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Am J Physiol Gastrointest Liver Physiol 284: G1084-G1092, 2003. First published January 29, 2003; doi:10.1152/ajpgi.00383.2002
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Vol. 284, Issue 6, G1084-G1092, June 2003

Electrical stimulation reveals complex neuronal input and activation patterns in single myenteric guinea pig ganglia

R. Bisschops1, P. Vanden Berghe1, E. Bellon2, J. Janssens1, and J. Tack1

1 Center for Gastroenterological Research and 2 Medical Image Computing (Radiology - ESAT/PSI), Katholieke Universiteit Leuven, 3000 Leuven, Belgium


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The myenteric plexus plays a key role in the control of gastrointestinal motility. We used confocal calcium imaging to study responses to electrical train stimulation (ETS) of interganglionic fiber tracts in entire myenteric ganglia of the guinea pig small intestine. ETS induced calcium transients in a subset of neurons: 52.2% responded to oral ETS, 65.4% to aboral ETS, and 71.7% to simultaneous oral and aboral ETS. A total of 41.3% of the neurons displayed convergence of oral and aboral ETS-induced responses. Responses could be reversibly blocked with TTX (10-6 M), demonstrating involvement of neuronal conduction, and by removal of extracellular calcium. omega -Conotoxin (5 × 10-7 M) blocked the majority of responses and reduced the amplitude of residual responses by 45%, indicating the involvement of N-type calcium channels. Staining for calbindin and calretinin did not reveal different response patterns in these immunohistochemically identified neurons. We conclude that, at least for ETS close to a ganglion, confocal calcium imaging reveals complex oral and aboral input to individual myenteric neurons rather than a polarization in spread of activity.

small bowel motility; calcium imaging; confocal microscopy; neuronal networks; fluo 3; calretinin; calbindin


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

AT THE TURN OF THE PREVIOUS century it was shown that the small intestine displays stereotypical reflexes in response to physiological stimuli (1, 2, 40). Current concepts in gastrointestinal physiology hypothesize a polarized model of neuronal control of intestinal reflex pathways. Descending interneurons, activated by oral stimulation, connect to inhibitory motor neurons resulting in circular muscle relaxation in the aboral direction. Ascending interneurons, activated by aboral stimulation, connect to excitatory motor neurons, causing a muscle contraction orally, leading to bolus propulsion in the aboral direction. Further support for this concept arose from electrophysiological (10, 15, 23, 25, 31, 33), immunohistochemical (12, 34), and retrograde labeling studies (3, 4, 24, 25, 31-33) or a combination of these (20) in the gastrointestinal tract of different animal models. In particular, the retrograde labeling experiments established the morphological polarity of projections of myenteric neurons in the guinea pig small intestine (33). Due to intrinsic technical limitations of tissue fixation, it is not entirely clear whether this morphological polarization is also reflected in the spread of neuronal activity in the myenteric plexus. Recently, Spencer et al. (35) demonstrated that the motor activity in the guinea pig small intestine does not necessarily display this polarization, because local distension caused a contraction in both the circular and the longitudinal muscle layer, both orally and anally to the site of stimulation. Moreover, other observations from classical electrophysiological studies revealed the presence of ascending inhibition and descending excitation to both the circular and longitudinal muscle (28).

Classical electrophysiology is less suitable for unraveling this problem, because impalement is generally confined to a single cell. Only one study (19) reports simultaneous recordings of electrical responses in different myenteric neurons. In recent years, new techniques have been developed to visualize neuronal activation either by voltage-sensitive dyes (21-23) or calcium indicators (7, 9, 27, 39, 41-45). Electrical stimulation of interneuronal fibers of cultured myenteric neurons induced a rise in intracellular calcium concentration ([Ca2+]i), which can be monitored by confocal recording of fluorescence changes in high-affinity calcium indicators, such as fluo 3. Because these responses require neuronal conduction and synaptic transmission, the optical monitoring of [Ca2+]i seems to offer a means to study the spread of neuronal activation in a myenteric neuronal network. This technique can also be used to record simultaneously from neurons in multilayer preparations (42).

The aim of the present study was to monitor the spread of neuronal activation to aboral or oral electrical stimulation in myenteric ganglia in situ. We wanted to investigate whether the hypothesized polarization of neuronal activation could be confirmed by using this technique. Subsequently we attempted to identify different activation patterns in different classes of immunohistochemically identified myenteric neurons.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Tissue preparation. Guinea pigs of either sex (250-700 g) were killed by cervical dislocation and exsanguinated by severing the carotid arteries, a method approved by the animal ethics committee of Katholieke Universiteit Leuven.

A portion of jejunum was removed and subsequently pinned out in a Sylgard-lined petri dish to be dissected into a longitudinal muscle myenteric plexus preparation (LMMP). Dissection was performed under continuous superfusion of a Krebs solution, which itself was continuously perfused with 95% O2-5% CO2 to keep the pH at 7.4. Tissue samples of ~1.5 × 1.5 cm were prepared and adhered on coverglass by using cyanoacrylate (Loctite Super Glue) as previously described (42). Tissues were then incubated with the fluorescent calcium indicator fluo 3-AM (30 × 10-6 M), for 45-60 min, at 37°C in a 95% O2-5% CO2 atmosphere in a tissue-specific medium (Ham's F-12 medium, 2% inactivated FBS, 1% penicillin/streptomycin, 0.5% gentamycin, and 10-6 M nifidipine). After being washed out (2 × 5 min) and recovered (at least 10 min), the tissues were transferred to a coverglass chamber mounted on an inverted confocal scanning microscope (Nikon TE 300-Noran Oz). Two platinum electrodes (50-µm diameter) were placed on an oral and/or aboral interganglionic fiber of the same myenteric ganglion and were connected to a Grass S88 electrostimulator. Electrical train stimulation (ETS) was applied orally, aborally, or simultaneously on both sides.

Images were recorded with a spatial resolution of 512 × 480 pixels and a temporal resolution of 2.5 Hz. Fluo 3-AM was excited at 488 nm (argon ion 100 mW multiline laser), and fluorescence was detected at 520/525 nm. Laser intensity never exceeded 20% of the maximal laser output to reduce phototoxicity and photobleaching.

One major problem of the use of LMMP is to avoid movement artifacts due to contraction of the underlying longitudinal muscle layer. In particular, movements in the x-y axis dimension can cause artifacts that resemble Ca2+ transients by shifting an area of higher baseline fluorescence into the region of interest. To reduce tissue movements induced by longitudinal muscle contractions, all experiments were performed in the presence of nifedipine (10-6 M) and at room temperature (22°C). Furthermore, a perfusion ring was applied to reduce movements mechanically. Specifically developed software was used to correct for residual minimal movements. Movements in the z-axis dimension in either direction were less likely to induce artifacts, because they caused a drop in Ca2+ fluorescence (n = 12). This is explained by the fact that neurons were focused on at the beginning of the recording. Distortion proximal or distal to the focal plane along the z-axis will result in a decrease in fluorescence.

Identification of myenteric neurons. We used a previously validated method (42-45) to identify the neurons in the ganglia of the myenteric plexus. Application of a high-potassium Krebs solution opens voltage-operated calcium channels and induces a subsequent calcium-induced Ca2+ release from intracellular calcium stores. This rise in [Ca2+]i leads to an increase in fluorescence as fluo 3 binds to Ca2+ (Fig. 1).


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  		Fig. 1.   Identification of the neurons by application of a high-potassium Krebs solution. Three consecutive confocal images (A-C) of a myenteric ganglion (outlined) were taken at different times during application of a high-potassium (75 mM) Krebs solution. A transient rise in calcium fluorescence is clearly visible in the neurons. Images were processed using specifically developed software, and changes in relative calcium fluorescence (RF; Fi/F0) in three representative neurons (region 1-3) are displayed in the graph.

Immunohistochemical staining. After electrostimulation experiments, tissues were incubated for 2 h or overnight in freshly prepared paraformaldehyde (2%) and picric acid (0.2%). After being washed in 0.1 M PBS, tissues were processed for permeabilization and blocking of nonspecific binding sites by a 2-h treatment in 0.1 M PBS with Triton X-100 and 4% goat serum. Subsequently, the tissues were exposed to primary antibodies that were also diluted in the blocking medium for a period of 48 h at 4°C. After incubation, tissues were rinsed in 0.1 M PBS (3 × 10 min) and subsequently were incubated with the secondary antibody in the blocking medium.

Primary antibodies and their dilutions used in this study were calbindin (Calb) mouse IgG MC (1/100; Sigma Immunochemicals) and calretinin (Calr) rabbit IgG PC (1/5,000; Chemicon International). Secondary antibodies and their dilutions used in this study were goat anti-rabbit FITC (1/50) and goat anti-mouse indocarbocyanin (Cy3) (1/500; both from Jackson ImmunoResearch Laboratories). Fluorescence was visualized on a Nikon Eclipse E600 inverted microscope. Photos were taken with an Olympus C-3040 digital camera.

Drugs and chemicals. omega -Conotoxin MVIIA and TTX were from Alomone Labs; Ham's F-12 medium, FBS, and antibiotics were from GIBCO; fluo 3-AM was from Molecular Probes.

Normal Krebs solution consisted of (in mM) 120.9 NaCl, 5.9 KCl, 1.2 MgCl2, 2.5 CaCl2, 11.5 glucose, 14.4 NaHCO3, and 1.2 NaH2Po4. Low-calcium/high-magnesium Krebs solution was (in mM) 122.9 NaCl, 5.9 KCl, 2.7 MgCl2, 0 CaCl2, 11.5 glucose, 14.5 NaHCO3, 1.2 NaH2PO4, and 2 EDTA. High- potassium Krebs solution was comprised of (in mM) 53.8 NaCl, 75 KCl, 1.2 MgCl2, 1.5 CaCl2, 11.5 glucose, 14.5 NaHCO3, and 1.2 NaH2PO4. All were from Merck.

Data analysis. Movie files were recorded during confocal scanning. Laser excitation always started 6 s before the beginning of the recording. At the end of the experiment, the files were converted to uncompressed Joint Photographic Experts Group (JPEG) file interchange format (JFIF) image files, which were transported via a file transfer protocol site to a personal computer. Images were then renamed to uncompressed JPEG files to be uploaded as a stack in an inhouse-developed computer application for manual analysis of the images. This program allowed us to scan through the different images and to draw regions of interest (ROIs). The frame with the ROIs was copied as a mask to the next images. In this mask, the ROIs could be slightly shifted alone or as a group to adapt for minimal residual movements. Image intensity histograms were calculated for each region, and each image and a list of average intensities was generated. These averages were subsequently copied to an Excel spreadsheet for further analysis. After filtering the averages by using a moving average filter (stepsize, n = 3), relative fluorescence (RF) was calculated as Fi/F0, with Fi being fluorescence and F0 being the base fluorescence at the beginning of the recording in the specific region of interest in a particular condition. A response to a stimulus was defined as a rise in RF that was transient and had an amplitude of at least twice the baseline noise.

All results are presented as averages ± SE. The proportion of responding neurons is expressed as a proportion of the number of neurons with a response to high K+ depolarization. Proportions of neurons responding to different stimuli were statistically analyzed by using a Fisher's exact test. P values <0.05 were considered statistically significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ETS experiments. To establish general characteristics of ETS responses, we performed unilateral ETS (15 or 30 V, 30 Hz, 3 s) in 18 different ganglia from 13 different animals. Application of high-potassium Krebs solution identified 191 myenteric neurons in these ganglia (10.6 neurons/ganglion), and the RF of fluo 3 rose to 1.66 ± 0.02 on average (139 neurons). The electrode was positioned either orally or anally on one of the fiber strands emerging from the ganglion of interest. The mean distance between the electrodes and the border of the ganglion was 137 ± 7 and 142 ± 7 µm for the oral and aboral electrodes, respectively.

The numbers of neuronal responses to different stimuli are summarized in Table 1. The overall response to either aboral or oral ETS was 73.3%. Relative fluo 3 fluorescence rose on average to 1.17 ± 0.01 (134 neurons). Although the observed responses were similar in the absence of tissue movement, it cannot fully be excluded that some responses were due to neuronal activation induced by tissue contraction.

                              
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  		Table 1.   Unilateral electrical stimulation of interganglionic fiber tract

In a second series of experiments, ETS was applied on the oral and aboral side of the same ganglion either subsequently or simultaneously on both sides (402 neurons in 23 ganglia; 14 different animals). The numbers of neuronal responses to different stimuli are summarized in Table 2. ETS elicited a response in 52.2 and 65.4% of the neurons, when applied orally and aborally, respectively (n = 402). Simultaneous stimulation on both sides of the ganglion (n = 322 neurons) caused a calcium transient in 71.7% of the neurons. The number of responding neurons was significantly higher during aboral and bilateral ETS compared with oral ETS [95% confidence interval (CI): 0.71-0.90 and 1.22-1.54, respectively; P < 0.001] but not between aboral and bilateral ETS.

                              
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  		Table 2.   Bilateral electrical stimulation of interganglionic fiber tract

To assess whether neurons responding to stimulation from one side could also be activated by ETS applied on the opposite side of the ganglion, we defined convergence of activation as the activation of a particular neuron by both unilateral oral and unilateral aboral ETS. Convergence was present in 41.3% of the total neuronal population; 79% of the neurons responding to oral ETS also displayed a calcium transient to a subsequent similar aboral stimulus. Conversely, 63.1% of the neurons responding to an aboral stimulation showed a response to a prior similar oral ETS.

To investigate whether some of the neurons that did not show a response to either oral or aboral ETS could be activated by bilateral stimulation, we defined spatial summation as activation of a neuron that did not respond to unilateral aboral or oral stimulation but that could be activated by simultaneous oral and aboral ETS. Spatial summation recruited another 4.7% of the total neurons.

Finally, in three experiments (n = 76 neurons) a voltage increment from 30 to 40 V was consecutively applied. This revealed an increase in responsiveness of 18, 13, and 9% of the neurons, respectively, during oral, aboral, and bilateral ETS. The proportion of the different response patterns is displayed in Fig. 2.


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  		Fig. 2.   Proportion of different responses during bilateral ETS of interganglionic fiber tracts. This bar graph shows the proportion of different responses during bilateral electrical train stimulation (ETS) of a ganglion. Oral, neurons responding to oral ETS only; aboral, neurons responding to aboral ETS only; convergence, neurons responding to both unilateral oral and aboral ETS; summation, neurons responding to simultaneous oral and aboral ETS only; nonresponding, neurons not responding to ETS.

Involvement of neuronal conduction and N-type Ca2+ channels. To test the involvement of neuronal conduction in eliciting Ca2+ transients to ETS, we performed similar experiments in the presence of TTX 10-6 M and after the removal of extracellular calcium. In 28 neurons (6 ganglia), responses to unilateral ETS (oral, n = 21; aboral, n = 7) were reversibly blocked by TTX (Table 1). Similarly, by using bilateral ETS (n = 48), a complete but reversible block of all responses to oral (33/48), aboral (28/48), or bilateral (31/48) stimulation was observed (Fig. 3A, Table 2).


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  		Fig. 3.   Effect of TTX, extracellular calcium removal, and omega -conotoxin on ETS-induced response. The effect of TTX 10-6 M (A), removal of extracellular calcium (B), and omega -conotoxin 5 × 10-7 M (C) is illustrated in this graph. Changes in RF (Fi/F0) are plotted against time. ETS is indicated by bars on the time axis. A: after superfusion with TTX for 5 min (as indicated by bars), orally applied ETS (30 V, 30 Hz, 3 s) in the presence of TTX did not cause any calcium transients. After washout for 10 min, this effect was reversed. B: after application of a low-calcium/high-magnesium solution (as indicated by the bar) (0 M Ca2+ Krebs) for 10 min, the ETS-induced calcium transient (30 V, 30 Hz, 3 s, orally applied) was completely abolished. This effect was reversed after 10 min of washout of the low-calcium/high-magnesium solution. C: after superfusion with omega -conotoxin (as indicated by the bar) for 5 min, orally applied ETS (40 V, 30 Hz, 3 s) in the presence of omega -conotoxin did not cause a calcium transient. This effect was partially in the total neuronal population and partially reversible, as demonstrated after washout for 10 min.

Removal of extracellular Ca2+ (n = 36) also abolished all responses to oral (8/36), aboral (29/36), or bilateral stimulation (31/36) (Fig. 3B, Table 2). Unilateral and bilateral ETS was applied in the presence of omega -conotoxin (5 × 10-7 M) (n = 53 and 121, respectively) (Fig. 3C, Tables 1 and 2). After incubation with omega -conotoxin for 5 min, a partial inhibition of responses was observed. In the presence of omega -conotoxin, RF of the residual Ca2+ transients rose to 1.07 ± 0.01 on average or 55.2 ± 0.1% of the response in control conditions.

The number of responses to oral ETS was significantly reduced from 67.6% under control condition to 20.7% in the presence of omega -conotoxin (95% CI: 1.99-4.00; P < 0.0001). Similarly, the number of responding neurons during aboral ETS was significantly diminished by omega -conotoxin from 76.0 to 34.7% (95% CI: 1.95-3.40; P < 0.0001). As in control conditions, the number of responding neurons was higher for aboral ETS (95% CI: 0.40-0.88; P < 0.01). The oral responses were, however, more sensitive to omega -conotoxin, because they dropped to one-third. The number of aboral responses only decreased by one-half (95% CI: 0.47-0.97; P < 0.05) (Fig. 4). Finally the responses to bilateral stimulation were also significantly decreased by omega -conotoxin from 85.1 to 64.5% (95% CI: 1.13-1.54; P < 0.001), but this proportion was significantly less compared with the blockage of oral and aboral responses (95% CI: 0.25-0.54 and 0.26-0.58, respectively, for oral and aboral proportion; P < 0.0001).


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  		Fig. 4.   Differential effect of omega -conotoxin on oral and aboral responses. The differential effect on the number of oral and aboral responses in the presence of omega -conotoxin 5 × 10-7 M is illustrated in this graph. The number of responding neurons to oral and aboral ETS (%total) in control conditions (gray bars) and in the presence of omega -conotoxin 5 × 10-7M (omega -CgTx) is displayed. Oral responses were more sensitive to omega -conotoxin because they dropped to 30%, whereas aboral responses only dropped to 54%. (95% CI: 0.47-0.96; P = 0.03).

ETS-induced activity in Calb- and Calr-positive neurons. To assess whether a different response pattern was present in different classes of neurons, we performed immunohistochemical staining for Calb and Calr, two nonoverlapping markers. Calb-positive (Calb+) neurons are believed to correlate with AH-type neurons, which are thought to be sensory neurons (18). Calr-positive (Calr+) neurons are likely to be excitatory motor neurons to the longitudinal muscle (6) or ascending interneurons (7).

Immunohistochemical staining was successfully performed in four ganglia. A total of 65 neurons were identified by high K+ depolarization. A total of 55% of these could be classified as either Calr+ (n = 20) or Calb+ (n = 16). Either orally or aborally applied ETS (30 V, 30 Hz, 3s) elicited a Ca2+ transient in 58% of these neurons (Fig. 5). The response ratio was the same for Calr+ (12/20) and Calb+ (9/16) neurons (95% CI: 0.61-1.87; P = 1.00). Convergence was present in 52% of the responding neurons: six Calr+ and five Calb+ neurons were activated by both oral and aboral ETS. Spatial summation recruited additional Calr+ (5%) and Calb+ neurons (6.2%). When comparing the responses to ETS between the neuronal population studied with immunohistochemistry and the population studied without subsequent immunohistochemistry, we found no statistical difference in response pattern regarding oral responses, bilateral responses, and summation of responses (95% CI: 0.73-1.16, 0.84-1.71, 0.18-1.02, respectively; P = 0.51, 1.00, and 0.07, respectively). Aboral responses (95% CI: 1.03-1.72; P = 0.018) and convergence of responses (95% CI: 1.032-2.41; P = 0.02) were, however, less frequent in the immunohistochemically studied population.


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  		Fig. 5.   Correlation between ETS and immunohistochemistry. This figure shows an example of a calbindin- (Calb+) and calretinin-positive (Calr+) neuron, both responding to oral ETS (30 V, 30 Hz, 3 s) as indicated by bars. C, top: immunohistochemical staining for Calb in red and Calr in green. The corresponding confocal image of the ganglion is displayed (C, bottom). The corresponding recordings of the changes in RF (Fi/F0) are displayed in A and B. A: changes in RF of a Calr+ neuron. B: changes in RF in response to the same stimulus in a Calb+ neuron.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, we demonstrated that electrical stimulation of interganglionic fibers induces a rise in intracellular calcium concentration in a subset of myenteric neurons. This phenomenon involves neuronal conduction and transmission as demonstrated by the effect of TTX, removal of extracellular calcium, and omega -conotoxin. We demonstrated a complex neuronal input of oral and aboral pathways to individual myenteric neurons, as shown by the presence of convergence and summation of responses. Moreover these interactions seem to be present in different classes of immunohistochemically identified neurons.

Technique. Previous calcium indicator studies demonstrated activity-dependent changes in intracellular calcium concentration in response to electrical stimulation both in S- and AH-type neurons (14, 15, 27, 39, 41, 46). Vanden Berghe et al. (45) showed that confocal Ca2+ imaging could be applied to monitor the spread of neuronal activation in a network of cultured myenteric neurons. In the present study, the myenteric network was studied in situ, leaving the intrinsic and original synapses and connections intact. Furthermore, the use of the in situ preparation rules out possible phenotypic changes due to culturing techniques. The technique used in our experimental setup was previously developed for multilayer preparations (42). We used a high K+ Krebs-induced depolarization to identify neurons within the ganglionic region. At least in culture conditions, there is a one-to-one correlation between neurons and cells displaying a K+-induced Ca2+ transient (45), and in multilayer preparation, this approach consistently identified neurons (42). Enteric glial cells do not express voltage-operated calcium channels (8, 13), nor does capacitative Ca2+ entry happen through these channels (26, 48). On the other hand, glial cells express serotonergic and purinergic receptors (11, 17). So it is not fully excluded that some glial cells were activated secondarily by neurotransmitter release from nearby neurons, which are depolarized either by high K+ or ETS.

Involvement of conduction and neurotransmission in generation of the Ca2+ transients. To test whether the registered Ca2+ transients were due to neuronal activation via conduction and neurotransmission, ETS was applied in the presence of TTX and in high-magnesium/low-calcium Krebs solution. Both conditions abolished, in a reversible manner, any response to ETS. The former implies that neuronal conduction via interganglionic fibers was responsible for inducing Ca2+ transients. The latter points to the requirement of extracellular calcium, most likely acting to induce calcium-induced release of calcium from intracellular inositol 1,4,5-trisphosphate or ryanodine-sensitive stores or to the involvement of neurotransmission in the generation of the Ca2+ transients. These observations support previous findings in vitro (45).

Although single pulses of electrical stimulation of interganglionic fibers produce fast excitatory postsynaptic potentials (EPSPs), which are not reflected in an increase of [Ca2+]i in S neurons (27), the ETS stimuli we used are known to generate multiple action potentials and a transient increase of fura 2 fluorescence. Because the affinity for calcium of fluo 3 is lower than the affinity of fura 2, it is unlikely that we would have picked up signals that do not represent action potentials. Furthermore, the temporal resolution of our system (2.5 Hz) did not allow registering fast events, such as fast EPSPs. The spatial resolution of our system and the magnification used (×20, air objective) does not allow us to record the possible localized Ca2+ transients that could be expected near the cell membrane when Ca2+ enters through a limited number of activated nicotinic receptor channels.

The N-type calcium-channel blocker, omega -conotoxin, reduced both the number of neuronal responses as well as the amplitude of the Ca2+ transients. N-type calcium-channels are believed to play an important role presynaptically in the release of neurotransmitters in different neuronal tissues (16, 38, 47). Observations of Ca2+ transients in cultured myenteric neurons by our group also indicated an inhibition of synaptic neurotransmitter release by omega -conotoxin (45). By analogy with these reports, the influence of omega -conotoxin on electrically evoked calcium transients in the present study suggests that a major part of the observed responses is synaptically driven.

Previous reports (27) also suggested the presence of N-type calcium channels on the soma of myenteric neurons, because the amplitude of Ca2+ transients induced by intracellular current injection was significantly reduced by 67%. So the omega -conotoxin-resistant responses are likely to represent antidromic activation of myenteric neurons, attenuated by blocking N-type calcium channels on the soma, thereby reducing the magnitude of the calcium influx into these cells.

Convergence and summation of responses point to a complex interaction of inputs to individual neurons, rather than to a polarization in activity. Observations of Bayliss (2) and Trendlenburg (40) at the turn of the previous century hypothesized a polarized spread of neuronal activity in the intestinal reflex pathways. Retrograde labeling of myenteric neurons established a morphological basis for this polarity (33). However, in this study, convergence of responses was present in a large proportion of the neurons, and spatial summation could be observed in a subset of myenteric neurons. These findings potentially point to a complex input of oral and aboral signals to individual neurons, rather than to a polarization in transmitting neuronal activity. We found a greater proportion of aboral responses than oral responses to ETS, although retrograde labeling studies have previously demonstrated (33) that the number of orally projecting neurons is smaller than the amount of anally projecting neurons in the guinea pig small intestine. Therefore, the higher number of responses to aboral ETS is rather unexpected. Because we stimulated relatively close to the ganglion, antidromic activation of descending neurons is a likely confounding factor. The occurrence of omega -conotoxin-resistant responses confirms that a subset of Ca2+ transients was antidromically activated. The finding that aboral responses were less sensitive to omega -conotoxin is consistent with the morphological observations of more aborally projecting neurons.

Interneurons pass through several ganglia in the course of their projections. About one-third of orally projecting interneurons, for instance, provide varicose side branches that loop around in the myenteric ganglia and presumably make contact with other myenteric neurons (5). Therefore, it is technically possible to activate one pathway from both sides of the ganglion. Notwithstanding the fact that interneurons constitute <19% of the total neuronal population (12) and these "loop around" connections are not abundant, it is possible that some converging Ca2+ transients were evoked by antidromic stimulation of one interneuron with collaterals in the ganglion of interest. In this particular case, the convergence of responses would not be due to converging ascending and descending pathways, because stimulation on either side of the ganglion would activate the same pathway.

However, several electrophysiological studies have also shown complex synaptic interactions in the myenteric plexus of the guinea pig and other species. Even with ETS of longitudinally orientated interganglionic fibers at sites up to 1.5 mm, Stebbing et al. (36) observed convergence of responses in 80% of impaled neurons in the guinea pig myenteric plexus.

Furthermore, morphological features do not necessarily translate directly into physiological observations; by using distension of guinea pig small intestine at the serosal site, Smith et al. (29) demonstrated a larger number of neurons responding to activation of ascending pathways (35%). Although the morphological features of S-type neurons showed more anally than orally projecting neurons (n = 28 and 17, respectively), activation of descending pathways only activated 26% of the neurons. They also observed a large amount of convergence of responses; 63% of the neurons responding to activation of descending pathways also responded to activation of ascending pathways. Conversely, descending pathways also activated 48% of the S neurons activated by ascending pathways. The majority of neurons displaying convergence were tertiary plexus neurons, but anally projecting and orally projecting neurons also showed convergence of responses.

With the use of the multisite optical recording technique, Peters et al. (23) demonstrated induction of fast EPSPs by fiber tract stimulation in 72 ± 12% of human submucosal plexus ganglia. Convergence of synaptic input occurred in 65-75%. They also observed different activation patterns with stimulation of different fiber tracts in guinea pig and mice enteric neurons (21, 23). Further experiments with increased distances of ETS application should be performed to investigate the contribution of antidromic and synaptic activation.

Finally, we studied ETS-induced responses in two groups of immunohistochemically identified neurons. We stained for Calb and Calr, two nonoverlapping markers (12). The group of immunohistochemically identified neurons displayed representative responses compared with the ETS-induced responses in earlier experiments. Calb+ neurons, which have a Dogiel type 2 morphology, are believed to correlate with electrophysiologically characterized AH-type neurons (18). Calr+ neurons are likely to be excitatory motor neurons to the longitudinal muscle (6) or ascending excitatory interneurons (7). With the use of this confocal calcium imaging technique and ETS in close vicinity of the myenteric ganglia, we could not identify a polarization in ETS-induced activity in these two classes of immunohistochemically identified neurons.

In conclusion, our data suggest a complex input of oral and aboral information to individual neurons of different immunohistochemically identified classes.


    ACKNOWLEDGEMENTS

This work was supported by a grant from Fund for Scientific Research, Flanders, Belgium (F.W.O.-Vlaanderen).


    FOOTNOTES

Address for reprint requests and other correspondence: R. Bisschops, Center for Gastroenterological Research, Katholieke Universiteit Leuven, 49 Herestraat, 3000 Leuven, Belgium (E-mail: raf.bisschops{at}med.kuleuven.ac.be).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published January 29, 2003;10.1152/ajpgi.00383.2002

Received 5 September 2002; accepted in final form 27 January 2003.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Gastrointest Liver Physiol 284(6):G1084-G1092
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