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
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ABSTRACT |
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.
-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
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INTRODUCTION |
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.
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MATERIALS AND METHODS |
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.
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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.
-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.
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RESULTS |
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.
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.
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.
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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
-conotoxin on ETS-induced response. The effect of TTX
10 6 M (A), removal of extracellular calcium
(B), and -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 -conotoxin (as indicated by the
bar) for 5 min, orally applied ETS (40 V, 30 Hz, 3 s) in the
presence of -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.
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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
-conotoxin (5 × 10
7 M) (n = 53 and 121, respectively)
(Fig. 3C, Tables 1 and 2). After incubation with
-conotoxin for 5 min, a partial inhibition of responses was
observed. In the presence of
-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
-conotoxin (95% CI: 1.99-4.00; P < 0.0001).
Similarly, the number of responding neurons during aboral ETS was
significantly diminished by
-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
-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
-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 -conotoxin on oral and aboral
responses. The differential effect on the number of oral and aboral
responses in the presence of -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 -conotoxin 5 × 10 7M ( -CgTx)
is displayed. Oral responses were more sensitive to -conotoxin
because they dropped to 30%, whereas aboral responses only dropped to
54%. (95% CI: 0.47-0.96; P = 0.03).
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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.
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DISCUSSION |
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
-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,
-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
-conotoxin (45). By analogy
with these reports, the influence of
-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
-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
-conotoxin-resistant responses
confirms that a subset of Ca2+ transients was
antidromically activated. The finding that aboral responses were less
sensitive to
-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.
 |
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