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1Department of Physiology, University of Oklahoma Health Sciences Center, 73190; and 2Oklahoma Foundation for Digestive Research, Basic Science Laboratories, Veterans Affairs Medical Center, Oklahoma City, Oklahoma 73104
Submitted 10 January 2003; accepted in final form 8 March 2003
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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,
1997; Rozen and Schulkin 1998;
Shi and Davis 1999). However,
studies designed to investigate the spinal mechanism(s) that influence or
initiate autonomic and somatic components of the fear and anxiety reactions to
a variety of stressors are limited. Several behavioral studies have shown that
manipulation of the amygdala modulates neuronal networks in the lumbosacral
spinal cord that process information of noxious spinal reflexes
(Helmstetter and Bellgowan
1993; Helmstetter et al.
1993; Manning and Mayer
1995a,b).
Furthermore, stereotaxic delivery of corticosterone onto the amygdala
increases indices of anxiety (Shepard et
al. 2000) and results in hypersensitivity to colorectal distension
(Greenwood-Van Meerveld et al.
2001). The increased colorectal responsiveness induced by
corticosterone applied to the amygdala is due, at least in part, to
hyperexcitability of lumbosacral spinal neurons receiving afferent inputs from
the colon and rectum (Qin et al.
2003).
The amygdala has a high density of both mineralocorticoid (MR) and
glucocorticoid (GR) receptors, which are known to bind adrenal steroids and to
produce a variety of effects on neuroendocrine and autonomic systems
(Chao et al. 1989;
De Kloet et al. 2000;
Herman et al. 1996;
Reul and De Kloet 1985;
Sakai et al. 2000). High
densities of MRs have been identified in the amygdala, and along with GRs, are
linked to anxiety and fear responses (Calvo
and Volosin 2001; Gesing et
al. 2001; Korte
2001; Korte et al.
1996; Shepard et al.
2000). Corticosterone binds with high affinity to both GRs and
MRs, whereas aldosterone selectively binds to MRs
(De Kloet et al. 2000;
Pavlides et al. 1996).
However, our understanding of how aldosterone acts on MRs within the amygdala
to modulate neuronal activity related to fear and anxiety responses is
incomplete. Since corticosterone activates both MRs and GRs in the amygdala,
the present study was designed to determine the importance of MRs in the
development of colorectal hypersensitivity and the descending modulation of
spinal neuronal activity through the use of stereotaxic delivery of
aldosterone onto the amygdala. Our results showed that activation of MRs in
the amygdala with aldosterone produced a marked increase in a visceromotor
behavioral response to mechanical distension of the colorectum and generally
produced descending facilitation to lumbosacral spinal neurons receiving
noxious input from the colon and rectum. A preliminary report of this work has
been published in abstract form
(Greenwood-Van Meerveld et al.
2002).
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Experiments were performed on 36 male Fischer-344 rats (230–380 g)
purchased from Charles River (Wilmington, MA). This strain of rat was chosen
for the present study because they are considered low-anxiety animals
(Glowa and Hansen 1994;
Gunter et al. 2000;
Pare 1992). To reduce the
stress associated with laboratory environment, rats were acclimated to the
animal facility at least for 1 wk. Prior to the experiment, the animal was
fasted 18–24 h with free access to water. Animals were randomly divided
into two groups: in aldosterone-implanted rats (n = 18), micropellets
containing aldosterone were stereotaxically implanted bilaterally at the
dorsal margin of the amygdala, whereas the control rats (n = 18) had
cholesterol micropellets implanted at the same sites
(Greenwood-Van-Meerveld et al.
2001; Shepard et al.
2000). Except for three additional control rats used to determine
neuronal responses to colorectal distension (CRD), cholesterol-implanted rats
overlap control animals used in previous studies
(Greenwood-Van-Meerveld et al.
2001; Qin et al.
2003). Briefly, animals were anesthetized with a combination of
ketamine (80 mg/kg ip) and xylazine (10 mg/kg ip). Rats were mounted in a
stereotaxic headholder. A small hole was made in the skull at the coordinates
2.5 mm posterior to bregma and 4.2 mm of the right and the left side of
midline. A 25-gauge stainless steel cannula containing a micropellet of
aldosterone (30 µg) or cholesterol (30 µg) was lowered 7.0 mm dorsally
from the dura matter to the dorsal margin of the central amygdaloid nucleus
(Paxinos and Watson 1986). The
micropellet was then expelled, the cannula was removed, and gel foam was
placed in the holes of the skull. Analgesic cream and antibiotic was spread
around the wound after the skin was closed. Animals were moved back to their
cages. The manipulation produces anxiety-like behavior in rats without obvious
spread of the aldosterone to other structures in the region of the amygdala
(Shepard et al. 2000). The
Institutional Animal Care and Use Committee of the University of Oklahoma
Health Sciences Center approved the experiments performed in this study.
Visceromotor recordings
For the 7 days following the amygdala implantation, rats were acclimated to
the laboratory and experimenter. On day 7 postimplantation, the level of
colorectal sensitivity was determined in response to mechanical distension
using the technique of Ness and Gebhart
(1988), in which a
visceromotor behavioral response (VMR) is recorded in unrestrained, freely
moving rats. The VMR is a reflex contraction of the abdominal musculature
induced by colorectal distension. To record the VMR, a strain gauge force
transducer (R.B. Products, Stillwater, MI) was positioned to follow the
direction of the right external oblique muscle and was carefully sutured (7
standard stitches, 3-0 silk) under anesthesia with isofluorane (2.5%). The
skin was sutured over the strain gauge, and the lead wires were looped around
the animal's flank and secured by a single skin suture. During the experiment,
the strain gauge was connected via a shielded cable to a chart recorder (Grass
Instruments, Quincy, CA) to monitor the number of abdominal muscle
contractions. A 5-cm latex balloon catheter was inserted via the anal canal 11
cm into the colon and secured with surgical tape wrapped around the tail. The
anesthesia and the surgical procedure lasted about 10 min, and the rats were
allowed to recover for 30–45 min before initiating the experiment.
Following recovery from the anesthesia, the number of abdominal muscle
contractions under basal conditions (colorectal balloon inserted but not
distended) were recorded for 10 min and visually displayed on a chart
recorder. The enhanced number of abdominal muscle contractions in response to
CRD were displayed on the chart and throughout the experiment the number of
abdominal contractions were determined manually directly from the chart
recording.
Spinal neuronal recording
Seven days following implantation, animals were initially anesthetized with sodium pentobarbital (60 mg/kg ip). The right carotid artery and left jugular vein were cannulated to monitor blood pressure or to inject saline and drugs during the experiment, respectively. A continuous intravenous infusion of pentobarbital (15–20 mg/kg/h) maintained anesthesia throughout the experiment. A volume-controlled respirator was used to provide artificial ventilation (50–55 strokes/min, 3.0–4.0 ml stroke volume). Animals were paralyzed with pancuronium bromide (0.4 mg/kg ip) and were given supplemental doses (0.2 mg/kg ip) during experiments. Colorectal temperature was kept at 36.7–37.3°C using a thermostatically controlled heating blanket and overhead infrared lamps.
A laminectomy was performed to expose lumbosacral spinal segments (L6–S1) for recording spinal neurons. Rats were mounted in a stereotaxic headholder and two spinal clamps were fixed on the thoracic (T10–T12) and on the sacral vertebrae to a metal frame. The dura mater of exposed spinal segments was removed. A small well was made with dental impression material and filled with agar (3–4% in saline) to improve recording stability and to protect the dorsal surface of the spinal cord from dehydration. Carbon-filament glass microelectrodes were used to record extracellular activity of single spinal neurons from midline to 2 mm lateral and 0–1.2 mm depth from dorsal surface of the spinal cord. In general, we searched for spinal neurons with spontaneous discharges with amplitudes that were large enough for analysis. Sometimes a burst of discharges that later disappeared could be recorded when the microelectrode was close to a neuron. This phenomenon made it possible to find and study responses of neurons that did not have spontaneous activity. Signals were displayed on and stored in a computer using Spike-2 software (Cambridge, UK). The data were analyzed after experiments.
CRD
To produce a colorectal stimulus, a 4–5 cm long latex balloon
connected to a sphygmomanometer was inserted into the descending colon and
rectum. CRD was induced by inflating the balloon with air. In the freely
moving rat model used to determine the levels of colorectal sensitivity, CRD
was performed using constant pressure distensions at 30 mmHg and the VMR was
recorded for 10 min. This stimulus induces the VMR that closely resembles that
seen in responses to a nociceptive distention pressure causing minimal
discomfort to the rat with a normosensitive and sensitized colon
(Gunter et al. 2000;
Plourde et al. 1997). In the
anesthetized preparation used for spinal neuronal recording, CRD was performed
at distension pressure of 80 mmHg for 20 s and was used as a noxious searching
stimulus (Ness and Gebhart
1987; Qin et al.
1999,
2003). Neurons responding to
CRD at 80 mmHg were tested with this stimulus two to three times to make sure
responses were consistent and repeatable. Then, graded distensions of 10, 20,
40, 60, and 80 mmHg pressure for 20 s at >1-min intervals were
administered. Stimulus-response curves of spinal neurons to graded CRD were
determined. Threshold pressure for the response of each neuron was calculated
by extrapolation of least-squares regression line derived from the
stimulus-response curve (Ness and Gebhart
1987,
1988).
Somatic fields
Neurons were characterized for cutaneous receptive fields with innocuous stimulation, using a camel-hair brush or light pressure from a blunt probe, and with noxious pinch of skin and muscles with blunt forceps. Neurons were classified as follows: wide dynamic range (WDR) cells responded to brushing the hair or light pressure of skin and had greater responses to noxious pinching of the somatic field; high-threshold (HT) cells responded only to noxious pinching of the somatic field; and low-threshold (LT) cells responded primarily to brushing stimuli. If a cutaneous receptive field was not found, movement of tail (MT) was tested.
Histology
After neurons responsive to CRD were studied, an electrolytic lesion (50
µA DC, anodal for 20 s, cathodal for 20 s) was made at the recording site
to mark its location. At the end of the experiment, the animal was killed with
an overdose of pentobarbital (>120 mg/kg). The lumbosacral spinal cord was
removed and placed in 10% buffered formalin solution. Frozen sections
(55–60 µm) of the lumbosacral cord were viewed to identify lesion
sites using the cytoarchitectonic scheme of Molander et al.
(1984).
Data analysis and statistical testing
The VMR to CRD was measured as the number of abdominal muscle contractions registered during the 10-min distension period. Three consecutive responses were measured in a single animal. Data are reported as the mean ± SE for each group (n = 8 rats). Comparisons between groups were made using the Student's unpaired t-test. Differences were considered significant at P < 0.05.
Neuronal activity was stored and evaluated on rate histograms (1 s/bin).
Spontaneous activity of neurons was determined by counting activity for 10 s
and then dividing by 10 to obtain impulses per second (imp/s). A CRD-evoked
response (imp/s) was calculated by subtracting the mean of 10 s of spontaneous
activity from the mean of 10 s of the maximal activity during CRD. Statistical
significance was assessed using Student's paired or unpaired t-test
and 
2 analysis. Differences were considered statistically
significant at P < 0.05. Slopes of stimulus-response curves
obtained from neurons examined for graded CRD were compared between
aldosterone- and cholesterol-implanted animals. Descriptive data are presented
as means ± SE.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Under basal conditions, with the colorectal balloon catheter inserted but not distended, the number of abdominal muscle contractions per distension period was not significantly different between cholesterol and aldosterone implanted rats (Fig. 1). Although 30 mmHg of colorectal balloon distention significantly increased the number of abdominal muscle contractions in both cholesterol- and aldosterone-implanted rats, aldosterone-implanted rats had significantly more abdominal muscle contractions during the 10-min distension period compared with rats with cholesterol implants (Fig. 1).
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Spinal neuronal recordings
A total of 349 spinal neurons recorded from L6–S1 spinal segments were examined for colorectal and somatic stimuli. Noxious CRD (80 mmHg) changed the activity of 68/182 (37%) spinal neurons recorded from aldosterone-implanted rats and from 56/165 (34%) neurons recorded from control rats with cholesterol implanted onto amygdala. Lesions made at the recording sites were identified histologically for 17 CRD-responsive neurons in aldosterone-implanted rats and 17 neurons in control rats. Distribution of these neurons was mainly located in laminae V, VI, VII, and X (Fig. 2).
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Response patterns
As described in our previous studies
(Qin et al. 1999, 2002), in
general, four patterns of neuronal responses to CRD were observed, and the
neurons were classified as excitation (E), inhibition (I),
excitation-inhibition (E-I), and I-E. Examples of different patterns of
responses to CRD are shown in Fig.
3. No significant difference between the proportions of
CRD-response patterns in aldosterone- and cholesterol-implanted animals was
found (Fig. 3, A and
B). A comparison of characteristics of spontaneous
activity and CRD-evoked responses of spinal neurons from aldosterone-implanted
and control animals are given in Table
1. The average magnitude and duration of responses in E and E-I
neurons recorded from aldosterone implanted rats was significantly greater and
longer than those recorded from control rats
(Table 1). Additionally, the
mean inhibitory responses to CRD in aldosterone-implanted groups were
significant greater than those in the cholesterol control group
(Table 1). The distribution of
the neuronal sites of the four patterns of CRD-responsive neurons within the
gray matter of the spinal cord in aldosterone-implanted animals was not
different from control groups (Fig.
2). Of neurons excited by CRD, the proportion of neurons with
higher spontaneous activity (>0.5 imp/s) in the aldosterone-implanted group
was significantly greater than in the control group (36/42 vs. 19/33,
P < 0.01).
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Short- and long-lasting responses
Based on the recovery time of cell activity to the control level following
noxious CRD (80 mmHg), E and I neurons responding to CRD were further
subdivided into two groups (Qin et al.
1999,
2003): neurons with recovery
time <5 s were classified as short-lasting excitatory (SL-E;
Fig. 4, A, B, and
G) or inhibitory (SL-I;
Fig. 4, C and
H), and neurons with recovery time >5 s were
classified as long-lasting excitatory (LL-E;
Fig. 4F) or inhibitory
(LL-I; Fig. 4I). The
proportion of these subgroups in aldosterone-implanted rats was not different
from those in control animals (Fig.
3C). A quantitative analysis of spontaneous activity and
excitatory responses to noxious CRD (80 mmHg) in SL-E and LL-E neurons is
shown in Table 2. SL-E neurons
in aldosterone-implanted rats had significantly greater magnitudes of
CRD-evoked responses than those of SL-E neurons in control rats (P
< 0.05, Table 2).
Furthermore, average durations of CRD-evoked responses in LL-E neurons of
aldosterone-implanted groups were significant longer than those in control
animals (P < 0.05, Table
2). In some neurons, responses to graded CRD (10, 20, 40, 60, 80
mmHg, 20 s for each distension) were then examined. The examples are shown in
Fig. 4. Slopes of
stimulus-response curves of SL-E neurons recorded from aldosterone-implanted
rats were significantly higher than those in control rats, although no
difference was found in LL-E neurons (Fig.
5, A and B).
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LT and HT responses
Based on the responsiveness of lumbosacral spinal neurons receiving input
from the colon, neurons were subdivided into two groups: LT neurons that
initially responded to intracolorectal pressure ≤20–30 mmHg; and HT
neurons that responded to ≥40 mmHg pressure of CRD
(Andrew and Blackshaw 2001;
Qin et al. 2003). Examples of
these neurons are shown in Fig.
4. LT neurons with excitatory responses to CRD were more
frequently encountered in aldosterone-implanted rats than in control rats
(35/39 vs. 18/31, P < 0.01,
Fig. 3D). The
relationship between neural responses and graded CRD in aldosterone-implanted
and control groups is shown in Fig. 5,
C and D. The slopes of stimulus-response curves
of LT-E or HT-E neurons in aldosterone-implanted rats were not different from
those in control rats (Fig. 5, C
and D). The LT-E and HT-E neurons were combined to
calculate the extrapolated threshold pressures. In rats with
aldosterone-implanted amygdala, mean threshold pressure for neuronal
excitatory responses to CRD (2.2 ± 0.7 mmHg, n = 39) was
significantly lower than mean threshold pressure in the cholesterol-implanted
group (4.6 ± 1.0 mmHg, n = 31, P < 0.05).
Responses to somatic inputs
Of 124 neurons responsive to CRD that were tested for somatic inputs, 62/68 (91%) neurons recorded from aldosterone-implanted rats received convergent inputs from cutaneous receptive fields and tail rotation. This percentage was not different from 52/56 (93%) neurons recorded in control animals. Cutaneous receptive fields were generally on the ipsilateral scrotum, perianal region, areas around tail root, and lower back part of the body (Fig. 6, A–C). However, of viscerosomatic convergent neurons, the number of WDR neurons recorded from aldosterone-implanted rats was significantly more than in control animals (P < 0.01, Fig. 6D), whereas HT neurons were less frequently encountered in aldosterone-implanted animals than in the control group (P < 0.05, Fig. 6D). Of spinal neurons without responses to CRD, a difference between proportions of HT and MT neurons was found in aldosterone-implanted and control groups (Fig. 6E).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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). Furthermore, we observed that long-term application of
aldosterone onto the amygdala enhanced the responsiveness of lumbosacral
spinal neurons to CRD. The results showed an increase in the average magnitude
and duration of excitatory responses of spinal neurons to noxious CRD and a
decrease in average threshold pressure for excitatory responses to CRD. In
general, these findings are consistent with changes in the properties of
lumbosacral spinal neurons produced by stereotaxic delivery of corticosterone
onto the amygdala (Qin et al.
2003). However, some significant quantitative differences were
found in the properties of subpopulations of spinal neurons responsive to CRD
between this study in which aldosterone was stereotaxically administered onto
the amygdala and our previous study with corticosterone
(Qin et al. 2003). These
differences likely reveal different roles for MRs and GRs within the amygdala
in triggering and maintaining primary central hyperexcitability accompanying
colorectal hypersensitivity. Modulation of the amygdala with glucocorticoids
Evidence exists to show that in addition to acting through classic
cytosolic receptors, mineralocorticoids may act directly on MRs on the
amygdaloid neuronal membrane to regulate cellular function
(De Kloet et al. 2000;
Sakai et al. 2000), resembling
that seen in kidney and vascular tissue. Specifically, the amygdala possesses
receptors that allow the mineralocorticoids to act through both genomic and
nongenomic mechanisms (membrane mode of action) to arouse salt appetite
(Sakai et al. 2000). Therefore
intact cell bodies in the amygdala appear to be necessary for the normal
expression of mineralocorticoid-induced effects when the steroids are given
locally or systemically. In the present study, to avoid damage of cell bodies,
micropellets containing aldosterone were stereotaxically and bilaterally
implanted at the dorsal margin of the central amygdala.
Neurotransmission in the amygdala related to fear and anxiety is not
understood completely. Some neurotransmitters, i.e., opiate, GABA, and
N-methyl-D-aspartate (NMDA), are involved in the
neurotransmission of inter-amygdala connections that transmit information to,
within, and out of the amygdala to regulate fear and anxiety responses
(Davis et al. 1994).
Currently, corticotropin releasing factor (CRF) is considered as a key
mediator and modulator of both anxiety and colorectal hypersensitivity
(Gray 1993;
Gray and Bingaman 1996;
Gue et al. 1997;
Heilig et al. 1994;
Holsboer 1999;
Owens and Nemeroff 1991).
Evidence supports the possibility that CRF may exert its effects by direct or
indirect activation of MRs in the amygdala
(Gesing et al. 2001). Since
MRs may be involved in stimulation of the
hypothalamic-pituitary-adrenocortical (HPA) axis, the interaction between CRF
and MRs presents a novel mechanism involved in the adaptation of the brain to
psychologically stressful events (Korte et
al. 1993; Oitzl et al.
1994). In support of this association, recent studies have shown
that stereotaxic administration of corticosterone to the amygdala increases
both the number of neurons expressing CRF as well as the level of CRF mRNA
within these neurons (Shepard et al.
2000). Moreover, MR occupancy by corticosterone is required for
the stimulatory effects of CRF on MR levels
(Gesing et al. 2001).
Therefore it is likely that colonic hypersensitivity induced by modulation of
the amygdala with glucocorticoids potentially involves MR-mediated release of
CRF. Thus activation of MRs by aldosterone implanted in the amygdala exerts
descending effects on spinal neurons responsive to CRD, at least in part,
through enhanced CRF expression in the amygdala.
Spontaneous activity
Spontaneous activity (>0.5 imp/s) was observed in 86% of the spinal
neurons excited by CRD in aldosterone-implanted rats compared with neurons in
control rats (58%). This result is different from our previous study
(Qin et al. 2003), in which
the ratio of silent and active neurons excited by CRD in
corticosterone-implanted rats was similar to those in control animals.
However, no significant difference in average spontaneous activity of
lumbosacral spinal neurons with colorectal inputs was observed between
aldosterone-implanted and control animals in the present study. This
observation is consistent with the previous study, which examined the
properties of lumbosacral spinal neurons in rats with corticosterone-implanted
amygdala (Qin et al.
2003).
In contrast to results in this and the previous study
(Qin et al. 2003), acute
inflammation of the colon with mustard oil or turpentine induces an increase
in spontaneous activity of postsynaptic dorsal column or spinal neurons
responsive to CRD (Al-Chaer et al.
1997; Ness and Gebhart
2001). However, after colon inflammation with dilute acetic acid
(0.6%), the spontaneous activity of spinal neurons with colorectal inputs
increased in only 1/12 neurons tested
(Olivar et al. 2000). In the
current study, the colon was neither inflamed nor infused with agents that
would cause primary peripheral hypersensitivity; the only challenge was
stereotaxic implantation of aldosterone onto the amygdala. It is thus
possible, based on our findings, that primary central sensitization triggered
and maintained by modulation of the amygdala with aldosterone has a different
effect on spinal neuronal responsiveness compared with secondary central
sensitization produced by peripheral inflammatory agents.
Responses to CRD
Based on the duration of responses to CRD, spinal neurons are categorized
as short- and long-lasting responses (Qin et al.
1999,
2003), which are similar to
the abrupt and sustained neurons described by Ness and Gebhart
(1987,
1988). Some of these neurons
have long ascending axonal projections to the brain, are inhibited by
analgesics, and are modulated by spinal inputs from distant segments
(Ness 2000; Ness and Gebhart
1987,
1988;
Qin et al. 1999). These
subpopulations may have different roles in the production and development of
CRD-related sensations and reflexes (Ness
and Gebhart 2001). In the present study, no difference in the
proportions of short- or long-lasting responses to CRD in
aldosterone-implanted and control animals was found. This observation differs
from a previous study, in which spinal neurons with SL-E responses were
encountered more frequently in the corticosterone-implanted rats compared with
the control group. Furthermore, the slope of the stimulus-response curve in
SL-E neurons increased in aldosterone-implanted rats compared with control
groups, whereas in the corticosterone implanted animals, the slopes of both
SL-E and LL-E neurons were higher than in control animals
(Qin et al. 2003). The higher
slopes of stimulus-response curves observed in either aldosterone- or
corticosterone-implanted rats suggested that neurons became more sensitive to
activation by colorectal afferent inputs at each intra-colonic pressure
measured. The difference observed in stimulus-response slopes for LL-E neurons
between aldosterone- and corticosterone-implanted rats may be due to the
selective occupation of MRs with aldosterone compared with the occupation of
both MRs and GRs with corticosterone.
Spinal neurons that respond to CRD in a graded fashion from the nonnoxious
range to noxious range of distending pressure are classified as LT and HT
neurons (Andrew and Blackshaw
2001). This study showed that the average threshold pressure for
excitatory responses of spinal neurons to CRD significantly decreased in rats
with aldosterone-implanted amygdala compared with control animals. Also, the
proportion of LT neurons with colorectal inputs in aldosterone-implanted
animals was significantly larger than those in control animals. The results
generally are consistent with observations in rats with corticosterone
implanted onto the amygdala (Qin et al.
2003). The decreased threshold for excitatory responses to CRD and
the higher proportion of LT neurons responding to CRD after aldosterone
implantation in the amygdala correlate well with a hypersensitive colon as
demonstrated by an exaggerated visceromotor response to innocuous colorectal
distension (Greenwood-Van Meerveld et al.
2001). These results are also consistent with a generalized
phenomena, i.e., nociceptive hypersensitivity to CRD can be produced by
colorectal pathological stimuli in animals with colorectal inflammation
(Cervero 1995;
Gebhart 2000) and patients
with functional bowel disorders such as irritable bowel syndrome where there
in no obvious inflammation (Mertz et al.
1995; Naliboff et al.
1997). Because no treatment was used to inflame the colon in the
current study, the decrease in average pressure threshold for neuronal
responses to CRD most likely is due to the changes of descending influences
from the amygdala rather than by alteration of the sensitivity of visceral
receptors and/or peripheral afferent fibers innervating the colon. Taken
together, our findings may explain why episodes of stress and anxiety worsen
symptoms in patients with functional bowel disorders.
Responses to somatic inputs
Both WDR and HT neurons in the spinal dorsal horn are considered as
nociceptive neurons for encoding the intensity of noxious cutaneous stimuli.
Administration of peripheral stimulation may change response properties of
spinal neurons or result in a reclassification of neural type from one
classification to another. In this study, significantly higher numbers of WDR
neurons and fewer HT neurons responsive to CRD were observed in
aldosterone-implanted rats than in control animals. This finding implies that
neurons with CRD inputs became more sensitive to nonnoxious cutaneous stimuli
in rats that had aldosterone implanted onto the amygdala. One possibility is
that HT neurons might be reclassified to WDR neurons because of changes in the
excitability of the HT neurons and/or an increase in the synaptic efficacy.
However, this phenomenon was not found in corticosterone-implanted rats
compared with cholesterol-implanted rats
(Qin et al. 2003). These
results suggest that aldosterone in amygdala is more likely to induce changes
in the modulation of somatic inputs compared with effects observed previously
with corticosterone in amygdala. Changes of somatic field properties in spinal
neurons with CRD inputs have been found in rats with colonic inflammation, in
which the number of WDR neurons significantly decreases after turpentine is
applied to the colon (Ness and Gebhart
2001). This differs from other studies, in which no significant
changes in the neuronal responses to cutaneous stimuli and in cutaneous
receptive fields were observed, even though colonic inflammation increased the
responses of lumbosacral spinal neurons and postsynaptic dorsal column neurons
to CRD in rats (Al-Chaer et al.
1997; Oliver et al. 2000). Clinical studies in patients with
irritable bowel syndrome have shown an enhancement of sensitivity that appears
to be limited to the gut, since they are not hypersensitive to hand immersion
in the ice water test or electrical stimulation of the hand
(Accarino et al. 1995;
Cook et al. 1987;
Zighelboim et al. 1995).
However, there are other studies in patients with irritable bowel syndrome who
present with both visceral and cutaneous hyperalgesia in the hand and foot
(Chang et al. 2000;
Mertz et al. 1995;
Naliboff et al. 1997;
Verne et al. 2001). The reason
for these differences is unclear.
Relative contribution of MRs and GRs
In general, both MRs and GRs process information relative to critical
adaptive behaviors and mediate neuroendocrine responses to fear, anxiety, and
stress in a coordinated manner. However, some differences exist in their
effects with respect to processing information related to stress. First, MRs
operate in a proactive mode determining the sensitivity of the stress response
system, whereas GRs facilitate recovery from stress in a reactive mode
(De Kloet et al. 2000).
Second, on the neuronal level, MR-mediated action maintains a stable
excitatory tone and attenuates the influence of modulatory signals. In
contrast, GR-mediated effects suppress excitability transiently raised by
excitatory stimuli (De Kloet et al.
2000). Third, MR sites are predominantly occupied under basal
serum corticosterone levels, whereas levels obtained during stress or
circadian peaks are necessary to saturate GR sites
(De Kloet et al. 1991;
Reul and De Kloet 1985;
Reul et al. 1987). Fourth, MRs
and GRs exert different effects on anxiety caused by various stressors. For
example, MRs could thus be involved in the expression of fear-induced freezing
behavior, whereas GRs could participate in the mechanisms underlying
generalization of anxiety (Korte et al.
1996). Forced swimming and novelty stress evoked an increase in MR
density, whereas cold exposure was ineffective
(Gesing et al. 2001). MRs and
GRs could thus regulate restraint-induced anxiety through effects on
perception and cognitive processes, respectively
(Calvo and Volosin 2001).
Finally, the amygdala has wide projections to autonomic-related visceral
centers in the brain and brain stem, including the lateral hypothalamus, the
periaqueductal gray, dorsal motor nucleus of the vagus, nucleus of the
solitary tract, parabrachial nucleus and raphe nuclei
(Davis et al. 1994). Our data
from the current study suggest that MRs and GRs may activate different
descending pathways to induce and develop primary central hyperexcitation in
spinal processing. Differences mentioned above in the functions of MRs and GRs
may be explained by the different effect of selective activation of MRs in the
amygdala with aldosterone compared with activation of both MRs and GRs with
corticosterone. Thus although the possibility exists that corticosterone may
produce its effects through activation of GRs and/or MRs, our findings in rats
with aldosterone-implanted amygdala suggest an effect mediated by MRs.
In summary, aldosterone implanted onto the amygdala to activate MRs selectively can enhance the responsiveness of lumbosacral spinal neurons to visceral inputs from the noninflamed colon and somatic receptive fields. These results suggested that MRs in the amygdala mediate the production and development of primary central hypersensitivity, which may result from a change of balance of descending inhibitory and facilitatory systems to modulate spinal neuronal activity. Findings in this study support a concept that primary central sensitization induced by chemical activation of the amygdala plays an important role in spinal neuronal processing for augmentation of visceromotor reflexes that, through correlative connections, may be linked to emotional and autonomic responses to anxiety and stress.
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-57028.
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FOOTNOTES |
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Address for reprint requests: C. Qin, Dept. of Physiology, Univ. of Oklahoma Health Sciences Center, P.O. Box 26901, Oklahoma City, OK 73190 (E-mail: chao-qin{at}ouhsc.edu).
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, neurons excited by
CRD. 
, neurons inhibited by CRD. 
, neurons excited/inhibited by
CRD. B: spinal laminae of gray matter of L6 segment
drawing from Molander et al. (1989). I-X, laminae; Liss, Liss's tract; LSN,
lateral spinal nucleus; Pyr, pyramidal tract; IM, intermedial nucleus.
C: neurons responsive to CRD in aldosterone-implanted animals.
