Functional alterations in jejunal myenteric neurons during
inflammation in nematode-infected guinea pigs
Jeffrey M.
Palmer1,
Margaret
Wong-Riley2, and
Keith A.
Sharkey3
1 Department of Biomedical
Sciences, Creighton University School of Medicine, Omaha, Nebraska
68178; 2 Department of Cellular
Biology and Anatomy, Medical College of Wisconsin, Milwaukee,
Wisconsin 53226; and 3 Department
of Physiology and Biophysics, University of Calgary, Calgary,
Alberta, Canada T2N 4N1
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ABSTRACT |
Intracellular recordings of jejunal myenteric
neurons with an afterspike hyperpolarization (AH) from
Trichinella spiralis-infected animals
showed enhanced excitability on days 3, 6, and 10 postinfection (PI) compared with uninfected animals. Lower
membrane potential, increased membrane input resistance, decreased
threshold for action potential discharge, decreased AH amplitude and
duration, and increased fast excitatory postsynaptic potential
amplitude and duration were characteristic of neuronal recordings from
infected animals. Concurrent with electrophysiological changes during
T. spiralis infection, increased
cytochrome oxidase activity, a marker of neuronal metabolic activity,
and the expression of nuclear c-Fos immunoreactivity, an
indicator of transcriptional-translational activity, were also observed
in myenteric ganglion cells. Double-labeling for
calbindin-immunoreactive myenteric neurons revealed that ~50% of
these neurons also expressed increased c-Fos immunoreactivity during
T. spiralis infection. Myeloperoxidase
activity was significantly higher in the jejunum of T. spiralis-infected guinea pigs on days 3, 6, and 10 PI vs.
uninfected counterparts. The expression of c-Fos in
calbindin-immunoreactive neurons together with enhanced neuronal
electrical and metabolic activity during nematode-induced intestinal
inflammation suggests the onset of excitation-transcription coupled
changes in enteric neural microcircuits.
enteric nervous system; excitation-transcription coupling; cytochrome oxidase; c-Fos immunoreactivity; calbindin immunoreactivity; Trichinella spiralis
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INTRODUCTION |
GASTROINTESTINAL inflammation caused by parasitic
nematode infection evokes functional disturbances in enteric
physiological effector systems, including the contractile activities of
the smooth muscle layers and their regulation by intrinsic nerves (6,
10). Altered intestinal motility is a prominent feature of the
integrated physiological response of the host animal to enteric
nematode infection, as well as contributing to symptoms of host
gastrointestinal distress and morbidity (6). Experimental enteric
infections with the nematodes Nippostrongylus
brasiliensis and Trichinella
spiralis are now well known to induce disturbances in
small intestinal motility in vivo that have been characterized by
increased transit rates of nonabsorbable markers (7, 15) and altered
smooth muscle myoelectric and contractile activities in parasitized
animals (13, 36). Altered small bowel motility during experimental
nematode infections is associated with significant functional
alterations of the individual components (i.e, smooth muscle and
intrinsic nerves) that comprise the intestinal neuromuscular apparatus
(10, 11, 32).
Intestinal inflammation associated both with enteric nematode parasites
and other causes leads to marked changes in the structure and
neurochemical content of intestinal nerves (40). Thus alterations in
intrinsic neuronal function in the intestine can be inferred and indeed
have been shown to exist. For example, several lines of evidence have
indicated that the release of endogenous stores of neurotransmitters
from both extrinsic and intrinsic nerve endings is impaired during
enteric nematode infection. Electrical field stimulation of intrinsic
inhibitory nerves was significantly reduced in its ability to cause
relaxation of jejunal circular smooth muscle from N. brasiliensis-infected rats (14). Collins and co-workers
(11, 43) have shown that stimulated release of the endogenous
radiolabeled neurotransmitters
[3H]acetylcholine and
[3H]norepinephrine
from jejunal longitudinal muscle-myenteric plexus (LMMP) preparations
was significantly decreased, whereas stimulated release of substance P
was significantly increased (42) in T. spiralis-infected rats compared with uninfected rats.
The hypothesis that a functional reorganization of the intrinsic neural
control of intestinal motility is triggered by mucosal inflammation in the nematode-parasitized mammalian intestine is supported in part by
the observations described above. To date, however, investigation of
functional alterations in single enteric neurons from the
nematode-parasitized intestine has not been performed.
Evidence obtained within the last several years has confirmed the role
of intrinsic neurons contained within the myenteric and submucosal
plexuses of the enteric nervous system (ENS) in the normal control and
coordination of local enteric sensorimotor reflexes evoked by luminal
stimuli (3, 5, 29, 31, 38). In particular, intestinal primary afferent
nerves have recently become a focal point for investigation because of
their proposed role in host defense against various noxious stimuli
(24). In addition to extrinsic primary afferent nerves, the ENS itself has primary afferent neurons located in both the submucosal (28) and
myenteric plexuses (3, 31). In the myenteric plexus of the guinea pig
small intestine, neurons acting as intrinsic primary afferent neurons
and contained in self-reinforcing networks have been identified
electrophysiologically as nerve cells having a significant afterspike
hyperpolarization (AH-type neuron) and a multipolar Dogiel type II
morphology (3-5, 17, 31, 41). The capacity of myenteric AH neurons
to respond electrophysiologically with heightened excitability to
changes in gut wall tension or tactile and chemical stimulation of the
mucosa has demonstrated the afferent sensory-like role of this neuronal
class in the ENS (3, 5, 18, 29, 31). AH neurons contained within local ganglionic networks and by virtue of their multipolar morphology have
been shown to not only make synaptic contacts with each other, but they
also can synaptically drive the activities of another class of
second-order neurons, designated as S-type neurons with a lamellar
unipolar morphology (Dogiel type I), that appear to function as
interneurons and motor neurons in the ENS (17, 46). These findings
strongly suggest a pivotal multirole capacity for the AH class of
myenteric neuron in the guinea pig small bowel. The effects of mucosal
inflammation on the properties and behavior of these intrinsic nerve
cells and the circuits of which they are a part are presently unknown.
The aim of our work was to investigate concurrently the electrical,
metabolic, and transcriptional activity of myenteric neurons in
response to experimental enteric T. spiralis infection in the guinea pig. Changes in the
electrical and synaptic behavior of myenteric AH neurons were selected
as the focal points for our intracellular electrophysiological studies
because of the inherent inexcitability of these neurons during basal
recording conditions (7, 33, 46), the high frequency of impalement of
this class of myenteric neuron in electrophysiological investigation
(7, 33, 46), and because of their proposed roles as key driver or
gating neurons and as primary afferent neurons in the ENS (3, 5, 29,
31, 38, 41, 46).
We also examined myenteric ganglia for indications of increased
metabolic activity using cytochrome oxidase (CO) histochemistry. Enhanced neuronal CO staining has been shown to be tightly coupled with
increased electrical activity of both central and peripheral neurons,
including myenteric neurons (25, 27, 33). Finally, the transcriptional
and translational activation of cellular immediate early gene products
was detected immunohistochemically in myenteric ganglion cells.
Cellular immediate early genes c-fos
and junB are sensitive markers whose
increased expressions have been correlated with enhanced neuronal
activation in the ENS (28, 37, 38). Our results suggest that mucosal
inflammation during enteric nematode infection significantly affects
the cellular neurophysiology of myenteric neurons manifested as
upregulated electrical, metabolic, and transcriptional activation. The
onset of excitation-transcription coupled changes might act as a signal
for long-term modulation of neuronal activity within intrinsic reflex
microcircuits.
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MATERIALS AND METHODS |
Animals and induction of inflammation.
All procedures involving the use of live animals for these studies were
reviewed and approved by the Institutional Animal Care and Use
Committee of the Medical College of Wisconsin (Milwaukee, WI), where
these studies were initiated, and by the Animal Research Committee of
Creighton University (Omaha, NE), where they were completed. Outbred,
male, Hartley strain guinea pigs, Cavia
porcellus (Harland Sprague Dawley,
Indianapolis, IN), weighing 300-500 g served as host animals and
were each orally inoculated with 8-10 × 103 T. spiralis muscle-stage larvae that had been isolated
using a previously described pepsin-hydrochloric acid digestion
technique (19), and were administered oropharyngally via feeding tube in a 0.2 ml 0.85% saline vehicle bolus. Other guinea pigs served as
age-matched uninfected controls and received only 0.2 ml of oropharyngally administered saline without the worms. All animals were
fed a standard guinea pig chow diet supplemented with vitamin C and had
free access to water during the course of the investigation.
Experimental protocol and design.
LMMP preparations were obtained from uninfected control guinea pigs and
T. spiralis-infected guinea
pigs on days 3, 6, and 10 postinfection (PI). Segments of the
proximal jejunum 5 cm distal to the ligament of Trietz and 20 cm in
length were removed from adult guinea pigs, which had been killed by a
stunning blow to the head and exsanguinated through a deep, sharp
ventral incision across the neck. Exposure of the myenteric plexus was
achieved with use of a previously described microdissection method (46) to prepare whole mounts of the longitudinal muscle layer and adherent myenteric plexus ganglia (LMMP) that were relatively free of overlying circular muscle. Jejunal LMMP preparations were then utilized for
either intracellular electrophysiological experiments, CO histochemistry, or c-Fos immunohistochemistry. For CO histochemistry and c-Fos immunohistochemistry, LMMP tissues from both uninfected guinea pigs and T. spiralis-infected
counterparts were processed concurrently on a given day PI to ensure
similar incubation and reaction conditions.
Electrophysiological and pharmacological methods.
The LMMP preparation was pinned to the Sylgard-coated bottom of a
small-volume (1.5 ml) tissue chamber and perfused with a circulating
Krebs solution warmed to 37°C and gassed with a mixture of 95%
O2-5%
CO2 (pH 7.3-7.4), at a rate
of 8-10 ml/min. The composition of the Krebs solution (in mM) was
120 NaCl, 5.0 KCl, 1.2 MgCl2, 1.35 NaH2PO4,
14.4 NaHCO3, 2.54 CaCl2, and 12.7 glucose. Myenteric ganglia were visualized with an inverted microscope (Diaphot, Nikon,
St. Louis, MO) equipped with Hoffman modulation contrast optics and
epi-illumination. Individual ganglia were immobilized between a pair of
electrolytically tapered L-shaped tru-chrome orthodontic steel wires
(100 µm length; Rocky Mountain Orthodontic, Denver, CO) that were
positioned parallel to the long axis of the ganglion and perpendicular
to the axis of the longitudinal muscle layer (46).
Transmembrane potentials of myenteric ganglion cells were recorded
intracellularly with sharp glass micropipettes fabricated with a
Brown-Flaming electrode puller (model P-87, Sutter Instruments, Novato,
CA) and filled with 3 M KCl with direct current (DC) resistances ranging from 50 to 80 M
. The microelectrode was coupled
to an electronic amplifier (Intra 767, World Precision Instruments, Sarasota, FL) that was equipped with a bridge circuit for simultaneous injection of electrical currents into the cell while recording the
transmembrane electrotonic responses of the neuron. After impalement
and stabilization of resting membrane potential, myenteric neurons were
classified according to their active and passive electrical membrane
properties. Electrical behavior, synaptically evoked responses, and
responses to pharmacological agents were recorded digitally on
videotape (Vetter Digital model 3000A PCM recorder, Vetter, Redersburg,
PA) for later playback and computerized analysis using a MacLab 4-s
data acquisition and analysis system (AD Instruments, Milford, MA) and
a Macintosh Power PC 7100/66 computer (Apple Computer, Cupertino, CA).
Neuronal resting membrane potentials were determined from the amplifier
electrometer once the voltage tracing displayed on the oscilloscope had
stabilized and remained unchanged for at least 10 min. After a stable
resting potential was obtained, injection of graded amplitude and
duration square-wave current pulses from an electronic stimulator
(model S48 stimulator, Astro-Med-Grass Instruments, Quincy, MA) through
the recording microelectrode was initiated to evoke depolarized or
hyperpolarized electrotonic membrane potentials to characterize the
passive and active membrane properties of the neuron. The membrane
input resistance of each neuron was calculated from the slope near the
origin of the best-fit line determined from computerized plots of the
current-voltage relationship for a given neuron during specific test
conditions. Current injection was controlled from a step-pulse
generator (Nihon Koden model SET-1201, Medical Systems, Greenvale, NY)
to deliver a sequence of six, 200-ms duration hyperpolarizing square
pulses with incremental increases of current at 1-s intervals. This
sequence of pulses was repeated three times at 10- to 20-s intervals
for control periods, during perfusion of applied neuroactive
pharmacological agents, and during washout of these specific test
agents from the tissue chamber.
Synaptic potentials were evoked by application of focal electrical
shocks of varying frequency (0.1-0.5 Hz), duration (300-600 µs), and intensity (6-14 V) to neuronal processes coursing
through interganglionic fiber tracts connecting the ganglion containing the impaled neuron with neighboring myenteric ganglia. Extracellular shocks applied to interganglionic connectives were delivered from the
tips of Teflon-coated platinum-iridium wires (20 µm diam; Medwire,
Mt. Vernon, NY) connected to an electronic stimulator (model S48
stimulator, Astro-Med-Grass Instruments).
Test agents for pharmacological studies of neuronal responses were
delivered to the tissue chamber by addition of the substance at the
desired concentration(s) to the perfusing Krebs solution. Alternatively, pharmacological agents were also delivered from fine
glass pipettes positioned directly over the ganglion in which the
neuron had been impaled that were connected to a picospritzer device
(General Valve, Fairfield, NJ) capable of ejecting defined amounts of
test agents from the pipettes with millisecond pulses of pressurized
nitrogen gas. Chemical agents used for these studies included ACh, TTX,
hexamethonium, and atropine, all obtained from Sigma Chemical (St.
Louis, MO).
CO histochemistry.
Preparations of LMMP from uninfected control guinea pigs and from
T. spiralis-infected counterparts were
concurrently processed histochemically for neuronal CO activity (21).
LMMP preparations were incubated for ~20 h in cold (4°C) 2%
(wt/vol) saponin without agitation to permeabilize the myenteric
ganglia. After saponin treatment, LMMP tissues were incubated in a
reaction medium consisting of 0.05% (wt/vol) 3,3'-
diaminobenzidine tetrahydrochloride and 0.03% (wt/vol) cytochrome C
type III dissolved in 0.1 M sodium phosphate buffer containing 4%
(wt/vol) sucrose at 37°C for 2 h with agitation. The reaction
was stopped by flushing the tissues with three successive changes of
cold (4°C) 0.1 M phosphate buffer. Tissues were then mounted on
chrome-alum gelatin-coated slides, dehydrated, and coverslipped.
Intensities of CO reaction product were analyzed by optical
densitometric measurements which were made with a Zeiss microscope fitted with a Zeiss PI-2 photometer and illuminated with a tungsten lamp. Comparisons were only made between the whole mount preparations of paired LMMP from uninfected and T. spiralis-infected guinea pigs that had been
concurrently incubated and treated with the same reaction medium for
the same lengths of time on the same day. Data were quantified in terms
of the number of neurons reaching the CO labeling threshold per 25 ganglia, as well as the intensity of light transmission through neurons
containing the reaction product. Densities of CO reaction product were
determined within single-labeled neurons using a ×40 objective
lens and a microdensitometry program as described previously (21). The
optical density of the reaction product in each cell (the inverse of
light transmission) provided a measure of the relative level of CO
activity present. Optical density values were expressed in terms of
arbitrary units, and they were then compiled and analyzed
statistically.
Immunohistochemistry.
Segments of proximal jejunum (4-5 cm in length) from groups of
uninfected control guinea pigs and T. spiralis-infected guinea pigs were
immunohistochemically stained for the expression of c-Fos as previously
described (37). The intestine was cut open longitudinally along the
mesenteric border and then pinned mucosa side up in cold (4°C)
Krebs solution containing 10 µM nifedipine onto the bottom of a
Sylgard-coated petri dish. This resulted in a full-thickness
rectangular sheet of intestine which was gently stretched to
a point at which firm resistance was encountered to further stretch.
Tissues were fixed overnight (16-18 h) in modified Zamboni's
fixative, permeabilized by washing in dimethylsulfoxide (3 changes for
10 min each), and subsequently washed in PBS (pH 7.4). Tissues were
then further microdissected, removing the mucosa-submucosa layers and
the overlying circular muscle to yield a LMMP preparation. Single- and
double-labeling methods were utilized with a monoclonal antibody
directed against the protooncogene protein product, c-Fos (antibody no.
TF161, generously provided by Dr. K. Riabowol, University of Calgary)
alone or together with an anti-calbindin polyclonal antibody (Swant).
Calbindin immunoreactivity has previously been shown to be present in
all of the AH-type neurons with Dogiel type II morphology in the guinea
pig small bowel (17, 18, 41). Tissues were incubated in primary
antibodies for 48 h at 4°C. After washing in PBS (3 changes for 10 min each) tissue preparations were incubated for 1 h at 22°C with
the fluorescent secondary antisera that consisted of goat anti-rabbit
antibody conjugated to fluorescein isothiocyanate, and sheep anti-mouse
antibody conjugated to CY3 (Sigma Chemical). After further washing
tissues were mounted in bicarbonate-buffered glycerol (pH 8.6) and
viewed with a Zeiss axioplan fluorescence microscope. The c-Fos-labeled
nerve cells were counted in at least five myenteric ganglia per animal
on days 3 (n = 2 animals) and
4 (n = 4) PI. Similarly, the number of c-Fos-labeled nerve cells that were
also immunoreactive for calbindin were identified and counted.
MPO assay.
Myeloperoxidase (MPO) activity was measured in extracts of
full-thickness sections (200-300 mg) of guinea pig jejunum as an index of the intensity of inflammation due to T. spiralis-induced injury using a previously described
spectrophotometric method (19). MPO levels were measured from gut
tissues obtained from the same animals in which electrophysiological,
CO, and c-Fos activities were investigated, providing us with a direct
assessment of inflammation status and intensity with any detectable
neuronal functional alterations due to nematode infection. Changes in
optical density per minute were measured spectrophotometrically at 475 nm in triplicate, and data were recorded and analyzed with a
computerized data acquisition and analysis system (MacLab, AD
Instruments). Results were determined from a standard
curve and expressed as units per milligram of wet tissue. A unit of MPO
activity was defined as one micromole of
H2O2
degraded per minute at 25°C.
Statistical analysis.
All data are reported as means ± SE, and the number of animals
studied is indicated in parentheses. Student's
t-test and single-factor ANOVA were
used to determine statistically significant differences among data
obtained from uninfected control and T. spiralis-infected guinea pigs at various time points
PI. Dunnett's post hoc multiple comparisons test was used to identify
significantly different means after ANOVA. Computerized optical
densitometric measurements of differences in the intensities of CO
reactivity of individual myenteric neurons of LMMP tissues from
uninfected and T. spiralis-infected guinea pigs were evaluated statistically by Student's
t-test. For all statistical analyses a
probability level of P < 0.05 was considered significant.
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RESULTS |
Neuronal electrical and synaptic behavior during jejunal T. spiralis
infection.
Intracellular electrophysiological recordings were obtained from
T. spiralis-infected guinea pigs on
days 3, 6, and
10 PI. Overall, a total of 201 neurons
in jejunal LMMP preparations from 52 T. spiralis-infected guinea pigs were examined at these
time points PI and compared with 64 neurons from 31 uninfected control animals. Ganglion cells were classified electrophysiologically according to previously accepted and published criteria for myenteric neurons from guinea pig small bowel. Four different types of neuronal electrical behavior were observed, including neurons that were classified as S, AH, type 3, and type 4 (see Refs. 4 and 46 for
description of different properties and categories). All four types
were encountered in ganglia from uninfected control guinea pigs,
whereas only S, AH, and type 4 were recorded from ganglia of
T. spiralis-infected animals. The
proportions (percent of total neurons studied) of the different
electrophysiological types of myenteric neurons studied in
T. spiralis-infected vs. uninfected animals were S, 14.9 vs. 12.5%; AH, 75.6 vs. 71.8%; type 3, 0 vs.
1.5%; and type 4, 9.9 vs. 14%.
Our investigation was focused on the effects of
T. spiralis-induced
inflammation on the AH class of myenteric neurons. Recordings from
these neurons during basal conditions are characterized by relatively
high resting membrane potentials, low membrane input resistances, a
TTX-insensitive action potential with a shoulder on the descending
phase, and a prominent AH. AH neurons normally fail to fire action
potentials repetitively throughout the duration of an injected current
stimulus of sufficient strength to surpass threshold. The prominent AH
that follows action potential discharge in these neurons is due to a
large Ca2+-activated
K+ conductance that is an
important regulator of excitability in these cells. Thus we thought
that the electrical properties of this neuronal class would be
advantageous for the detection and assessment of changes triggered by
the enteric inflammatory response to the worms due to their
1) resistance to repetitive action
potential discharge (33, 46), 2)
high frequency of impalement in intracellular studies of enteric neural
function (4, 46), and 3) recently identified role as intrinsic primary afferent neurons (3, 5, 17, 29,
31, 38, 41). The other commonly encountered neuronal type (S neurons)
possesses a high basal activity level characterized by stimulus-evoked
repetitive action potential discharge, spontaneous action
potential discharge, and high membrane input resistances that could
make it difficult to determine neuronal behavioral changes due to
parasitic nematode-evoked inflammation.
Forty-six of the 64 neurons (71.8%) recorded from uninfected guinea
pigs were AH neurons. In comparison, 152 of 201 cells (75.6%) recorded
from T. spiralis-infected guinea pigs
were of this same neuronal classification. Resting membrane potentials of AH neurons from T. spiralis-infected guinea pigs were significantly more
depolarized on each of the days that were examined PI than those
recorded from age-matched uninfected animals (Table
1). Membrane input resistances determined
from the slopes of current-voltage plots were also significantly
increased in AH neurons from T. spiralis-infected animals as early as
day 3 PI (Table 1) and remained
elevated through day 10 PI compared
with AH neurons from uninfected animals. This finding indicated that
membrane conductance was markedly decreased in these neurons during
T. spiralis infection. Membrane input
resistances were measured at least three times during the recording
period to determine stability for a given neuron. In functional studies
of myenteric AH neurons from normal animals, membrane depolarization
accompanied by increased input resistance usually occurs only in
response to excitatory agonist receptor-activated
K+ channel closure (46) or
Cl
channel opening (2).
Together, these results suggested that an enhanced excitability state
existed for AH myenteric neurons during the inflammatory response due
to T. spiralis infection.
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Table 1.
Electrical and synaptic properties of AH-type myenteric neurons in
guinea pig jejunum during Trichinella spiralis infection
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Further confirmation of this finding was obtained when a significant
increase in the number of action potentials was evoked by intrasomal
injection of square-wave depolarizing current pulses of comparable
amplitudes in AH neurons from T. spiralis-infected animals compared with uninfected
controls (Table 1). In neurons from uninfected guinea pigs (Fig.
1, A and
B), it was difficult to evoke action
potential discharge, often necessitating intrasomal injection of
increasingly larger depolarizing current pulses through the recording
microelectrode to evoke one to three action potentials at the highest
current amount passed. In contrast, AH neurons from T. spiralis-infected guinea pigs (Fig. 1,
C and
D) showed a remarkable lack of
accommodation at threshold (repetitive discharge), and an increase in
the amplitude of the depolarization increased the number of evoked
action potentials further. This response of AH neurons in
T. spiralis-parasitized guinea pigs
was a consistent finding throughout the enteric phase of infection and
occurred at much lower current strengths compared with uninfected
animals. This finding is evidence for a lowered threshold for
excitation in AH myenteric neurons from T. spiralis-inflamed LMMP.

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Fig. 1.
Electrical excitability of myenteric afterspike hyperpolarization (AH)
neurons recorded from Trichinella
spiralis-infected guinea pigs on day
6 postinfection (PI).
A: electrotonic potentials and single
action potential discharge evoked by intrasomal injection of
increasingly larger steps of depolarizing current through the recording
electrode in a neuron from an uninfected guinea pig.
B: electrotonic potentials evoked by
intrasomal injection of increasingly larger steps of hyperpolarizing
current in a neuron from an uninfected guinea pig.
C: repetitive action
potential discharge evoked by intrasomal injection of a single step of
depolarizing current into a neuron from a T. spiralis-infected guinea pig.
D: anodal-break excitation (i.e.,
action potential discharge at offset of stimulus) evoked by intrasomal
injection of increasingly larger steps of hyperpolarizing current into
a neuron from a T. spiralis-infected
guinea pig. All injected square-wave current pulses were 200-ms
duration and 0.2-0.8 nA magnitude.
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The occurrence of anodal-break excitation (action potential discharge)
at the offset of intrasomal injection of square-wave hyperpolarizing
current pulses was another sign of the increased excitability in
myenteric AH neurons of T. spiralis-infected guinea pigs. The incidence of
anodal-break excitation in recordings of AH neurons from
T. spiralis-infected animals increased
through the duration of the enteric phase of infection peaking by
day 6 PI (Table 1). Electrotonic
potentials evoked by injection of increasingly larger steps of
hyperpolarizing current through the microelectrode into a neuron from
an uninfected guinea pig are shown in Fig.
1B. In comparison, Fig.
1D shows the same current injection
protocol utilized in a recording from a AH neuron from a
T. spiralis-infected guinea pig on
day 6 PI that resulted in hyperpolarizing electrotonic potential changes of larger amplitude evoked at lower current strengths than neurons from uninfected controls.
Alterations in the waveform of the action potential as well as the
amplitudes and durations of the AH were also observed in recordings of
myenteric AH neurons during enteric T. spiralis infection (Table
2; Fig. 2).
Comparative analysis of action potential waveforms showed a small but
significant decrease in action potential duration as early as
day 3 PI and persisting through
day 10 PI compared with those from
uninfected animals (Fig. 2, A and
B). However, maximum action
potential amplitudes were not significantly different between
uninfected and T. spiralis-parasitized
guinea pigs throughout the course of infection. The rate of change of voltage vs. time of the action potential downstroke (calculated as the
average slope) of AH neurons from T. spiralis-infected animals showed a more rapid rate of
descent compared with those from uninfected animals at all time points
examined PI (Table 2). These data suggest that the inward
Ca2+ current that occurs during
the action potential and which is represented by the
Ca2+ shoulder of the downstroke is
significantly reduced in AH neurons from ganglia in T. spiralis-inflamed tissues or that other channels responsible for outward rectification are blocked.
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Table 2.
Characteristics of AP and AH of AH-type myenteric neurons in guinea pig
jejunum during T. spiralis infection
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Fig. 2.
Alterations in action potential waveform and amplitude of AH of
myenteric AH neurons recorded from T. spiralis-infected guinea pigs on
day 6 PI.
A: stimulus-evoked (depolarizing
pulse: 20 ms, 0.5 nA) action potential showing prominent shoulder on
falling phase (top filled arrowhead)
and undershoot (bottom filled
arrowhead) in a neuron from an uninfected guinea pig.
B: stimulus-evoked (depolarizing
pulse: 20 ms, 0.5 nA) action potential with absent or reduced shoulder
(top open arrowhead) and more rapid
undershoot (bottom open arrowhead)
in a neuron from a T. spiralis-infected guinea pig.
C: increasingly larger steps of
injected depolarizing current (200 ms, 0.2-1.2 nA) resulted in a
maximum discharge of only 3 action potentials with a summated AH
(bottom filled arrowhead) in a
neuron from an uninfected guinea pig; return to resting potential after
first action potential (top filled arrowhead).
D: increasingly larger steps of
injected depolarizing current (200 ms, 0.1-1.0 nA) resulted in a
maximum discharge of 12 action potentials with a correspondingly
summated AH (bottom open arrowhead)
in a neuron from a T. spiralis-infected guinea pig; return to resting
potential after first action potential (top open
arrowhead).
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The peak amplitudes and durations of the AH after stimulus-evoked
action potential discharge were significantly decreased as well in AH
neurons from myenteric ganglia in T. spiralis-infected animals compared with uninfected
animals (Table 2; Fig. 2, C and
D). This was determined using the
same duration of injected current (20 ms) but variable current
strengths based on the threshold potential of the neuron so that only a
single action potential would be discharged. Summated AHs were also
evoked with use of a 200-ms depolarizing current pulse, and in a few AH
neurons from uninfected guinea pigs a maximum of three action
potentials could be evoked but only at large current strengths
reflective of the relatively low excitability state of these neurons
under normal conditions (Fig. 2C).
In marked contrast, a summated AH could be evoked in nearly all AH
neurons recorded from ganglia in T. spiralis-inflamed tissues corresponding in its
amplitude with increased numbers of action potentials discharged in
response to increasingly greater current strengths (Fig.
2D). Activation of antidromic action
potentials with focal shocks applied to axons ramifying in
interganglionic connective fibers also generated summated AHs in AH
neurons from T. spiralis-parasitized
animals but not uninfected controls (Fig.
3, A and
B). This observation was additional
confirmation that somal excitability of these neurons had been
dramatically increased during nematode infection. Significant reduction
of AH amplitude and duration (Fig.
3B), a key regulatory mechanism
limiting action potential discharge (46), allows repetitive action
potential discharge in these cells.

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Fig. 3.
Antidromically activated action potentials were evoked in jejunal
myenteric AH neurons with focal extracellular shocks (300-500 ms
duration, 4-8 V, 0.1-1 Hz) applied to interganglionic
connective fiber tracts entering the ganglion in which the neuron was
impaled. A: graded increases in
frequency and strength of applied shocks failed to evoke more than one
action potential discharge with an AH in an AH neuron from an
uninfected guinea pig (filled dot beneath each event represents number
of spikes discharged in a one-to-one relationship with the number of
shocks applied). B: graded increases
in frequency of applied shocks at a given stimulus strength produced
corresponding increases in number of action potentials discharged
together with a summation in the amplitude and duration of the AH in an
AH neuron recorded from a T. spiralis-infected guinea pig on day
6 PI (filled dots beneath each event represent the
number of spikes discharged in a one-to-one relationship with the
number of shocks applied; horizontal bar, 4.8 s; vertical bar, 10 mV).
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Enhanced electrical excitability of myenteric AH neurons in
T. spiralis-inflamed jejunal LMMP was
also evident in the high proportion of spontaneously active neurons
(unstimulated action potential discharge) that were observed in our
recordings at every time point examined PI compared with uninfected
controls (Table 1; Fig. 4,
A-C).
This high level of spontaneous excitability does not occur in normally
quiescent AH neurons. Spontaneous action potential discharge in these
neurons was TTX-insensitive (Fig. 4B). Furthermore, spontaneous
activity did not appear to be synaptically driven because exposure to
0.3 µM TTX, a neurotoxin that blocks axonal action potential
conduction and neurotransmitter release, only slightly attenuated the
discharge rate but did not abolish unstimulated firing of action
potentials (Fig. 4B). With use of intrasomal injection of constant hyperpolarizing DC current,
spontaneous action potential discharge was completely abolished at a
current-clamped membrane potential around
85 mV (data not
shown).

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Fig. 4.
Spontaneous action potential discharge in AH myenteric neurons recorded
in jejunal ganglia from T. spiralis-infected guinea pigs on
day 6 PI.
A: spontaneous action potential
discharge and action potential waveform before addition of TTX to Krebs
buffer solution superfusing longitudinal muscle-myenteric plexus (LMMP)
preparation in recording chamber. B:
TTX-insensitive spontaneous action potential discharge and waveform
after a 10-min exposure to TTX (0.3 µM).
C: spontaneous action
potential discharge and waveform after 10-min washout of TTX from
recording chamber. Arrowheads, effect of TTX on
Na+-dependent component of action
potential upstoke.
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|
Twelve of 46 AH neurons in uninfected guinea pigs (26%) and 81 of 152 AH neurons from T. spiralis-infected
guinea pigs (53%) had fast excitatory postsynaptic potentials (EPSP).
The ability of myenteric AH neurons to produce suprathreshold fast EPSP
after synaptic activation was also enhanced in ganglia obtained from T. spiralis-infected guinea pigs on
days 3-10 PI compared with uninfected controls. Data presented in Table 1 show that significant increases were detected in both the amplitude and duration of fast EPSP
evoked in AH neurons from T. spiralis-inflamed LMMP. Fast EPSP were evoked in AH
neurons using graded-strength focal electrical shocks applied
extracellularly to interganglionic fiber tracts (Fig.
5, A and
B) and were hexamethonium sensitive
(200 µM). Application of ACh microejected from a fine-tipped pipette onto the impaled neuron in the presence of hexamethonium still evoked a
rapid postsynaptic depolarization. Thus hexamethonium reversibly
decreased the amplitudes of fast EPSP (data not shown), confirming that
they involved presynaptically released ACh acting at postsynaptic
nicotinic cholinergic receptors. Fast EPSP evoked in AH neurons of
myenteric ganglia from uninfected guinea pigs routinely failed to reach
suprathreshold depolarization triggering action potential discharge
(Fig. 5A). In contrast, fast EPSP
evoked in AH neurons of ganglia from T. spiralis-infected animals on day
6 PI as shown in Fig.
5B routinely achieved suprathreshold depolarization and action potential discharge.

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Fig. 5.
Enhanced fast excitatory synaptic transmission in myenteric AH neurons
recorded from T. spiralis-infected
guinea pigs on day 6 PI.
A: fast excitatory postsynaptic
potentials (EPSP) recorded at current clamped membrane potential of
59 mV. Extracellular stimulus pulse parameters: 600 µs
duration, 0.1 Hz frequency, and increasing stimulus strength (numbered
arrows) as shown at 8.2 (1), 8.7 (2), and 9.2 V
(3).
B: fast EPSP recorded at
current clamped membrane potential of 60 mV. Extracellular
stimulus pulse parameters: 500 µs duration, 0.1 Hz frequency, and
increasing stimulus strength (numbered arrows) as shown at 6.4 (1), 6.8 (2), and 7.0 (3) V.
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Neuronal CO activity during jejunal T. spiralis infection.
Histochemical staining of LMMP whole mounts for detection of changes in
neuronal CO activity demonstrated a significant increase in staining
intensity due to enteric T. spiralis
infection on days 3, 6, and
10 PI. Photomicrographs of myenteric
ganglia showed not only an increased staining intensity of individual
ganglion cells in tissues from T. spiralis-infected guinea pigs compared with uninfected
counterparts (Fig. 6,
A and
B) but also an increase in the
number of neurons with high CO activity. These findings were widespread
throughout the myenteric plexuses of T. spiralis-infected vs. uninfected animals (data not
shown).

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Fig. 6.
Cytochrome oxidase (CO) histochemical reactivities of jejunal myenteric
ganglion cells in whole mount longitudinal muscle-myenteric plexus
(LMMP) preparations from uninfected
(A) and T. spiralis-infected
(B) age-matched guinea pigs on
day 6 PI (magnification ×100).
Note markedly increased CO reaction product densities of compared
ganglion cells (indicated by filled arrowheads) in LMMP whole mount
from T. spiralis-infected guinea pig
vs. uninfected counterpart.
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|
Measurements of the optical densities of 50 individual ganglion cells
selected in a blinded and randomized fashion from each whole mount
preparation were used to quantify CO reactivity changes due to
T. spiralis infection.
Comparisons were only made between preparations that had been
concurrently incubated and stained over the same 24-h period from a
pair of age-matched uninfected and T. spiralis-infected guinea pigs examined on a specific
day PI. Significant increases in the number of myenteric ganglion cells
with higher reaction product densities and hence higher optical density
readings occurred in T. spiralis-infected animals on each day examined PI
compared with uninfected counterparts (i.e., for each time point, no.
of uninfected animals, n = 3; no. of
infected animals, n = 3). These
results were reproduced in tissues obtained and densitometrically
analyzed from three different sets of uninfected and
T. spiralis-infected animals. Figure
7 shows results expressed in terms of
arbitrary optical density units obtained from paired
uninfected-infected LMMP tissues examined on day
3 (Fig. 7A, uninfected
0.666 ± 0.013 vs. infected 0.812 ± 0.0147;
P < 0.0001),
day 6 (Fig.
7B, uninfected 0.756 ± 0.0104 vs.
infected 1.022 ± 0.0194; P < 0.0001), and day 10 (Fig.
7C, uninfected 0.734 ± 0.012 vs.
infected 0.968 ± 0.0147; P < 0.0001) PI. These data indicate that overall metabolic activity in
myenteric neurons is significantly elevated during the mucosal inflammatory response to T. spiralis.
The findings of increased CO reactivity in myenteric ganglion cells
from T. spiralis-infected guinea pigs
correlated with parasite-induced neuronal electrophysiological changes
and elevations in jejunal tissue MPO activity (see below). These
results are consistent with the coupling of enhanced electrical excitability to increased neuronal metabolic activity (21, 22, 27, 33)
and serve as corroborative evidence that excitability of AH neurons is
enhanced during T. spiralis-induced
inflammation.

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Fig. 7.
Changes in optical density of CO reaction product in jejunal myenteric
ganglion cells in LMMP whole mount preparations from paired uninfected
and T. spiralis-infected guinea pigs
examined at each time point. Three separate pairs of simultaneously
processed tissues (i.e., uninfected × T. spiralis-infected) were examined at
day 3 (A), day
6 (B), and
day 10 PI
(C). Significant increase (paired
Student's t-test) in intensity of CO
reaction product of myenteric ganglion cells occurred on each day PI
compared with respective uninfected control.
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Neuronal nuclear protooncogene expression during jejunal T. spiralis
infection.
Changes in the expression of neuronal immediate early gene products in
myenteric ganglia were examined using standard fluorescence microscopy
of LMMP whole mounts immunostained with specific antibodies directed
against c-Fos on days 1, 3, 4, and
6 PI. In control animals, no c-Fos
immunoreactivity was detected in the myenteric plexus at any time after
inoculation with saline vehicle alone (Fig. 8A). The c-Fos immunoreactivity was
detected on days 3-6 PI in neuronal nuclei of the myenteric plexus from parasitized guinea pigs
(Fig. 8, A and
B) but not at the other time points
studied. The quality of this staining differed slightly from that seen in other circumstances. In myenteric ganglia from T. spiralis-infected animals, c-Fos immunoreactivity had a
slightly punctate appearance, whereas normally it tends to have a
homogenous smooth appearance in the nucleus (with an unstained
nucleolus). The reason for the change in character of the staining is
unclear at this time. An immediate early gene response in myenteric
neurons appears likely to occur during the enteric phase of
T. spiralis infection in association with the early and peak stages of jejunal mucosal injury
and inflammation that attends epithelial invasion and occupation by the
worms. The marked increase in c-Fos immunoreactivity observed in
myenteric plexus preparations from infected animals on
days 3-6 PI was coincident with
enhanced neuronal electrical excitability, metabolic activity, and peak
levels of tissue MPO activity (see below) during jejunal
T. spiralis infection in guinea pigs.
Quantitative estimates of the peak c-Fos immunoreactivity at
days 3 and
4 revealed that ~50% of the
ganglion cells were c-Fos positive (45 ± 5 cells/ganglion, n = 28 ganglia from six animals,
assuming about 100 neurons/ganglia) (24, 35).

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Fig. 8.
c-Fos immunoreactivity (IR) in LMMP whole mounts from jejunal myenteric
plexus from uninfected (A) and
T. spiralis-infected guinea pig
(B). Calbindin IR shown in
C is from same preparation as shown in B on
day 3 PI. c-Fos IR was absent from
myenteric plexus in control animals
(A). After infection, nuclear
neuronal c-Fos IR was found in about 45-50% of myenteric neurons
(B). Double-labeling with antibodies
raised against calbindin revealed extensive overlap (arrows) between
c-Fos and calbindin IR in myenteric neurons. Scale bar, 50 µm.
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Double-labeling studies for calbindin immunoreactivity (Fig.
8C), a marker of AH cells in the
myenteric plexus of the guinea pig, revealed that 52 ± 7% of the
calbindin immunoreactive neurons expressed c-Fos, whereas 12 ± 2%
of the c-Fos nerve cells were calbindin positive. Thus these findings
suggest that a significant proportion (~50%) of those intrinsic
neurons that could be potentially considered to be primary afferent
neurons due to their calbindin immunoreactivity (17, 18, 41) expressed
an increased transcriptional and translational activity. We should
stress that it is also apparent from our results that the increase in
c-Fos immunoreactivity was a widespread phenomenon that most likely
included other classes of myenteric plexus neurons, possibly motor
neurons and interneurons. Thus these data are comparable to the
widespread increase we observed in CO staining intensity of neurons in
the myenteric plexus of the T. spiralis-inflamed jejunum that also more than likely
included other types of neurons.
MPO activity during T. spiralis-induced jejunal inflammation.
Determination of tissue MPO activity as a measure of granulocyte
infiltration is a standard measure of acute inflammatory reactions in the mucosa or in full-thickness specimens of
gastrointestinal tissue (19), and correlates with the intensity of the
inflammatory response. The activity of MPO in extracted tissues from
the jejunum of guinea pigs during the enteric phase of
T. spiralis infection was
significantly elevated as early as day
3 PI (P < 0.05),
peaked by day 6 PI
(P < 0.001), and was still increased
on day 10 PI (P < 0.01) compared with jejunal
tissues from uninfected guinea pigs (Fig.
9). A maximum increase of ~10-fold
occurred in the mean MPO activity in tissues from T. spiralis-infected guinea pigs on day
6 PI compared with the mean MPO activity detected in
jejunal tissues from uninfected control animals.

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Fig. 9.
Myeloperoxidase (MPO) activity (means ± SE) in jejunal tissue
measured as index of inflammation intensity evoked by enteric infection
in guinea pigs with T. spiralis on
days 3, 6, and 10 PI
compared with uninfected controls (number of animals from which tissues
were obtained for MPO assay is indicated at base of each bar).
* P < 0.05. ** P < 0.01. *** P < 0.001.
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 |
DISCUSSION |
Our results demonstrate that the electrical and synaptic behavior of
myenteric AH neurons is upregulated toward an increased state of
excitability during invasion of the jejunal mucosa by enteric stages of
the parasitic nematode T. spiralis.
Functional excitability of jejunal AH neurons recorded from
T. spiralis-infected guinea pigs on
days 3, 6, and
10 PI was shown by lower mean resting membrane potentials, increased membrane input resistances, decreased duration of action potentials, and decreased amplitude and duration of
postspike hyperpolarizing afterpotentials in these nerve cells compared
with those recorded from uninfected animals. Enhanced excitability was
also evidenced by an increased incidence of spontaneous action
potential discharge and anodal break excitation in AH-type neurons.
Additionally, when focal stimulation was applied to interganglionic fiber tracts entering the ganglion in which the impaled AH neuron was
located, we detected increased amplitude and duration of evoked fast
EPSP that attained threshold for action potential discharge. There was
also a marked increase in axonal activation of antidromic action
potential invasion of AH neurons observed in infected animals.
In the present investigation, the increased excitability observed in AH
neurons from nematode parasitized jejunum was temporally associated
with both histopathological changes (data not shown) and significant
elevation of MPO activity in extracted jejunal tissues. These findings
were consistent with previous work using this particular animal model
for enteric host-parasite interactions (1, 8) and verified the inflamed
state of the T. spiralis-infected small bowel. Quantification of MPO activity is a reliable index of
inflammation intensity in mucosal, submucosal, and smooth muscle tissues during T. spiralis infections
(19, 32). MPO activity was used as an indicator of the influx of
increased numbers of granulocytic myeloid inflammatory cells into the
mucosa-submucosa and deeper intestinal tissue that release
proinflammatory mediators. The response to T. spiralis infection in our study occurred as early as
day 3 PI, with a peak in MPO activity
detected on day 6 PI. Increased MPO
levels on days 3, 6, and 10 PI with
T. spiralis are well correlated
temporally with the occurrence of functional disturbances in intestinal
motility and secretion in vivo and in vitro (1, 6, 7, 13, 19, 36),
altered contractile behavior of isolated smooth muscle strips in vitro
(32), and altered release of neurotransmitters from the myenteric
plexus in vitro (11, 42, 43) in rat and guinea pig hosts.
We corroborated our electrophysiological results with a histochemical
method designed to gauge the presence of increased metabolic activation
of neurons in jejunal myenteric ganglia from T. spiralis-infected guinea pigs. CO has been shown to be
a sensitive and reliable marker for increased activity in central and
peripheral neurons (25, 27, 33). Colchicine, cholera toxin, and the
neurotoxins, 6-hydroxydopamine, 5,7-dihydroxytryptamine, and
veratridine have all been shown to increase the intensity of CO
reactivity in enteric neurons (27, 33). Mawe and Gershon (33)
successfully used this technique to demonstrate that a functional
heterogeneity in the state of neuronal activation exists in myenteric
plexus ganglia of the guinea pig small bowel under normal conditions and that veratridine-stimulated increases in the intensity (i.e., optical density) of CO reaction product in myenteric ganglion cells
correlated closely with increased electrical excitability of myenteric
AH neurons. Thus enhanced electrical excitability in AH neurons, as
well as in other central and peripheral neurons (25, 27, 33), is
tightly coupled to a corresponding increase in aerobic metabolism. Our
results are consistent with a correlation between increased electrical
and metabolic activation of AH neurons during enteric
T. spiralis infection. The largest
increase in CO intensity occurred on day
6 PI and coincided with the peak increase detected in
MPO activity of jejunal tissues from infected animals. Our findings
also suggest that during the inflammatory process there is a
generalized increase in neuronal activation within the plexus
indicative of a decreased functional heterogeneity in the jejunal
myenteric ganglia. Wong-Riley and co-workers have demonstrated that
stimulated increases in neuronal CO activity are dependent primarily on
changes induced in the regulation of the amount of enzyme protein
present in the cell (21) and that transcriptional activation of both
nuclear and mitochondrial subunit genes for CO is regulated by the
level of neuronal electrical activity (22).
Based on the results presented above, we can speculate that the
functional consequences of upregulated excitability of AH neurons
during T. spiralis infection in guinea
pig jejunum could involve altered somal gating properties of these
multipolar and potentially multifunctional nerve cells. This
electrophysiological class of neuron is a key component of both the
sensory and motor limbs of local reflex arcs in the intestine (3, 5,
29, 31). This class of neuron has been proposed to regulate and facilitate the buildup and spread of excitation throughout the myenteric plexus based on stimulus-evoked modulation of the inherent inexcitability of the neuronal soma (46). Sustained membrane depolarization of AH neurons, similar to that occurring during the slow
EPSP and dependent on K+ channel
closure (30, 46), permits incoming excitation from the synaptically
activated neurites or soma to propagate to outgoing neurites that
target other neurons or drive effector tissue activity. The lower
resting membrane potentials we observed in AH neurons from
T. spiralis-infected jejunum would
seem to assure that threshold for action potential discharge will be
attained and propagated and could increase synaptic effectiveness and
amplification vital for coordination of effector tissues responding to
the noxious presence of the worms in the mucosal epithelium.
Another key finding of our work consisted of significant decreases
observed in the amplitude and duration of AHs in AH neurons from
T. spiralis-inflamed LMMP. This
finding clearly demonstrated that a key mechanism governing
excitability of these nerve cells, involving a receptor-operated
Ca2+-activated
K+ conductance that acts to limit
action potential discharge frequency (30, 46), was altered during
enteric T. spiralis infection. This
interconversion of the functional state of AH neurons from one of high
action potential accommodation (i.e., low excitability) to low
accommodation (i.e., high excitability) of action potential discharge
resulted in increased firing frequency of stimulus-evoked spikes.
Our data are consistent with that presented by Kunze and colleagues
(31) who showed that under conditions of variable stretch AH neurons in
the myenteric plexus of the guinea pig ileum are also able to convert
from a low state to a high state of excitability. This functional
property of AH neurons in the ENS is reminiscent of the intrinsic
integrative properties of mammalian central and peripheral autonomic
neurons and invertebrate sensory neurons that adapt their rate of spike
discharge in response to the tone or strength of a conditioning
stimulus that has altered the postspike hyperpolarizing afterpotential
in these cells (12, 39, 45). For example, modulatory changes in the
hyperpolarizing afterpotential are known to occur as a result of
long-term potentiation in mammalian hippocampal neurons (12) and in
conditioned learning and training in neurons of the marine invertebrate
Aplysia (23), which reflect changes in
neuronal sensory plasticity. Walters and co-workers have characterized
inflammation-induced changes in the electrical and synaptic behavior of
Aplysia sensory neurons in response to axonal injury (20, 45) and periaxonal inflammation (9) that are nearly
identical to our results in mammalian myenteric neurons.
Inflammation-induced changes in neuronal properties in Aplysia included lower resting
membrane potential, decreased threshold for action potential discharge,
decreased amplitude of AH, decreased accommodation of stimulus-evoked
spike discharge, increased membrane input resistance, and increased
amplitude and duration of evoked fast EPSP. We also observed an
enhancement of fast excitatory synaptic transmission in LMMP from
T. spiralis-inflamed guinea pigs. This
finding suggests an upregulated sensitivity of the postsynaptic
neuronal membrane to neurotransmitters and other neuroactive factors
released during nematode-induced inflammation that prime the cellular
regulatory mechanisms involved in promoting neuronal responsiveness to
excitatory stimuli.
It is possible that the alterations we observed in the
electrophysiology of myenteric AH neurons might reflect, in whole or in
part, the effects of the nematode-induced intestinal inflammation. Neuronal damage, injury, or stress could likely be due to the release
and presence of reactive oxygen metabolites (19), the formation of
nitric oxide-dependent nitrating species like peroxynitrite (34), and
other proinflammatory and anti-parasitic factors that are known to be
released by the increased numbers of inflammatory and immune effector
cells recruited to the nematode-infected small bowel (6, 16). However,
other findings obtained utilizing physiological approaches suggest that
this is not the only possibility that could account for the spectrum of
functional alterations manifested by the nematode- parasitized
intestine. For example, evidence presented by Alizadeh et al. (1)
clearly demonstrated the occurrence of highly organized and coordinated
net aboral propulsive movements of isolated jejunal segments from
T. spiralis-infected guinea pigs on
days 10-20 PI, whereas segments
from uninfected counterparts did not demonstrate such activity.
Isolated gut segments capable of generating propulsive complexes in
vitro would require an intact, viable intrinsic nervous system.
Furthermore, nematode-induced inflammation has been shown to include
the production and release of factors such as histamine, serotonin,
neuropeptides, prostanoids, and other arachidonic acid-derived
mediators, all of which have been shown to have significant neuroactive
properties (6, 46) and would not necessarily exert deleterious effects
on neuronal function. Thus it seems reasonable to infer that such
highly organized propulsive events present only in the
T. spiralis-infected jejunum are
unlikely to be mediated by damaged intrinsic nerve cells whose function
would be compromised. Instead, the observations of Alizadeh et al. (1)
together with our present data strongly implicate an adaptive, plastic
response leading to an increased level of intrinsic neurally activated
intestinal motor organization and coordination during nematode
infection and inflammation.
Electrical and metabolic activation of AH myenteric neurons persisted
throughout the enteric phase of T. spiralis infection in the guinea pig jejunum. Marked
changes in the parameters of excitability we examined, but most
significantly in the postspike hyperpolarizing afterpotential which is
an important regulatory event, led us to investigate the potential
basis for these long-lasting changes in neuronal activity. Events such
as the AH are known to be modulated by stimuli acting via
receptor-induced intracellular second messengers that ultimately
activate protein phosphorylation and changes in gene expression (46).
It is now well established that excitation-transcription coupling in
spinal sensory neurons induces changes in nociceptive processing that
can produce significant functional changes for action potential
activation (35). Protein products of immediate-early genes such as
c-fos are involved in cellular
stimulus-transcription coupling effectively acting as "third"
messengers regulating transcription of target genes that affect
neuronal phenotypic expression in response to extracellular stimuli
(35, 44). Traub et al. (44) have reported a large induction of Fos-like
immunoreactivity in rat lumbrosacral spinal cord neurons after noxious
colorectal distension. Other models of nerve injury and stimulation
have also been shown to result in the differential induction of Fos and
Jun protein expression (35, 38, 40). Our results showing induction of
c-Fos immunoreactivity as early as day
3 PI are consistent with the notion that
excitation-transcription coupling also occurs in myenteric neurons
during T. spiralis-induced inflammation. Nearly one-half of the total neurons in the myenteric plexus showed nuclear Fos immunoreactivity in infected animals. Because
the typical neuronal induction and expression of c-Fos immunoreactivity
usually has a rapid onset (i.e., within 60 min of stimulation) and a
relatively short duration (i.e., 2-4 h) (35, 38, 40, 44), the
persistent increased immunoreactivity may be due to changes other than
electrical excitability. When calbindin immunoreactivity was also
examined in these same tissues we found that slightly more than 50% of
the c-Fos immunoreactive neurons were also calbindin immunoreactive.
Because neuronal calbindin immunoreactivity in the guinea pig myenteric
plexus has been shown to be a marker of AH-type neurons that can act as
primary afferent neurons (17, 18, 41), our data suggest the possibility
that a significant number of those AH neurons serving in a
multifunctional capacity as primary afferent neurons within the
myenteric plexus of T. spiralis-inflamed tissues could have become activated
during infection.
Our data are different from those of Ritter et al. (38) who were unable
to demonstrate increased expression of c-Fos in calbindin
immunoreactive neurons stimulated by distension, peristalsis, and
forskolin application. The reasons for this discrepancy are unclear but
might be reflective of the nature of the stimuli, possibly the actions
of released inflammatory mediators in our T. spiralis-infected animals that affect the excitability
of the AH-calbindin immunoreactive neurons. We believe these results strongly support our hypothesis that a functional reorganization of
intrinsic neural function is initiated during the enteric phase of
T. spiralis infection involving
transcriptional and translational activation of neuronal
immediate-early genes.
In conclusion, our intracellular electrophysiological investigations
have revealed the occurrence of functional alterations in single
enteric neurons during intestinal inflammation induced by enteric
parasitism. Our results clearly demonstrate that the electrical and
metabolic activities of jejunal myenteric AH neurons are significantly
increased during nematode infection. These changes are associated with
markedly elevated levels of the enzyme MPO, an index of granulocyte
infiltration into gut tissues. Furthermore, we have presented evidence
of the occurrence of excitation-transcription coupling in a significant
proportion of myenteric plexus neurons during T. spiralis infection, as well as in a large percentage of
those cells that are calbindin immunoreactive and possibly intrinsic
primary afferent neurons. Collectively, these findings suggest that a
functional reorganization of intrinsic neural function is initiated
during small intestinal nematode infection that could be involved in
the activation of intestinal motor responses that may subserve adaptive
or host defense purposes.
 |
ACKNOWLEDGEMENTS |
We express gratitude to Kara Doffek, Wayne Kaboord, and Winnie Ho
for expert technical assistance. We also thank Gerald Schneider, Bioimaging Facility, Department of Biomedical Sciences, Creighton University School of Medicine, for expert assistance with computerized graphics and imaging, and Dr. Wolfgang Kunze, Department of Physiology, University of Melbourne, for valuable comments on the manuscript.
 |
FOOTNOTES |
This work was supported in part by a Creighton Health Futures
Foundation Faculty Development Award and the National Institute of
Diabetes and Digestive and Kidney Diseases Grant DK-39647 to J. M. Palmer, the National Institute of Neurological Disorders and
Stroke Grant NS-18122 to M. Wong-Riley, and Research Funds from the
Medical Research Council of Canada, and the Crohn's and Colitis
Foundation of Canada to K. A. Sharkey. K. A. Sharkey is an Alberta
Heritage Foundation for Medical Research Senior Scholar.
Address for reprint requests: J. M. Palmer, Dept. of Biomedical
Sciences, Creighton Univ. School of Medicine, 2500 California Plaza,
Omaha, NE 68178.
Received 30 December 1997; accepted in final form 2 July 1998.
 |
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