Analysis of purinergic and cholinergic fast synaptic transmission to identified myenteric neurons

Neuroscience 116 (2003) 335–347

ANALYSIS OF PURINERGIC AND CHOLINERGIC FAST SYNAPTIC TRANSMISSION TO IDENTIFIED MYENTERIC NEURONS K. NURGALI, J. B. FURNESS* AND M. J. STEBBING

reserved.

Department of Anatomy and Cell Biology, University of Melbourne, Parkville, Vic. 3010, Australia

Key words: enteric nervous system, fast excitatory postsynaptic potential, ATP, acetylcholine, myenteric neurons, neurotransmitter.

Abstract—Types and projections of neurons that received cholinergic, purinergic and other fast excitatory synaptic inputs in myenteric ganglia of the guinea-pig distal colon were identified using combined electrophysiological recording, application of selective antagonists, marker dye filling via the recording microelectrode, and immunohistochemical characterisation. Fast synaptic inputs were recorded from all major subtypes of uniaxonal neurons including Dogiel type I neurons, filamentous interneurons, circular muscle motor neurons and longitudinal muscle motor neurons. Fast excitatory postsynaptic potentials were completely blocked by the nicotinic receptor antagonists hexamethonium or mecamylamine in 62% of neurons tested and were partially inhibited in the remaining neurons. The P2 purine receptor antagonist, pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid, reduced the amplitudes of fast excitatory postsynaptic potentials in 20% of myenteric neurons. The 5-hydroxytryptamine3 receptor antagonist granisetron reduced the amplitude of fast excitatory postsynaptic potentials in only one of 15 neurons tested. In five of five neurons tested, the combination of a nicotinic antagonist, pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid, granisetron and 6-cyano-7-nitroquinoxaline-2,3-dione did not completely block the fast excitatory postsynaptic potentials. Immunohistochemical studies of the neurons that had been identified electrophysiologically and morphologically imply that P2X2 receptors may mediate fast transmission in some neurons, and that other P2X receptor subtypes may also be involved in fast synaptic transmission to myenteric neurons of the guinea-pig distal colon. Neurons with nicotinic and pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid-sensitive fast excitatory postsynaptic potentials were present in both ascending and descending pathways in the distal colon. Thus, neither cholinergic nor mixed cholinergic/purinergic synaptic responses are confined to a particular class of neuron. The results indicate that acetylcholine and ATP are the major fast excitatory neurotransmitters in guinea-pig distal colon myenteric ganglia. © 2003 IBRO. Published by Elsevier Science Ltd. All rights

A role of acetylcholine (ACh) in fast synaptic transmission in the enteric nervous system is well established. Fast excitatory postsynaptic potentials (EPSPs) recorded from myenteric plexus neurons are substantially reduced in amplitude by nicotinic ACh receptor (nAChR) antagonists (Nishi and North, 1973; Hirst et al., 1974). Recently, it was found that the majority of fast EPSPs in myenteric neurons in the ileum have a noncholinergic component (Galligan et al., 2000), implying that substances other than ACh act as fast neurotransmitters in myenteric neurons. ATP, 5-hydroxytryptamine (5-HT) and glutamate may each contribute to non-nicotinic fast excitatory neurotransmission in the myenteric ganglia in the guinea-pig ileum (Galligan and Bertrand, 1994; Kirchgessner and Liu, 1996; Zhou and Galligan, 1996, 1998; Lepard et al., 1997; Lepard and Galligan, 1999; Galligan et al., 2000). The largest noncholinergic component appears to be mediated by ATP and is blocked by suramin and pyridoxal phosphate-6-azophenyl2',4'-disulfonic acid (PPADS) (Galligan and Bertrand, 1994; Lepard et al., 1997; Lepard and Galligan, 1999). Fast EPSPs with a purinergic component were found throughout the length of the gut but they were most prominent in the ileum and were rare in the gastric corpus (Lepard and Galligan, 1999). In non-enteric neurons, ATP also appears to mediate fast synaptic transmission at ligand-gated P2X receptors (Edwards et al., 1992; Bardoni et al., 1997). At present, seven mammalian P2X receptor subunits (P2X1–P2X7) have been identified by molecular cloning and most of these can form both homomeric as well as heteromeric assemblies (for reviews: Ralevic and Burnstock, 1998; MacKenzie et al., 1999; No¨renberg and Illes, 2000; Dunn et al., 2001; Khakh et al., 2001). Pharmacological studies in cultured myenteric neurons of the guinea-pig ileum revealed that applied ATP predominantly activated P2X receptors that were insensitive to ␣,␤-methylene ATP (␣,␤-meATP) and were slowly desensitising (Zhou and Galligan, 1996). The authors concluded that these receptors were composed of P2X2 subunits. However, a role for other P2X receptor subtypes in myenteric neurons has not been excluded (Barajas-Lo´pez et al., 1996). P2X2 receptor immunoreactivity has been found in myenteric neurons of the guinea-pig and rat small intestine (Vulchanova et al., 1996; Castelucci et al., 2002). P2X2 receptor subunits are expressed by specific types of my-

*Corresponding author. Tel: ⫹61-3-83448859; fax: ⫹61-3-93475219. E-mail address: [email protected] (J. Furness). Abbreviations: ACh, acetylcholine; AH, designation of neurons with broad action potentials and long-lasting after hyperpolarisations; AHP, after hyperpolarising potential; ␣,␤-meATP, ␣,␤-methylene ATP; AMPA, aminomethyl phosphonic acid; AP, action potential; Cin, input capacitance; ChAT, choline acetyltransferase; CNQX, 6-cyano7-nitroquinoxaline-2,3-dione; EPSP, excitatory postsynaptic potential; FITC, fluorescein isothiocyanate; 5-HT, 5-hydroxytryptamine; IC50, concentration causing 50% inhibition; mRNA, messenger RNA; nAChR, nicotinic acetylcholine receptor; NOS, nitric oxide synthase; PPADS, pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid; Rin, input resistance; RMP, resting membrane potential.

0306-4522/03$30.00⫹0.00 © 2003 IBRO. Published by Elsevier Science Ltd. All rights reserved. doi:10.1016/S0306-4522(02)00749-2

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enteric plexus neurons, including inhibitory motor neurons and intrinsic primary afferent neurons in the guinea-pig myenteric plexus (Castelucci et al., 2002). P2X3 receptors are also expressed by enteric neurons, although the populations of neurons are different from those expressing P2X2 receptor immunoreactivity (Poole et al., 2002). Microsurgical lesion experiments and physiological studies on reflex pathways have demonstrated that ATP contributes to excitatory neuro-neuronal transmission in descending pathways in the guinea-pig ileum (Johnson et al., 1999; Lepard and Galligan, 1999; Bian et al., 2000). In addition, a pharmacological analysis of reflex responses in vitro demonstrated that ATP is an excitatory transmitter in ascending pathways (Spencer et al., 2000). In the peripheral autonomic nervous system, ATP has been reported to be a cotransmitter with one or more other transmitters such as noradrenaline, ACh, nitric oxide, neuropeptide Y, 5-HT, calcitonin gene-related peptide, substance P and vasoactive intestinal peptide (Burnstock, 1999). Corelease of ATP with ACh has been detected in the autonomic nervous system and at the somatic neuromuscular junction (Burnstock, 1999). Moreover, many peripheral neurons coexpress nicotinic and P2X receptors (Silinsky and Gerzanich, 1993; Nakazawa, 1994). In the myenteric plexus, all enteric neurons, except nitric oxide synthase (NOS)-immunoreactive inhibitory muscle motoneurons, also contain choline acetyltransferase (ChAT) (Furness, 2000), suggesting that myenteric interneurons are cholinergic and that ATP and ACh might be coreleased at synapses between myenteric neurons. Molecular studies have revealed that P2X and nAChRs are structurally independent (Khakh, 2001). However, a functional interaction between these receptor types has been found in guinea-pig cultured myenteric, submucosal and sympathetic neurons (Barajas-Lo´pez et al., 1998; Searl et al., 1998; Zhou and Galligan, 1998; Khakh et al., 2000). There is evidence that 5-HT also acts as a fast excitatory neurotransmitter at enteric synapses. In the guineapig ileum, 11% of myenteric neurons receive fast synaptic input mediated through receptors blocked by 5-HT3 receptor antagonists (Zhou and Galligan, 1999). Glutamate, the major excitatory neurotransmitter in the CNS, was recently also reported to be an excitatory neurotransmitter in the enteric nervous system. It has been shown that glutamate mediates fast EPSPs via aminomethyl phosphonic acid (AMPA) receptors in AH/Dogiel type II neurons in the myenteric plexus of the guinea-pig ileum (Liu et al., 1997). Although the populations of myenteric neurons in the distal colon are similar to those in the small intestine, some morphological and immunohistochemical differences have been found between neurons in these regions. For example, three types of descending and one type of ascending interneurons have been identified in the ileum on immunohistochemical and morphological grounds (Costa et al., 1996; Furness, 2000), whereas in the distal colon four types of descending and three types of ascending interneurons have been distinguished (Lomax and Furness, 2000). The majority of uniaxonal neurons (70%) in the guinea-pig small intestine have descending projections

(Song et al., 1996), whereas the numbers of ascending and descending neurons are nearly equal in the distal colon (Lomax et al., 1999; Tamura et al., 2001). Moreover, filamentous descending somatostatin-immunoreactive interneurons, present in the ileum and duodenum (Portbury et al., 1995; Song et al., 1997; Clerc et al., 1998) were not found in the distal colon (Lomax et al., 1999). These differences imply that the reflex pathways are differently organised in the distal colon compared with the ileum, which presumably reflects functional differences between the two regions (Lomax et al., 1999; Lomax and Furness, 2000). The aim of this study was to identify the types and projections of neurons that receive cholinergic, purinergic and other types of fast synaptic inputs in myenteric neurons of the guinea-pig distal colon using intracellular electrophysiological recordings combined with marker dye filling via the recording microelectrode, followed by immunohistochemical characterisation.

EXPERIMENTAL PROCEDURES All experiments were performed on segments of distal colon removed from guinea-pigs (150 –275 g) (inbred Hartley strain colony of the Department of Anatomy and Cell Biology at the University of Melbourne) after they were stunned by a blow to the head and killed by cutting the carotid arteries and severing the spinal cord. The University of Melbourne Animal Experimentation Ethics Committee approved all procedures. All efforts were made to minimise animal suffering and numbers of animals used. The segments (2–3 cm) were taken from between 2 and 5 cm oral to the pelvic brim, the oral end was marked, and the segments were placed in physiological saline (composition in mM: NaCl, 118; KCl, 4.8; NaHCO3, 25; NaH2PO4, 1.0; MgSO4, 1.2; glucose, 11.1; CaCl2, 2.5; equilibrated with 95% O2/5% CO2) and initially kept at room temperature. The solution contained 3-␮M nicardipine and 1-␮M hyoscine (both from Sigma-Aldrich, Sydney, Australia) to inhibit muscle movement. The mucosa, submucosa and circular smooth muscle were carefully removed to expose the myenteric plexus. The recording dish (volume 1 ml) was then placed on the stage of an inverted microscope and continuously superfused (4 ml/min) with physiological saline preheated to yield a bath temperature of 35–37 °C. The tissue was equilibrated with perfusate for 1–2 h before recording commenced.

Electrophysiology Neurons were impaled with conventional borosilicate glass microelectrodes (100 –210 M⍀) filled with 1% biocytin (Sigma-Aldrich) in 1-M KCl. Recordings were made using an Axoclamp 2B amplifier (Axon Instruments, Foster City, CA, USA) in bridge and discontinuous current clamp mode. Signals were digitised at 1–10 kHz, using a Digidata 1322A interface (Axon Instruments), and stored using personal computer-based data acquisition software (Axoscope 8.2, Axon Instruments). Measurements of electrophysiological properties were made after allowing the impalements to stabilise for at least 15 min without applying intracellular holding current. At this time the ability of the cell to fire an action potential upon intracellular current injection was assessed. Only neurons that were able to fire an action potential and had resting membrane potentials (RMPs) more negative than ⫺40 mV were included in the electrophysiological analyses. Electrical stimuli were delivered to internodal strands using a fine tungsten stimulating electrode (10 –50-␮m tip diameter), insulated except at the tip. Stimuli were delivered via an ISO-Flex stimulator controlled by a Master-8 programmable pulse generator

K. Nurgali et al. / Neuroscience 116 (2003) 335–347 (both from AMPI, Jerusalem, Israel). Fast EPSPs were evoked by pulses of 0.1-ms duration and 0.3– 0.5-mA intensity at 10-s intervals. This interval was chosen as optimal in the range 100 ms to 20 s because every stimulus evoked fast EPSPs of approximately the same amplitude. The strength of stimuli was increased until no further increase in response amplitude was seen (maximal stimulus strength). For analysis of fast EPSPs we measured the average amplitude and width at half amplitude of 10 fast EPSPs recorded whilst the membrane potential was held at ⫺90 mV by injecting current through the recording microelectrode, to prevent action potential initiation by the fast EPSPs. Reductions in amplitude caused by antagonists were included in the data if the reduction was 5% or more of the control amplitude. To determine input resistance (Rin), small hyperpolarising current pulses (duration 100 ms, intensity 0.02– 0.05 nA), resulting in deflections of less than 10 mV, were injected. RMP, Rin, cell input capacitance (Cin), action potential (AP) amplitude, AP width at half amplitude, and AP threshold were determined using in-house analysis routines written in Igor Pro 4.0 analysis software (WaveMetrics, Lake Oswego, OR, USA).

Drugs Hexamethonium chloride, mecamylamine, PPADS, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and tetrodotoxin were obtained from Sigma-Aldrich. Granisetron was obtained from Smith Kline Beecham (Harlow, UK). All drugs were applied by addition to the superfusion solution. Drugs were present in the extracellular solution for at least 20 min prior to measuring the amplitude of the evoked response.

Neuron identification and immunohistochemistry Biocytin diffused from the recording electrodes into the neurons during the impalement, after which the ganglion and cell position were drawn and the recording electrode was moved to a fresh ganglion to avoid ambiguity of cell identity. At the end of each experiment, the tissue was fixed overnight in 2% formaldehyde plus 0.2% picric acid in 0.1-M sodium phosphate buffer (pH 7.0), cleared in three changes of dimethylsulphoxide, and washed in phosphate-buffered saline (3⫻). The tissue was then reacted with streptavidin coupled to Texas Red to reveal the biocytin and processed for the immunohistochemical demonstration of the P2X2 receptor subunit, NOS, ChAT or calretinin (Clerc et al., 1998). To localise P2X2 immunoreactivity we used a rabbit antiserum raised against amino acid sequence 457– 472 of the rat P2X2 receptor, with a single Cys extension at the N terminal (AB5244 from Chemicon, Temecula, CA). Incubation was for 48 h at 4 °C at a dilution of 1:120. Other primary antisera used were: rabbit anti-NOS (BS Masters, 1:200) rabbit anticalretinin (Swant, Bellizona, Switzerland) at 1:1000 and goat anti-ChAT 1:50 (AB114P from Chemicon). Incubation was for 24 or 48 h at room temperature. The secondary antibodies were donkey antirabbit immunuglobulin, coupled to fluorescein isothiocyanate (FITC), 1:50 (Amersham, Melbourne, Australia) or donkey antisheep FITC, 1:50 (Jackson ImmunoResearch Laboratories, PA). Preparations were examined on a Zeiss Axioplan microscope equipped with the appropriate filter cubes for discriminating between FITC and Texas Red fluorescence. To analyse the morphologies and projections of the impaled neurons, preparations in which impaled nerve cells had been identified were removed from the slides and washed in phosphatebuffered saline, prior to conversion of the streptavidin, bound to the biocytin, to a permanent deposit (Clerc et al., 1998). This was achieved using goat antistreptavidin antiserum coupled to biotin (Vector Laboratories, Burlingame, CA, USA), diluted 1:50 at room temperature. The biotin was in turn localised using an avidin– biotin– horseradish peroxidase kit (Vectastain, Vector Laborato-

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ries). The horseradish peroxidase was reacted with diaminobenzidine and hydrogen peroxide to yield a permanent deposit. Cell shapes, positions and projections were evaluated on an Olympus BH microscope under positive-low phase contrast optics, and drawn with the aid of a camera lucida drawing tube at ⫻400 or ⫻1000 magnification.

Statistics Electrophysiological data are presented as mean⫾S.E.M. Means were compared with a Student’s t-test (paired and unpaired data, as appropriate, two tailed). Differences were considered statistically significant at Pⱕ0.05.

RESULTS Electrophysiological properties and morphology of neurons with fast EPSPs Neurons in the myenteric plexus of guinea-pig distal colon were characterised electrophysiologically and then identified by shape and axonal projection after intracellular injection of biocytin. Recordings were taken from a total of 181 neurons. Of these, 156 were uniaxonal neurons and 25 had Dogiel type II morphology. All uniaxonal neurons responded to internodal strand stimulation with fast EPSPs, but none of the Dogiel type II neurons had fast synaptic inputs. Passive and active electrophysiological properties of neurons with fast EPSPs are presented in Table 1. Most of these neurons (139/156) had typical S-type electrophysiology, that is, they received large-amplitude fast EPSPs and had narrow APs with no late after-hyperpolarising potential (AHP) following the AP (Hirst et al., 1974; Bornstein et al., 1994). Seventeen S neurons had late AHPs that followed the AP. Fast EPSPs recorded from uniaxonal neurons with late AHPs were of similar amplitude and time course to those recorded from neurons with typical S-type electrophysiology. S/uniaxonal neurons were identified by their morphologies and the targets they innervated. There were 21 circular muscle motor neurons (13% of uniaxonal neurons); some of these had Dogiel type I morphology and some had a mixture of short filamentous and lamellar dendrites (Lomax et al., 1999). The other groups were longitudinal muscle motor neurons (n⫽3, 2%) and filamentous ascending interneurons (n⫽30, 19%). Dogiel type I neurons whose axons were not traced to the muscle were 102 (65%) of 156 adequately filled uniaxonal neurons. Most (75/102) of the Dogiel type I neurons that were not identified as motor neurons had anally projecting axons, 26/102 had orally projecting axons and 1/102 projected locally to another ganglion. Circular muscle motor neurons had descending (11/21), ascending (6/21) and local (4/21) projections. Longitudinal muscle motor neurons had local (2/3) and descending (1/3) projections. All filamentous neurons (30/156) had a single axon that projected orally. Their axons provided side branches in myenteric ganglia, confirming that these are interneurons (Lomax et al., 1999). RMP, Rin, AP amplitude, AP width at half amplitude and threshold current to evoke an AP were not significantly

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Table 1. Electrophysiological properties of neurons with fast excitatory postsynaptic potentials (EPSPs) in the guinea-pig distal colon Projection of neurons

Electrophysiological type

Resting membrane potential (mV)

Input resistance (M⍀)

Input capacitance (pF)

Action potential amplitude (mV)

AP width at half amplitude (ms)

AP threshold (nA)

Fast EPSP amplitudea (mV)

Fast EPSP width at half amplitudea (ms)

Presence of spontaneous fast EPSPs (number of neurons)

Dogiel type I (102/156)

Ascending (26/102); descending (75/102); local (1/102) Ascending (6/21); descending (11/21); local (4/21) Ascending (0/3); descending (1/3); local (2/3) Ascending (n ⫽ 30)

S (92/102) and AH with fast EPSP (10/102)

⫺48⫾0.6, n⫽87

183⫾14, n⫽81

14⫾1.3, n⫽82

54⫾1.1, n⫽81

1.41⫾0.06, n⫽81

0.18⫾0.02, n⫽68

18.5⫾1.2, n⫽57

17.7⫾1.1, n⫽56

33/102

S (16/21) and AH with fast EPSP (5/21)

⫺47⫾1.7, n⫽20

220⫾39, n⫽16

10⫾2.2, n⫽16

54⫾2.6, n⫽17

1.28⫾0.12, n⫽17

0.16⫾0.03, n⫽14

16.2⫾2.0, n⫽12

16.3⫾3.1, 16.4 n⫽13

5/21

S (3/3)

⫺42⫾0.9, n⫽3

243⫾22, n⫽3

14⫾10, n⫽3

53⫾5.1, n⫽3

1.53⫾0.58, n⫽3

0.07⫾0.02, n⫽3

16.7⫾0.6, n⫽2

14.9⫾1.7, n⫽2

2/3

S (28/30) and AH with fast EPSPs (2/30)

⫺51⫾1.3, n⫽29

162⫾17, n⫽26

21⫾2.2, n⫽26 (a, b)

57⫾2.7, n⫽26

1.19⫾0.07, n⫽26

0.18⫾0.02, n⫽23

26.5⫾2.3, n⫽16 (a, b)

19.1⫾2.2, n⫽18

16/30

Circular muscle motor neurons (21/156) Longitudinal muscle motor neurons (3/156) Filamentous (30/156) a

For each recording the average of 10 fast EPSPs recorded at ⫺90 mV was determined. (a) Pⱕ0.05, significantly different from Dogiel type I neurons. (b) Pⱕ0.005, significantly different from circular muscle motor neurons.

K. Nurgali et al. / Neuroscience 116 (2003) 335–347

Morphological type

K. Nurgali et al. / Neuroscience 116 (2003) 335–347

different between the morphological subgroups of uniaxonal neurons (Table 1). However, filamentous neurons had greater Cin, implying that they have larger surface areas compared with the Dogiel type I neurons (Pⱕ0.05) and circular muscle motor neurons (Pⱕ0.005). To prevent generation of APs by EPSPs, measurements of EPSP amplitudes were taken at holding potentials of ⫺90⫾5 mV. At this potential, nonregenerative proximal process potentials were sometimes observed, but APs were not evoked. Measurements were not taken if a proximal process potential obscured the peak of the fast EPSP. Fast EPSPs were elicited by stimulation of nerve fibres in connectives adjacent to the ganglia, and were blocked by tetrodotoxin (1 ␮M). The maximum amplitudes of fast EPSPs were greater in filamentous ascending interneurons compared with the Dogiel type I neurons (Pⱕ0.05) and circular muscle motor neurons (Pⱕ0.005; Table 1). Effect of nicotinic receptor antagonists on fast EPSPs The pharmacological properties of fast EPSPs were tested in 74 myenteric neurons. All fast EPSPs recorded from myenteric neurons were partly or completely inhibited by the nAChR antagonists hexamethonium (100 ␮M) or mecamylamine (10 ␮M). nAChR antagonists reduced the amplitudes of fast synaptic EPSPs by 90 –100% (average 94.6%) of control in 62% (46/74) of neurons (Fig. 1A, B). In the remaining 38% (28/74) of neurons, fast EPSPs were only partly (⬍90%) inhibited by nAChR antagonists, suggesting the involvement of other transmitters in mediating fast synaptic excitation. In these cells the fast EPSPs were inhibited by 5–30% of control amplitude in 9/28 neurons, by 30 – 60% in 16/28 neurons and by 60 –90% in 3/28 neurons (Fig. 1B). The three neurons in this last group were exposed to antagonist for less than 20 min and it is possible that their fast EPSPs would have eventually been blocked completely. In 59/74 experiments, nicotinic antagonists were applied before the application of any other antagonist. In 15/74 experiments hexamethonium or mecamylamine was used after superfusion of antagonists for other receptors that had little or no effect on the amplitude of fast EPSP. The inhibitory effect of nicotinic blockers on fast EPSPs was similar regardless of the sequence of antagonist application. Mecamylamine-sensitive fast EPSPs in the guinea-pig distal colon had reversal potentials of about 0 mV, which is similar to findings in the ileum (Galligan and Bertrand, 1994). Neurons with fast EPSPs that were inhibited by 90 – 100% by nicotinic antagonists had the following morphologies and projections: 32/46 neurons had Dogiel type I morphology with ascending (n⫽7) and descending (n⫽25) projections; 7/46 cells were circular muscle motor neurons with ascending (n⫽1), descending (n⫽4) and local (n⫽2) projections; 6/46 cells had filamentous ascending morphology and one neuron was a descending longitudinal muscle motor neuron (Fig. 2). Altogether 30/46 neurons had descending axons, 14/46 had ascending axons and 2/46 had local projections.

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Of cells with only cholinergic fast EPSPs, 16 descending neurons were tested for NOS immunoreactivity and seven of these were NOS-immunoreactive (Fig. 1C). All filamentous ascending interneurons tested were positive for calretinin (Fig. 1D), as previously described (Lomax et al., 1999). Spontaneous fast EPSPs were recorded in 56/156 uniaxonal neurons (Table 1), which is consistent with previous findings in the distal colon (Wade and Wood, 1988). However, spontaneous fast EPSPs were not seen in the presence of nicotinic antagonists. PPADS (10 ␮M), applied prior to nAChR antagonists, did not block spontaneous fast EPSPs. Effect of PPADS The P2 receptor antagonist PPADS was used to test for a purinergic component of the residual, hexamethonium-/ mecamylamine-resistant, fast EPSPs. It has been shown that PPADS used at concentration of 1–10 ␮M effectively blocks PPADS-sensitive P2 receptors with little or no inhibition of ecto-nucleotidases (Lambrecht, 2000). In myenteric neurons in the guinea-pig ileum, the concentration causing 50% inhibition (IC50) for PPADS to inhibit fast EPSP amplitude was 3 ␮M (Lepard and Galligan, 1999). In 21 experiments PPADS (10 ␮M) was applied to neurons with hexamethonium- or mecamylamine-resistant fast EPSPs and the effect of PPADS was calculated relative to the amplitude of fast EPSPs after nAChR block. The average amplitude of hexamethonium-/mecamylamine-resistant fast EPSPs in 21 neurons was 12.8⫾2.6 mV and the width at half amplitude was 18.7⫾3.2 ms. In 14 of these neurons, PPADS (10 ␮M) reduced the amplitude of hexamethonium-/mecamylamine-resistant fast EPSPs by 90 –100% (average 93.5% of nicotinic antagonist-resistant amplitude) (Fig. 3A, B). In three cells PPADS inhibited the amplitude of hexamethonium-resistant fast EPSPs by 60 – 90%, in two cells by 5–30% and the other two cells did not have a PPADS-sensitive component. Increases in stimulus strength did not affect the amplitude of fast EPSPs in the presence of PPADS. When PPADS was used prior to nAChR antagonists (eight neurons) it had no effect on the amplitude of fast EPSPs in seven neurons and inhibited the compound fast EPSP by 62% of control amplitude in one neuron. Subsequent application of nicotinic receptor antagonists completely blocked the fast EPSP in these neurons. This one neuron with PPADS-sensitive component together with 14 neurons with 90 –100% inhibition of hexamethonium-resistant component by PPADS were considered as neurons with mixed cholinergic/purinergic fast EPSPs. They were 20% (15/74) of all tested cells. Neurons in which PPADS had little effect were further investigated with other antagonists. The 15 neurons with mixed cholinergic/purinergic inputs were of the following morphological types: Dogiel type I with descending (8/15 neurons) and ascending (2/15) projections, a circular muscle motor neuron with an ascending axon (1/15) and filamentous ascending interneurons (4/15) (Fig. 4). Overall, in the guinea-pig distal colon,

Fig. 1. Effect of nicotinic acetylcholine receptor (nAChR) antagonists on fast excitatory postsynaptic potentials (EPSPs) in identified distal colon myenteric neurons. (A) Example of a fast EPSP that was reduced in amplitude by hexamethonium (100 ␮M). Hexamethonium or mecamylamine (10 ␮M) reduced fast EPSP amplitude by 90 –100% in 62% (46/74) of neurons. Traces represent an average of 10 fast EPSPs recorded at a holding membrane potential of ⫺90 mV (F⫽stimulus artefact). (B) Frequency distribution of the percentage inhibition of fast EPSP amplitude by nAChR antagonists, hexamethonium (100 ␮M) or mecamylamine (10 ␮M). Values were calculated relative to fast EPSP amplitude in the absence of the receptor blocker. All neurons from which recordings were made were filled with biocytin during impalement. Biocytin was revealed with streptavidin coupled to Texas Red and examined by fluorescence microscopy (C1, D1). Neurons were further processed for immunohistochemical staining with appropriate antibodies. (C1-3) A nitric oxide synthase (NOS)-immunoreactive Dogiel type I neuron that had a nicotinic fast EPSP. The neuron was filled with biocytin during recording, which was revealed with streptavidin–Texas Red (STR, C1, filled neuron at arrow). NOS immunoreactivity is shown in C2 (arrow). The label was converted to a permanent deposit and the neuron was drawn using a camera lucida (C3). This neuron had an anally projecting axon. (D1-3) A filled calretinin-immunoreactive filamentous ascending interneuron, which had a nicotinic fast EPSP, that was revealed by streptavidin–Texas Red linked to the intracellularly injected biocytin (D1), by calretinin immunoreactivity (D2) and by camera lucida drawing made after the intracellular marker was made permanent (D3).

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Fig. 2. Examples of neurons with fast excitatory postsynaptic potentials inhibited by 90 –100% of control amplitude by hexamethonium (100 ␮M) or mecamylamine (10 ␮M) drawn using a camera lucida. The neurons labelled 1, 2, 3, 4, 6, 9, 10, 12 are Dogiel type I neurons with descending projections of their axons. Neuron 8 is an ascending Dogiel type I neuron. Neurons 5 and 7 are circular muscle motor neurons with local (5) and ascending (7) axon projections; 11 is a filamentous ascending interneuron, 13 is a local longitudinal muscle motor neuron.

8/15 neurons with a PPADS-sensitive fast EPSP had descending and 7/15 had ascending projections. All descending Dogiel type I neurons with a PPADSsensitive component of the fast EPSP which were tested for NOS were NOS-immunoreactive (Fig. 3C). Likewise, all PPADS-sensitive neurons tested for ChAT were ChAT-immunoreactive. Five descending Dogiel type I neurons with PPADS-sensitive fast EPSPs were processed for P2X2 receptor immunoreactivity. 1/5 of these was strongly immunoreactive whilst 4/5 were weakly immunoreactive (Fig. 3D). Other neurons, without PPADS-sensitive fast EPSPs, were also weakly immunoreactive for P2X2 receptors. With this antiserum, receptor immunoreactivity separates neurons into weakly and strongly staining groups; in both cases absorption experiments indicate that labelling is specific (Castelucci et al., 2002).

Effect of granisetron In our experiments, the 5-HT3 receptor selective antagonist granisetron (1 ␮M) was used in eight neurons with nicotinic antagonist and PPADS-resistant fast synaptic inputs in the presence of hexamethonium or mecamylamine after washout of PPADS. In another seven neurons granisetron was used prior to other antagonists. In all but one experiment granisetron did not affect the amplitude of control or hexamethonium-/ PPADS-resistant fast EPSPs. For the one neuron that was affected, the amplitude of the fast EPSP in the absence of any antagonists was 21.3 mV and the width at half amplitude was 14.8 ms. Hexamethonium reduced the fast EPSP amplitude to 9.8 mV and the width at half amplitude to 7.3 ms. Granisetron reduced the amplitude of the hexamethonium-/PPADS-resistant fast EPSP by

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Fig. 3. Effect of pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) on the nicotinic acetylcholine receptor (nAChR) antagonist-resistant components of fast excitatory postsynaptic potentials (EPSPs). (A) Example of a fast EPSP that was partly blocked by hexamethonium (100 ␮M) and further reduced in amplitude by PPADS (10 ␮M). (B) PPADS (10 ␮M) applied to neurons with hexamethonium-resistant fast EPSPs reduced the amplitude of residual fast EPSPs by 90 –100% in 14/21 neurons, and to lesser extents in other neurons. Values were calculated relative to the amplitude of nAChR antagonist-resistant components of fast EPSPs (100%). (C1-3) A neuron with mixed cholinergic and purinergic fast EPSPs. The neuron was filled with biocytin during recording and the biocytin was revealed with streptavidin–Texas Red (STR, C1, filled neuron at arrow). The neuron was immunoreactive for nitric oxide synthase (C2, upper arrow; a second neuron was also impaled in this ganglion, lower arrow). (C3) The neuron drawn using a camera lucida. This neuron had an anally projecting axon. (D1-3) Neuron with mixed input that was immunoreactive for the P2X2 purine receptor subunit. (D1) STR image of the filled neuron. (D2) P2X2 purine receptor immunoreactivity. (D3) Camera lucida drawing. The neurons had Dogiel type I morphology and an anally directed axon.

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Fig. 4. Examples of neurons in which pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid reduced the amplitude of the nicotinic acetylcholine receptor antagonist-resistant component of the fast excitatory postsynaptic potential by 90 –100%. Neurons were drawn using a camera lucida. Neurons labelled 1 and 9 are filamentous ascending interneurons. Those labelled 2, 3, 5, 6, 8 are descending Dogiel type I neurons, neuron 7 is an ascending Dogiel type I neuron, and neuron 4 is an ascending (excitatory) circular muscle motor neuron.

58%, without reducing its duration (Fig. 5A). This neuron had Dogiel type I morphology with an ascending axon and large cell body (Fig. 5B). In one neuron granisetron reduced the time-course of the fast EPSP. Effect of CNQX The effect of the AMPA receptor antagonist CNQX (20 ␮M) was tested in 5/74 uniaxonal neurons in which a hexamethonium- or mecamylamine-resistant component of the fast EPSP was not fully blocked by PPADS and/or granisetron. CNQX was applied in the presence of hexamethonium, after washout of PPADS or granisetron. It did not reduce the amplitude of hexamethonium-/mecamylamine-resistant fast EPSPs. In the five neurons tested that had fast EPSPs resistant to nicotinic antagonists, PPADS and granisetron, the amplitudes of

fast EPSPs increased to about the amplitude seen after hexamethonium following addition of CNQX (Fig. 6A). These results suggest that other neurotransmitters, but not glutamate acting at AMPA receptors, contribute to the fast synaptic transmission in uniaxonal myenteric neurons in the distal colon. Neurons with fast EPSPs resistant to nicotinic antagonists, PPADS, granisetron and CNQX had morphologies of Dogiel type I neurons with ascending (2/5) and descending (2/5) projections and a circular muscle motor neuron with ascending projection (1/5) (Fig. 6B).

DISCUSSION The present study has investigated the electrophysiological properties, immunohistochemical characteristics, mor-

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Fig. 5. Effect of granisetron on fast synaptic input to a distal colon myenteric neuron. Left, granisetron (1 ␮M) inhibited the amplitude of the fast excitatory postsynaptic potential that had been reduced but not blocked by hexamethonium (100 ␮M) and pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (10 ␮M). The neuron was a Dogiel type I neuron with ascending axonal projection (camera lucida drawing at right).

phologies and projections of neurons with different pharmacologies of fast synaptic input. Our results provide evidence that ACh and ATP are the major neurotransmitters mediating fast EPSPs in guinea-pig distal colon myenteric neurons. Only 12% of neurons had fast EPSPs that were not blocked (⬎90% reduction) by a nicotinic receptor antagonist or a nicotinic receptor antagonist plus PPADS. The results of immunohistochemical studies of the electrophysiologically and morphologically identified neurons that are discussed below imply that the P2X2 purine receptor subunit is involved in purinergic transmission, but that other receptor types also contribute to purinergic transmission in the myenteric neurons in the guinea-pig distal colon. Fast EPSPs were completely blocked by nAChR antagonists in 62% of neurons that were tested and were

inhibited partially in the remaining neurons. A PPADSsensitive component was confined to 20% of myenteric neurons. The 5-HT3 receptor antagonist granisetron reduced the amplitude of the fast EPSP in only 1 of 15 myenteric neurons; in other neurons, already blocked by an nAChR antagonist or PPADS, granisetron was not tested. Fast EPSPs resistant to nAChR antagonists, PPADS, granisetron and the glutamate receptor antagonist, CNQX, were revealed. This could be due to the involvement of a novel neurotransmitter, or to actions of ATP, ACh, glutamate or 5-HT at receptor subtypes not sensitive to hexamethonium, PPADS, granisetron or CNQX. In the distal colon, cholinergic and mixed cholinergic/ purinergic inputs were not confined to a particular class of myenteric neuron. Nicotinic and PPADS-sensitive fast EPSPs were recorded in both ascending and descending neurons. P2X receptors The fast rise time and short duration of the non-nicotinic fast EPSPs recorded in this study and their inhibition by the P2 receptor antagonist PPADS (10 ␮M) indicate the involvement of ligand-gated P2X receptors. Studies on cultured myenteric neurons of guinea-pig small intestine suggest that applied ATP acts predominantly at P2X2 receptors (Zhou and Galligan, 1996). The majority of receptors in cultured myenteric neurons of guinea-pig small intestine (92%) had properties similar to those of P2X2 or P2X5 receptors in expression systems, as they were slowly desensitised by ATP and only weakly activated by ␣,␤-meATP (Zhou and Galligan, 1996). These responses were blocked by PPADS. Because P2X2 receptor messenger RNA (mRNA) is found throughout the peripheral nervous system whilst P2X5 receptor mRNA has been found only in the CNS (Collo et al., 1996), it has been

Fig. 6. Example of a fast excitatory postsynaptic potential (EPSP) that was resistant to nicotinic acetylcholine receptor antagonists, pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), granisetron and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Resistant responses were recorded from five neurons. (A) Antagonists were applied in the following order: hexamethonium (100 ␮M), granisetron (1 ␮M), PPADS (10 ␮M) and CNQX (20 ␮M). Hexamethonium and PPADS caused reductions in fast EPSP amplitude, whereas granisetron reduced fast EPSP duration in this neuron. Addition of CNQX after washout of PPADS increased the fast EPSP amplitude. (B) Camera lucida drawings of two neurons that had fast EPSPs resistant to all four antagonists: ascending Dogiel type I neuron (above) and ascending circular muscle motor neuron (below).

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concluded that ATP in myenteric neurons acts predominantly at P2X2 receptors (Zhou and Galligan, 1996). The results of our investigation in distal colon myenteric neurons imply the presence of fast EPSPs mediated by PPADS-sensitive receptors other than P2X2 and also possibly PPADS-resistant P2X receptors. We localised P2X2 receptors on pharmacologically and morphologically characterised neurons in intact myenteric ganglia. Our experiments revealed that only one neuron tested was strongly immunoreactive for P2X2 receptors, whereas other neurons with PPADS-sensitive fast EPSPs were weakly immunoreactive for P2X2 receptors, suggesting the presence of subunits other than P2X2 on these cells. The possibility of the marker dye affecting the immunohistochemical reaction cannot be excluded. However, recent immunohistochemical studies on the distribution of P2X2 receptors throughout the guinea-pig intestine have shown that they are confined to about 25% of myenteric neurons (Castelucci et al., 2002). Furthermore, there is evidence for the presence of non-P2X2, but PPADS-sensitive receptors in enteric neurons. It was found that a small proportion of cultured myenteric neurons of the guinea-pig ileum responded to ATP with pharmacological properties of P2X1 or P2X3 receptors. These receptors were activated by both ATP and ␣,␤-meATP, and responses desensitised rapidly (Zhou and Galligan, 1996). P2X3-immunoreactivity has been detected in neurons of the myenteric and submucosal plexuses in the human and guinea-pig colon (Yiangou et al., 2001; Poole et al., 2002). Some neurons without PPADS-sensitive fast EPSPs were also weakly immunoreactive for P2X2 receptors. Castelucci et al. (2002) reported that 92% of neurons in the distal colon with strong P2X2 receptor immunoreactivity were also NOS-immunoreactive. These are Dogiel type I neurons with descending axons (Lomax and Furness, 2000). However, in this study only 8/15 neurons with PPADS-sensitive fast EPSPs had descending axons. It is also possible that a minor component of transmission is mediated through PPADS-insensitive purine receptors. PPADS blocks rat recombinant homomeric P2X1, P2X2, P2X3 and P2X5 receptors and heteromeric P2X2/3 and P2X1/5 receptors with IC50 values in the range 0.1–5 ␮M and P2Y1 receptors with similar potencies (for reviews: Ralevic and Burnstock, 1998; Lambrecht, 2000; North and Surprenant, 2000; Khakh et al., 2001). PPADS is a weak or ineffective antagonist (IC50ⱖ10 ␮M) at rat recombinant homomeric P2X4, P2X6 and P2X7 receptors, at heteromeric P2X4/6 receptors, and also at a number of P2Y receptors (Ralevic and Burnstock, 1998; Lambrecht, 2000; North and Surprenant, 2000; Khakh et al., 2001). Immunoreactivity for the PPADS-resistant P2X7 receptor has been found in cell bodies in both myenteric and submucosal plexuses and in association with nerve fibres in the guinea-pig small intestine (Hu et al., 2001). Thus, residual fast EPSPs that were not blocked by PPADS and nAChR antagonists might be due to ATP acting at PPADS-insensitive P2X-type purine receptors. Although properties of recombinant P2X receptors resemble closely those from native tissue (MacKenzie et al.,

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1999), pharmacological differences between native and recombinant P2X receptors do exist (No¨renberg and Illes, 2000). Moreover, interspecies differences have been shown for both native and recombinant receptors in terms of their sensitivity to agonists and antagonists (for review: No¨renberg and Illes, 2000). Therefore, there may be limitations in extrapolating data from rat native and recombinant receptors to studies in guinea-pig. Effects of nicotinic receptor antagonists and PPADS on fast EPSPs The proportion of fast EPSPs (62%) that were blocked by nicotinic antagonists in the distal colon was greater than in the ileum, where 25% of neurons exhibited fast EPSPs that were more than 90% blocked by nAChR antagonists (Galligan et al., 2000). Conversely, the proportion of electrically evoked fast EPSPs inhibited by PPADS in the myenteric neurons of the distal colon (20%) was much less than in the ileum (67%) (Lepard et al., 1997; Galligan et al., 2000). Our results are thus in agreement with LePard et al (1997) who reported that at maximal stimulus strength the proportion of fast EPSPs with a purinergic component in the distal colon was less than in the ileum. These differences in types of synaptic transmission together with morphological and immunohistochemical differences found between neurons in these regions imply that the neuronal circuits are differently organised in the distal colon compared with the ileum. Spontaneous fast EPSPs were observed in about onethird of S neurons in the control, but were not seen in the presence of the nicotinic antagonists, hexamethonium or mecamylamine. This suggests that the dominant transmission from neurons that are spontaneously active is cholinergic. However, we cannot exclude the possibility that there is a purinergic component of spontaneous transmission that was not found in the present work. Cotransmission of ATP with ACh In the ileum and colon myenteric plexus all enteric neurons, except the NOS-immunoreactive inhibitory muscle motoneurons, are ChAT-immunoreactive (Furness, 2000; Lomax and Furness, 2000). The axons of NOS-immunoreactive inhibitory muscle motoneurons do not make synapses with other neurons as they project only to muscle (Furness, 2000). This indicates that all neurons with cell bodies in the myenteric plexus whose axons make synapses with other neurons in the plexus express a cholinergic phenotype. Therefore, purinergic inputs of myenteric origin presumably come from neurons that are cholinergic, suggesting that ACh and ATP are likely to be cotransmitters to myenteric neurons. Types of neurons with cholinergic and mixed cholinergic/purinergic inputs In the guinea-pig ileum, microsurgical lesion experiments and physiological studies on reflex pathways demonstrated that ATP mediates excitatory neuro-neuronal transmission in descending pathways (Johnson et al., 1999;

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Lepard and Galligan, 1999; Bian et al., 2000). In the ileum, some myenteric neurons with suramin-sensitive fast EPSPs were identified morphologically as inhibitory motor neurons with anally projecting axons (Johnson et al., 1999). However, experimental data on reflexes from the mucosa demonstrated that both ACh and ATP act as excitatory neuro-neuronal transmitters in ascending and descending pathways (Spencer et al., 2000). The results of the present study of distal colon myenteric neurons demonstrate that neither purinergic nor cholinergic inputs are confined to a particular class of enteric neurons. In our experiments, cells with both ascending and descending projections had cholinergic or PPADS-sensitive fast EPSPs. These findings are consistent with data from the ileum reported by Spencer et al. (2000). Other transmitters There is evidence that 5-HT acts as a fast excitatory neurotransmitter at 5-HT3 receptors, albeit at very small number of synapses. In the ileum it has been shown that 11% of myenteric neurons receive fast synaptic input mediated by 5-HT acting at 5-HT3 receptors (Zhou and Galligan, 1999). In our experiments in the distal colon, granisetron (1 ␮M) reduced fast EPSP amplitude in only 1/8 neurons with hexamethonium-resistant fast EPSP that was tested. The lack of 5-HT-mediated fast synaptic inputs can be related to the small number of 5-HT-containing neurons and terminals in the myenteric plexus (Wardell et al., 1994). The population of 5-HT-containing neurons comprises about 2–3% of neurons in the myenteric plexus in both the guinea-pig ileum (Costa et al., 1982; Costa et al., 1996) and distal colon (Wardell et al., 1994), and these neurons all project anally. All 5-HT-containing neurons are immunoreactive for ChAT, suggesting that 5-HT and ACh could be cotransmitters (Furness, 2000). Glutamate was also recently reported to be an excitatory neurotransmitter in the enteric nervous system. Enteric neurons express the neuronal glutamate transporter EAAC1 and both N-methyl-D-aspartate and AMPA receptor subtypes (Liu et al., 1997). It has been reported that glutamate acting at AMPA receptors can contribute to fast synaptic excitation in AH/Dogiel type II myenteric neurons in the guinea-pig ileum (Liu et al., 1997). However, none or very few Dogiel type II cells in the distal colon have fast synaptic inputs (Tamura et al., 2001; Nurgali et al., 2002), so it has not been possible to investigate the involvement of glutamate in these neurons. In our experiments, an AMPA glutamate receptor antagonist, CNQX, did not reduce the amplitude of hexamethonium-/mecamylamineresistant fast EPSPs recorded from S neurons. These results suggest that other neurotransmitters, or PPADSresistant P2X receptors, contribute to the fast synaptic transmission in myenteric neurons in the distal colon.

CONCLUSION The present study shows that ACh and ATP both act as excitatory neurotransmitters in ascending and descending pathways in the myenteric plexus of the distal colon. Nei-

ther cholinergic nor purinergic signalling was confined to a particular class of enteric neurons. The findings suggest that cholinergic and purinergic pathways are differently organised in the distal colon compared with the ileum, which is consistent with the distinct functional properties of these two regions. The results also suggest that receptor subtypes other than P2X2 are involved in purinergic transmission to distal colon myenteric neurons. Acknowledgements—These studies were funded by the National Health and Medical Research Council (Australia). Melanie Coffey is thanked for her excellent assistance with the processing for cell morphology and immunohistochemistry and preparation of figures.

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(Accepted 25 September 2002)