Patch-clamp study of neurons and glial cells in isolated
myenteric ganglia
M.
Hanani1,
M.
Francke2,
W.
Härtig2,
J.
Grosche2,
A.
Reichenbach2, and
T.
Pannicke2
1 Laboratory of Experimental Surgery, Hebrew
University-Hadassah Medical School, Jerusalem 91240, Israel; and
2 Paul Flechsig Institute for Brain Research,
Leipzig University, D-04109 Leipzig, Germany
 |
ABSTRACT |
Most of the physiological information on
the enteric nervous system has been obtained from studies on
preparations of the myenteric ganglia attached to the longitudinal
muscle layer. This preparation has a number of disadvantages, e.g., the
inability to make patch-clamp recordings and the occurrence of muscle
movements. To overcome these limitations we used isolated myenteric
ganglia from the guinea pig small intestine. In this preparation
movement was eliminated because muscle was completely absent, gigaseals were obtained, and whole cell recordings were made from neurons and
glial cells. The morphological identity of cells was verified by
injecting a fluorescent dye by micropipette. Neurons displayed voltage-gated inactivating inward Na+ and Ca2+
currents as well as delayed-rectifier K+ currents.
Immunohistochemical staining confirmed that most neurons have
Na+ channels. Neurons responded to GABA, indicating that
membrane receptors were retained. Glial cells displayed
hyperpolarization-induced K+ inward currents and
depolarization-induced K+ outward currents. Glia showed
large "passive" currents that were suppressed by octanol,
consistent with coupling by gap junctions among these cells. These
results demonstrate the advantages of isolated ganglia for studying
myenteric neurons and glial cells.
enteric nervous system; electrophysiology; sodium channels; calcium
channels
 |
INTRODUCTION |
KNOWLEDGE OF THE ENTERIC nervous system has benefited
greatly from studies using isolated tissues. Particularly important were studies utilizing the longitudinal muscle-myenteric plexus (LMMP)
preparation, in which the physiology and pharmacology of myenteric
neurons were characterized (for reviews see Refs. 9, 12, 30, 34).
Still, a few disadvantages limit the usefulness of LMMP preparation:
1) substances released from the muscle tissue may interfere
with the interpretation of biochemical and pharmacological experiments;
2) muscle movements can dislodge microelectrodes during
electrophysiological recordings and disturb optical measurements; and
3) the muscle may reduce the visibility of the neurons. To overcome these difficulties, Yau et al. (35) developed a new preparation consisting of isolated myenteric ganglia from the guinea
pig, which they used for studying acetylcholine release. This method
yielded a large number of ganglia and was employed to measure cAMP
synthesis in response to various agonists (36) and to study the
regulation of substance P release by adenosine (28) and of vasoactive
intestinal peptide release by nitric oxide (17).
Fiorica-Howells et al. (7) used isolated ganglia to investigate the
pharmacological mechanism by which serotonin induces cAMP synthesis in
myenteric ganglia. The availability of isolated myenteric ganglia has
opened up interesting possibilities for culturing myenteric ganglia
from adult animals. These cultures proved to be helpful in biochemical
(6, 16) and electrophysiological (1, 18, 23, 38) experiments.
A serious drawback of the LMMP preparation is the presence of a
connective tissue layer (basal lamina) over the ganglia, which prevents
the use of patch electrodes. Therefore, patch-clamp studies have been
done only on cultured myenteric neurons (1, 3, 8, 24) and cultured
myenteric glia (4). A major disadvantage of culture preparations is the
disruption of the organization of the tissue, which underlies neuronal
connectivity, relations between glia and neurons, and interactions
within the glial network. In central nervous system (CNS) research this
problem has been partly overcome by the use of slices, which enable
recordings with patch electrodes. Patch-clamp recordings
have also been made in intact sympathetic ganglia (14). In this study,
we show that isolated myenteric ganglia are suitable for patch
recordings from neurons and also from glia, whose small size has so far
precluded their systematic electrophysiological investigation in LMMP
preparations. In this paper we describe the isolation and recording
techniques and present an account of the basic electrophysiological
properties of myenteric neurons and glial cells.
 |
MATERIALS AND METHODS |
The isolation of myenteric ganglia was done according to the method of
Yau et al. (35), with some modifications (23). Guinea pigs of either
sex weighing 250-300 g were stunned and bled. The small intestine
was removed and placed in cold Krebs solution containing (in mM): 120.9 NaCl, 5.9 KCl, 14.4 NaHCO3, 2.5 MgSO4, 1.2 Na2HPO4, 2.5 CaCl2, and 11.5 glucose. The longitudinal muscle with the attached myenteric plexus
preparation was stripped and cut into 5-mm segments, which were
incubated in an enzyme mixture containing (in mg/100 ml Krebs solution)
125 collagenase type IA, 100 protease type IX, and 2.5 DNase I (all
from Sigma). The incubation was at 37°C for 35 min with
continuous shaking. After incubation, the ganglia were allowed to
settle and the supernatant was discarded. After two washes in Krebs
solution, the ganglia were collected with a micropipette under a
dissecting microscope and were washed for 20 min in medium 199 (Sigma)
containing 500 µg/ml streptomycin sulfate, 500 U/ml penicillin G
sodium, and 0.25 µg/ml amphotericin B (all from Sigma). The ganglia
were mounted on a polycarbonate filter (7) or on a glass coverslip. The
filters or coverslips were placed in a petri dish with medium 199 containing 10% fetal calf serum, 100 µg/ml streptomycin, 100 U/ml
penicillin G sodium, and 0.05 µg/ml amphotericin. The ganglia were
kept in an incubator at 37°C with 5% CO2 and were used
after 3-48 h.
For the electrophysiological experiments the tissues were placed on the
bottom of a chamber that was attached on the stage of a Zeiss Axioskop
microscope equipped with fluorescence illumination. The chamber was
superfused with Krebs solution at room temperature; the solution was
bubbled with 95% O2-5% CO2. Drugs were
applied by bath perfusion. The preparation was viewed with a water
immersion ×40 objective using Nomarski optics. Patch pipettes
were made from borosilicate glass capillaries using a Narishige puller. The pipettes were filled with a solution containing (in mM) 10 NaCl,
130 KCl, 1.0 CaCl2, 2.0 MgCl2, 10 HEPES, and 10 EGTA, pH 7.1. The electrode resistance was 5-10 M
. Lucifer
yellow (0.1%) was added to the pipette solution to enable
morphological identification of recorded cells.
Whole-cell voltage clamp recordings were made using an Axopatch 200A
amplifier. Currents were low-pass filtered at 1 kHz and digitized using
a 12-bit analog-to-digital converter. Data acquisition and analysis and
generation of voltage command protocols were performed with ISO2
software (MFK, Niedernhausen, Germany). Input resistances were measured
with a 10-mV hyperpolarizing step from a holding potential of
80
mV. For all measurements series resistance compensation was used up to
30% to minimize voltage errors. At the end of the recording from each
cell, its identity was established by observing its morphology with
fluorescence illumination.
Lucifer yellow-injected cells were first observed and photographed with
a Zeiss fluorescence microscope. For further study, they were inspected
without fixation with a confocal microscope (Zeiss LSM510). In
immunohistochemical experiments, isolated ganglia were fixed in 4%
paraformaldehyde for 2 h and subsequently rinsed with 0.1 M
Tris-buffered saline (TBS), pH 7.4. Nonspecific binding sites were
blocked with 5% normal goat serum in TBS containing 0.3% Triton X-100
(NGS-TBS-T) for 1 h. The tissue was next incubated in 5 µg/ml affinity-purified rabbit antibodies directed against Na+ channels (type II or Pan, Alomone Labs, Jerusalem,
Israel) for 16 h at room temperature. After several rinses in TBS,
immunoreactivity was visualized by incubation in Cy3-tagged goat
anti-rabbit IgG (Jackson Laboratories), 20 µg/ml in TBS containing
2% bovine serum albumin for 1 h. For double immunofluorescence
labeling of sodium channels and the Ca2+-binding protein
calbindin, ganglia were fixed and blocked as described for single
staining. Afterwards, mouse anti-calbindin (clone Cl-300, Sigma), 1:200
in NGS-TBS-T, was applied simultaneously with rabbit antibodies to
Na+ channels (5-10 µg/ml). Rinsed tissue was then
treated with a cocktail consisting of Cy2-goat anti-mouse IgG (Jackson)
and Cy3-tagged goat anti-rabbit IgG, both at 20 µg/ml for 1 h.
Finally, all tissues were extensively rinsed with TBS, briefly washed
in distilled water, air dried, and coverslipped with Entellan (Merck).
The omission of primary antibodies resulted in the absence of any cellular staining.
Mean values are given with standard deviations. We used the
t-test for unpaired samples and the Wilcoxon test for paired
samples. Analysis was done with SPSS for Windows software.
 |
RESULTS |
Morphological observations.
Figure 1A shows a preparation
typical of those used in this study, as seen during the experiments.
The preparation consists of several ganglia interconnected by fiber
tracts. With the use of Nomarski optics, single neurons were clearly
visible. Glial cells could not be seen under these conditions, although
they outnumber the neurons. A Lucifer yellow-stained neuron is shown in
Fig. 1B; this cell was characterized electrophysiologically as
a neuron. Although the morphological identification of the neurons was
unequivocal, distinction between subtypes of neuronal morphologies was
not possible in most cases. Figure 1C shows a glial cell
stained with Lucifer yellow; the small size of this cell and the fine,
short processes are typical for glia (21). This cell had the
electrophysiological properties of a glial cell.

View larger version (67K):
[in this window]
[in a new window]
|
Fig. 1.
Morphological features of isolated myenteric ganglia and the 2 major
cell types found in them. A: micrograph showing 4 interconnected myenteric ganglia attached to glass coverslip as seen
during electrophysiological experiments, 24 h after being isolated.
Ganglia are marked with asterisks; 1 of the fiber bundles connecting
ganglia is indicated with an arrow. B: morphology of a
myenteric neuron injected with Lucifer yellow from recording electrode.
C: myenteric glial cell injected with Lucifer yellow.
|
|
Electrophysiology of neurons.
Patch-clamp recordings in the whole cell configuration were made from
82 neurons, which were identified by their morphology after filling
with Lucifer yellow (Fig. 1B). Because neurons could not be
clearly classified in most cases, we pooled the data for all the cells.
Input resistance was measured with a 10-mV hyperpolarizing step from a
holding potential of
80 mV and was 392 ± 203 M
(mean ± SD, n = 63); the resting membrane potential was
46 ± 13 mV (n = 58). Figure 2A
shows current recordings from neurons. The neuronal current pattern was
characterized by outward rectification. Hyperpolarizations evoked only
small inward currents. Depolarizing steps to
30 mV or higher
evoked fast-inactivating inward currents. In some neurons, the current
response was repetitive (Fig. 2B). In most cells, the inward
currents had two time constants of inactivation, a fast early time
constant and a slower late one, suggesting the involvement of two types
of ionic channels. The inward inactivating currents were followed by
slower activating, sustained outward currents (presumably
delayed-rectifier K+ current).

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 2.
Current responses recorded from neurons in isolated myenteric ganglia
using patch-clamp recordings. A: depolarizing and
hyperpolarizing voltage steps between 140 and +60 mV in 20-mV
increments were applied from a holding potential of 80 mV.
Hyperpolarizing pulses evoked only small currents, which might be due
to inwardly rectifying K+ channels. Depolarizing potentials
elicited fast-inactivating inward currents and sustained outward
currents. Inset: fast inward currents at potentials of
10, +10, and +30 mV at a faster time scale after removal by
subtraction of capacitative artifacts. B: recordings from
another neuron. Depolarizing pulses between 40 and +20 mV were
applied and evoked multiple inward current responses. Holding potential
was 80 mV.
|
|
We next performed a series of experiments to learn the ionic nature of
the inward currents. Figure 3A
shows current responses under control conditions. Replacing the
extracellular Na+ with choline eliminated the fast
component of the inward current (n = 28, Fig. 3B).
Tetrodotoxin (1 µM) blocked this component as well (n = 3, data not shown), consistent with a major contribution of
Na+ channels. In most neurons, after the Na+
current was blocked, a slower inward current was still recorded. This
current was inhibited by the Ca2+ channel blocker
Cd2+ (1 mM, n = 19, Fig. 3C). The
difference between the traces in Fig. 3, C and B, is
displayed in Fig. 3D and represents the net voltage-activated
inward Ca2+ current. Likewise, eliminating Ca2+
from the external solution suppressed the slow inward current (n = 2). To obtain information on the type of the
voltage-dependent Ca2+ channels we first incubated the
tissue in Na+-free solution (Fig.
4A) and then added the L-type
Ca2+ channel blocker nimodipine (5 µM). Nimodipine
inhibited strongly (but not completely) the inward currents in all five
cells tested (Fig. 4, B and C), indicating the presence
of L-type Ca2+ channels in the myenteric neurons. In some
cells, blocking Ca2+ currents inhibited the late outward
currents, which were apparently due to K+ flow (Fig.
4B). Thus it appears that the neurons also have
Ca2+-dependent K+ channels.

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 3.
Current responses recorded from a myenteric neuron in response to
depolarizing voltage steps under altered extracellular medium. Voltage
steps were applied between 80 and +20 mV in 20-mV increments
from 80 mV. A: fast-inactivating current responses were
evoked under control conditions. B: when Na+ was
replaced by choline, fast early currents disappeared but a slower
component of inward current persisted. C: addition of
Cd2+ (Ca2+ channel blocker, 1 mM) completely
inhibited remaining inward currents. D: difference between
currents in B and C yielded Cd2+-sensitive
current, i.e., net Ca2+ current.
|
|

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 4.
Effects of L-type Ca2+ channel blocker nimodipine on
currents recorded in a myenteric neuron. A: voltage steps were
applied between 80 and +40 mV at 20-mV increments; holding
potential was 80 mV. Recordings were made in
Na+-free solution to eliminate Na+ currents.
Under these conditions, no fast inward currents were detected because
of large outward K+ currents (A). Application of 5 µM nimodipine reduced sustained outward current (B).
Difference between A and B, i.e., nimodipine-sensitive
current, is shown in C. Nimodipine blocked a fast inward
current and also reduced outward current. Early part of C is
displayed at a faster time scale in inset, showing currents at
potentials of 10, 0, +10, and 20 mV. Inward currents appear to
be due to flow through L-type Ca2+ channels, whereas
sustained outward currents are apparently due to
Ca2+-dependent K+ channels.
|
|
We concluded from these experiments that all neurons displayed both
Na+ and Ca2+ inward currents. In some of the
experiments, we identified the morphological type of the cells and
found that this conclusion holds for both Dogiel type I (n = 8)
and Dogiel type II (n = 5) neurons. (In the rest of the cases
the morphology of the neurons could not be determined with certainty.)
However, the ratio between the Na+ and Ca2+
inward currents varied among cells. In some cells the Ca2+
current was barely measurable, whereas in others it was prominent.
To learn the distribution of Na+ channels in myenteric
neurons we stained the ganglia with an antibody against Na+
channels. The ganglia were also stained for the
Ca2+-binding protein calbindin, which is a marker for most
AH-type neurons (10). Action potentials in these neurons have a large component of voltage-induced inward Ca2+ current
("Ca2+ spikes"; Refs. 29, 34). Figure
5 shows a confocal image of the staining
for the two proteins. It was evident from this and other such
experiments that there is a subpopulation of calbindin-positive neurons. It also appeared that most of the neurons were immunopositive to the Na+ channel antibody, but the intensity of the
staining varied among cells. Some of the calbindin-positive neurons
showed weak staining for the Na+ channels, whereas others
were brightly stained for both proteins. Thus it seems that there is a
continuum of intensity of staining for Na+ channels. In
most studies on the distribution of calbindin in myenteric neurons, the
guinea pig ileum has been used. In the present work the ganglia were
isolated from both the ileum and jejunum. To compare between these two
parts we performed double immunostaining for calbindin and for
Na+ channels in the myenteric plexus of the jejunum. The
results showed that a subpopulation of neurons were positive for
calbindin and had the morphology of Dogiel type II neurons. Many more
cells were positive for Na+ channels, but there was a
partial overlap between these two populations, as described above. Thus
it appears that the use of calbindin as a marker for Dogiel type II
cells holds for the entire guinea pig small intestine.

View larger version (42K):
[in this window]
[in a new window]
|
Fig. 5.
Confocal image of immunohistochemical staining of myenteric ganglia for
Na+ channels and calbindin. Ganglia were fixed 20 h after
being isolated. Immunoreactivity for calbindin is shown in green and
for Na+ channels is shown in red. Many neurons appear
orange or yellow, indicating costaining for both Na+
channels and calbindin. Only few neurons are green, indicating absence
of Na+ channels. Colored arrows indicate examples of
corresponding type of staining.
|
|
To find out whether neurotransmitter receptors were retained in the
isolated ganglia we recorded responses to GABA. As demonstrated in Fig.
6, GABA (50 µM) evoked an inward current,
which partly inactivated in the presence of the transmitter. Similar
responses were obtained in 8 of 10 neurons tested.

View larger version (8K):
[in this window]
[in a new window]
|
Fig. 6.
Response of a myenteric neuron to GABA (50 µM), applied as indicated
by line. Note inactivation of GABA-evoked current. Holding potential
was 80 mV.
|
|
Electrophysiology of glial cells.
Recordings were made from 69 myenteric glial cells. These cells were
characterized by low input resistances (90 ± 42 M
, n = 33)
and by mostly "passive" currents, as shown in Fig.
7A; the resting membrane potential
was
55 ± 10 mV (n = 40). Ba2+ (1 mM), a
K+ channel blocker, had only a small effect on input
resistance (110 ± 47 M
, n = 8). This difference is not
significant (t-test, P > 0.2). The passive currents
may be, at least partly, the result of coupling among glial cells,
which is believed to be via gap junctions (22, 27). To
test this possibility we added the gap junction blocker octanol (27),
at 0.5 mM, to the bathing solution to block gap junctions. In most
cells, octanol reduced the passive currents and caused the appearance
of voltage-dependent outward currents (typical for delayed-rectifier
K+ current). In the presence of octanol, strong
hyperpolarizing voltages evoked time-dependent inactivation of inward
currents (typical for inward-rectifier K+ current; see Fig.
7B). Input resistance was only slightly increased by octanol
(from 59 ± 23 to 71 ± 21 M
, n = 5). This increase was
also not significant (Wilcoxon test, P > 0.06). Addition of 1 mM Ba2+ in the presence of octanol strongly reduced the
inward currents (Fig. 7C) and increased the input resistance
from 72 ± 18 to 490 ± 497 M
(n = 9). This difference was
significant (P < 0.02, Wilcoxon test). This result indicated
the presence of Ba2+-sensitive, inwardly rectifying
K+ channels in myenteric glia. Incubating the tissues in
medium with low pH (6.55), which also blocks gap junctions (27),
affected input resistance and membrane currents similarly to octanol
(data not shown).

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 7.
Current responses of glial cells in isolated myenteric ganglia. In
A-C, voltage steps were applied in 20-mV increments
between 180 and 0 mV; holding potential was 80 mV. Scale
bars in C correspond to A-C. A: currents
under control conditions showed no time or voltage dependence.
B: responses in presence of 0.5 mM octanol, which blocks
coupling via gap junctions. "Passive" currents were reduced,
which revealed a voltage-dependent outward current (delayed rectifier)
and time-dependent inactivation of inward current at strong
hyperpolarizing voltages (inward rectifier K+ current).
C: addition of 1 mM Ba2+ (which blocks
K+ currents) strongly reduced inward currents. Results
indicate presence of voltage-activated K+ channels in
myenteric glia. D: fast-inactivating inward currents were
recorded in another glial cell. Voltage steps to 40, 20,
0, and +20 mV were applied after a 500-ms prepulse of 120 mV.
Currents are difference between recording in extracellular solution
containing 1 mM Ba2+ and recording in a solution containing
Ba2+ and 1 µM tetrodotoxin; thus they represent currents
through voltage-dependent Na+ channels.
|
|
Large passive currents were observed in 41 of 61 myenteric glial cells.
In the other 20 cells voltage- and/or time-dependent currents were
observed in normal Krebs solution. Fast-inactivating inward currents
were found in a small number of glial cells (n = 7, Fig.
7D). The amplitude of these currents was much smaller than that
of those recorded in neurons. In one glial cell that was tested,
tetrodotoxin (1 µM) blocked these currents.
Cells displaying low input resistance and electrophysiological
properties as shown in Fig. 7 had the morphological features of enteric
glial cells (Fig. 1C). In most cases (37 of 45), glial cells
were found to be dye-coupled to 2-25 other glial cells (Fig. 8). When the tissue was incubated with
octanol before the recording, there was no dye coupling (n = 4). Under control conditions, only 8 of 45 cells were not dye coupled
and 7 of them displayed voltage-dependent currents. These eight glial
cells had an input resistance of 156 ± 64 M
(n = 8), which
is significantly higher than the resistance of coupled glia (P < 0.001, t-test). This value was significantly lower than the
resistance of neurons (392 ± 203 M
, P < 0.001, t-test) and supports the identification of these cells as glia rather than neurons.

View larger version (131K):
[in this window]
[in a new window]
|
Fig. 8.
Dye coupling among myenteric glial cells. Single glial cell was
injected with Lucifer yellow from recording pipette. About 25 neighboring glia were stained because dye spread, apparently through
gap junctions.
|
|
 |
DISCUSSION |
Investigations of cells in isolated preparations such as brain slices
and sympathetic ganglia have been extremely useful in the study of
these systems. A great advantage of these preparations is the ability
to make patch-clamp recordings from neurons and glial cells while these
cells retain many of their original connections. This is in contrast to
tissue culture preparations, in which cell interactions, when they
occur, probably do not accurately reflect the original connectivity.
The experiments described here clearly demonstrate the utility of
isolated, intact myenteric ganglia for investigation of the physiology
and pharmacology of myenteric neurons and glial cells in their original
environment. A major advantage of this method is that it enables patch
recording from these cells. Optical recordings of Ca2+
signals in neurons are also facilitated in this preparation (19).
Myenteric neurons have been studied extensively using intracellular
recording, and much information on their pharmacology has been obtained
(9, 34). These studies have provided important knowledge of this system
and have contributed to neuropharmacology in general. However, only
limited progress has been made in understanding the membrane properties
of myenteric neurons and glial cells. Studies using patch recordings
were done on cultured myenteric neurons (1, 3, 8, 24, 33, 38, 39).
Patch recordings have not been done on myenteric neurons in the intact
ganglia because the basal lamina covering the ganglia prevents the
formation of a gigaseal with a patch pipette. In the present work we
show that patch recordings can be made after isolating the ganglia, apparently because the enzymatic digestion breaks the basal lamina and
allows access of the pipette to the cells. A possible criticism of this
method is that cells may be damaged by the proteolytic enzymes.
However, electron microscopic observations (7, 35) showed no damage to
the ganglia after the isolation procedure. To minimize cell damage we
used a lower concentration of the enzymes than those used by previous
workers for the same incubation time and kept the ganglia for 3 h or
more in an incubator before the recordings, to allow tissue recovery.
Despite these precautions we cannot rule out that the isolation of the
ganglia caused a certain degree of damage or abnormalities in the
cells. This may account for the difficulty in classifying neurons
according to morphology and for the variations in the ratio in
Na+ and Ca2+ currents among cells.
In comparison to myenteric neurons, very little is known about the
physiology and pharmacology of myenteric glia, which may function like
CNS astrocytes (13, 22). The small size of these cells (cell body
diameter 8 µm; Ref. 22) has so far precluded a systematic
investigation of these cells with intracellular microelectrodes. In two
studies, some physiological properties of cultured myenteric glia were
investigated. Broussard et al. (4) made patch-clamp recordings from the
glial cells, and Zhang et al. (37) used Ca2+ imaging to
investigate their responses to endothelin. In the present study we
demonstrated that patch recordings from glial cells in isolated
myenteric ganglia were feasible. We found that myenteric glia have low
input resistance, consistent with their being connected by gap
junctions (11, 22, 27). Indeed, by injecting Lucifer yellow into these
cells, we found that they were dye coupled and that this coupling was
blocked by the gap junction blocker octanol. In accord with these
observations, most myenteric glia displayed large passive currents in
response to voltage steps. In the presence of octanol these passive
currents were reduced, allowing us to observe inward-rectifying
currents, which were blocked by Ba2+, and also outwardly
directed delayed currents. Thus it appears that these cells have at
least two types of voltage-dependent K+ channels. Broussard
et al. (4) recorded delayed-rectifying K+ currents in
cultured myenteric glia but not inward-rectifying K+
channels. They also recorded inward Na+ currents in 4 of 20 glial cells, as found by us in a subpopulation of the glial cells. Most
of the glial cells that were not dye coupled under control conditions
displayed time- and voltage-dependent currents, similar to those
obtained when octanol was used to uncouple the cells. It thus appears
that the coupling among glia masks the voltage-dependent ionic
currents. The physiological significance of these ionic channels is not
yet clear, and they may not contribute to the behavior of the cells
when they are strongly coupled. However, under conditions favoring the
uncoupling of the cells (e.g., when intracellular pH is low), these
channels may be functionally important. Glial cells are believed to
regulate many neuronal functions (26), and thus alterations in glial
properties may in turn affect myenteric neurons, which are in close
contact with the glial cells (11).
Our electrophysiological results show that myenteric neurons in the
isolated ganglia display several types of voltage-sensitive ion
channels. We recorded fast inward currents in all the cells identified
morphologically as neurons. In all the neurons examined, these inward
currents consisted of both Na+ and Ca2+
currents. Ca2+ currents were identified in
Na+-free solution, using the Ca2+ channel
blocker Cd2+. Ca2+ currents were smaller than
Na+ currents and had slower decay times. The L-type
Ca2+ channel blocker nimodipine strongly inhibited the
Ca2+ currents, indicating the existence of L-type
Ca2+ channels in myenteric neurons. This blockade was not
complete, suggesting the presence of other types of Ca2+
channels. The presence of L-type Ca2+ channels has been
reported in rat myenteric neurons (8, 24). In contrast, Grafe et al.
(15) reported that the L-type Ca2+ channel blocker D-600
had no effect on the electrophysiological properties of AH-type
myenteric neurons of guinea pigs. A possible explanation for this
discrepancy is that D-600 is a verapamil derivative and not a
dihydropyridine like nimodipine. Baidan et al. (2) recorded
Ca2+ currents in cultured myenteric neurons and found that
nifedipine was not very effective in blocking these currents.
Nevertheless, they concluded that both L- and N-type currents were
present. The quantitative difference from our results may be caused by the fact that we used intact, isolated ganglia rather than cultures. Using a Ca2+ imaging technique, Simeone at al. (31) found
that acetylcholine caused large increases in intracellular
Ca2+ concentration
([Ca2+]i) in cultured myenteric
neurons from guinea pigs, which were completely blocked by 10 µM
nifedipine. They concluded that the [Ca2+]i increases were caused by
Ca2+ influx through voltage-gated L-type Ca2+
channels, which is in accord with our observations. It is interesting to note that in many studies of myenteric neurons in the LMMP preparation Ca2+ blockers of the dihydropyridine family
were used to prevent muscle contraction (see, e.g., Ref. 32).
Obviously, it was assumed that these drugs have no direct effects on
myenteric neurons, although the discussion above suggests that these
drugs may, at least partly, block L-type Ca2+ channels in neurons.
The observation of voltage-gated Ca2+ currents in virtually
all myenteric neurons is consistent with the findings of Hanani and
Lasser-Ross (20), who measured Ca2+ transients in single
myenteric neurons simultaneously with intracellular recordings. They
showed that both S- and AH-type myenteric neurons had voltage-activated
Ca2+ currents. Previous studies on myenteric neurons showed
that S-type neurons have pure Na+ spikes, whereas spikes in
AH neurons have a prominent Ca2+ component (25, 29, 34).
Our results do not contradict, but modify, these observations. We
showed that all myenteric neurons studied have voltage-gated
Na+ and Ca2+ channels. The
contributions of these channels to inward currents vary among cells.
These conclusions are supported by immunohistochemical staining for
Na+ channels, which showed that most neurons possess these
channels, but to varying degrees. Many calbindin-positive neurons,
which were shown to be mostly AH-type neurons (10), were immunopositive for Na+ channels. These conclusions are supported by
Ca2+-imaging studies showing that both neuron types have
voltage-activated Ca2+ channels (5, 20). Thus the
distinction between AH- and S-type neurons on the basis of the nature
of the action potentials is not as sharp as thought previously and is
more a matter of degree.
The fast inward currents were followed by currents that appeared to be
delayed-rectifying K+ currents, as found in cultured
myenteric neurons (38). We also obtained evidence for the presence of
Ca2+-induced K+ currents, as found in studies
using intracellular recordings (34). We expect that the ability to make
patch recordings will lead to studies in which the ionic and molecular
mechanisms of neurotransmitter and drug effects on myenteric neurons
will be investigated in detail.
The recordings from myenteric glia can shed new light on the role of
these cells in the enteric nervous system. Glial cells in the CNS have
been shown to possess voltage-sensitive ion channels and
neurotransmitter and hormone receptors, and they are able to synthesize
and release a variety of neuroactive compounds (26). It is now widely
accepted that glia have more than a mechanical role in the nervous
system and may have key physiological functions in normal and
pathological conditions. Our observations indicate that myenteric glia
share some physiological properties with central glia. We found that
these cells possess a number of voltage-gated ion
channels
inward-rectifier and delayed-rectifier K+
channels as well as voltage-activated Na+ channels. In view
of the complex pharmacology of myenteric neurons, it will be
interesting to investigate the effect of putative neurotransmitters on
myenteric glia. We expect that future studies using the isolated ganglia will reveal how myenteric neurons and glia interact to produce
the overall integrative ability of this system.
 |
ACKNOWLEDGEMENTS |
The authors thank A. Diener for technical assistance.
 |
FOOTNOTES |
This work was supported by the United States-Israel Binational Science
Foundation (95-00568 and 98-00185) and the Israel Science Foundation administered by the Israeli Academy of Sciences and Humanities (to M. Hanani) and by the Deutsche Forschungsgemeinschaft (to A. Reichenbach).
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: M. Hanani,
Laboratory of Experimental Surgery, Hadassah Univ. Hospital, Mount
Scopus, Jerusalem 91240, Israel (E-mail: hananim{at}cc.huji.ac.il).
Received 28 May 1999; accepted in final form 12 November 1999.
 |
REFERENCES |
1.
Baidan, LV,
Zholos AV,
Shuba MF,
and
Wood JD.
Patch-clamp recording in myenteric neurons of guinea pig small intestine.
Am J Physiol Gastrointest Liver Physiol
262:
G1074-G1078,
1992[Abstract/Free Full Text].
2.
Baidan, LV,
Zholos AV,
and
Wood JD.
Properties of calcium currents determined by patch clamp recording in myenteric neurones from adult guinea pig small intestine (Abstract).
Gastroenterology
102:
A420,
1992.
3.
Barajas-Lopez, C,
Peres AL,
and
Espinosa-Luna R.
Cellular mechanisms underlying adenosine actions on cholinergic transmission in enteric neurons.
Am J Physiol Cell Physiol
271:
C264-C275,
1996[Abstract/Free Full Text].
4.
Broussard, DL,
Bannerman PG,
Tang CM,
Hardy M,
and
Pleasure D.
Electrophysiologic and molecular properties of cultured enteric glia.
J Neurosci Res
34:
24-31,
1993[ISI][Medline].
5.
Christofi, FL,
Guan Z,
Lucas JH,
Rosenberg-Scahffer LJ,
and
Stokes BT.
Responsiveness to ATP with an increase in intracellular free Ca2+ is not a distinctive feature of calbindin-D28 immunoreactive neurons in myenteric ganglia.
Brain Res
725:
241-246,
1996[ISI][Medline].
6.
Christofi, FL,
Hanani M,
Maudlej N,
and
Wood JD.
Enteric glial cells are major contributors to formation of cyclic AMP in myenteric plexus cultures from adult guinea-pig small intestine.
Neurosci Lett
159:
107-110,
1993[ISI][Medline].
7.
Fiorica-Howells, E,
Wade PR,
and
Gershon MD.
Serotonin-induced increase in cAMP in ganglia isolated from the myenteric plexus of the guinea-pig small intestine: mediation by a novel 5-HT receptor.
Synapse
13:
333-349,
1993[ISI][Medline].
8.
Franklin, JL,
and
Willard AL.
Voltage-dependent sodium and calcium currents of rat myenteric neurons in cell culture.
J Neurophysiol
69:
1264-1275,
1992[Abstract/Free Full Text].
9.
Furness, B,
and
Costa J.
The Enteric Nervous System. Edinburgh, UK: Churchill Livingstone, 1987.
10.
Furness, JB,
Trussel DC,
Pompolo S,
Bornstein JC,
and
Smith TK.
Calbindin neurons of the guinea-pig small intestine: quantitative analysis of their numbers and projections.
Cell Tiss Res
260:
261-272,
1990[ISI][Medline].
11.
Gabella, G.
Ultrastructure of the nerve plexuses of the mammalian intestine: the enteric glial cells.
Neuroscience
9:
425-436,
1981.
12.
Gershon, MD,
Kirchgessner AL,
and
Wade PR.
Functional anatomy of the enteric nervous system.
In: Physiology of the Gastrointestinal Tract, edited by Johnson LR. New York: Raven, 1994, p. 381-422.
13.
Gershon, MD,
and
Rothman TP.
Enteric glia.
Glia
4:
195-204,
1991[ISI][Medline].
14.
Gola, M,
Niel JP,
Bessone R,
and
Fayolle R.
Single-channel and whole-cell recordings from non-dissociated sympathetic neurones in rabbit coeliac ganglia.
J Neurosci Methods
43:
13-22,
1992[ISI][Medline].
15.
Grafe, P,
Mayer CJ,
and
Wood JD.
Synaptic modulation and calcium-dependent potassium conductance in myenteric neurones in the guinea-pig.
J Physiol (Lond)
305:
235-248,
1980[Abstract].
16.
Grider, JR,
and
Bonilla OM.
Differential expression of substance P, somatostatin, and VIP in neurons from cultured myenteric ganglia.
Am J Physiol Gastrointest Liver Physiol
267:
G322-G327,
1994[Abstract/Free Full Text].
17.
Grider, JR,
and
Jin JG.
Vasoactive intestinal peptide release and L-citrulline production from isolated ganglia of the myenteric plexus: evidence for regulation of vasoactive intestinal peptide release by nitric oxide.
Neuroscience
54:
521-526,
1993[ISI][Medline].
18.
Hanani, M.
Neurons and glial cells of the enteric nervous system: studies in tissue culture.
J Basic Clin Physiol Pharmacol
4:
157-179,
1993[Medline].
19.
Hanani, M,
Francke M,
Katchalsky S,
Rahamimoff R,
and
Reichenbach A.
Patch recordings and confocal microscopic Ca2+ imaging in isolated myenteric ganglia.
Neurogastroenterol Motil
10:
74,
1998.
20.
Hanani, M,
and
Lasser-Ross N.
Activity-dependent changes in intracellular calcium in myenteric neurons.
Am J Physiol Gastrointest Liver Physiol
273:
G1359-G1363,
1997[Abstract/Free Full Text].
21.
Hanani, M,
and
Reichenbach A.
Morphology of HRP-injected glial cells in the myenteric plexus of the guinea-pig.
Cell Tissue Res
278:
153-160,
1994[ISI][Medline].
22.
Hanani, M,
Zamir O,
and
Baluk P.
Glial cells in the guinea-pig myenteric plexus are dye coupled.
Brain Res
497:
245-249,
1989[ISI][Medline].
23.
Hanani, M,
Xia Y,
and
Wood JD.
Myenteric ganglia from the adult guinea-pig small intestine in tissue culture.
Neurogastroenterol Motil
6:
103-118,
1994[ISI].
24.
Hirning, LD,
Fox AP,
and
Miller RJ.
Inhibition of calcium currents in cultured myenteric neurons by neuropeptide Y: evidence for direct receptor/channel.
Brain Res
532:
120-130,
1990[ISI][Medline].
25.
Hirst, GDS,
Johnson SM,
and
van Helden DF.
The calcium current in a myenteric neurone of the guinea-pig ileum.
J Physiol (Lond)
361:
297-314,
1985[Abstract].
26.
Kettenmann, H.,
and
Ransom BR
(Editors).
Neuroglia. New York: Oxford Univ. Press, 1995.
27.
Maudlej, N,
and
Hanani M.
Modulation of dye coupling among glial cells in the myenteric and submucosal plexuses of the guinea pig.
Brain Res
578:
94-98,
1992[ISI][Medline].
28.
Moneta, NA,
McDonald TJ,
and
Cook MA.
Endogenous adenosine inhibits evoked substance P release from perifused networks of myenteric ganglia.
Am J Physiol Gastrointest Liver Physiol
272:
G38-G45,
1997[Abstract/Free Full Text].
29.
Nishi, S,
and
North RA.
Intracellular recording from the myenteric plexus of the guinea-pig ileum.
J Physiol (Lond)
231:
471-491,
1973[ISI][Medline].
30.
North, RA.
Electrophysiology of the enteric nervous system.
Neuroscience
7:
315-325,
1982[ISI][Medline].
31.
Simeone, DM,
Kimball BC,
and
Mulholland MW.
Acetylcholine-induced calcium signaling associated with muscarinic receptor activation in cultured myenteric neurons.
J Am Coll Surg
182:
473-481,
1996[ISI][Medline].
32.
Smith, TK,
Furness JB,
Costa M,
and
Bornstein JC.
An electrophysiological study of the projections of motor neurones that mediate non-cholinergic excitation in the circular muscle of the guinea-pig small intestine.
J Auton Nerv Syst
22:
115-28,
1988[ISI][Medline].
33.
Willard, AL.
Substance P mediates synaptic transmission between rat myenteric neurones in cell culture.
J Physiol (Lond)
426:
453-71,
1990[Abstract].
34.
Wood, JD.
Physiology of the enteric nervous system.
In: Physiology of the Gastrointestinal Tract, edited by Johnson LR. New York: Raven, 1994, p. 423-482.
35.
Yau, WM,
Dorsett JA,
and
Parr EL.
Characterization of acetylcholine release from enzyme-dissociated myenteric ganglia.
Am J Physiol Gastrointest Liver Physiol
256:
G233-G239,
1989[Abstract/Free Full Text].
36.
Xia, Y,
Baidan LM,
Fertel RH,
and
Wood JD.
Determination of levels of cAMP in myenteric ganglia of guinea-pig small intestine.
Eur J Pharmacol Mol Pharmacol
225:
21-27,
1992[Medline].
37.
Zhang, W,
Sarosi J,
Barnhart D,
Yule DI,
and
Mulholland MW.
Endothelin-activated calcium signaling in enteric glia derived from neonatal guinea pig.
Am J Physiol Gastrointest Liver Physiol
272:
G1175-G1185,
1997[Abstract/Free Full Text].
38.
Zholos, AV,
Baidan LV,
Starodub AM,
and
Wood JD.
Potassium channels of myenteric neurons of guinea-pig small intestine.
Neuroscience
89:
603-618,
1999[ISI][Medline].
39.
Zhou, X,
and
Galligan JJ.
P2X purinoceptors in cultured myenteric neurons of guinea-pig small intestine.
J Physiol (Lond)
496:
719-729,
1996[Abstract].
Am J Physiol Gastrointest Liver Physiol 278(4):G644-G651
0193-1857/00 $5.00
Copyright © 2000 the American Physiological Society