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Keywords:

  • enteric nervous system;
  • extracellular stimulation;
  • neuronal network;
  • ω-conotoxin

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENT
  8. References

The enteric nervous system controls most of the gastrointestinal functions. We applied confocal microscopy and the Ca2+ indicator Fluo-3 as an optical approach to study synaptic activation in cultures of myenteric neurones. The optical recording of [Ca2+]i (the intracellular Ca2+ concentration) was used to monitor activation, since [Ca2+]i is crucial in the coupling between neuronal excitation and the activation of several intracellular events. Extracellular fibre tract stimulation (2 s, 30 Hz) caused a transient [Ca2+]i rise in a subset of neurones (50%). These transients lasted for 5.2 s (n=36), with an average amplitude of 3.4 ± 1.3 times the basal concentration. The removal of extracellular Ca2+ (n=15) or the application of 10–6M tetrodotoxin (n=16) blocked this response. The N-type Ca2+-channel blocker ω-conotoxin (5 × 10 –7M) abolished the [Ca2+]i increase, while blockade of L-type and P/Q type Ca2+ channels had no effect. Single stimuli evoked a [Ca2+]i rise in the processes. ω-conotoxin-sensitive postsynaptic events required repetitive stimulation. Cholinergic blockade did not inhibit the [Ca2+]i rise in all neurones, suggesting that, besides acetylcholine, other neurotransmitters are involved. Optical imaging of [Ca2+]i can be used to study synaptic spread of activation in enteric neuronal circuits expressed in culture.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENT
  8. References

The enteric nervous system (ENS) controls various gastrointestinal functions, including motor activity, secretion, absorption and local blood perfusion. Classical electrophysiological techniques using microelectrode impalements are widely used as a means to study the electrical and synaptic properties of neurones in the ENS.1 Micro-electrode impalements, however, yield relatively low experimental efficiency, especially in larger species. Moreover, the single-cell recordings do not allow the study of synaptic interactions between multiple neurones in the myenteric plexus, since they only provide information at the single neurone level. The intracellular calcium concentration ([Ca2+]i) is important in the control of cell excitability. It triggers neurotransmitter release and regulates protein synthesis and gene expression.2, 3 Fluorescent Ca2+ indicators allow the optical measurement of [Ca2+]i changes4 providing a noninvasive tool to study multiple cells simultaneously and to gather information on cell to cell interactions. Cultures of myenteric neurones are a valid model for the study of myenteric neurones. These cultures consist of several morphological and electrophysiological classes of neurones, reflecting the diversity of the myenteric neurones in situ.5[6][7]–8 Myenteric neurones in culture display [Ca2+]i signalling when stimulated by neuroligands, indicating that the expression of functional receptors is preserved in culture.9[10][11][12]–13 Action potential discharge causes a step-wise increase in the [Ca2+]i in myenteric neurones.14 These data suggest that neuronal activation is reflected in changes in [Ca2+]i in cultured myenteric neurones.

The aim of the present study was to validate an optical approach to study the spread of synaptic activity in the myenteric neural network, hence overcoming some of the limitations of a classical intracellular approach.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENT
  8. References

Myenteric neurone cultures

Primary cultures of myenteric neurones were prepared from adult guinea-pig small intestine according to a previously described method.8, 15 The Principles of Laboratory Animal Care were followed as well as the specific national laws of the Ministerie van Landbouw, Belgium.

In brief, the longitudinal muscle and the adherent myenteric plexus (LMMP) were dissected from the circular muscle and the mucosa; the LMMP was digested in an enzymatic solution containing protease (1 mg mL–1) and collagenase (1.25 mg mL–1) (both from Sigma Chemical Co., Bornem, Belgium). After a 30-min incubation at 37 °C, the suspension was placed on ice and spun at 500 g, thereby stopping the enzymatic reaction. Ganglia were picked up and plated in Lab-Tek® culture dishes (Nalge-NUNC International, Naperville, IL, USA) in which they adhered to the coverglass bottom. After a few days, neurones started growing in network-like structures reminiscent of the ganglionated plexus. The culture medium was changed every 2 days. The growth medium was Medium 199 (GIBCO, Merelbeke, Belgium) enriched with 10% foetal bovine serum (FBS; GIBCO) and 50 ng mL–1 7sNGF (7s nerve growth factor; Alomone Laboratories, Jerusalem, Israel). The glucose concentration was elevated to 30 m M and the Ca2+ concentration was adjusted to 2.5 m M by adding CaCl2. Antibiotics were added to the medium in the following concentrations; 100 μg mL–1 streptomycin, 100 U mL–1 penicillin, 1 μg mL–1 amphotericin B, and 50 μg mL–1 gentamycin (all from GIBCO). Culture chambers were kept in an incubator at 37 °C continuously gassed with 5% CO2. 10 μM arabinose-C-furanoside was added in order to prevent the proliferation of dividing cells such as glial cells and fibroblasts. Experiments were performed on 7–10-day-old cultures.

Immunochemical staining

Cultured cells were fixed in a freshly prepared phosphate-buffered (pH 8.0) 2%/0.2% paraformaldehyde/picric acid solution. Permeabilization (2 h) and blocking of the nonspecific binding sites (1 h) occurred in a 0.1 M phosphate buffered saline (PBS) solution with 0.5% (w/v) Triton X-100 and 4% (v/v) goat serum. An antibody raised in rabbit against neurone specific enolase (NSE) was used as the primary antiserum (Polysciences Inc., Warrington, PA, USA; 1:1000, 20 h), the secondary antibody was an FITC-labelled antibody against rabbit IgG (Jackson Immunores Laboratories Inc., West Grove, PA, USA; 1:500, 2 h). The preparations were visualized under a NIKON microscope equipped with a fluorescence unit and filtercube (B-2 A: EXBP470–490, DM 505, EM BA 510/20). To prevent bacterial growth NaN3 was added to the incubation solutions (0.3% w/v).

Experimental medium

Experiments were performed at room temperature in a Krebs solution containing: 150 m M NaCl; 6 m M KCl; 1.5 m M CaCl2; 1 m M MgCl2; 10 m M glucose; 10 m M HEPES. The pH of the solution was adjusted to 7.38 with NaOH (5 M). The zero Ca2+ solution contained 2.5 m M MgCl2, and 2 m M EDTA. High K+ medium used to depolarize the cell membrane contained 75 m M K+. Osmolarity was adjusted by lowering the Na+ concentration to 81 m M.

Extracellular stimulation

A Pt electrode (diameter 50 μm) was placed on the interconnecting strands 0.3–0.7 mm away from the neurones of interest. Frequency, duration and amplitude of the stimulus could be altered (GRASS instruments, Quincy, USA). Single and prolonged high frequency (2 s, 30 Hz) stimuli were used to evoke fast and slow excitatory post synaptic potentials, respectively.

Intracellular Ca2+ measurements To measure changes in intracellular Ca2+ the acetoxy-methylester form of the long wavelength Ca2+ indicator Fluo-3 (Kd= 450 n M) was used. The neurones were loaded during a 30–45 min incubation in the experimental Krebs solution containing 2.5 μL mL–1 pluronic acid F-127 (25% wt/wt) and 7.5 μM Fluo-3-AM (both from Molecular Probes, Leiden, the Netherlands). Upon entering the cytosol the ester is cleaved by cytoplasmatic esterases and the free Fluo-3 molecule is trapped in the cell. The coverglass chambers were mounted on an inverted confocal laser-scanning microscope (CLSM; Leica Lasertechnik GMBH, Heidelberg, Germany) equipped with a photomultiplier for detection and quantification of the fluorescence. The use of a confocal microscope allowed us to avoid indirect light from other focal planes and it also reduced phototoxicity due to the selective point-scanning illumination. The Fluo-3 molecule was illuminated with the 488 nm line of a 50 mW Argon laser. The detection pinhole was set between 90 and 110 μm. Images (125 μm/125 μm) were recorded (40x oil objective, Fluotar, NA 1.3) with a resolution of 256 × 256. The temporal resolution was set at 0.83 Hz. The number of neurones within one microscopic field ranged from 2 to 20. Image analysis was performed on an IBM UNIX R/S 6000 workstation. Using a computer program developed specifically for this study, an arbitrary shaped region of interest was indicated in the image on screen and average pixel values over this region were calculated for a stack of images consecutive in time. The individual neurones could easily be distinguished and regions were drawn around the soma of the neurones. For the neurite experiments small rectangular regions were used.

Quantification of Ca2+ transients

The changes in [Ca2+]i are reflected in the changes in fluorescence of the Fluo-3 indicator. The fluorescence of Fluo-3 is expressed as a relative value, being the fluorescence (F) of a certain region minus the fluorescence of the background (Fbck) divided by the fluorescence of that region at time zero minus the background fluorescence at time zero:

inline image

Since RF is directly proportional to [Ca2+]i the change in RF can be used to indicate the changes in [Ca2+]i. The most important property of these changes is the fact that they are transient, therefore they will also be referred to as Ca2+ transients. The amplitude and the duration of the [Ca2+]i signals were calculated with software developed for personal computer. The results are expressed as average values ± SEM and statistical differences were calculated using a Student’s t-test.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENT
  8. References

Membrane depolarization by means of high potassium

The application of high K+ (75 m M) evoked a rise in [Ca2+]i in a subset (±80%) of cultured cells. The myenteric neurone cultures were not completely void of fibroblasts and glial cells, but phase-contrast microscopy allowed us to distinguish between neurones and other cell types due to the differences in morphological appearance. In contrast to neurones with their pronounced thick somata, fibroblasts and glial cells have flat cell bodies, sticking close to the coverglass base. The morphological identification was confirmed by a subsequent immuno-chemical staining for NSE ( Fig. 1). The morphological and immunochemical identification revealed a 1:1 correlation between neurones and the cells displaying a K+-induced [Ca2+]i response (n=11). The fluorescence of Fluo-3 was elevated by a factor 5.43 (±2.2) upon addition of 75 m M K+. No rise in [Ca2+]i was detected in glial cells and fibroblasts. In the absence of extracellular Ca2+, no Ca2+ transients could be evoked, either in the processes (n=12) or in the cell bodies themselves (n=8).

image

Figure 1 Cultured myenteric neurones. (A) Phase-contrast picture of myenteric neurone cultures. Other cell types, such as fibroblasts ([DOWNWARDS DOUBLE ARROW]) and glial cells are also present. The neurones (arrows) and neuronal processes (arrowheads) can easily be observed (bar=1. 00 μm). (B) Immunochemical staining for neurone specific enolase (NSE). The neuronal cell bodies (arrow) and processes (arrowheads) identified in (A) are immunoreactive for NSE.

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Depolarization experiments were repeated in the presence of Ca2+ channel blockers. In each of the 22 neurones tested, high K+-induced transient [Ca2+]i changes. Nifedipine (1.0 μM), a potent L-type Ca2+ channel blocker, reduced the amplitude of the rise in [Ca2+]i (1.87 vs. 1.39, P < 0.01). N-type Ca2+ channels can be reversibly blocked by the marine snail toxin ω-conotoxin (MVIIA)(5 × 10–7 M). The addition of ω-conotoxin did not block the response, but significantly decreased the amplitude of the transients (4.08 vs. 3.17, P < 0.01). Additional application of 10–8 Mω-agatoxin, a reversible blocker of the P/Q-type Ca2+ channel, reduced the [Ca2+]i transients further to 2.61, which is 64% of the original amplitude (n=5). The K+-induced changes in [Ca2+]i were not significantly altered under cholinergic blockade (10–6 M atropine, 10–4 M hexamethonium (Sigma), n=9). The combination of cholinergic blockade and N-type Ca2+ channel blockade, did not inhibit the response to K+ depolarization (n=14).

Electrical fibre tract stimulation

Response to extracellular stimulation Focal extracellular stimulation (ECS) of the interconnective strands caused a rise in [Ca2+]i in about 50% of the neurones within the microscopic field. In total 40 out of 79 neurones displayed a rise in [Ca2+]i upon electrical stimulation of the connecting fibres. The highest response rate was obtained using a 30-Hz stimulus lasting for 2–3 s. The fluorescence increased on average by a factor of 3.47 (±1.29). The duration of the response, defined as the width at half height of the Ca2+ transient, was 5.22 s (±1.48) (n=36 transients from 10 cells). The [Ca2+]i changes could be observed in the images recorded immediately after the onset of ECS. The shape of the transients matched the shape of the transients observed in other cell types after chemical stimulation; a steep rise followed by a exponential decay lasting for a few seconds.16 The occurrence of the response was an all or nothing effect. When present, the shape of the response was independent of the properties of the stimulus.

Role of extracellular Ca2+ To investigate the contribution of extracellular Ca2+ concentration ([Ca2+]e) to the observed [Ca2+]i changes, Ca2+ free Krebs solution was superfused. In each of the 15 neurones tested, the Ca2+ transients were completely abolished when Ca2+ was omitted from the extracellular medium and reappeared after the original extracellular Ca2+ concentration ([Ca2+]e) was restored ( Fig. 2a).

image

Figure 2 Neuronal [Ca2+]i changes induced by extracellular stimulation of fibre tracts ([DOWNWARDS ARROW]). (A) These responses are completely abolished when Ca2+ was omitted from the extracellular solution and reappear after restoring the [Ca2+]e (n=15). (Be) Similarly, the blockade of neuronal conduction with TTX (10–6 M), prevents all ECS-induced [Ca2. +]i rises (n=16).

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Involvement of neuronal conduction In a separate set of experiments, the extracellular electrode was positioned in close vicinity (<100 μm) of the cells or interconnective fibres without making actual contact. Stimulation could not evoke responses in the neurones. Hence, actual physical contact between the fibre and the electrode was essential to induce a rise in [Ca2+]i. In the presence of 10–6 M tetrodotoxin (TTX; RBI, Natick, MA, USA) the ECS of the network branches failed to evoke Ca2+ transients in the target neurones (n=16). The effect of TTX was completely reversible ( Fig. 2b).

Involvement of voltage-dependent Ca2+ channels The importance of extracellular Ca2+ suggests the involvement of membrane Ca2+ channels in the regulation of the [Ca2+]i transients. In the central nervous system as well as in the gastrointestinal tract, several neuronal voltage-operated Ca2+ channels (VOCC) have been described. Therefore, the possible involvement of L-, N-and P/Q-type Ca2+ channels was studied. The responses were not significantly altered in the presence of 10–6 M nifedipine (n=4). In seven out of eight neurones the responses were completely blocked by the action of the N-type channel blocker, ω-conotoxin (5 × 10–7 M) ( Fig. 3); in only one cell did a reduced response persist. The responses recovered completely after a few minutes of wash-out. In four other cells, the addition of 10–8 Mω-agatoxin, a P/Q type channel blocker, did not affect the responses.

image

Figure 3.  The N-type Ca2+ channel blocker ω-conotoxin (5 × 10–7 M) completely blocks the ECS-induced ([DOWNWARDS ARROW]) [Ca2+]i rise (seven out of eight neurones).

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[Ca2+]i signalling in processes Directly stimulated neuronal processes displayed slightly different [Ca2+]i signalling during ECS; the amplitude and the duration of the response were dependent on the stimulus properties, while for cell bodies this was an on/off effect once the stimulus parameters reached a critical threshold. While no detectable changes in [Ca2+]i were generated in the cell bodies during single ECS, measurable transients with an average amplitude of 2.3 lasting for 1.92 s (n=10) could be observed in the processes. In the absence of [Ca2+]e no responses were detected in the processes (n=9). The N-type Ca2+ channel blocker ω-conotoxin (5 × 10–7 M) reduced the amplitude of the signal in the processes to 78%.

Apart from single stimuli, three different stimulus frequencies (10, 20 and 30 Hz) were tested. The average amplitude of the response was 5.12 ± 0.75 (n=6). No difference in amplitude of the [Ca2+]i rise in the processes was detected when evoked at 10, 20 or 30 Hz (n=2). On the other hand, increasing the stimulus amplitude from 5 V to 10 V, raised the amplitude of the response with 31.6 ± 5.9% (n=10). This phenomenon was independent of the stimulus frequency, since at 10, 20 and 30 Hz a similar increase was observed (n=2). We observed that brief ECS was able to generate a transient rise in the stimulated fibre tract without causing a change in [Ca2+]i in the postsynaptic neuronal cell bodies. However, when the duration of the stimulus was increased, a rise in [Ca2+]i was observed both in the process and in the cell body. The application of ω-conotoxin (5 × 10–7 M) reversed this effect (n=2), abolishing the rise in [Ca2+]i in the cell body without inhibiting the response in the axon ( Fig. 4).

image

Figure 4.  Brief (1 s) ECS ([DOWNWARDS ARROW]) induces a rise in [Ca2+]i in the processes, represented by the dashed line, without evoking a response in the cell body. When the duration of the ECS is prolonged (3 s, [DOWNWARDS ARROW]), a response can be observed both in the processes and in the cell body (full line). The addition of ω-conotoxin (5 × 10–7 M) blocks the rise in [Ca2+]i in the cells without affecting the [Ca2+]i rise in the processes.

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Postsynaptic receptors

In intracellular recordings, the single and high frequency stimulation of fibre strands has been extensively used to evoke fast and slow excitatory postsynaptic potentials (EPSPs).1, 8 Several neurotransmitters are involved in these synaptic events. Since the majority of the myenteric neurones of the guinea pig small intestine is cholinergic,17 particular interest was paid to the role of nicotinic and muscarinic receptors.

Hexamethonium In six (33%) out of 20 neurones tested, Ca2+ transients evoked by extracellular stimulation were reversibly blocked by addition of 10–4 M hexamethonium (Hex). In 14 other neurones (66%), Hex was not able to block Ca2+ transients, although the amplitude of the Ca2+ transient in some of these neurones was decreased (average reduction in five cells: 25.8%) ( Fig. 5).

image

Figure 5.  The nicotinic receptor blocker hexamethonium (1 m M) has a dual effect on the ECS-induced ([DOWNWARDS ARROW]) [Ca2+]i rise. Hexamethonium blocks the [Ca2+]i rise in 33% of the cells, represented by the full line, while in 66% only a reduction in the amplitude of the [Ca2+]i rise is observed (dotted line). This differential effect implies that the nicotinic receptor is not the only receptor involved in the neurotransmission.

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Atropine Simeone et al. reported that the acetylcholine (ACh)-induced rise in [Ca2+]i of myenteric neurones in culture was mainly due to the activation of muscarinic receptors,11 therefore, ECS-induced Ca2+ transients were studied during superfusion of atropine (10–6 M). In two out of the seven cells, the Ca2+ transients were not affected in the presence of 10–6 M atropine. In two other cells the response was reduced by approximately 30%, while in three other neurones a 10% increase in amplitude was detected. The induced transients were completely blocked by TTX (10–6 M).

Noncholinergic neurotransmitters In four neurones, Ca2+ transients were studied in the presence of both atropine (10–6 M) and Hex (10–4 M). Complete cholinergic blockade was not able to inhibit the electrically induced [Ca2+]i transients. In contrast, the amplitude of the response was increased in two neurones. Again, TTX blocked every response.

Signalling in the processes The responses in the axons, directly connected with the stimulation site, were not blocked either by Hex (10–4 M, n=8), or by atropine (10–6 M, n=6). Simultaneous administration of Hex and atropine, reduced the amplitude of the responses in the processes from 1.45 ± 0.18–1.21 ± 0.08 (not significant, n=8).

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENT
  8. References

We demonstrated the feasibility of the optical recording of [Ca2+]i signalling as a means to study the spread in activation in the myenteric neuronal network. Our results clearly show that electrical activation of interneuronal connectives induces a rise in [Ca2+]i. This phenomenon requires neuronal conduction, as shown by the influence of TTX. It also requires synaptic transmission as demonstrated by the effect of low Ca2+ and by the differential effect of ω-conotoxin, both on directly stimulated fibres and target neurones, and on ECS and high K+-induced responses.

The K+-induced [Ca2+]i rise, resulting from the opening of VOCCs, can be used to physiologically distinguish between neurones and other cell types such as glial cells and fibroblasts, since the latter do not seem to express VOCCs. Immunohistochemical staining for NSE confirmed the reliability of this technique and the optical phase-contrast inspection. Our data suggest the presence of multiple types of Ca2+ channels in the membrane of myenteric neurones, because specific blockade of each of the Ca2+ channel types leads to a reduced [Ca2+]i rise induced by high K+. Unlike Trouslard et al. who optically recorded spontaneous fast synaptic events in single cells,12 we were able to simultaneously monitor synaptically-mediated [Ca2+]i transients in multiple neurones. Moreover, the use of confocal microscopy allowed us to avoid indirect light from underlying cells, which was indispensable for the study of neuronal processes. The arbitrarily chosen stimulation point implies that our data are only relevant for the neurones responding to the ECS. Non-responding neurones might be in a nonexcitable condition or, more likely, they may lack a direct or a synaptic connection to the stimulation site. ECS fails to induce a [Ca2+]i rise when there is no contact between the electrode and the fibres. In addition, TTX blocks all ECS-induced responses.

Although single stimuli, which elicit fast synaptic events in cultured myenteric neurones,8 are an adequate means to evoke transient [Ca2+]i rises in the processes of the neuronal network, they failed to induce a detectable response in cell bodies. Prolonged high frequency stimulation, which induces either a train of fast synaptic events or a slow synaptic event, was most effective in generating responses. Due to the limited temporal and spatial resolution of the system we cannot exclude the presence of local or very brief membrane events. Detailed scanning close to the membrane of single neurones, injected with a Ca2+ indicator, has shown that both single fast synaptic events and action potential generation cause changes in [Ca2+]i in myenteric neurones.14, 18

In the absence of [Ca2+]e no transient changes in [Ca2+]i could be induced, and in normal [Ca2+]e conditions ω-conotoxin mimicked this zero [Ca2+]e effect. N-type Ca2+ channels located in the presynaptic nerve terminals play an important role in the regulation of neurotransmitter release in various neuronal tissues.19[20][21][22]–23 Our observation that ω-conotoxin inhibits transients in neuronal cell bodies but not in the processes, supports the hypothesis that in our model too, N-type Ca2+ channels are involved in the control of neurotransmitter release. Single stimuli, although increasing [Ca2+]i in the processes, are probably too short to release significant numbers of neurotransmitter quanta in the synaptic cleft. When both the frequency and the duration of the stimulus are increased a transient response can be seen in the postsynaptic cell. Addition of ω-conotoxin reverses this effect.

None of the other Ca2+ channel blockers mimicked the all or nothing effect observed with ω-conotoxin. The high K+ data, however, suggest the presence of L-type Ca2+ channels. Apparently, these are not involved in either neurotransmitter release or the action of the transmitter, since nifedipine did not alter the ECS-induced signalling. The latter agrees with studies performed on whole mount LMMP preparations where nifedipine is used as a muscle relaxant because of its limited effect on neuronal function. In other studies, however, nifedipine was reported to block the acetylcholine (ACh) and substance P (SP)-induced Ca2+ signalling in myenteric neurones.11, 24 The [Ca2+]i rise in neuronal cell bodies was not affected by ω-agatoxin. The insignificant role of P/Q-type Ca2+ channels and the importance of N-type Ca2+ channels in isolated autonomic neuroeffector preparations was previously shown by Lundy and Frew.25

Our data suggest that the ECS-induced signalling does not exclusively depend on nicotinic neurotransmission, since Hex did not abolish all ECS-induced responses. The amplitude of the Hex-resistant [Ca2+]i signals was either partially reduced or unaltered, which reflects a multiple, combined cholinergic and noncholinergic synaptic input ( Fig. 6). Although we cannot fully exclude the involvement of antidromic activation, a major role for synaptic transmission is supported by the differential effect of single ECS on processes and cell bodies and by the observation that ω-conotoxin blocks all cell body responses. In contrast to the findings of Simeone et al. where the induction of [Ca2+]i signalling by ACh was completely blocked by the selective M1 antagonist, pirenzipine,11 the ECS-induced responses were preserved or became even larger under muscarinic blockade. The increased amplitude might be due to the inhibition of a presynaptic inhibitory muscarinic receptor.26, 27 Similar results were observed under complete cholinergic blockade, providing further evidence for the involvement of noncholinergic neurotransmitters besides ACh. Both serotonin (5-HT; 5-hydroxy-tryptamine) and SP have been proposed as neurotransmitters. Patch clamp experiments showed that SP is involved in the synaptic transmission in rat neuronal cultures28 and SP-induced Ca2+ signalling was reported in guinea-pig myenteric neurone cultures.24 We also obtained evidence that SP and 5-HT are probably involved in the control of the ECS-induced [Ca2+]i.29, 30

image

Figure 6.  This scheme summarizes the main findings of the ECS-induced [Ca2+]i signalling in myenteric neurones in culture. ECS applied to one of the fibre strands evokes a rise in [Ca2+]i in subset of the myenteric neurones. TTX blocks all responses. Multiple fibre stimulation might explain the different effects of Hex on the response amplitude. Only presynaptic N-type Ca2+ channels are represented, while somal N- and L-type Ca2+ channels have been omitted for reasons of clarity.

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In summary, we demonstrated that electrical stimulation of the interconnecting fibre strands in myenteric neurones in culture results in a [Ca2+]i rise in neighbouring neurones. These changes are TTX- and ω-conotoxin-sensitive and require synaptic transmission. A subset of the responses persisted under specific nicotinic and muscarinic blockade. This finding implies the involvement of cholinergic and noncholinergic neurotransmission. We conclude that optical recording of [Ca2+]i can be used to monitor synaptic activation and that, in combination with confocal microscopy, it may provide a novel tool to study of neuronal activation in the myenteric plexus in situ.

ACKNOWLEDGMENT

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENT
  8. References

Parts of this study were already published in abstract form in Gastroenterology 1997; 112 (4): A842.

References

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENT
  8. References
  • 1
    Wood JD. Application of classification schemes to the enteric nervous system. J Auton Nerv Syst 1994; 48: 17 29.
  • 2
    Kennedy MB. Regulation of neuronal function by calcium. Trends Neurosci 1989; 12: 417 20.
  • 3
    Berridge MJ. Neuronal calcium signalling. Neuron 1998; 21: 13 26.
  • 4
    Tsien RY. Fluorescence measurement and photochemical manipulation of cytosolic free calcium. Trends Neurosci 1998; 11: 419 24.
  • 5
    Willard AL & Nishi R. Neurons dissociated from rat myenteric plexus retain differentiated properties when grown in cell culture. II. Electrophysiological properties and responses to neurotransmitter candidates. Neuroscience 1985; 16: 201 11.
  • 6
    Hanani M & Burnstock G. Synaptic activity of myenteric neurons in tissue culture. J Auton Nerv Syst 1985; 14: 49 60.
  • 7
    Nishi R & Willard AL. Neurons dissociated from rat myenteric plexus retain differentiated properties when grown in cell culture. I. Morphological properties and immunocytochemical localization of transmitter candidates. Neuroscience 1985; 16: 187 99.
  • 8
    Hanani M, Xia Y, Wood JD. Myenteric ganglia from the adult guinea-pig small intestine in tissue culture. Neurogastroenterol Mot 1994; 6: 103 18.
  • 9
    Kimball BC & Mulholland MW. Neuroligands evoke calcium signaling in cultured myenteric neurons. Surgery 1995; 118: 162 70.
  • 10
    Kimball BC, Yule DI, Mulholland MW. Extracellular ATP mediates Ca2+ signaling in cultured myenteric neurons via a PLC-dependent mechanism. Am J Physiol 1996; 270: G587 93.
  • 11
    Simeone DM, Kimball BC, Mulholland MW. Acetylcholine-induced calcium signaling associated with muscarinic receptor activation in cultured myenteric neurons. J Am Coll Surg 1996; 182: 473 81.
  • 12
    Trouslard J, Mirsky R, Jessen KR, Burnstock G, Brown DA. Intracellular calcium changes associated with cholinergic nicotinic receptor activation in cultured myenteric plexus neurones. Brain Res 1993; 624: 103 8.
  • 13
    Christofi FL, Guan Z, Wood JD, Baidan LV, Stokes BT. Purinergic Ca2+ signaling in myenteric neurons via P2 purinoceptors. Am J Physiol 1997; 272: G463 73.
  • 14
    Hanani M & Lasser-Ross N. Activity-dependent changes in intracellular calcium in myenteric neurons. Am J Physiol 1997; 273: G1359 63.
  • 15
    Jessen KR, Saffrey MJ, Burnstock G. The enteric nervous system in tissue culture. I. Cell types and their interactions in explants of the myenteric and submucous plexuses from guinea pig, rabbit and rat. Brain Res 1983; 262: 17 35.
  • 16
    Goldbeter A, Dupont G, Berridge MJ. Minimal model for signal-induced Ca2+ oscillations and for their frequency encoding through protein phosphorylation. Proc Natl Acad Sci USA 1990; 87: 1461 5.
  • 17
    Steele PA, Brookes SJ, Costa M. Immunohistochemical identification of cholinergic neurons in the myenteric plexus of guinea-pig small intestine. Neuroscience 1991; 45: 227 39.
  • 18
    Smith TK & Shuttleworth CWR. A critical role for ryanodine sensitive calcium stores in myenteric AH neurons. Dig Dis Sci 1998; 43: A67A67 (Abstract).
  • 19
    Hall ZW. The Nerve Terminal In: Hall ZW, ed. An Introduction to Molecular Neurobiology Sunderland, Massachusetts: Sinauer Associates Inc., 1992: 148–78.
  • 20
    Hirning LD, Fox AP, McCleskey EW, Olivera BM, Thayer SA, Miller RJ, Tsien RW. Dominant role of N-type Ca2+ channels in evoked release of norepinephrine from sympathetic neurons. Science 1988; 239: 57 61.
  • 21
    Wiley JW, Gross RA, Lu YX, Macdonald RL. Neuropeptide Y reduces calcium current and inhibits acetylcholine release in nodose neurons via a pertussis toxin-sensitive mechanism. J Neurophysiol 1990; 63: 1499 507.
  • 22
    Wessler I, Dooley DJ, Werhand J, Schlemmer F. Differential effects of calcium channel antagonists (omega-conotoxin GVIA, nifedipine, verapamil) on the electrically-evoked release of [3H]acetylcholine from the myenteric plexus, phrenic nerve and neocortex of rats . Naunyn Schmiedebergs Arch Pharmacol 1990; 341: 288 94.
  • 23
    Takahashi T, Tsunoda Y, Lu Y, Wiley J, Owyang C. Nicotinic receptor-evoked release of acetylcholine and somatostatin in the myenteric plexus is coupled to calcium influx via N-type calcium channels. J Pharmacol Exp Ther 1992; 263: 1 5.
  • 24
    Sarosi GAJr , Kimball BC, Barnhart DC, Zhang W, Mulholland MW. Tachykinin neuropeptide-evoked intracellular calcium transients in cultured guinea-pig myenteric neurons. Peptides 1998; 19: 75 84.
  • 25
    Lundy PM & Frew R. Effect of omega-agatoxin-IVA on autonomic neurotransmission. Eur J Pharmacol 1994; 261: 79 84.
  • 26
    Dzieniszewski P & Kilbinger H. Muscarinic modulation of acetylcholine release evoked by dimethylphenylpiperazinium and high potassium from guinea-pig myenteric plexus. Eur J Pharmacol 1978; 50: 385 91.
  • 27
    Morita K, North RA, Tokimasa T. Muscarinic presynaptic inhibition of synaptic transmission in myenteric plexus of guinea-pig ileum. J Physiol Lond 1982; 333: 141 9.
  • 28
    Willard AL. Substance P mediates synaptic transmission between rat myenteric neurones in cell culture. J Physiol Lond 1990; 426: 453 71.
  • 29
    Vanden Berghe P, Tack J, Coulie B, Missiaen L, Janssens J. The 5-HT3 receptor mediates the effect of serotonin on intracellular calcium concentration in cultured myenteric neurons. Neurogastroent Mot 1998; 10: 107107 (Abstract).
  • 30
    Vanden Berghe P, Tack J, Coulie B, Missiaen L, Janssens J. Substance P evokes a rise in intracellular calcium concentration in cultured myenteric neurons via an inositoltrisphosphate pathway. Gastroenterology 1998; 114: A1187A1187 (Abstract).