Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada 89557-0046
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ABSTRACT |
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Hillsley, K., J. L. Kenyon, and T. K. Smith. Ryanodine-Sensitive Stores Regulate the Excitability of AH Neurons in the Myenteric Plexus of Guinea-Pig Ileum. J. Neurophysiol. 84: 2777-2785, 2000. Myenteric afterhyperpolarizing (AH) neurons are primary afferent neurons within the gastrointestinal tract. Stimulation of the intestinal mucosa evokes action potentials (AP) that are followed by a slow afterhyperpolarization (AHPslow) in the soma. The role of intracellular Ca2+ ([Ca2+]i) and ryanodine-sensitive Ca2+ stores in modulating the electrical activity of myenteric AH neurons was investigated by recording membrane potential and bis-fura-2 fluorescence from 34 AH neurons. Mean resting [Ca2+]i was ~200 nM. Depolarizing current pulses that elicited APs evoked AHPslow and an increase in [Ca2+]i, with similar time courses. The amplitudes and durations of AHPslow and the Ca2+ transient were proportional to the number of evoked APs, with each AP increasing [Ca2+]i by ~50 nM. Ryanodine (10 µM) significantly reduced both the amplitude and duration (by 60%) of the evoked Ca2+ transient and AHPslow over the range of APs tested (1-15). Calcium-induced calcium release (CICR) was graded and proportional to the number of APs, with each AP triggering a rise in [Ca2+]i of ~30 nM Ca2+ via CICR. This indicates that CICR amplifies Ca2+ influx. Similar changes in [Ca2+]i and AHPslow were evoked by two APs in control and six APs in ryanodine. Thus, the magnitude of the change in bulk [Ca2+]i and not the source of the Ca2+ is the determinant of the magnitude of AHPslow. Furthermore, lowering of free [Ca2+]i, either by reducing extracellular Ca2+ or injecting high concentrations of Ca2+ buffer, induced depolarization, increased excitability, and abolition of AHPslow. In addition, activation of synaptic input to AH neurons elicited a slow excitatory postsynaptic potential (sEPSP) that was completely blocked in ryanodine. These results demonstrate the importance of [Ca2+]i and CICR in sensory processing in AH neurons. Activity-dependent CICR may be a mechanism to grade the output of AH neurons according to the intensity of sensory input.
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INTRODUCTION |
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The enteric nervous system is comprised of the
myenteric and submucosal plexi, which are interconnected and control
complex reflexes within the gastrointestinal tract. Intracellular
microelectrode recordings have revealed two broad electrophysiological
classes of myenteric neurons, S/Type I and AH/Type II neurons
(Bornstein et al. 1994; Hirst et al.
1974
; North 1973
; Wood 1994
). S
neurons lack a slow afterhyperpolarization
(AHPslow), have a prominent fast synaptic input
(Furukawa et al. 1986
; Hirst and McKirdy
1974
), and comprise both interneurons and motorneurons
(Bornstein et al. 1991
; Smith et al.
1992
). Afterhyperpolarizing (AH) neurons are named for the
characteristic AHPslow (4-20 s), which follows action potential (AP) firing in these neurons (Hirst et al.
1974
; Nishi and North 1973
). AH neurons
are primary afferent neurons in the guinea-pig
intestine (Furness et al. 1990
; Kunze et al. 1995
; Regina et al. 1993
; Song et al.
1994
) and are found in both the myenteric and submucosal plexi.
They are multipolar and have projections to the mucosa. Stimulation of
the mucosal villi results in the activation of AP firing in these
mucosal processes, which results in an AP that can be detected in the
cell soma, that is followed by a prominent
AHPslow in the soma (Kunze et al.
1995
; Smith 1994
). This gating of the soma by
the AHPslow thereby reduces neurotransmission
from these neurons to second-order neurons in the reflex pathways that
regulate motility.
In AH neurons, addition of tetrodotoxin (TTX) only partially ablates
APs, and residual events are blocked by the removal of extracellular
Ca2+ ions (Hirst et al. 1985;
North 1973
). The Ca2+ current
contributing to AH neuron APs has been characterized (Hirst et
al. 1985a
), but the identity of the channel is unclear (Vogalis et al. 2000b
). Ca2+
influx that occurs during AP firing ultimately leads to activation of a
Ca2+-activated K+
conductance, resulting in the AHPslow
(Hirst et al. 1985
). Preliminary studies using fura-2 to
image Ca2+ transients in AH neurons have
demonstrated that AP firing transiently increases intracellular free
calcium ([Ca2+]i), with a
time course that closely matches the time course of AHPslow (Tatsumi et al. 1988
;
Vogalis et al. 2000a
). This suggests that intracellular
Ca2+ is an important mediator of
AHPslow.
However, the mechanism by which Ca2+ influx leads
to AHPslow is not understood in AH neurons.
Prolonged Ca2+ influx across the neuronal
membrane may contribute to AHPslow (Hirst
et al. 1985a), although activation of internal stores has also
been proposed (North and Tokimasa 1987
). In several
other types of neurons, an analogous AHPslow is
also observed. Activation of the AHPslow is at
least partially dependent on intracellular Ca2+
stores in vagal afferent (Moore et al. 1998
), vagal
dorsal motor (Sah and McLachlan 1991
), sympathetic
(Kawai and Watanabe 1989
), and parasympathetic neurons
(Yoshizaki et al. 1995
). In particular, Ca2+ influx associated with APs triggers
Ca2+ release from ryanodine-sensitive stores, and
it is this calcium-induced calcium release (CICR) that underlies
AHPslow.
The aim of this study was to perform an analysis of changes in [Ca2+]i with changes in electrical activity in AH neurons, and in particular, to investigate the role of ryanodine-sensitive intracellular Ca2+ stores in the generation of AHPslow in myenteric AH neurons.
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METHODS |
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General
Guinea pigs of either sex (250-350 g) were killed by CO2 asphyxiation followed by severing of the carotid arteries and exsanguination. This procedure was authorized by the Institutional Animal Care and Use Committee at the University of Nevada, Reno. A 15-cm length of ileum was taken 10-12 cm above the caecum and the luminal contents were flushed away with modified Krebs-Ringer Buffer (KRB) injected into the lumen with a syringe inserted into the oral end of the segment. A segment of ileum (~40-mm-long) was then opened along its mesenteric border and pinned mucosa uppermost to a Sylgard® dish and the mucosa, submucosa, and circular muscle layer were removed to expose the ganglia of the myenteric plexus. After dissection, the preparation was transferred to the Sylgard floor of an electrophysiological chamber (diameter 3 cm). In the central region of the chamber, the sylgard was removed and the preparation stretched over a thin glass coverslip window and pinned at either end and held firm and flat against the glass coverslip by drawing it under two elastic bands. The recording chamber was then mounted on the stage of an inverted microscope (Nikon Diaphot) and perfused with warmed KRB (~35°C) at a rate of approximately 5 ml/min. The KRB contained (mM) 120.35 NaCl, 5.9 KCl, 2.5 CaCl2, 1.2 MgCl2, 15.5 NaHCO3, 1.2 NaH2PO4, and 11.5 dextrose. This solution had a pH of 7.4 when bubbled to equilibrium with 97% O2-3% CO2. Nicardipine and atropine (both 2 µM) were present throughout all experiments to minimize muscle movements that would dislodge recording microelectrodes.
Recording methods
Standard microelectrode techniques (Shuttleworth and
Smith 1999) were used to impale neurons within the myenteric
plexus. Microelectrodes were fabricated from borosilicate glass
capillary (1.0 mm od, 0.7 mm id 1B100F-4; WPI Corp., Sarasota, FL) on a Brown and Flaming Microelectrode Puller (Sutter Instruments, San Francisco, CA) and filled with a solution consisting of bis-fura-2 (1-5 mM) in 1 M KCl. These electrodes had resistances ranging from 150 to 240 M
. Electrical activity was sampled using an Axoprobe 2B
dual-channel high-impedance preamplifier (Axon Instruments, Foster
City, CA). Depolarizing or hyperpolarizing current pulses were injected
via a bridge circuit, and the pulse duration and frequency regulated
using an output from a programmable eight-channel pulse generator
(Master-8, AMPI, Jerusalem, Israel). Current pulses varied from 1-500
ms in duration and 0.05-0.95 nA in intensity. The intracellular
voltage and current were displayed on a two-channel digital storage
oscilloscope (Tektronix 468R, Beaverton, OR). Data were digitized and
analyzed using Ionoptix software (Milton, MA). Synaptic inputs were
stimulated using a bipolar tungsten stimulating electrode (diameter 100 µm) applied to internodal strands of the myenteric plexus. The
stimulating electrode was controlled using an Isoflex (AMPI) controller
connected to a 90V DC battery, and duration and frequency were timed
with the Master 8.
When a stable recording was obtained, bis-fura-2 was injected into the cell using hyperpolarizing current pulses (0.25-0.5 nA, 2 Hz, 100-200 ms for 5-10 min). bis-Fura-2 signals were monitored using an Ionoptix imaging system, and an increase in cell fluorescence typically occurred within 5-10 min after impalement. In this system, bis-fura-2 excitation was achieved using a mercury arc lamp shuttered between 340- and 380-nm excitation filters and delivered to an inverted Nikon Diaphot microscope via a flexible liquid light guide. Ganglia were viewed using a 40× oil immersion objective (Nikon). Emission signals were passed through a 510-nm cutoff emission filter and collected by a charge coupled device (CCD) video camera (Ionoptix). The images were acquired and analyzed using IonWizard software (v4.4). bis-Fura-2 filling was monitored at approximately 2 min intervals, and after 5-10 min, cells which did not display clear increases in fluorescence emission above background were discarded. Neurons that displayed clear increases in E500, from both 340- and 380-nm excitation, were then left unstimulated for approximately 5 min before experiments were begun. No changes in cell behavior occurred due to bis-fura-2 when the electrical properties of AH neurons were compared before and after cell filling. In addition, once a cell was filled with bis-fura-2, impalements could be maintained for up to 2 h, similar to impalements made with electrodes filled solely with KCl. In all experiments, the [Ca2+]i was measured from the whole-cell body of neurons as opposed to specific regions, and so [Ca2+]i refers to the global cytoplasmic Ca2+ concentration. The degree of bis-fura-2 filling was not uniform among the cells in this study, and this may be due in part to differences in microelectrode resistance in different impalements. [Ca2+]i was estimated using IonWizard software from the ratio of E500 following excitation at 340 and 380 nm. Calibration constants were determined using in vitro calibration solutions.
Statistics
Data are expressed in means ± standard error (SE). Linear regression and exponential decay analysis were performed using Graphpad Prism software. Student's paired t-tests were used to assess significant differences between calculated means; P < 0.05. Unless specified, n is the number of neurons, where one neuron was sampled from each animal.
Drugs and solutions
Atropine, caffeine, nicardipine, and ryanodine were all from Sigma Chemical Co (St. Louis, MO). bis-Fura-2-pentapotassium salt was from Molecular Probes (Eugene, OR).
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RESULTS |
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Simultaneous intracellular recordings and calcium imaging
measurements were obtained from 34 myenteric AH/Type II neurons, which
were characterized according to previously established criteria (Bornstein et al. 1994; Hirst et al.
1974
; Nishi and North 1973
). The size and shape
of the neuronal cell body was revealed with bis-fura-2 filling,
although neuronal process emanating from the cell body were rarely
discerned. AH neurons were elliptical with a maximum diameter of
31.7 ± 2.1 µm and a minimum diameter of 19.4 ± 1.8 µm
(n = 14), a size consistent with other studies of AH
neurons (Bornstein et al. 1991
; Furness et al.
1988
; Hanani et al. 1998
; Smith et al.
1999
; Vogalis et al. 2000
).
Evoked and spontaneous Ca2+ transients
Myenteric AH neurons had a mean resting membrane potential (RMP)
of 65 ± 2.2 mV (n = 21) and a mean resting
[Ca2+]i of
203.6 ± 8.4 nM (n = 12). Depolarizing current
pulses stimulated APs (1-15), which were followed by an
afterhyperpolarization (AHPslow) with a mean
amplitude of 8.6 ± 0.5 mV and a mean duration of 12.6 ± 1 s (n = 60 trial in 14 neurons). The voltage and
[Ca2+]i responses of a
typical neuron to the injection of a depolarizing current pulse (0.25 nA, 400 ms) are shown in Fig. 1.
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Phasic increases in [Ca2+]i were also detected following depolarizing current pulses, as shown in Fig. 1A. In 73 trials (n = 14 neurons), the increase in [Ca2+]i ranged from 33 nM (following a single AP) to 720 nM (following 15 APs), with an average increase of 203.5 ± 21.1 nM. The duration of evoked Ca2+ transients ranged from 2 to 35 s and had a mean duration of 10.6 ± 0.6 s.
The magnitude of evoked Ca2+ transients was not dependent on the duration or intensity of depolarizing current pulses per se, but on the number of APs that were elicited by any given stimulus, as shown in Fig. 2. In 73 trials from 14 neurons analyzed, there was a positive linear correlation between the number of APs and the amplitude of the Ca2+ transient evoked (R2 = 0.66, P < 0.001). Data from an individual neuron are plotted in Fig. 2B. This correlation suggests that Ca2+ influx associated with AP firing is necessary for the Ca2+ transient. The change in [Ca2+]i was calculated to be 47 nM/AP.
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As is demonstrated in Fig. 1, the durations of AHPslow and the Ca2+ transient were similar in all AH neurons. In 32 trials from seven neurons, the correlation of these two parameters was assessed by measuring the time at which 90% of the response had decayed (duration 90%). There was a strong correlation between the duration of the Ca2+ transient and the duration of AHPslow (R2 = 0.92, P < 0.001), indicating an interdependence between the two parameters, as shown in Fig. 3A. The slope of this relationship was >1, such that the duration of AHPslow was somewhat longer than the Ca2+ transient.
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In addition, the amplitude of the Ca2+ transient related to the amplitude of AHPslow, as can be observed in Fig. 2A. Figure 3B shows a significant correlation between the amplitude of the Ca2+ transient and the amplitude of AHPslow (R2 = 0.84, P < 0.05) in 15 trials from four neurons.
Ca2+ transient profile
[Ca2+]i began to
rise during or immediately after the first AP and rose to its peak in
299 ± 16 ms after the onset of the depolarizing current pulse
(n = 70 trials in 14 neurons). In 11 neurons, there was
a linear correlation between the timing of the peak
[Ca2+]i and the maximum
AHPslow (R2 = 0.73, P < 0.001), although the
Ca2+ peak always preceded the maximum
AHPslow. The peak
[Ca2+]i was only
sustained briefly, as the
[Ca2+]i was within 10%
of the peak value for 281 ± 68 ms (range = 30-670 ms,
n = 12 neurons) during maximal stimulation. The decay
of the Ca2+ transient was well fit by a
single-phase exponential curve (mean R2 = 0.78 ± 0.04), with a mean
time constant of decay () of 1.93 ± 0.34 s
(n = 17 trials from 10 neurons). An example of a single exponential decay fit is shown in Fig. 1.
Spontaneous APs
Spontaneous APs in AH neurons were occasionally observed in this study (6/34 neurons) typically when the cell became depolarized. Spontaneous AP firing was followed by small brief afterhyperpolarizations (2.5 ± 0.7 mV amplitude, 2.2 ± 0.2 s duration). In 3/6 neurons, small and brief Ca2+ transients were also evoked by spontaneous APs with a mean increase in [Ca2+]i of 38.3 ± 18.1 nM. Examples of spontaneous events are shown in Fig. 4.
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Inexcitable cells
Several studies have previously reported data from inexcitable
cells that are of unknown cell type. Recordings were made from six
large inexcitable cells with relatively negative RMPs (75 to
85 mV, n = 6). Depolarizing current pulses (0.01 to
5.0 nA, 100-500 ms) could not evoke an AP or any change in
[Ca2+]i and all six of
these cells remained inexcitable for up to 60 min following impalement.
In three of these cells, focal electrical stimulation (0.2 ms, 10 Hz,
for 0.5 s) produced a burst of antidromic APs that was accompanied
by a robust Ca2+ transient (see Fig.
5) and a small sEPSP, suggesting that
these cells were neurons. Repetitive synaptic stimulation for up to 30 min converted all inexcitable cells to "typical" AH neurons. After
the conversion, the cells had RMPs in the range of
65 to
75 mV and
APs could be elicited by a stimuli of
0.25 nA.
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Cytoplasmic [Ca2+]i changes
In seven preliminary experiments, electrodes were filled with 5 mM
bis-fura-2 in a 1 M KCl solution. Using this higher concentration of
bis-fura-2, relatively inexcitable AH neurons, with a resting membrane
potential of approximately 70 mV, were rapidly converted (within
5-10 min) into excitable AH neurons (RMP ~
50 mV) and AHPslow characteristic of these neurons was
virtually abolished, as shown in Fig.
6A. As changes in electrical
properties were never observed with electrodes filled with 1 mM
bis-fura-2, which was used in all other experiments in this study, it
is probable that high concentrations of bis-fura-2 acted as a buffer of
[Ca2+]i. As prominent
Ca2+ transients are still evoked with high
bis-fura-2 electrodes (see Fig. 6A), this suggests that
Ca2+ is completely buffered before it can
activate membrane-bound channels.
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Depletion of [Ca2+]i by perfusing the tissue with KRB containing zero Ca2+ also resulted in a rapid shift of the properties of AH neurons, as tested in three AH neurons. As is illustrated in Fig. 6B, neurons depolarize (17.4 ± 4 mV), become more excitable, and AHPslow is blocked. In addition, the resting level of [Ca2+]i is reduced (by 112 ± 27.1 nM) and evoked Ca2+ transients are virtually abolished. These experiments indicate the importance of the level of free [Ca2+]i in regulating the excitability of myenteric AH neurons.
Effect of ryanodine and caffeine
To determine the role of ryanodine-sensitive intracellular Ca2+ stores in the activity of myenteric AH neurons, recordings were made in the presence of caffeine and ryanodine.
The effects of relatively low concentrations of caffeine (1-5 mM) were
investigated in six AH neurons in the presence of nicardipine (2 µM)
and atropine (2 µM). Depolarizing current pulses were injected every
30 s and the changes in electrical properties and
[Ca2+]i were monitored.
In 4/6 experiments, the impalement of the AH neuron was lost within 5 min due to vigorous contractions of the smooth muscle evoked by
caffeine. However, within this limited time frame, a modest but
significant hyperpolarization of AH neurons was detected from
63.3 ± 3.8 to
69.1 ± 2 mV (P < 0.05, n = 6). This change in the RMP induced by caffeine was
coincident with an increase in the resting
[Ca2+]i of 21.8 ± 6.2 nM, and an increase in the amplitude of evoked Ca2+ transients of 136.7 ± 27.2 nM. These
observations suggest that the release of Ca2+
from intracellular stores is enhanced by caffeine which is reflected in
the elevation of resting
[Ca2+]i and an increase
in Ca2+ transient amplitude.
The effect of ryanodine perfusion (10 µM) was tested in nine AH neurons. Ryanodine had no significant effect on the resting [Ca2+]i, the RMP, or on input resistance in four neurons where hyperpolarizing current pulses were applied throughout the drug perfusion (0.15 nA, 200 ms, 0.2 Hz). Depolarizing current pulses, to elicit APs and Ca2+ transients, were applied in control conditions and in the presence of ryanodine (Fig. 7).
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Evoked Ca2+ transients were decreased by
ryanodine, with the amplitude changing from a ratio of 0.3 to 0.12 (P < 0.05, n = 9), a change of
91.1 ± 23.1 nM. The duration of the Ca2+
transients decreased from 11.82 ± 2.03 to 5.21 ± 1.17 s (P < 0.05, n = 9).
AHPslow was also reduced from 9.17 ± 0.85 to 3.73 ± 0.63 mV (P < 0.01, n = 9) in the presence of ryanodine. The duration of
AHPslow decreased from 16.39 ± 2.7 to
5.61 ± 1.6 s (P < 0.01, n = 9) in ryanodine. An example of all of these effects of ryanodine (10 µm) on an AH neuron is illustrated in Fig. 7. These data imply that
the release of Ca2+ from internal stores by CICR
via ryanodine receptors is responsible for part of the
Ca2+ transient and AHPslow.
As described earlier, a relationship exists between the number of action potentials and the amplitude of the Ca2+ transient. The effect of ryanodine on this relationship was tested in five AH neurons, with the results summarized in Fig. 8. In control conditions, the slope of the linear relationship was 47 nM/AP over the range of 1-4 APs. Ryanodine significantly reduced the amplitude of evoked Ca2+ transients to 16 nM/AP (34% of control). Thus, this suggests that CICR is activated by a single AP. However, the change in the slope implies that CICR is not fully activated by a single AP, i.e., successive APs trigger additional CICR. The difference between the two slopes, 29 nM/AP, provides an estimate of the contribution of CICR through ryanodine receptors to the Ca2+ transient evoked by each AP.
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In the presence of ryanodine, more APs were necessary to evoke a given change in [Ca2+]i. In 11 experiments in seven different neurons, the amplitudes of a control and ryanodine Ca2+ transient were matched and the amplitude of the resultant AHPslow was recorded. The Ca2+ transient amplitudes were well matched in control (90.2 ± 14.4 nM) and ryanodine (90 ± 13 nM, P = 0.95) conditions. The amplitude of AHPslow evoked by these changes in [Ca2+]i were not significantly different in control (4.9 ± 0.6 mV) and in ryanodine (5.0 ± 1.7 mV, P = 0.61) conditions. However, the number of APs necessary to evoke the same changes in control and ryanodine conditions was significantly different (control median = 2 APs, ryanodine median = 6 APs, P < 0.001). That is, the same changes in Ca2+ and AHPslow were elicited by two APs in control and six APs in ryanodine. Therefore, these data demonstrate that membrane potential is controlled by [Ca2+]i, regardless of the source of the change in [Ca2+]i. This conclusion is supported by data in Fig. 8B, which shows that the relationship between the durations of AHPslow and the Ca2+ transient is not significantly different in the presence of ryanodine.
Ryanodine blocks slow synaptic transmission
Electrical stimulation of internodal strands with high-frequency trains (20 Hz, 1 s) evoked sEPSPs, which gave rise to Ca2+ transients during AP firing. Evoked sEPSPs were completely abolished in the presence of ryanodine in 8/8 AH neurons tested. An example of the complete blockade of sEPSPs by ryanodine is shown in Fig. 9. This blockade typically took 10-20 min after ryanodine application to become effective. The sEPSP blockade was not dependent on any changes in RMP.
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DISCUSSION |
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There are two principal results of this study. 1) Myenteric AH neurons have a mechanism for amplifying [Ca2+]i following an AP. Ca2+ influx associated with an AP triggers ryanodine-sensitive CICR, which elevates [Ca2+]i that underlies the AHPslow. CICR also plays some role in the generation of slow synaptic events. 2) AHPslow in myenteric AH neurons is regulated by bulk changes in [Ca2+]i, regardless of whether CICR is functional or not.
Initial experiments, where high concentrations of bis-fura-2 were used
in the electrode, caused AH neurons to depolarize and become very
excitable. bis-Fura-2 is a calcium buffer related to
bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic
acid (BAPTA), and so high [bis-fura-2]i buffers
all of the free [Ca2+]i.
Depolarization and blockade of AHPslow by high
concentrations of calcium buffers has previously been reported in
nodose (Moore et al. 1998) and hippocampal neurons
(Lancaster and Zucker 1994
). Thus, it is clear that
cytoplasmic-free [Ca2+]i
is important in regulating the low RMP and relative inexcitability of
myenteric AH neurons.
Electrodes filled with 1 mM bis-fura-2 did not alter any of the
characteristics of AH neurons, and the RMP of AH neurons reported in
this study fall within the normal range of these cells
(Bornstein et al. 1994). At
65 mV, the mean resting
[Ca2+]i in AH neurons was
approximately 200 nM. Shuttleworth and Smith (1999)
used
an almost identical experimental approach and found that highly
excitable S neurons had a mean resting
[Ca2+]i of approximately
100 nM, half that of AH neurons. It is of interest to note that when
[Ca2+]i is depleted to
levels recorded in S neurons using zero Ca2+ KRB,
AH neurons assume many of the electrical characteristics of S neurons.
Thus, the high resting
[Ca2+]i is proposed to be
an important factor in determining the differences in RMP and
excitability between AH and S neurons. However, the mechanism behind
establishing and maintaining the high Ca2+ levels
in AH neurons is unclear.
Ryanodine perfusion had no effect on the RMP, input resistance, or on
the resting [Ca2+]i of
cells. This lack of effect of ryanodine on the resting properties of
neurons has been reported in superior cervical ganglia (Davies et al. 1996) and in cultured myenteric neurons (Kimball
et al. 1996
). This indicates that the high levels of
[Ca2+]i are independent
of CICR. Further comparisons between the present study and that of
Kimball et al. (1996)
are limited by differences in the
nature of the stimuli used and the populations of cells studied. It is
of interest to note however that virtually all cultured myenteric
neurons responded to 10 mM caffeine (Kimball et al.
1996
), indicating that virtually all cultured myenteric neurons
have ryanodine-sensitive stores. In the present study, caffeine evoked
an increase in the resting level of
[Ca2+]i, which was
accompanied by a hyperpolarization of the RMP. These results suggest
that although there is no measurable spontaneous CICR, the RMP can be
modulated by elevations in
[Ca2+]i induced by CICR.
The RMP was not altered by ryanodine at a time when
AHPslow was attenuated. Davies et al.
(1996) similarly found in superior cervical ganglia that
AHPslow was inhibited by ryanodine with no effect
on RMP. It has been proposed previously, based on the differential
effects of barium, that the gKCa underlying the
AHPslow and the RMP in AH neurons is the same
channel but operated by different Ca2+ pools
(Hirst et al. 1985
; North and Tokimasa
1987
). The simplest explanation of our data is that
gKCa is activated by the high resting levels of
[Ca2+]i (200 nM) to keep
the RMP low. During AP firing, CICR further elevates the global
Ca2+ leading to activation of these same channels
to evoke AHPslow. Therefore,
gKCa appears to be regulated solely by
[Ca2+]i, rather than
being regulated by separate Ca2+ pools.
Ca2+ transients were evoked by APs, as has
previously been reported in myenteric AH (Tatsumi et al.
1988; Vogalis et al. 2000a
), myenteric S
(Shuttleworth and Smith 1999
), nodose (Cohen et
al. 1997
), sympathetic (Hua et al. 1993
), and
hippocampal neurons (Lancaster and Zucker 1994
). There
was a linear relationship found between the number of APs evoked and
the amplitude of the Ca2+ transient. The maximum
stimulus in this study elicited 15 APs and no saturation of the
amplitude of the Ca2+ transient was recorded.
Cohen et al. (1997)
reported a quasi-linear relationship
between the number of action potentials and Ca2+
transient amplitude in nodose neurons up to a 20 AP stimulus, at which
point the Ca2+ transient amplitude/CICR became saturated.
There is a low activation threshold for induction of CICR in AH
neurons, as compared with the activation threshold that has been
reported in Purkinje (Llano et al. 1994), sympathetic
ganglia (Hua et al. 1993
), and dorsal root ganglion
(DRG) neurons (Shmigol et al. 1995
).
Subthreshold stimuli do not evoke AHPslow and
hence CICR, as has previously been reported (North 1973
)
in AH neurons. However, slow AHPs in celiac neurons, which were
subsequently shown to be inhibited by ryanodine (Jobling et al.
1993
), are activated by subthreshold depolarizing pulses
(Cassell and McLachlan 1987
) and thus have an even lower
activation threshold than AH neurons. In myenteric AH neurons, a single
AP is a sufficient stimulus to trigger CICR, but does not maximally
activate CICR. This is quite different from CICR in cardiac myocytes,
where each single AP triggers a maximal global
Ca2+ release event (Niggli 1999
)
and may reflect a greater degree of processing control in AH sensory neurons.
As in nodose neurons (Cohen et al. 1997), in myenteric
AH neurons there is a decrease in the slope of the linear relationship between the number of APs and the amplitude of the
Ca2+ transient in the presence of ryanodine.
Hence, as the number of spikes is increased, CICR is increased. This
may be due to either an increase in the activity of ryanodine receptors
or a recruitment of more ryanodine receptors. Estimates from this study suggest that CICR from ryanodine receptors contributes ~30 nM of the
~50 nM change in
[Ca2+]i triggered by each
AP. Thus, CICR amplifies the change in
[Ca2+]i by two- to
threefold, whereas in nodose neurons a 10-20 fold amplification of
Ca2+ transients induced by CICR was found
(Cohen et al. 1997
).
As reported previously in myenteric AH (Tatsumi et al.
1988; Vogalis et al. 2000a
) and nodose neurons
(Cohen et al. 1994
), there was a correlation between the
durations of the Ca2+ transient and
AHPslow. The duration of the
Ca2+ transient was consistently shorter than the
duration of AHPslow, as has previously been
reported (Hanani and Lasser-Ross 1997
). The relationship
between the durations was not significantly altered by ryanodine. The
ionic channel that is responsible for AHPslow in
myenteric AH neurons was identified as a
Ca2+-activated potassium channel (Hirst et
al. 1974
, 1985b
; North 1973
). Thus, a prolonged
elevation in [Ca2+]i
provides the sustained activation of gKCa which
produces AHPslow.
The contribution of ryanodine-sensitive intracellular
Ca2+ stores to AHPslow was
assessed using ryanodine and caffeine. The size of both the evoked
Ca2+ transients and the afterhyperpolarization
were significantly attenuated to similar degrees by ryanodine.
Ryanodine reduced the amplitude (40% of control) and duration (44% of
control) of the Ca2+ transients and reduced the
amplitude (41% of control) and duration (34% of control) of the
afterhyperpolarization. In addition, caffeine (1 mM) increased the
amplitude of the evoked Ca2+ transients by 50%.
Therefore, at least 60% of the afterhyperpolarization in AH neurons is
mediated through CICR via ryanodine receptors. Ryanodine has been
reported to have a variety of effects in different neurons. A partial
block of AHPslow was reported in CA pyramidal neurons (Pineda et al. 1999; Tanabe et al.
1998
) and celiac ganglia (Jobling et al. 1993
),
while a complete block was reported in nodose neurons (Moore et
al. 1998
), dorsal motor nuclei (Sah and McLachlan
1991
), and otic ganglia (Yoshizaki et al. 1995
).
No ryanodine block was reported in hippocampal CA1 neurons
(Zhang et al. 1995
) or sympathetic neurons (Goh
et al. 1992
). The mechanism through which the remainder of
AHPslow is mediated in myenteric AH neurons
remains a source of speculation. This remnant of
AHPslow may be mediated by
Ca2+ influx associated with spike firing,
Ca2+ release from different intracellular
Ca2+ stores, or a signal transduction cascade.
Ryanodine completely abolished sEPSPs in myenteric AH neurons,
indicating that CICR is critical in either the synaptic transmission or
transduction of sEPSPs in these cells. Although previous studies in
bullfrog sympathetic ganglia have indicated that ryanodine-sensitive stores can have only a modulatory effect on slow synaptic transmission (Cao and Peng 1999), this is the first report to our
knowledge of a blockade by ryanodine of synaptic transmission. The
mechanism responsible for this blockade may be a mix of both
presynaptic and postsynaptic effects and clearly requires further investigation.
The characteristic properties of AH neurons are dramatically influenced
by the levels of free
[Ca2+]i. The results of
this study indicate that AH neurons have a high resting level of
[Ca2+]i compared with
myenteric S neurons. Experimental perturbations that effectively
diminish the amounts of free
[Ca2+]i depolarize AH
neurons and alter their electrical properties, making them virtually
indistinguishable from S neurons.
[Ca2+]i rises in response
to AP firing and activates a Ca2+-activated
potassium conductance to "gate" sensory neuron output. Furthermore,
the results of this study indicate that the source of any changes in
[Ca2+]i do not influence
the close relationship between
[Ca2+]i and RMP. In the
presence of ryanodine, if the amplitude of a Ca2+
transient is matched to one in control conditions, the amplitude of
AHPslow evoked is the same. In addition, the
strong correlation found between AHPslow and
Ca2+ transient durations is not affected by
ryanodine. Therefore, although other intracellular messengers
undoubtedly influence their behavior (Bertrand and Galligan
1995; Zafirov et al. 1985
), the level of free
[Ca2+]i clearly is an
important modulator of the activity of AH neurons.
In summary, CICR modulation of [Ca2+]i plays a crucial role in regulating the excitability of myenteric AH neurons. These neurons appear to be most similar to nodose ganglia neurons, as CICR is activated by every action potential. Blockade of CICR by ryanodine has two major effects, an inhibition of AHPslow and blockade of sEPSPs. Hence, CICR modulation of [Ca2+]i plays a critical role in gating the transmission of sensory information to the output processes of myenteric AH neurons.
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ACKNOWLEDGMENTS |
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This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Program Project Grant PO1 DK-41315.
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FOOTNOTES |
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Address for reprint requests: T. K. Smith (E-mail: tks{at}physio.unr.edu).
Received 6 June 2000; accepted in final form 7 August 2000.
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REFERENCES |
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