Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada 89557
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ABSTRACT |
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Vogalis, Fivos,
Kirk Hillsley, and
Terence
K. Smith.
Diverse Ionic Currents and Electrical Activity of Cultured
Myenteric Neurons From the Guinea Pig Proximal Colon.
J. Neurophysiol. 83: 1253-1263, 2000.
The aim of this
study was to perform a patch-clamp analysis of myenteric neurons from
the guinea pig proximal colon. Neurons were enzymatically dispersed,
cultured for 2-7 days, and recorded from using whole cell patch clamp.
The majority of cells fired phasically, whereas about one-quarter of
the neurons fired in a tonic manner. Neurons were divided into three
types based on the currents activated. The majority of tonically firing
neurons lacked an A-type current, but generated a large fast transient outward current that was associated with the rapid repolarizing phase
of an action potential. The fast transient outward current was
dependent on calcium entry and was blocked by tetraethylammonium. Cells
that expressed both an A-type current and a fast transient outward
current were mostly phasic. Depolarization of these cells to
suprathreshold potentials from less than 60 mV failed to trigger action potentials, or action potentials were only triggered after a
delay of >50 ms. However, depolarizations from more positive potentials triggered action potentials with minimal latency. Neurons that expressed neither the A-type current or the fast transient outward
current were all phasic. Sixteen percent of neurons were similar to
AH/type II neurons in that they generated a prolonged afterhyperpolarization following an action potential. The current underlying the prolonged afterhyperpolarization showed weak inward rectification and had a reversal potential near the potassium equilibrium potential. Thus cultured isolated myenteric neurons of the
guinea pig proximal colon retain many of the diverse properties of
intact neurons. This preparation is suitable for further biophysical and molecular characterization of channels expressed in colonic myenteric neurons.
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INTRODUCTION |
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Myenteric neurons comprise a heterogeneous
population of cells, including sensory neurons, motor neurons, and
interneurons (Costa et al. 1996; Furness et al.
1990
), which coordinate the contractile activities of the
muscle. Intracellular microelectrode recordings have revealed two broad
electrophysiological classes of myenteric neuron in both the small and
large intestine, called S/Type 1 and AH/Type 2 neurons
(Bornstein et al. 1994
). Fast excitatory postsynaptic
potentials (fEPSPs) can be readily evoked in S neurons (S for
synaptic), whereas they are rarely evoked in AH neurons (Hirst
et al. 1974
; Nishi and North 1973
). AH neurons
are named for their characteristically slow onset and long-lasting
calcium-dependent afterhyperpolarization (3-20 s duration), which
follows action potential firing in these neurons. S neurons comprise
the interneurons and motor neurons within the reflex pathways
(Brookes et al. 1997
; Smith et al. 1992
),
whereas AH neurons include the primary afferent neurons (Kunze
et al. 1995
; Smith 1994
).
In both the small and large intestine, most S neurons (80-90%) are
phasic or rapidly adapting, with a minority of tonic or slowly adapting
cells (Kunze et al. 1997; Wade and Wood
1988
). Highly excitable tonic S neurons can be more readily
impaled in the corners of ganglia near the junction with internodal
strands (Smith et al. 1999
). Differences in the
electrical activities and connections between myenteric neurons may
well account for the different neurally mediated motility patterns that
are generated in different regions of the gastrointestinal tract
(Spencer et al. 1999
; Stevens et al.
1999
).
At the cellular level, the phasic firing pattern of AH neurons has been
attributed to the expression of a Ca2+-activated
K+ conductance
(GK-Ca) (Hirst et al.
1985; Morita and North 1985
). The slow
afterhyperpolarization in AH neurons is abolished by blocking
Ca2+ entry (Hirst et al. 1974
,
1985
; Nishi and North 1973
) and is generated by an as yet to be identified tetraethylammonium
(TEA)-resistant (Hirst et al. 1985
) and partially
charybdotoxin-sensitive K+ channel (Kunze
et al. 1994
). Neurons in which this conductance is inhibited,
by stimulation of muscarinic (North and Tokimasa 1983
)
or tachykinin receptors (Morita and North 1985
), can
fire in a tonic manner. Although the expression of
GK-Ca may account for the phasic
firing pattern in AH neurons, in S neurons a phasic firing
pattern probably depends on the differential expression of conductances
other than GK-Ca, because a large proportion of S neurons also fires phasically (Bornstein et al. 1994
;
Kunze et al. 1997
) but lack
GK-Ca, and a subpopulation of S neurons that do
express a GK-Ca (Sk-type) are
tonically active (Shuttleworth and Smith 1999
;
Smith et al. 1999
).
In the rat and guinea pig sympathetic ganglia, the phasic firing
pattern of neurons has been attributed to the higher expression of a
large M-type K+ channel current (Cassell et al.
1986; Wang and McKinnon 1995
). Such M-type
currents, however, have not been reported in myenteric neurons
(Galligan et al. 1989
; Morita and North
1985
). In addition to the absence of an M-type current,
repetitive firing pattern may be favored by the expression of an
anomalously rectifying inward (Ih) current,
as in A-type cells in the rat nodose ganglion (Doan and Kunze
1999
).
In the present study the relationship between firing activity and the
expression of ionic currents, principally K+-channel
currents, in cultured myenteric neurons from the guinea pig proximal
colon was investigated. Intact myenteric neurons from the proximal
colon exhibit a diversity of firing patterns (Messenger et al.
1994). The aim of this study was to investigate whether
cultured neurons from this tissue retain their heterogeneity in
culture, and whether the different firing patterns can be attributed to
the expression of particular ionic conductances.
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METHODS |
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Dissociation of myenteric plexus
Male guinea pigs (Simonsen Laboratories, Gilroy, CA; 200-300 g) were killed by asphyxiation with CO2 in a specially constructed chamber and then exanguinated, in compliance with the requirements of the Animal Ethics Committee at the University of Nevada. A 3-cm length of proximal colon was removed and cleaned of its contents by flushing physiological saline through the lumen. The tissue was then sectioned longitudinally and pinned out flat in a dish containing sterile Ca2+-free Hanks solution. The mucosa and most of the circular muscle layer were dissected away using fine forceps and scissors under a binocular microscope. The myenteric plexus-longitudinal muscle preparation was cut into small pieces and transferred to a test tube containing 0.2% collagenase (Worthington) dissolved in Ca2+-free Hanks solution. The tissues were incubated in this solution for 12 min at 37°C and then washed four times with enzyme-free Ca2+-free Hanks solution and gently triturated through a fire-polished glass Pasteur pipette for 10-15 min. The suspension was then centrifuged at 800 rpm for 10 min after which the supernatant was discarded and the pellet resuspended in 2 ml of Ca2+-free Hanks solution. Aliquots of this solution were added to 35-mm plastic dishes fitted with glass coverslip bottoms that had been previously coated with polyornithine and laminin. The dishes contained 2.5 ml each of cell culture medium consisting of Dulbecco's modified Eagle's medium (DMEM no. 11885; GIBCO, Gaithersburg, MD) plus 10% fetal bovine serum. The medium was supplemented with 1% L-glutamine, 0.075% fluorodeoxyuridine, 0.175% uridine, and 2% antibiotics/antimycotics [penicillin (10,000 units/ml), streptomycin (10 mg/ml), and amphoterecin B (0.5 mg/ml)] to suppress infection. In addition 50 ng mouse nerve growth factor (Alomone Labs, Jerusalem, Israel) was added to each dish. The dishes were maintained in a humidified incubator with 5% CO2 at 37°C for 2-7 days before use. The culture medium in the dishes was changed every 2 days.
Appearance of cultured myenteric neurons and patch clamping
Processes began to appear on neurons usually after 24-48
h in culture and extensive networks of neurons were apparent after several days, with many neurons being organized into clumps (see Fig.
1). Conventional patch-clamp techniques
were used to record ionic currents and membrane potential changes from
these neurons. The cells were continuously bathed with a solution
containing (in mM) 160 NaCl, 2 KCl, 1 MgCl2, 5 CaCl2, 10 HEPES, and 11 glucose, pH adjusted to
7.4 with NaOH. The bathing solution in the dish was maintained at
32-34°C. The majority of neurons that were patched lay
isolated away from the clumps of cells, and the tip of the patch
electrode was lowered onto the center of the cell body (Fig. 1).
Membrane currents and membrane potentials were recorded using an EPC-9
patch-clamp amplifier (Heka Instruments, Lambrecht, Germany) and Pulse software running under Windows 95. Currents were usually filtered on-line at 2-5 kHz and digitized at 10 kHz. Current-clamp recordings were digitized at various frequencies (0.5-20 kHz). Patch
pipettes were drawn from thin-walled fiber-filled capillary glass
(Clark, GF-115011) to have resistances of 2-5 M when filled with
the following standard internal solution: 160 KCl, 1 MgCl2, 2 Na2ATP, 0.5 NaGTP, 10 HEPES, and 0.1 EGTA. The pH of this
solution was adjusted to 7.2 with KOH. Where indicated, recordings were also obtained with pipettes containing high-EGTA (10 mM) intracellular solution and 1 mM CaCl2. Series resistance compensation was
usually employed (60-70%), but, as illustrated in Fig. 2,
this was insufficient to faithfully space clamp the cell and resulted
in unclamped action potentials being triggered. Capacitance transients
were cancelled using a P/4 procedure. In the majority of recordings,
the current-voltage (I-V) relationship was linear at
potentials between
80 and
110 mV. In some cells, however, the
"leak" current rectified inwardly (especially when
[K+]o was raised to 5 mM) and the P/4
procedure was not employed, but the linear leak-current component was
subtracted off-line.
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All drugs used were bought from Sigma (St. Louis, MO). Stock solutions
of the following drugs were made up: tetraethylammonium-Cl (TEA; 1 M), 4-aminopyridine (4-AP; 0.5 M, pH 7), and tetrodotoxin (TTX; 1 mM).
The latter was stored at 20°C, whereas TEA and 4-AP were stored at
2-8°C.
Analysis of data
I-V plots were constructed for the current traces by
averaging the current over the last 25 ms of 100- to 200-ms test pulses (IOUT-SUST) and in the case of rapidly
inactivating currents, measuring the maximum outward current generated
within the initial 25 ms of the test pulse. Where illustrated,
conductance-voltage (G-V) curves were constructed
by dividing the current by driving force assuming a reversal potential
close to 106 mV for our transmembrane [K+]
gradient. For statistical comparison and averaging, currents were
normalized to the peak current recorded at 0 mV.
Passive membrane resistance was determined from the slope of the
I-V relationship of the nonleak subtracted currents in the region of 60 to
80 mV by fitting linear regression lines through the data points representing
IOUT-SUST. Usually this value closely matched the input resistance determined automatically by the amplifier in the whole cell mode. The resting membrane potential (RMP) was measured as the interpolated "zero current potential" at which the
I-V of the end-of-pulse current crossed the voltage axis. The capacitance of the soma was usually read off the capacitance cancellation circuitry on the amplifier in the whole cell mode, or it
was estimated by measuring the area under the capacitance current
evoked by a 20-mV hyperpolarizing step and dividing by this value by 20 mV (Armstrong and Gilly 1992
). Student's
t-test was used to test for statistical significance between means.
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RESULTS |
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Electrical characteristics of cultured myenteric neurons
Whole cell current recordings were obtained from a total of
132 cells that had been kept in culture for 2-7 days. Cells that were
patch clamped had smooth agranular cell bodies with one or more axonal
processes (Fig. 1). Neuronal cells were identified as those generating
fast inward currents (IIN-FAST) at the
onset of a depolarizing voltage step. Based largely on the
configuration of the outward currents that followed
IIN-FAST, neuronal cells were divided
into three groups. In group I, cells generated a fast transient outward
current
(IOUT-FAST), but
they also expressed a subthreshold A-type transient outward current
(IA) at depolarizations positive of
40 mV. In group II, cells lacked a subthreshold
IA but generated the
IIN-FAST/IOUT-FAST
complex and an outward current that was sustained for the duration of
test pulses (IOUT-SUST), which was
present in all cells. Neuronal cells that generated neither
IOUT-FAST nor
IA, but only expressed
IOUT-SLOW made up group III. Eight
cells were inexcitable and lacked
IIN-FAST and were assumed not to be
neurons. Typical currents recorded from representative cells from each
group are shown in Fig. 2. Also shown are
the responses of these cells to depolarizing current pulses, recorded
under current clamp.
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Although these groups did not clearly distinguish between cells with different firing properties, a larger proportion of tonically firing cells were classified in group II. Some basic properties of cells in each group are given in Table 1. Notable differences between cell groups included the more negative "resting" potentials (or zero current voltages, VI=0), the larger cell capacitance of group II cells, and the longer duration action potentials of cells in group III. Overall, the majority of neurons fired in a phasic manner (64%), but tonically firing neurons (20%) and AH neurons (16%) were also recorded. The outward currents generated by these cells were then analyzed in more detail.
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Properties of IOUT-FAST and role in the action potential
The fact that IIN-FAST was
activated over a narrow voltage range (30 to
20 mV), in an
all-or-none manner, suggested strongly that it was generated by action
potentials that were triggered in a region of the cell that had escaped
voltage control. Similarly, the close temporal relationship between
IOUT-FAST and
IIN-FAST suggested that
IOUT-FAST was action potential
dependent and that it represented current flow during the
repolarization/fast afterhyperpolarization. This conclusion is
supported by the observation that
IOUT-FAST was absent in cells treated
with TTX (1-3 µM, n = 3), which blocked IIN-FAST.
The identity of the conductance that was responsible for IOUT-FAST was investigated using known K+-channel blockers. External TEA ions (5 mM, n = 5) abolished IOUT-FAST (Fig. 3A1) and increased the duration of the action potential (Fig. 3A2). In two cells tested, addition of Cd2+ (0.1 mM) to the bathing solution also abolished IOUT-FAST without affecting the IIN-FAST (Fig. 3B1), and also broadened the action potential. These data indicate that IOUT-FAST was dependent on Ca2+ influx through voltage-gated Ca2+ channels and that it is generated by the opening of TEA-sensitive Ca2+-dependent BK-type K+ channels that aid in action potential repolarization. This is further supported by the fact that the addition of 4-AP (1-4 mM, n = 4) to the bathing solution had little effect on IOUT-FAST (Fig. 3C1) or on action potential firing (Fig. 3C2). This suggests that IOUT-FAST was not mediated by the opening of A-type or delayed-rectifier K+ channels.
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Properties of A-type outward current and its role in AP firing
The voltage dependence of the subthreshold
IA was determined in five cells that
had been treated with TTX to remove contamination by
IIN-FAST and the associated
IOUT-FAST. As shown for one of these
cells in Fig. 4A,
IA was typically activated by step
depolarizations positive to 40 mV and was inactivated almost to
completion at holding potentials positive of
40 mV (Fig.
4A2). The corresponding conductance-voltage (G-V)
relationships of IA and of the
noninactivating IOUT-SUST recorded
from this cell are plotted in Fig. 3B to illustrate that the
conductance underlying IA activated
~20 mV more negative than that which generates
IOUT-SUST. This suggests that these two currents are generated by different voltage-gated
K+ channels.
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The availability of IA as a function
of voltage suggests that ~20% of the current in the cell depicted in
Fig. 4C is available for activation at 60 mV. In six cells
analyzed by fitting a single Boltzmann function to plots of the
available current versus conditioning (holding) potential (as in Fig.
4C), the mean voltage at which IA was half-maximally inactivated was
72.5 ± 4.8 mV, with an average slope factor of
7.3 ± 0.9 mV. The inactivating portion of the current, obtained by digital
subtraction of the currents elicited from holding potentials of
65
and
40 mV (see Fig. 4A3), revealed that
IA peaked within 5 ms at potentials
positive of 0 mV. IA inactivated
almost to completion, with a mean time constant of 24.3 ± 2.6 ms
at 0 mV (n = 5).
Classic A-type K+ channel currents are blocked by
millimolar 4-AP. We therefore tested the effect of 4-AP (1-4 mM) on
cells expressing IA. Figure
5 shows that in a phasically firing cell, 4-AP (1 mM) had little effect either on the resting potential or on the
action potential configuration triggered by a depolarizing current step
(Fig. 5A1), although the afterhyperpolarization appeared to
be blunted. The facilitation of action potential firing by 4-AP was
only apparent when the membrane potential was held at potentials
negative of about 60 mV, where a significant fraction of
IA becomes available (see Fig.
4C). At these holding potentials, depolarizing current steps
in the absence of 4-AP failed to trigger action potentials in the soma,
and the resultant electrotonic potential displayed a characteristic
creep. In the presence of 4-AP (1 mM), however, the same depolarizing
current step immediately triggered an action potential, and the
"creep" was absent.
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Under voltage clamp, it was revealed that 4-AP (1 mM) blocked a large fraction of the subthreshold IA (Fig. 5, B1 and B2). This concentration of 4-AP was sufficient to block IA by ~50% (Fig. 5A2). A similar reduction in the magnitude of IA by 4-AP (1-4 mM) was seen in five other cells.
Properties of IOUT-SUST and its role in electrical excitability
The voltage dependence of the sustained outward current
(IOUT-SUST) recorded from cells
lacking a net subthreshold IA (groups II and III) was determined by plotting the mean end-of-pulse currents as a function of test potential (pulse duration of 100-200 ms). The
I-V relationship constructed from averaged data from 29 cells is plotted in Fig. 6B
() and shows that IOUT-SUST under
net-current conditions activated at potentials positive to
30 mV. The
I-V relationship of
IOUT-SUST was also determined in eight
cells treated with TTX (1-3 µM) to eliminate contamination by the
IIN-FAST/IOUT-FAST complex. Typical outward currents recorded under these conditions from
one such cell are shown in Fig. 6A, whereas the averaged data are plotted in Fig. 6B (
). The I-V
relationship of IOUT-SUST in TTX
indicates that this current was generally larger and activated at more
negative potentials (approximately
50 mV) in the presence of TTX. The
corresponding G-V relationship constructed from these data
means was well described by a single Boltzmann function with a voltage
of half-maximal activation of
10.3 mV and a voltage dependence of +15
mV (Fig. 6C). These values predict that ~5% of the
conductance generating ISUST is
activated at
60 mV.
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On average, externally applied TEA (5 mM) inhibited
IOUT-SUST by 66 ± 8%
(mean ± SE, n = 8 cells from groups I
and II; Fig. 7A). In a
tonically firing cell with a subthreshold
IA, TEA (5 mM) reduced the firing frequency
from 39 to 33 Hz and also decreased the peak afterhyperpolarization
from 44 to
34 mV (Fig. 7B). In addition, TEA
increased the peak of the action potential and almost doubled its
duration (Fig. 7C). In two other cells, however, both
lacking IA, TEA (5 mM) had no effect on
IOUT-SUST (see Fig. 3A),
suggesting that this current may be generated by a heterogeneous population of delayed rectifier-type K+ channels.
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Neurons with a slow afterhyperpolarization
A characteristic feature of AH/type 2 myenteric neurons is the
development of a slow afterhyperpolarization (AH) lasting several seconds following one or more action potentials (Hirst et al. 1974; Nishi and North 1973
). In the present
study we found nine neurons that generated slow afterhyperpolarizations
after firing one or several action potentials in response to short (50 ms) depolarizing current steps. Recordings from one such cell are shown
in Fig. 8A and demonstrate
that the excitability of the cell is decreased during the AH; injection
of the suprathreshold current pulse during the recovery phase of
the AH failed to trigger another action potential (pulse b
in Fig. 8A). The duration of action potentials in these
cells averaged 3.2 ± 0.4 ms (n = 9), and in six
cells a characteristic shoulder on the repolarization phase was
evident.
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Under whole cell voltage-clamp conditions, step depolarizations
in AH neurons revealed the presence of either
IOUT-FAST (7/9 cells) or
IA (2/9 cells), and so the majority of
AH neurons were classified into group II because they lacked an
apparent subthreshold IA. To determine
the voltage dependence of the current underlying the slow AH, the
current that was generated after a 50-ms stimulus step to +40 mV was
recorded at several potentials (Fig. 8B). This current
peaked within 1-2 s after the stimulus and was outward at potentials
positive to 80 mV. To isolate the current underlying the AH
(IAH), the background current recorded
at the same potentials was subtracted from the slowly developing
poststimulus current (averaged over 1 s at its peak). All three
currents, including IAH are plotted in
Fig. 8C, which shows that the I-V relationship of
IAH is linear between
30 and
110
mV and appears to weakly rectify inwardly. The extrapolated reversal
potential (Erev) of IAH lies negative of
110 mV and
close to
120 mV. A similarly negative extrapolated
Erev for
IAH was calculated for the other two cells.
Inward rectification in colonic myenteric neurons
The absence of any appreciable inwardly rectifying
currents in the majority of neurons described in the preceding sections may have been due to the use of low (2 mM)
[K+]o in the bathing
solution. In a series of experiments in which [K+]o was raised to 5.4 mM, we found that
inwardly rectifying current was present in 20 of 32 cells, at
potentials negative of 60 mV (Fig. 9).
In all but three of these cells, the inwardly rectifying current
(IIR) lacked time dependence (Fig.
9A1), whereas in 14 of 15 cells that expressed
IIR also expressed
IA (Fig. 9A1). The nature of
IIR was not investigated further in the
present study. However, we noted that cells expressing
IIR fired phasically when depolarized (Fig.
9A2), and their resting potentials averaged
41 ± 4 mV compared with
49 ± 4 mV (n = 14) for
cells lacking this current. A slow AH was not observed in any cell
exhibiting IIR. In eight cells that fired
tonically or showed slow adaptation over 500-ms depolarizations, a
time-dependent inwardly rectifying current was evident at potentials
negative of
60 mV (Fig. 9, B1 and B2).
In one cell tested, this current was inhibited by Cs+ (1 mM) added to the bathing solution, suggesting that it was an
Ih-like current.
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DISCUSSION |
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Retention of diverse firing properties of cultured myenteric neurons
In the present study we have shown that myenteric neurons from the
proximal colon of the guinea pig retain the same electrophysiological behaviors in culture that have previously been demonstrated in intact
neurons from the same tissue (Messenger et al. 1994).
Our findings contrast with those in cultured myenteric neurons from the
rat small intestine (Nishi and Willard 1985
), over 95%
of which fired phasically, and none of which exhibited prolonged afterhyperpolarizations. At present, it is unclear whether any of these
differences are due to varying culture conditions, or in the case of AH
cells, the length of time in culture (Nishi and Willard
1985
). However, differences in the proportion of neuronal phenotypes probably also reflects regional differences in neuronal excitability, because microelectrode studies have shown that the ratio
of phasic to tonic S cells varies from 9:1 in the guinea pig ileum
(Bornstein et al. 1994
; Kunze et al.
1997
) to 4:1 in the distal colon (Wade and Wood
1988
).
Therefore the most meaningful comparison for the neurons in this study
is to neurons from intact preparations of the guinea pig proximal colon
(Messenger et al. 1994). The proportions of tonic and
phasic S neurons and AH neurons in the present study were very similar
to that found in intact preparations. Cultured neurons were comprised
of 16% AH neurons, and of the remaining S neurons, 80% were phasic
and 20% tonic. Fresh intact neurons were similarly comprised of 18%
AH neurons, and of the remaining S neurons, 79% were phasic and 21%
tonic (Messenger et al. 1994
). Thus, although concerns
about changes in phenotypic expression of cultured neurons cannot be
completely allayed, in this preparation the proportion of different
neurons demonstrates a high degree of correlation with intact neurons.
However, some minor differences were observed between cultured and intact myenteric colonic neurons, primarily, the RMP of cultured neurons was more positive than intact neurons. Because whole cell patch clamping causes less of a nonspecific leak current than using sharp intracellular electrodes, this may be reflected in the different RMPs. The differences in the electrode and/or extracellular solutions are also likely to reflect differences in the RMP. For example, in the present study, EGTA was present in varying concentrations in the electrode solution, thus altering the levels of free intracellular calcium and modulating calcium-activated conductances. The RMP was consistently more positive if higher concentrations of EGTA were present (see Table 1); therefore if no EGTA was present, then the RMP would probably be similar to the levels seen in intact neurons. In addition, the extracellular HEPES solution contained lower potassium and higher calcium concentrations than the Kreb's solution used in intact preparations. This solution was used to maximize the driving force for the expression of IAHP, to ensure that no AH neurons were missed.
Neuronal classification
The classification of neurons employed in the present study was based on the features of the outward current generated in cultured neurons to classify cells into three groups: 1) those with a subthreshold IA-like current, 2) those with an action potential-associated fast transient outward current (IOUT-FAST), and 3) a slowly activating outward current. This was not a complete characterization of channels expressed in these neurons, and it is likely that the firing properties of neurons are a reflection of a variety of channels active at different potentials. In light of this, it is perhaps not surprising that the firing properties of the cultured myenteric neurons in this study do not absolutely correlate with the classification scheme employed. However, certain trends were observed between the firing properties and channels expressed. Tonic S neurons were predominantly (73%) those cells that expressed IOUT-FAST but lacked IA. Phasic neurons were found in all three groups, but all neurons that lacked both IOUT-FAST and IA were phasic. Interestingly, although all AH neurons expressed IAH, the majority of AH neurons (78%) also expressed IOUT-FAST but lacked IA.
Role of IOUT-FAST currents
The inhibition of IOUT-FAST and
the consequent broadening of the action potential by external TEA and
by Cd2+ suggests that it was mediated by the
opening Ca2+-dependent K+
channels of the BK-type that are involved in action potential repolarization (Davies et al. 1996). The fact that
IOUT-FAST was also TTX sensitive
suggests that the rapid upstroke of the
Na+-dependent action potential opens N-type
voltage-gated Ca2+ channels, which in turn leads
to Ca2+ entry and the opening of BK-type channels
to aid in the termination of the action potential. The transient nature
of this current is consistent with the
Ca2+-dependent K+ channels
being activated directly by Ca2+ entering through
co-localized voltage-gated Ca2+ channels, as
described in rat hippocampal neurons for N-type Ca2+ channels and BK channels (Marrion and
Tavalin 1998
). Because this outward current was only seen when
an unclamped action potential was elicited suggests that the
Ca2+-dependent K+ channels
are situated in a region of the cell where action potentials are
initiated and that cannot be properly voltage clamped.
Role of IA outward currents
The presence of IOUT-FAST in many
myenteric neurons may have obscured an underlying
IA suggesting that we may have
underestimated the proportion of cells expressing
IA. A-type currents have been reported
previously in intact myenteric neurons, both in AH type neurons
(Hirst et al. 1985; Hoffman et al. 1997
)
and also in tonically firing S-type neurons (Brookes et al.
1997
; Smith et al. 1999
) in the guinea pig
ileum. Therefore the expression of IA
does not seem to determine the phasic/tonic firing pattern of myenteric neurons. The relatively negative activation range of
IA suggests that
IA may be important in inhibiting or
delaying action potential firing at potentials slightly negative of the
resting level and may influence the ability of these cells to integrate
incoming signals.
Difference and roles of delayed rectifier-type outward currents
The slowly inactivating or sustained outward current
(IOUT-SUST) recorded in myenteric
neurons is probably generated by a mixture of different delayed
rectifier-type K+ channels. For example,
IOUT-SUST in cells expressing
IA activated at more positive
potentials (approximately 20 mV) than
IOUT-SUST in cells lacking
IA. Thus cells may express different
levels of at least two different delayed-rectifier type channels, and
the pharmacological sensitivity and voltage dependence of
IOUT-SUST may depend on the
relative contributions of the whole cell current.
Functionally, the negatively activating
IOUT-SUST in cells lacking
IA suggests that this current may
contribute to the resting potential in these cells and in generating
the late repolarization phase of the action potential. The low-voltage
threshold of IOUT-SUST is similar to
the M-type current in phasic neurons in the superior mesenteric
ganglion (Wade and Wood 1988), suggesting that
IOUT-SUST may contribute to the phasic
firing pattern of myenteric neurons. The role of the more positively
activating IOUT-SUST in
IA-expressing cells may be to regulate
excitability at more positive potentials and prevent excessive depolarization.
Myenteric AH neurons
Unlike Nishi and Willard (1985), we found that 16%
of neurons were able to generate a slow AH after a single or a few
action potentials. This slow AH had many properties in common with the slow AH in the intact myenteric neurons (Hirst et al.
1985
). Examination of the underlying current revealed that it
had a similar time course to the AH and showed weak inward
rectification characteristic of the corresponding current in intact AH
neurons (North and Tokimasa 1983
). The current decreased
in amplitude as membrane potential approached
EK, suggesting that it was carried by
K+ (Hirst et al. 1985
;
North and Tokimasa 1983
).
More recently, Zholos et al. (1999) have suggested that
the slow AH in guinea pig myenteric neurons may be generated in part by
an inwardly rectifying K+ conductance. Such
currents have been reported previously in intact AH neurons
(Galligan et al. 1989
; Hirst et al.
1985
). In the present study, however, in cells that generated a
slow AH, there was little evidence of inward rectification at
potentials negative of
80 mV. Inwardly rectifying currents were
evident, however, when external K+ was raised to
5.4 mM. But in the majority of cells, this inwardly rectifying current
showed no obvious time dependence and was inward at potentials negative
of
60 mV.
In summary, this study has shown that cultured isolated myenteric neurons of the guinea pig proximal colon retain many of the electrophysiological properties, including diverse firing patterns, of intact neurons. Therefore this preparation is suitable for further characterization of the ion channels expressed in different functional classes of myenteric neurons, which together with single-cell polymerase chain reaction, will allow us to assign molecular entities to the ionic events underlying these different firing patterns.
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ACKNOWLEDGMENTS |
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We thank I. Janiak for neuronal cell cultures.
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants PO1 DK-41315 to T. K. Smith and K. Hillsley and DK-50137 to F. Vogalis.
Present address of F. Vogalis: Dept. of Anatomy and Cell Biology, University of Melbourne, Parkville, VIC 3042, Australia.
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FOOTNOTES |
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Address for reprint requests: T. K. Smith, Dept. of Physiology and Cell Biology, Anderson Medical Sciences Building/352, University of Nevada School of Medicine, Reno, NV 89503.
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.
Received 18 June 1999; accepted in final form 5 November 1999.
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REFERENCES |
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