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. 2001 Nov 1;536(Pt 3):727–739. doi: 10.1111/j.1469-7793.2001.00727.x

Glycine activates myenteric neurones in adult guinea-pigs

Michel Neunlist *,†, Klaus Michel *, Dania Reiche *, Gisela Dobreva *, Korinna Huber *, Michael Schemann *
PMCID: PMC2278892  PMID: 11691868

Abstract

  1. We studied the effects of glycine on myenteric neurones and muscle activity in the colon and stomach of adult guinea-pigs.

  2. Intracellular recordings revealed that myenteric neurones responded to local microejection of glycine (1 mm) with a fast, transient membrane potential depolarisation (57 % of 191 colonic neurones and 26 % of 50 gastric neurones). Most glycine-sensitive neurones had ascending projections and were choline acetyltransferase immunoreactive. Glycine preferentially activated neurones with a late afterhyperpolarisation (AH-neurones) and tonic spiking neurones with fast synaptic inputs (tonic S-neurones) but less frequently phasic S-neurones and inexcitable (non-spiking) neurones. The depolarisation had a reversal potential at −19 ± 13 mV, which was increased by 18 ± 10 % upon lowering extracellular chloride concentration and decreased by 38 ± 14 % in furosemide (frusemide, 2 mm).

  3. Strychnine (300 nm) reversibly abolished the glycine-induced depolarisation and the Cl− channel blocker picrotoxin (100 μm) reduced the amplitude of the depolarisation by 55 ± 5 %. The glycine effect was a postsynaptic response because it was not changed after nerve blockade with tetrodotoxin (1 μm) or blockade of synaptic transmission in reduced extracellular [Ca2+]. The effect was specific since the response was not changed by the nicotinic antagonists hexamethonium (200 μm) and mecamylamine (100 μm), the GABAA receptor antagonist bicuculline (10 μm), the NMDA antagonist MK-801 (20 μm) or the 5-HT3 antagonist ICS 205930 (1 μm).

  4. Glycine (1 mm) induced a tetrodotoxin- and strychnine-sensitive contractile response in the colon; the contractile response in the stomach was tetrodotoxin insensitive.

  5. Glycine activated myenteric neurones in the adult enteric nervous system through strychnine-sensitive mechanisms. The glycine-evoked depolarisation was caused by Cl− efflux and the maintenance of relatively high intracellular chloride concentrations involved furosemide-sensitive cation-chloride co-transporters.

The concept that the classical excitatory transmitter glutamate and the inhibitory transmitters γ-aminobutyric acid (GABA) and glycine act primarily in the central nervous system has been challenged over recent years. Some of the studies that have demonstrated the effects of these transmitters in the peripheral nervous system were performed in the enteric nervous system (ENS). The ENS is an integrative nervous system within the wall of the gut and regulates major gastrointestinal functions (for review see Wood, 1994). In the ENS, glutamatergic neurotransmission involving N-methyl-d-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and metabotropic receptors has been described (Liu et al. 1997; Hu et al. 1999; Ren et al. 2000). GABA effects on myenteric neurones are primarily excitatory and mediated via bicuculline-sensitive and -insensitive mechanisms (Cherubini & North, 1984a,b). In agreement, both GABA and glutamate have been shown to influence various gastrointestinal functions such as motility (Ong & Kerr, 1982; Tonini et al. 1989), electrolyte and water transport (MacNaughton et al. 1996), gastric acid secretion (Tsai et al. 1987) or gallbladder emptying (Gielkens et al. 1997) via their effects on the enteric nervous system.

Unlike the effects of glutamate and GABA, the effect of glycine on enteric neurones has rarely been studied and the few studies available report contradictory results (Mayer et al. 1982; Cherubini & North, 1984a; Liu et al. 1997). Mayer et al. (1982) showed that glycine induced a fast membrane depolarisation while Cherubini & North (1984a) observed no such effect. Data on the effects of glycine on gastrointestinal functions are very limited. Glycine caused a contractile effect in the guinea-pig taenia coli (Ishizawa & Miyazaki, 1979) and strychnine, an antagonist of the inhibitory glycine receptor, increased non-adrenergic inhibitory postsynaptic potentials in colonic smooth muscle (Vladimirova & Shuba, 1978). Indirect evidence for neurally mediated glycine effects has been provided by a study which showed that glycine caused a release of serotonin (Gross & Sturkie, 1975), one of the most important neuromodulators in the enteric nervous system. Recently, expression of the high affinity glycine transporter GLYT1 has been demonstrated in the basolateral membrane of enterocytes and there has been some speculation as to whether basolateral glycine uptake is involved in terminating the neuromodulatory activity of glycine (Christie et al. 2001).

Glycine is the major inhibitory neurotransmitter in the spinal cord and hindbrain areas of vertebrates (Bechade et al. 1994). Activation of the glycine receptor results in the opening of a chloride channel that can be specifically blocked by strychnine (Curtis et al. 1971). The concept that glycine acts solely as an inhibitory neurotransmitter has been revised over the past few years. Studies have revealed that in neonatal neurones of the central and peripheral nervous system glycine has excitatory effects that are also mediated via strychnine-sensitive mechanisms (Ito & Cherubini, 1991; Boehm et al. 1997). In addition, glycine serves as an allosteric activator of NMDA receptors which mediate glutamatergic excitation in the central nervous system (Johnson & Ascher, 1987).

The aim of this study was to investigate the effects of glycine in the enteric nervous system and to characterise its mechanisms of action on myenteric neurones of the guinea-pig proximal colon and stomach. In addition, we studied the effects of glycine on isolated circular muscle strips from the guinea-pig colon and gastric corpus.

METHODS

Preparations

The experiments were performed on isolated segments of proximal colon and gastric corpus of the guinea-pig. Male guinea-pigs, weighing 200–500 g, were killed by cervical dislocation and the proximal colon and stomach were quickly removed. All experiments were approved and carried out according to the guidelines of the Animal Care Centre at the School of Veterinary Medicine in Hannover. For recordings from colonic myenteric neurones a 4–7 cm long segment of colon (about 5 cm distal to the caeco-colic junction) was opened along the mesenteric border, pinned out flat in a dissection dish and continuously superfused with ice-cold Krebs solution (for composition see below). For recordings from gastric myenteric neurones, the stomach was opened, flushed with Krebs solution and the parietal part of the corpus was used for further dissection. In both the gastric and colonic preparations the mucosa and circular muscle were removed using small forceps in order to expose the myenteric plexus. For intracellular recordings, a 4 cm × 2 cm segment of the colon or a 1.5 cm × 2.0 cm segment of the gastric corpus was pinned in a Sylgard (Dow Corning)-covered recording chamber (5.5 cm × 4 cm) and continuously perfused at a flow of 13 ml min−1 with oxygenated Krebs solution at 35–37 °C. The Krebs solution contained (mm): 117 NaCl, 4.7 KCl, 1.2 MgCl2, 1.2 NaH2PO4, 25 NaHCO3, 2.5 CaCl2, 11 glucose and was continuously gassed with 95 % O2 and 5 % CO2. To reduce smooth muscle activity, nifedipine was added to the Krebs solution at a final concentration of 1–3 μm.

Electrophysiology

Intracellular recordings from myenteric neurones were performed as described previously in detail (Neunlist et al. 1999). The experiments were done under visual control using an Olympus IX 50 inverted microscope (Olympus, Hamburg, Germany). Unless otherwise stated, intracellular recordings were performed with borosilicate glass microelectrodes (WPI, Sarasota, FL, USA) filled with 0.5 % neurobiotin in 0.5 m KCl and 5 mm potassium acetate. The electrodes had resistances of between 150 and 250 MΩ. Signals were amplified (Intra 767, WPI, Newhaven, CT, USA or MEZ 8300, Nihon Kohden, Japan), displayed on an oscilloscope (DSO 420, Gould Instruments, Dietzenbach, Germany) and a chart recorder (Gould TA 11, Gould Instruments) and stored using a DAT recorder (DTR-1202, Biologic Science Instruments, Claix, France). Data were analysed and displayed off-line using a Macintosh computer and a MacLab system (MacLab 4 s/e with Chart software 3.5.1, ADInstruments, Castle Hill, Australia).

In the colon, neurones were grouped into four different classes based on their response to intracellular current injection (depolarising rectangular pulses, < 0.15 nA; duration, 300 ms; Messenger et al. 1994). AH-neurones responded with one or two action potentials followed by a late long lasting hyperpolarisation (up to 10 s). Tonic S-neurones fired action potentials throughout the pulse. Phasic S-neurones fired action potentials only at the onset of the pulse. Inexcitable neurones never generated action potentials but received fast excitatory post-synaptic potentials (fEPSPs). Neurones were filled with neurobiotin using current pulses of 0.3 nA with a pulse width of 300 ms delivered at 0.3 Hz for 3 min.

Synaptic inputs to cells were evoked by electrical stimulation of the interganglionic fibre tracts with a 25 μm Teflon-coated platinum electrode connected to a constant voltage isolation unit. Electrical pulses had a duration of 300 μs and amplitudes varying between 1 and 15 V. Synaptic inputs were studied either after a single extracellular pulse or following a train of pulses (50–100 pulses delivered at 20 Hz).

Pharmacology

Substances were applied either by adding them to the superfusing Krebs solution or via a glass pipette filled with the substance dissolved in Krebs solution. The glass pipette was connected to a constant pressure application system (35 p.s.i.) and placed in close proximity (∼100 μm) to the impaled neurones. Fast Green (0.5 mm) was used for visual control of drug ejection. For the microejection studies, solutions were delivered at a rate of 15.4 ± 11.6 nl s−1 (n = 4 pipettes tested) leading to an ejection volume of 1–50 nl depending on the duration of the ejection pulse.

In order to determine the ionic mechanisms that may be involved in the glycine-induced changes of membrane potential, different solutions were used. Low Cl− Krebs solution contained (mm): 117 sodium isethionate, 4.7 KCl, 1.2 MgCl2, 1.2 NaH2PO4, 25 NaHCO3, 2.5 CaCl2 and 11 glucose. To block synaptic potentials we used Krebs solution that contained decreased [Ca2+] (1.25 mm) and elevated [Mg2+] (16 mm). In order to verify that the glycine-induced depolarisation was not due to leakage of KCl into the intracellular compartment, control experiments were performed with intracellular electrodes filled with 0.5 m potassium acetate instead of KCl.

All drugs were dissolved in Krebs solution. The following drugs were purchased from Sigma (Deisenhofen, Germany): glycine, d-serine, nifedipine, hexamethonium, mecamylamine, tetrodotoxin, strychnine, bicuculline and furosemide. The 5-HT3 receptor antagonist ICS 205930 was a gift from Novartis (Basel, Switzerland). The NMDA receptor antagonist MK-801 was purchased from RBI (Sigma).

Immunohistochemistry

The immunohistochemistry was performed as described previously in detail (Neunlist et al. 1999). At the end of the electrophysiological experiments the tissues were fixed overnight at 4 °C with 4 % paraformaldehyde containing 0.2 % picric acid in 0.1 m phosphate buffer. After three washes in 0.1 m phosphate buffer tissues were pre-incubated for 1 h in 0.1 m phosphate-buffered saline solution (PBS) containing 4 % horse serum and 0.5 % Triton X-100. The tissue was then exposed for 18 h at room temperature to primary antisera diluted in PBS-containing serum and Triton X-100. The following primary antisera were used: anti-choline acetyltransferase (ChAT) raised in rabbits (P3YEB, 1:2000; Schemann et al. 1993), goat anti-ChAT (AB144P, 1:100; Chemicon, Temecula, CA, USA), or rabbit anti-nitric oxide synthase (NOS; 210–501–5025, 1:3000; Alexis, Grünberg, Germany). After three washes in PBS, the preparation was incubated for 2 h in buffer solution containing streptavidin-Texas Red (1:1000, Gibco BRL) or streptavidin- fluorescein isothiocyanate (FITC, 1:1000; Dianova, Hamburg, Germany) to label the neurobiotin-filled cells. In addition the following fluorophore-coupled secondary antibodies were used: goat or donkey anti-rabbit IgG conjugated to 7-amino-4-methylcoumarin-3-acetic acid (AMCA, 1:50, Dianova) or donkey anti-goat IgG conjugated to indodicarbocyanin (Cy5, Dianova). The tissues were further rinsed 3 times in PBS and mounted in a glycerol solution (AF1, Citifluor, Canterbury, UK). The fluorescence microscope used for immunohistochemical studies was an Olympus IX 70 equipped with appropriate filter blocks to separately view the fluorophores (Neunlist et al. 1999). Pictures were acquired with a black and white video camera (Mod. 4910, Cohu Inc., San Diego, CA, USA) connected to a Macintosh computer and controlled by IPLab Spectrum 3.0 software (Signal Analytics, Vienna, VA, USA). The long processes of individual neurones were traced to define their projections as previously described (Schemann & Schaaf, 1995). Neurones with ascending axonal projections were thus distinguished from those with descending axonal projections.

Organ bath experiments

To record contractions of the circular muscle, mucosa-free strips of the proximal colon and parietal gastric corpus (5 mm × 10 mm) were placed in an organ bath filled with 50 ml Krebs solution that was continuously gassed with 95 % O2 and 5 % CO2 and maintained at 37 °C. The basal tension was set to 1.6 g, and the tissue was equilibrated for 90 min. Changes in tension were recorded with an isometric force transducer (Experimetria, Budapest, Hungary), and acquired and analysed using a Mac Performa 475 computer with the MacLab 4 s/e system (WissTech, Spechbach, Germany).

Statistics

All values are given as means ±s.d. Mean values were compared using Student's paired or unpaired t test or Spearman's rank test for data not normally distributed. One-way analysis of variance (Student-Newman-Keuls test) was performed for multiple comparisons. The χ2 test was used to test for significant differences in proportions. Differences were considered as significant for P < 0.05.

RESULTS

Glycine activates myenteric neurones in the colon

Intracellular recordings were obtained from 191 myenteric neurones of the proximal colon. Based on their electrophysiological characteristics, 26 % were classified as AH-neurones, 42 % as tonic S-neurones, 18 % as phasic S-neurones and 14 % as inexcitable neurones. The resting membrane potential (Vrest) from 169 colonic myenteric neurones was −69 ± 9 mV for AH-neurones (n = 47), −52 ± 9 mV for tonic S-neurones (n = 70), −51 ± 9 mV for phasic S-neurones (n = 31) and −62 ± 8 mV for inexcitable neurones (n = 21). AH- and inexcitable neurones had a significantly more negative resting membrane potential than tonic and phasic S-cells, which agrees with previous data (Messenger et al. 1994). The input resistances of the cells were calculated by measuring the membrane response (voltage change) during constant current hyperpolarising pulses which were applied through the intracellular electrode. The values were not different between the cell types (see also Messenger et al. 1994) and were therefore pooled. The average input resistance was 328 ± 142 MΩ.

Microejection of 1 mm glycine (n = 191 cells) induced in 57 % of all colonic myenteric neurones a transient depolarisation (Fig. 1; see below for further characterisation of the response); 43 % did not respond to glycine application. We observed in only one neurone a transient membrane hyperpolarisation in response to glycine. The proportion of neurones responding to glycine was dependent on the cell type. Thus, a large proportion of AH-neurones (78 %; 39 of 50) or tonic S-neurones (71 %; 57 of 80), yet a significantly smaller proportion (P = 0.0007) of phasic S-neurones (23 %; 8 of 35) and inexcitable neurones (19 %; 5 of 26), responded to glycine. Intracellular neurobiotin injection revealed the projection preferences of 69 colonic myenteric neurones. The majority of colonic neurones that responded to glycine had ascending projections (79 %; 33 of 42) while neurones insensitive to glycine projected preferentially in the descending direction (53 %; 14 of 27). Immunohistochemistry for ChAT or NOS was performed in 74 colonic myenteric neurones. Of 46 neurones responding to glycine, 81 % were ChAT immunoreactive and 12 % were NOS immunoreactive; in the remaining neurones analysis was not possible due to insufficient immunoreactivity. Of 28 neurones insensitive to glycine, 63 % were ChAT immunoreactive and 25 % were NOS immunoreactive; in the remaining neurones analysis was not possible due to insufficient immunoreactivity. The putative function of colonic myenteric neurones was analysed in 43 neurobiotin-filled cells where it was possible to identify some of their terminal endings. Neurones with processes in the circular muscle or expansion bulbs as a result of removing the circular muscle during tissue dissection were considered to be motor neurones and neurones with varicose or basket-like endings in other myenteric ganglia were considered to be interneurones (see Schemann & Schaaf 1995). Of 16 tonic S-neurones that responded to glycine four were descending and four ascending motor neurones, two were descending and six ascending interneurones. One phasic S-neurone that responded to glycine was an ascending interneurone. Previously, we showed that colonic myenteric AH-neurones had multipolar Dogiel Type II morphology and most probably function as intrinsic primary afferent neurones; their long processes projected to the mucosa or to other ganglia (Neunlist et al. 1999). In agreement with this study, all neurobiotin-filled AH-neurones had multipolar morphology and at least one of their long processes projected within the myenteric plexus (n = 10 with glycine response; n = 8 without glycine response). Successful identification of neurobiotin-filled processes was possible in seven glycine-insensitive neurones (4 phasic and 3 tonic S-neurones); these were all interneurones which branched into other myenteric ganglia.

Figure 1. Glycine evoked a rapid, transient depolarisation in colonic myenteric neurones.

Figure 1

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A, intracellular injection of neurobiotin revealed the numerous filamentous dendrites of a tonic S-neurone. This neurone responded to a 500 ms microejection of glycine (marked by arrow) with a depolarisation, which was associated with spike discharge (B). C, neurobiotin-filled AH-neurone with multipolar morphology and smooth cell body. This neurone responded to a 500 ms microejection of glycine (marked by arrow) with a depolarisation (D).

Characteristics of the excitatory glycine response in colonic myenteric neurones

Glycine induced a rapid transient depolarisation. Multiple applications of glycine (200–500 ms pulses) given at short intervals induced a pronounced desensitisation of the depolarising response (Fig. 2B). The time intervals between successive glycine applications needed to be at least 60 s in order to evoke reproducible glycine responses. Therefore, it was not possible to perform concentration-response curves using long-term perfusion of glycine. The average amplitude of the depolarisation and the half-maximum duration were calculated for microejections ranging between 300 and 500 ms duration. Both parameters were not different between the four cell types and the data were therefore pooled. The membrane potential depolarisation induced by glycine had an amplitude of 8.0 ± 5 mV (n = 34) and a half-maximum duration of 2 ± 1 s (n = 18). The amplitude and duration of the depolarising response to glycine increased as a function of the microejection duration (Fig. 2A). During the glycine-evoked depolarisation the membrane response to constant current hyperpolarising pulses decreased in amplitude, indicating that the input resistance decreased (26 ± 18 %, n = 12, P = 0.002, Fig. 3A). During repolarisation the input resistance gradually increased again and once the voltage had returned to baseline the membrane resistance returned to pre-glycine levels (Fig. 3A).

Figure 2. Dose dependency of the glycine response and its desensitisation in colonic myenteric neurones.

Figure 2

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A, the amplitude of the depolarisation and the spike discharge increased with increased duration of glycine microejection (marked by arrows) in a tonic S-neurone. The half-maximum duration and the amplitude of the depolarisation were plotted as a function of the duration of glycine microejection (•, half-maximum duration; ○, amplitude of depolarisation). B, microejections of glycine (marked by arrows) applied at relatively short intervals induced a desensitisation of the response in a gastric myenteric neurone. Increasing the application interval (Δt) induced larger amplitude responses.

Figure 3. Glycine-evoked depolarisation was associated with a decrease in membrane resistance and reversed at potentials more positive than the resting membrane potential.

Figure 3

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A, glycine (500 ms) microejection to a tonic S-neurone evoked a depolarisation. The membrane resistance decreased as indicated by the decrease in the membrane response to constant current hyperpolarising pulses (current trace not shown). B, in a different tonic S-neurone glycine was applied while clamping the membrane potential at different levels. The amplitude of the depolarisation increased with more negative membrane potential. The results from this neurone are shown in C. The amplitude was plotted against the membrane potential and a linear fit was applied. The line intersects the x-axis at −18 mV, which is the calculated reversal potential (KCl-filled intracellular electrode). Glycine applications are marked by arrows.

To calculate the reversal potential of the glycine response we applied glycine while clamping the membrane potential to values that were more positive and more negative than the resting membrane potential (Fig. 3B). The amplitude of the glycine-induced depolarisation decreased linearly with more positive membrane potentials within the range −95 to −30 mV. The amplitudes of the glycine responses were plotted against the membrane potential and the reversal potential was calculated using a linear fit (Fig. 3C). With electrodes containing 0.5 m KCl the glycine response was calculated to reverse at a membrane potential of −19 ± 13 mV (n = 10; Fig. 3C). In order to verify that the leakage through the microelectrode did not modify the intracellular chloride concentration, recordings were made with electrodes containing 0.5 m potassium acetate. Under these conditions, application of glycine also induced a fast, transient membrane depolarisation with amplitudes similar to the those obtained with KCl-filled microelectrodes (9 ± 4 mV at a Vrest of −60 ± 10 mV; P = 0.76; n = 5). In addition the reversal potential was not different from that obtained with KCl-filled electrodes (−12 ± 19 mV; P = 0.4; n = 7).

In order to test whether glycine evoked a direct, postsynaptic effect on the neurones, the preparations were perfused with Krebs solution containing reduced [Ca2+] and elevated [Mg2+]. In this solution all synaptically induced slow or fast EPSPs were abolished but the glycine response was not affected (amplitude: 8.3 ± 4.6 versus 8.6 ± 4.5 mV in low [Ca2+]; P = 0.49; n = 4; Fig. 4). In addition, blockade of nerve conduction by perfusion of 1 μm tetrodotoxin (TTX) did not modify the glycine-evoked depolarisation (amplitude: 12.8 ± 2.4 versus 12.3 ± 2.3 mV in TTX; P = 0.24; n = 4; Fig. 4).

Figure 4. The excitatory glycine effect on colonic myenteric neurones was a postsynaptic response.

Figure 4

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A, glycine (400 ms) microejection to an AH-neurone evoked a depolarising response in normal Krebs solution. B, neither the amplitude nor the duration of the response was changed in the presence of a solution containing reduced Ca2+ and high Mg2+ concentrations, which blocked all synaptic input. C, in a tonic S-neurone, 350 ms glycine microejection evoked a depolarising response associated with spike discharge. D, blockade of nerve conduction by the Na+ channel blocker tetrodotoxin (0.3 μm) did not block the response. As expected in S-neurones, the sodium-carried action potentials were blocked. Glycine applications are marked by arrows.

Pharmacology of glycine responses

Glycine receptors in the central and peripheral nervous system are reversibly blocked by the plant alkaloid strychnine. In enteric neurones, perfusion of strychnine (300–600 nm) totally abolished glycine-induced depolarisation (n = 15; Fig. 5). Glycine reponses fully recovered following 15 min washout.

Figure 5. Excitatory glycine response in colonic myenteric neurones was blocked by strychnine and was picrotoxin sensitive.

Figure 5

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A, a 200 ms glycine microejection evoked a depolarising response in an AH-neurone, which was totally abolished by bath application of 600 nm strychnine (2 min into strychnine application, B) and recovered 45 min after washout of strychnine (C). D, a 200 ms glycine microejection evoked a depolarising response in a tonic S-neurone which was associated with spike discharge. This response was reduced by 100 μm picrotoxin (E) and recovered 30 min after washout (F). Glycine applications are marked by arrows.

To further characterise the pharmacology of the excitatory glycine responses, we used 100 μm picrotoxin, which previously has been used to distinguish glycine receptor isoforms containing α2 picrotoxin-sensitive and α2/β picrotoxin-insensitive receptors (Elster et al. 1998). In enteric neurones of the colon, bath perfusion of 100 μm picrotoxin reversibly reduced the amplitude of the glycine-induced depolarisation by 55.0 ± 5.0 % (P = 0.003; n = 3; Fig. 5).

GABA-induced depolarisation in guinea-pig myenteric neurones via GABAA receptors (Bertrand & Galligan, 1992) had characteristics similar to those observed for glycine. Perfusion of 10 μm bicuculline did not affect the glycine-induced depolarisation (P = 0.9; n = 3; Fig. 6A and B), which ruled out any non-specific effects of glycine on GABAA receptors.

Figure 6. The excitatory glycine effect in colonic myenteric neurones was specific and not mediated by GABAA, NMDA, nicotinic or 5-HT3 receptors.

Figure 6

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A, C, G and I, microejection of glycine (500, 400 and 200 ms, and 1 s) evoked an excitatory response. This response was not blocked by the GABAA receptor antagonist bicuculline (10 μm, tonic S-neurone, B), the NMDA-receptor blocker MK-801 (20 μm, phasic S-neurone, D), the nicotinic receptor blocker hexamethonium (200 μm, tonic S-neurone, H) or the 5-HT3 receptor antagonist ICS 205930 (1 μm, AH-neurone, J). E, a 400 ms microejection of glycine evoked a depolarisation; however, 400 ms microejection of d-serine in the same neurone did not evoke any response (AH-neurone, F). Glycine applications are marked by arrows.

To determine whether NMDA receptors were involved in mediating the effects of glycine, the effect of the NMDA receptor antagonist MK-801 was studied. The glycine-mediated depolarisation was not changed in the presence of 20 μm MK-801 (P = 0.99; n = 6; Fig. 6C and D). Furthermore, in neurones that exhibited the excitatory glycine response, microejection of d-serine (1 mm; n = 3; Fig. 6E and F), a full agonist at the strychnine-insensitive glycine co-agonist site of the NMDA receptor, did not induce any membrane potential changes.

The glycine receptor is a member of the superfamily of ligand-gated ion channels and the response characteristics resembled fast responses in myenteric neurones which are mediated by 5-HT3 or nicotinic receptors. However, the response to glycine was not affected by the nicotinic antagonists hexamethonium (200 μm) or mecamylamine (100 μm) (P = 0.77; n = 5; Fig. 6G and H) or by the 5-HT3 antagonist ICS 205930 (1 μm) (P = 0.42; n = 3; Fig. 6I and J).

In order to determine whether glycine is involved in synaptic transmission in myenteric neurones, we induced fast or slow EPSPs by electrical stimulation of interganglionic fibre tracts. We then studied whether strychnine (n = 8) or desensitisation of glycine receptors following multiple glycine microejections (n = 9) modified the synaptically induced responses. Under these conditions fast and slow EPSPs remained unchanged.

Ionic mechanisms involved in glycine-induced depolarisation

Experiments were performed in order to determine whether chloride fluxes mediated glycine-induced depolarisation. In initial experiments we did not observe any significant change in the glycine-induced response when we replaced chloride in the extracellular bathing solution with sodium isethionate. This was probably due to a depletion of intracellular chloride over time as previously shown for GABA-induced chloride fluxes (Adams & Brown, 1975). In order to prevent this effect, we lowered the chloride concentration in the microejection pipette only. Most of the chloride in the microejection pipette (control, 129.1 mm) was replaced by isethionate solution (low-chloride, 12.1 mm). In this way we could apply the glycine in a low chloride solution by local microejection without changing the chloride concentration in the superfusing Krebs solution. Under these conditions, the amplitude and the half-maximum duration of the glycine responses were increased by 18 ± 10 % and 55 ± 55 %, respectively (n = 7; Fig. 7). This result and the calculated reversal potential suggested a relatively high intracellular chloride concentration in myenteric neurones. To analyse further how myenteric neurones maintain such a high intracellular chloride concentration, we used the loop diuretic furosemide to block the Na+-K+-2Cl− and K+-Cl− transporters. Furosemide has previously been shown to block the inward Cl− transport mechanism in immature neurones (Owens et al. 1996). Following bath perfusion of 2 mm furosemide, the amplitude of the glycine-induced depolarisation was reduced by 38 ± 14 % compared with control (17.6 ± 7.8 versus 10.9 ± 4.9 mV in furosemide; P = 0.005; n = 7; Fig. 7). Furthermore, in the presence of furosemide, the reversal potential of the glycine-induced depolarisation was shifted significantly by 20 ± 12 mV towards more negative membrane potentials compared with control (P = 0.04; n = 4). This suggested the presence of a net inward furosemide-sensitive chloride flux in myenteric neurones.

Figure 7. Changes in chloride conductance were involved in the glycine-evoked depolarisation in colonic myenteric neurones.

Figure 7

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A, a 500 ms microejection of glycine evoked an excitatory response in a tonic S-neurone. B, the amplitude of the depolarisation as well as the spike discharge were increased when the extracellular chloride concentration was reduced to 12.1 mm. C, 200 ms glycine microejection evoked a depolarisation with spike discharge in a tonic S-neurone. D, the spike discharge was blocked and the depolarisation was reduced after blockade of cation-chloride transporters with 2 mm furosemide. Glycine applications are marked by arrows.

Excitatory glycine response in gastric myenteric neurones

In order to exclude regional specificity of the glycine response we also performed intracellular recordings from myenteric neurones in the stomach. All gastric myenteric neurones responded to glycine microejection with a membrane depolarisation (mean amplitude of 12 ± 5 mV, n = 13), although the proportion of neurones that responded to glycine was significantly smaller (26 %; 13/50) than in the colon. The reversal potential of the glycine-induced depolarisation in gastric myenteric neurones was −18 ± 12 mV (n = 3), which was not different from that in colonic myenteric neurones (P = 0.9). The depolarisation was associated with a 38 ± 17 % decrease in the cell input resistance (n = 6).

Effects of glycine on colonic and gastric motility

In order to evaluate a putative functional role for glycine, its effect on the basal contractile activity of isolated colonic and gastric circular muscle strips was evaluated in organ bath experiments. Circular muscle strips showed spontaneous contractile activity. After the equilibration period, the basal muscle tone in the colon and stomach was 1.6 ± 0.4 and 0.9 ± 0.2 g, respectively. Application of 1 mm glycine induced a biphasic contractile response in 75 % of the colonic preparations (n = 25). This response consisted of a fast onset, transient contraction (181 ± 78 % increase in basal tone), reaching its maximum after 14 ± 5 s, and a late onset contraction (145 ± 112 % increase in basal tone), reaching its maximum after 69 ± 21 s (n = 13; Fig. 8A).

Figure 8. Glycine evoked contractile responses in colonic muscle preparations.

Figure 8

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A, addition of 1 mm glycine to the organ bath evoked a biphasic contractile response which consisted of a fast and slow onset increase in muscle tone. B, in the presence of 10 μm strychnine, both components were significantly reduced. C, the glycine response almost recovered after washout of strychnine. D, in the presence of tetrodotoxin (0.3 μm) both components were abolished, leaving a very slowly developing contraction. This indicated that most of the glycine response was neurally mediated. Application of glycine is indicated by the arrows. Glycine was continuously present after addition to the bath. The muscle recordings are all from the same preparation.

In the presence of 0.5 μm TTX the basal tone in colonic tissues was reduced by 13 ± 10 % (n = 7) and the spontaneous contractions were abolished. With TTX present in the bath, application of glycine did not induce the biphasic contractile response, only a slow 24 ± 12 % increase in tone, which reached a maximum 143 ± 50 s after addition of glycine (Fig. 8D).

Bath application of strychnine at a concentration of 300 nm, which abolished glycine effects in myenteric neurones, did not significantly modify glycine-induced motility responses (n = 6). At a concentration of 3 μm, strychnine significantly reduced the amplitude of the fast and late onset contraction by 60 ± 40 and 42 ± 26 %, respectively (n = 7; Fig. 8B). Strychnine at a concentration of 30 μm further reduced the amplitude of the fast and late onset contraction by 70 ± 27 and 66 ± 23 %, respectively (n = 7). We did not observe an effect of strychnine itself on basal tone or phasic contractile activity.

In gastric muscle preparations, glycine induced only a slow, late onset increase in tone (26 ± 18 % above basal level, n = 4), reaching a maximum within 3 min of glycine application. This increase was not neurally mediated because it persisted in 0.5 μm TTX (28 ± 24 % increase in basal level, n = 4, P = 0.90).

DISCUSSION

The results of the present study showed that (i) glycine evoked a strychnine-sensitive depolarising response in the adult enteric nervous system, (ii) this excitatory glycine response was due to the efflux of Cl− ions and the high intracellular Cl− concentration needed for this efflux was maintained by a furosemide-sensitive mechanism, and (iii) the glycine-evoked increase in colonic motility, but not the increase in gastric motility, was neurally mediated and this probably involved the enteric nervous system. We conclude from these results that glycine activated neurones in the adult enteric nervous system through a mechanism which is pharmacologically similar to glycine-evoked inhibitory responses in the brain and spinal cord. One possible functional implication we identified was the glycine-induced neurally mediated increase in colonic muscle activity.

Glycine excited predominantly AH- and tonic S-neurones; only a relatively small proportion of phasic S-neurones and inexcitable neurones responded to glycine. Like their counterparts in the ileal myenteric plexus (Brookes, 2001), AH-neurones in the the colon have been suggested to function as intrinsic primary afferent neurones (Neunlist et al. 1999) and their processes branch into many adjacent ganglia (Messenger et al. 1994; Neunlist et al. 1999). In agreement with previous studies, tonic S-neurones act either as motor neurones or interneurones (Messenger et al. 1994; Brookes, 2001). Therefore it has to be concluded that glycine excited intrinsic primary afferent neurones as well as motor and interneurones in the colonic myenteric plexus. The glycine-insensitive neurones were electrophysiologically phasic S-neurones or inexcitable neurones. So far the function of these two cell populations has not been identified. Our results, which are based on a relatively small number of neurobiotin-filled cells, would indicate that insensitivity to glycine occurs in a particular class of interneurones.

Excitatory effects of glycine in the enteric nervous system

We detected excitatory responses to glycine in the adult enteric nervous system. It is noteworthy that GABA, which, like glycine, acts as an inhibitory transmitter in the central nervous system, also has excitatory effects in the enteric nervous system (Cherubini & North, 1984a). Glycine effects did not involve GABAA receptors because they remained unchanged in the presence of 10 μm bicuculline, which blocked GABA-mediated activation of myenteric neurones through GABAA receptors (Zhou & Galligan, 2000). Our data suggested that the excitatory effect of glycine was coupled to a relatively high intracellular chloride concentration in enteric neurones. The reversal potential of the glycine-induced response (around −20 mV) was similar to that observed for the excitatory effect of GABA, histamine or the NK-3 agonist senktide on myenteric neurones and was consistent with an increase in chloride conductance which underlies these depolarising responses (Cherubini & North, 1984a; Bertrand & Galligan, 1992, 1994; Starodub & Wood, 2000). In our study the reversal potential of the glycine-induced response did not differ between potassium chloride- and potassium acetate-filled electrodes. This is in contrast to a previous study on myenteric neurones which demonstrated that the reversal potential of GABA-induced depolarisation was shifted with potassium chloride-filled electrodes in a way which would indicate that Cl− leaks into the cell (Cherubini & North, 1984a). The differences between the two studies are probably due to the fact that we used higher resistance electrodes and lower concentrations of potassium chloride in the microelectrode. This might explain why leakage of Cl− into the neurones appeared to be negligible under our experimental conditions. Since our bathing solution contained 160 mm Cl−, the intracellular Cl− concentration was calculated to be about 55 mm, a value which was close to that found in myenteric neurones during whole-cell perforated patch clamp experiments (Starodub & Wood, 2000). We could show that furosemide-sensitive mechanisms were involved in maintaining the high intracellular Cl− concentrations in enteric neurones. In rat sympathetic neurones, Ballanyi & Grafe (1985) have also shown that the Na+-K+-Cl− cotransporter was responsible for intracellular Cl− accumulation. We cannot rule out the possibility that other transporters might be additionally involved: among the candidates are the Na+-dependent Cl− transport driven by the transmembrane gradient for Na+, the K+-Cl− cotransporter or the Cl−-HCO3− exchange system driven by intracellular production of CO2/HCO3−.

Excitatory effects of glycine have been reported in embryonic or during early postnatal life in neurones of the central and peripheral nervous system (Ito & Cherubini, 1991; Reichling et al. 1994; Boehm et al. 1997; Ehrlich et al. 1999). In neurones of the central nervous system responses to glycine change from depolarisation to hyperpolarisation during the second week after birth; this shift is coupled to the developmental increased expression of the neurone-specific chloride-extruding K+-Cl− cotransporter KCC2 and a decreased expression of the Na+-K+-Cl− contransporter (Plotkin et al. 1997; Lu et al. 1999; Rivera et al. 1999; Kakazu et al. 1999). Furosemide sensitivity of these cation-chloride cotransporters has been described in central nervous system neurones (Kakazu et al. 1999; DeFazio et al. 2000). If developmentally regulated expression patterns of cation-chloride transporters are responsible for the switch from neonatal Cl− efflux to the mature Cl− influx in the brain, it may be concluded that this developmental change is not occurring in enteric neurones and that mechanisms of neuronal Cl− extrusion might be poorly developed in enteric neurones. It is noteworthy that the switch occurring in neurones of the central nervous system not only affects synaptic behaviour but also has broader implications for gene expression, neurotrophic effects and receptor clustering (Wang et al. 1994; Kirsch & Betz, 1998). It remains unknown why there is no switch from a depolarising to a hyperpolarising response in the enteric nervous system. The enteric nervous system has an enormous repertoire of excitatory transmitters and thus there is no evidence that maintaining the excitatory effects of GABA or glycine would be crucial for the normal functioning of enteric nerves. Therefore, it is more likely that enteric neurones have to maintain a relatively high intracellular Cl− concentration during their entire life. An increased Cl− conductance is involved in the effect of a number of excitatory neurotransmitters and neuromodulators on enteric neurones, for example Cl− efflux is involved in substance P- and histamine-mediated depolarisation of myenteric neurones (Bertrand & Galligan, 1994; Starodub & Wood, 2000).

Glycine evoked a transient fast depolarisation in colonic and gastric myenteric neurones. Glycine-mediated excitatory effects were due to a direct activation of enteric neurones since blockade of axonal spike propagation or synaptic transmission did not modify the response. The response was also specific because strychnine reversibly blocked the response, which, however, remained unchanged after blockade of GABAA, nicotinic, NMDA or 5-HT3 receptors.

The available data indicate that glycine may not act as a neurotransmitter in the enteric nervous system since bath perfusion of strychnine did not modify electrically induced fast or slow EPSPs. Furthermore, fast and slow EPSPs remained unchanged during desensitisation evoked by multiple applications of glycine. This was different for GABA, which has been reported to decrease the amplitudes of fast and slow EPSPs (Cherubini & North, 1984b). If glycine is not synthesised and released by enteric nerves, the other possible source may be dietary glycine, which is absorbed by epithelial cells and released into the blood. Based on the finding that an anti-glycine serum does not stain epithelial cells in the rat gut (Davanger et al. 1989), it is unlikely that endocrine cells might be a source for glycine.

It has previously been shown that glycine currents are mediated by two types of channel, which differ in their picrotoxin sensitivity (Yoon et al. 1998). Picrotoxin appeared to affect α homo-oligomeric, but not α/β hetero-oligomeric glycine receptors (Rajendra et al. 1997). The glycine-evoked depolarisation in enteric neurones was picrotoxin sensitive and it may be concluded that the glycine receptor on enteric neurones contains α subunits. In the newborn animal, glycine receptors are made of α2 homomers while in the adult the receptor is formed by α1/β heteromers (Rajendra et al. 1997). This neonatal glycine receptor may be expressed in the enteric nervous system; this is supported by the excitatory effect of glycine, by the picrotoxin sensitivity of the glycine response and by the finding that the glycine-evoked Cl− efflux was coupled to a relatively high intracellular Cl− concentration. However, at the moment a definitive conclusion is not possible and the identity of the receptor subunits involved in the excitatory glycine effects on enteric neurones awaits further studies.

Functional role of glycine responses

Since myenteric neurones are mainly involved in the regulation of smooth muscle activity, we determined the putative role of glycine as a regulator of colonic and gastric motility. Our study revealed that only the colonic muscle responded to glycine application with a neurogenically mediated contraction. This result is in agreement with the observation that in the stomach only a minority of neurones were glycine sensitive. In the stomach, the secretomotor neurones are located in the myenteric plexus (Reiche & Schemann, 1999) and we can therefore not rule out the possibility that the glycine-sensitive myenteric neurones in the stomach might influence the activity of epithelial cells rather than smooth muscle cells. The neurogenic contractile response in the colon agrees with our finding that the vast majority of glycine sensitive neurones are ChAT immunoreactive and hence would release acetylcholine, which contracts smooth muscle in the gut.

The different strychnine sensitivities observed in the electrophysiological studies (full block at 300 nm) and the motility studies (no significant block at 300 nm) could result from a shift in the inhibitory properties of strychnine. Indeed, it was shown that when glycine and strychnine were applied together the half-maximum inhibition value of strychnine could be 10 times larger than when strychnine was applied before glycine (Boehm et al. 1997). Although we incubated the tissue with strychnine 10 min prior to the application of glycine, it is unknown whether strychnine was able to block all receptors within the 10 min period. Although we applied strychnine for long periods we cannot rule out the possibility that higher concentrations of strychnine are required to overcome the diffusion barrier caused by the thickness of the preparation. An alternative explanation for the strychnine-resistant glycine responses in muscle preparations might be the activation of NMDA receptors. Indeed, it has been shown that glutamate evoked contractions of the longitudinal muscle in the guinea-pig ileum (Campbell et al. 1991). This response was potentiated by glycine and glycine site antagonists caused glycine-sensitive inhibition of glutamate-evoked contractions (Campbell et al. 1991). Strychnine would not block this NMDA-mediated component of the glycine-evoked contraction.

In conclusion, this study revealed that glycine acted as an excitatory amino acid in the adult enteric nervous system. The excitation was mediated via strychnine-sensitive mechanisms and involved Cl− efflux. Furosemide-sensitive mechanisms participated in the maintenance of chloride homeostasis.

Acknowledgments

This study was supported by DFG Sche 267/4–3, 4–4 and SFB 280. M.N. received a Marie Curie stipend from the EC. We thank Susanne Hoppe and Baerbel Leppich for excellent technical support.

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