprenant, North & Katayama, 1987; Wade & Wood, 1988). When tested on eleven neurones at concentrations up to 25 gm, neither of these compounds significantly altered slow EPSPs or neuronal responses to SP

(data not shown).

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Anti-substance P antisera Because the synthetic peptides that block responses to SP in other systems did not block either responses to SP or slow EPSPs in these cultured neurones, it was necessary to find an alternative means of selectively antagonizing postsynaptic A

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Fig. 7. Comparison of actions of SP and VIP. A, responses of a myenteric neurone to pressure ejection of SP (2 /M) and VIP (300 nM). B, during bath application of 5 /ZM-SP (left) or 300 nM-VIP (right), pressure ejection of VIP or SP failed to evoke a detectable postsynaptic response. The small hyperpolarizations are pressure artifacts. Calibration: 30 s, 10 mV. C, V-I relations of a neurone at rest (control, A) and during responses to

superfusion of 5 ,uM-SP (0) or 300 nM-VIP (O). Curves obtained during responses to SP and VIP intersect the control curve at about -85 mV, suggesting a common reversal potential. responses to particular peptides. Anti-SP has proven useful for antagonizing neurally evoked responses in both peripheral and central nervous systems (Angel, Go & Szurszewski, 1984; Kaufman, Rybicki, Kozlowski & Iwamato, 1986; Randic, Ryu & Urban, 1986). Accordingly, anti-SP antisera were tested for their ability to antagonize slow EPSPs. When applied at dilutions ranging from 1: 10 to 1: 50, anti-SP blocked or reduced slow EPSPs evoked by eight of thirteen neurones tested (examples in Fig. 8). Complete blockade was achieved in three cases (e.g. Fig. 8A) while only partial reduction of the slow EPSPs occurred in the other five cases (e.g. Fig. 8B), even at 1:10 dilutions. Antisera to VIP, which antagonize slow EPSPs evoked by dualfunction myenteric neurones (Willard, 1990), and normal rabbit serum did not alter slow EPSPs significantly. Antagonistic actions of anti-SP were maximal within

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10 min of adding antisera to cultures, suggesting that antibody molecules had relatively good access to neurally released SP. Randic et al. (1986) observed that antiSP caused a 5-10 mV depolarization of neurones during long incubations (50-100 min). I observed no such depolarization of either pre- or postsynaptic neurones, perhaps because of the much shorter incubation times. A

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Fig. 8. Effects of different sera on slow EPSPs. During the times indicated by horizontal lines, presynaptic neurones were stimulated at 15 Hz. Cultures were incubated in antisera for 10 min before attempting to evoke slow EPSPs. A, anti-SP (1: 20), but neither antiVIP (1:10) nor normal rabbit serum, reduced the slow EPSP. After washing out the antiSP for 10 min, the slow EPSP had partially recovered. Note that the duration of stimulation of the presynaptic neurone was increased 3-fold during exposure to anti-SP. B, anti-SP (1:20) caused only partial reduction of slow EPSPs evoked by another neurone. C, slow EPSPs evoked by a third neurone were unaffected by anti-SP (1:20) or anti-VIP (1:10). None of the sera used had detectable effects on presynaptic resting potentials, action potentials or input resistance. Panels A, B and C were obtained from neurones in three different cultures. Figure 3B-D contains photomicrographs of the neurones that yielded the recordings in this figure. Calibration: 30 s, 15 mV.

All eight of the neurones whose postsynaptic actions were reduced or blocked by anti-SP were SP-positive when tested immunohistochemically (examples shown in Fig. 3B and C). In contrast, none of four neurones that evoked EPSPs that were unaffected by anti-SP contained detectable amounts of SP-LIR (the fifth neurone was lost during fixation). This difference in the number of SP-positive neurones that were sensitive and insensitive to anti-SP was significant (P < 0 04). DISCUSSION

Evidence that substance P mediates slow transmission between enteric neurones Four lines of evidence suggest that an SP-like molecule mediates at least some of the slow EPSPs evoked by cultured myenteric neurones. (1) Subsets of neurones that evoked slow EPSPs contained SP-LIR. (2) Responses to SP mimicked the slow EPSPs. (3) Desensitization to SP prevented the slow EPSPs and responsiveness to

SUBSTANCE P-MEDIATED SLOW EPSPs 465 SP was reduced during slow EPSPs. (4) Antisera against SP antagonized slow EPSPs evoked by SP-positive but not those evoked by SP-negative neurones. The weakest evidence is the mimicry and blockade of slow EPSPs by SP. Such data are necessary but insufficient to argue that SP mediates slow EPSPs. They are especially weakened by the fact that VIP, a transmitter candidate that was not detected in identified drivers of slow EPSPs, was at least as potent as SP at mimicking and desensitizing the slow EPSPs. The ability of anti-SP antisera to antagonize slow EPSPs provides perhaps the most compelling argument in favour of SP as a transmitter of slow EPSPs in these cultures. The simplest interpretation of the effects of anti-SP is that the immunoglobulins prevented neurally released SP from reaching or effectively interacting with postsynaptic receptors. The ready reversibility of the antagonism by anti-SP argues against antagonism due to binding of immunoglobulins to membrane-bound molecules involved in presynaptic release mechanisms or postsynaptic response mechanisms. Likewise, the lack of effects of normal rabbit serum or of anti-VIP, which selectively antagonizes slow EPSPs evoked by dual-function cholinergic myenteric neurones in these cultures (Willard, 1990), argues against non-specific deleterious effects of serum components on slow synaptic transmission. Anti-SP antisera failed to cause complete blockade of slow EPSPs evoked by five of eight SP-positive neurones (e.g. Fig. 8B). Two possible interpretations of this result are: (1) the presynaptic neurone releases more SP than the antisera can neutralize. Testing of this hypothesis will require a more quantitative approach using precisely known concentrations of affinity-purified antibodies. (2) SP-positive neurones release additional slow transmitterss. Individual myenteric neurones can contain multiple pharmacologically active peptides derived either from the same or different precursors (see reviews by Furness & Costa, 1987; Furness et al. 1988). A likely co-transmitter with SP is neurokinin A (NKA). Substance P derives from a-, ,- or y-preprotachykinin (PPT). a-Preprotachykinin encodes only SP but ,- and yPPT also encode NKA (Krause, Chirgwin, Carter, Xu & Hershey, 1987). Neurokinin A is present in extracts of rat gastrointestinal tissues (Theodorsson-Norheim, Brodin, Norheim & Rosell, 1984) and, in extracts of guinea-pig small intestine, vesicles containing SP and NKA migrate identically in density gradients, suggesting that SP and NKA may co-exist in secretary vesicles (Deacon, Agoston, Nau & Conlon, 1987). If such vesicles release their peptides, it is reasonable to hypothesize that SP and NKA are co-released and that NKA might mediate that anti-SP-resistant component of slow EPSPs. This hypothesis is supported further by the finding of this study that NKA can evoke responses similar to the slow EPSPs. To date, however, I am unaware of any studies that have reported evoked release of NKA from enteric neurones. Thus, future experiments should investigate the possible role of NKA as a co-transmitter with SP. The strength of any arguments based upon experiments with antisera depends critically upon the specificity of the antisera used. The IncStar antiserum crossreacts with several tachykinins (Henken, Tessler, Chesselet, Hudson, Baldino & Murray, 1988). The Peninsula antiserum is claimed by the vendor to have less than 1 % cross-reactivity with either neurokinin A or B. Given the similarity of the results obtained with both antisera, it seems likely that a molecule very similar to authentic

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SP is contained in the myenteric neurones studied here. However, unambiguous identification of the slow transmitters) will require detailed chemical analysis of the SP-like molecule or molecules that are released by cultured myenteric neurones. What other substances mediate slow EPSPs? Both VIP and 5-HT can evoke responses similar to those evoked by SP (Willard & Nishi, 1985b). However, to date no identified drivers of slow EPSPs have contained detectable levels of VIP- or 5-HT-LIR. Vasoactive intestinal polypeptidecontaining neurones are abundant in these cultures (Nishi & Willard, 1985) and VIP-mediated slow EPSPs can be evoked by dual-function (ACh/VIP) neurones (Willard, 1990). Thus it is significant that no VIP-positive drivers of slow EPSPs were found. Two possible explanations for the apparent absence of VIP-positive drivers of slow EPSPs are (1) that they project for distances greater than a microscopic field of view before making functional contacts with other neurones and (2) that repetitive stimulation causes depletion of somatic VIP to levels below threshold for detection by immunocytochemistry. The latter possibility seems somewhat unlikely because VIP-LIR was readily detected in heavily stimulated dual-function neurones (Willard, 1990) as well as in neurones that had failed to evoke slow EPSPs and that had been stimulated repetitively while testing them. Two relatively trivial reasons may account for the failure to detect 5-HT-LIR in identified drivers. First, only about 5% of neurones in these cultures are 5-HTpositive (Nishi & Willard, 1985, 1988). Secondly, the 5-HT-positive neurones are among the very smallest in the cultures and therefore are less likely to have been selected for making recordings. Thus, detection of 5-HT-mediated synaptic connections would require either extensive examination of small neurones in these cultures or growth of neurones under culture conditions that support the survival of significant numbers of large 5-HT-containing neurones (Nishi & Willard, 1988). Other possible candidates that are found in subsets of neurones in these cultures include calcitonin gene-related peptide (CGRP) (A. L. Willard, unpublished observations), gastrin-releasing peptide (GRP; also called mammalian bombesin) and cholecystokinin (CCK) (Nishi & Willard, 1985). In addition, ATP or other purines could be considered as possible candidates (e.g. Mihara et al. 1985). The actions of CCK or GRP on neurones in these cultures have not been examined. However, these substances are found only in a small number of small neurones in these cultures (Nishi & Willard, 1985) and thus neurones containing them are unlikely to have been studied. In preliminary experiments, ATP and CGRP have failed to evoke responses that mimic slow synaptic excitation of myenteric neurones (A. L. Willard, unpublished observations). Thus, identification of non-tachykinin mediators of slow EPSPs in these cultures remains to be achieved.

Postsynaptic mechanism of slow synaptic excitation Three lines of evidence suggest strongly that the slow EPSPs observed in this study result from decreased resting potassium conductance. (1) The increased resistance that occurred during slow EPSPs suggests that an ionic conductance decreases. (2) The linear voltage-current curves between -90 and -50 mV argue in favour of suppression of 'resting' rather than voltage-sensitive channels. (3) The

SUBSTANCE P-MEDIATED SLOW EPSPs 467 reversal of slow EPSPs at potentials near EK and the ability of the Nernst equation to predict accurately the shifts in reversal potential caused by altered extracellular concentrations of potassium argue that the synaptically suppressed channels are selective for potassium ions. Decreased resting potassium conductance caused both direct depolarization of postsynaptic neurones and also indirect excitation due to the effects of increased input resistance. However, pharmacological actions of SP are often more complex than simply causing decreased resting potassium conductance. Both rapidly and slowly rising responses and both increases and decreases in conductance occur in response to application of SP to cultured rat (Willard & Nishi, 1985 b) and guinea-pig (Hanani & Burnstock, 1984, 1985) myenteric neurones or to rat sympathetic neurones (Konishi, Song, Ogawa & Kanazawa, 1989). Substance P can also increase the excitability of subsets of myenteric neurones without altering resting input resistance or membrane potential (Willard & Nishi, 1985b). Voltage-clamp analysis of neurones in 3- to 15day-old cultures has revealed at least six voltage-gated currents that activate at potentials more positive than -50 mV, including at least two types of voltage-dependent potassium currents whose suppression could enhance postsynaptic excitability (Franklin & Willard, 1987). If the SP-like mediator of the slow EPSPs also altered one or more classes of voltage-gated channels that regulate properties of action potentials, this could further enhance postsynaptic excitability. Further studies will be necessary to determine whether voltage-gated conductances are altered during slow synaptic excitation and how they might contribute to the spiking activity observed during some of the slow EPSPs (e.g. Figs 2 and 4C). Comparison to tachykinin-mediated transmission in other systems Although there have been no previous studies of synaptic potentials evoked by individual SP-containing neurones, there are three well-characterized systems that have provided strong evidence of tachykinin-mediated slow synaptic transmission between neurones: the inferior mesenteric ganglion (1MG) of the guinea-pig (Konishi, Tsunoo & Otsuka, 1979; Konishi & Otsuka, 1985) the guinea-pig enteric nervous system (ENS) (Katayama & North, 1978; Johnson et al. 1981; Bornstein et al. 1984; Surprenant, 1984; Mihara et al. 1985), and the dorsal horn of the rat spinal cord (Randic et al. 1986, 1988). In each of these systems, there are abundant SP-positive presynaptic processes, calcium-dependent release of SP can be detected, and presynaptic stimulation evokes slow EPSPs that can be mimicked by responses to SP and blocked by desensitization to SP. In addition, in the guinea-pig IMG and the rat dorsal horn, synthetic SP antagonists block responses to SP and antagonize slow EPSPs. In the guinea-pig ENS, the SP antagonists have not been very useful because they both fail to block actions of SP and because they cause release of noradrenaline (Surprenant et al. 1987; Wade & Wood, 1988).

Conductance changes The decreased resting potassium conductance caused by slow EPSPs in the present study is very similar to conductance changes observed during slow EPSPs and responses to SP in neurones of the guinea-pig ENS (Katayama et al. 1979; Surprenant, 1984; Surprenant et al. 1987) and to slow EPSPs evoked by dual-

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function myenteric neurones (Willard, 1990). Substance P also evokes slow depolarizations accompanied by decreased conductance in neurones of the adult rat myenteric plexus (Brookes, Ewart & Wingate, 1988). In contrast, a much more complex set of changes in ionic conductances occurs during slow EPSPs and responses to tachykinins in neurones of the guinea-pig IMG (Dun & Minota, 1981; Griffith, Hills & Brown, 1988) and the rat dorsal horn (Murase, Ryu & Randic, 1989). The present finding that responses to another transmitter candidate, VIP, can also reduce resting potassium conductance and mimic slow EPSPs parallels the finding of Mihara et al. (1985) that responses to several different transmitter candidates can mimic slow EPSPs in neurones of the guinea-pig submucous plexus. Tachykinin receptor subtypes Studies of binding of radiolabelled tachykinins suggest that gastrointestinal tissues possess at least three types of tachykinin receptor, each of which binds one of the three naturally occurring mammalian tachykinins preferentially (reviewed in Jacoby, 1988). NK-1, NK-2 and NK-3 receptors are proposed to have highest affinity for SP, neurokinin A and neurokinin B (neuromedin K), respectively. Previous studies have concluded that guinea-pig enteric neurones have mainly NK- 1 receptors (Galligan et al. 1987; Burcher & Bornstein, 1988). The results of the present study, which suggest that NK-1 receptors mediate slow EPSPs between rat myenteric neurones, agree with those conclusions. However, more detailed pharmacological studies of the receptors mediating these slow EPSPs must be done.

Apparent lack of a 'classical' co-transmitter The presynaptic neurones in this study evoked slow EPSPs exclusively; none evoked fast EPSPs. The most straightforward interpretation of this result is that these neurones do not release a 'classical' transmitter that causes fast EPSPs. If this interpretation is correct, this would be the first clear demonstration of neurones that communicate with other neurones exclusively via transmitters that evoke only slow EPSPs. There have been reports of neurones that appear to receive only slow synaptic inputs, but it has not been possible to test whether the presynaptic neurones lack a classical co-transmitter. For example, subsets of AH (type 2) myenteric neurones in the guinea-pig receive robust slow synaptic potentials (Wood & Mayer, 1978; Johnson, Katayama & North, 1980) but appear to receive no fast EPSPs (Nishi & North, 1973; Hirst, Holman & Spence, 1974; Grafe, Wood & Mayer, 1979). It is not known whether drivers of slow EPSPs in AH neurones lack a co-transmitter that evokes fast EPSPs because the somata of AH neurones lack nicotinic receptors for ACh (North & Nishi, 1976; Tokimasa, Cherubini & North, 1983), the only known transmitter of fast EPSPs in the myenteric plexus. Thvs it is possible that drivers of slow EPSPs in guinea-pig AH neurones also release ACh but that the somata of the postsynaptic AH neurones do not respond to it. (Grafe et al. (1979) reported that some AH neurones receive small (1-3 mV) nicotinic EPSPs, but those EPSPs appear to occur on distal dendrites.) In contrast to AH neurones of the guinea-pig, all of identified postsynaptic neurones that were tested in this study had nicotinic responses to ACh and many of them received fast nicotinic EPSPs from other neurones in the cultures. Thus, if the

469 SUBSTANCE P-MEDIATED SLOW EPSPs drivers of slow EPSPs release a classical co-transmitter to which the postsynaptic neurones do not respond, that co-transmitter is unlikely to be ACh, because this would require that the presynaptic neurones release it only onto insensitive areas of postsynaptic membrane. Furthermore, although choline acetyltransferase-LIR and SP-LIR co-exist in subsets of submucous neurones in the guinea-pig (Furness, Costa & Keast, 1984), SP-LIR has not been detected in physiologically identified cholinergic enteric neurones in culture (Willard & Nishi, 1987; Willard, 1990). An alternative interpretation of the absence of fast EPSPs is that the slow EPSPs result from diffusion of an SP-like transmitter away from a synaptic contact at which it and a classical co-transmitter are released onto a different neurone. Such a phenomenon occurs in the bull-frog sympathetic nervous system: axons of the 7th and 8th spinal nerves release ACh and a luteinizing hormone-releasing hormone (LHRH)-like peptide onto type C postganglionic neurones in the 9th and 10th paravertebral ganglia. The LHRH then diffuses away from the C neurones to evoke late slow EPSPs in nearby type B neurones not contacted directly by the LHRHreleasing terminals (Jan & Jan, 1982). Because of the relatively large volume of extracellular fluid in the cultures, diffusion of sufficient transmitter to evoke detectable slow EPSPs is unlikely to occur over distance of more than a few micrometres. However, this possibility cannot be fully excluded in the absence of electron microscopic examination and reconstruction of identified pre- and postsynaptic neurones. This work was supported by NIH grant NS24362. I thank Ms Laura Fleck for excellent technical assistance. REFERENCES

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