Download Raphe Magnus Neurons Respond to Noxious Colorectal Distension

J Neurophysiol 89: 2506 –2515, 2003.
First published November 13, 2002; 10.1152/jn.00825.2002.
Raphe Magnus Neurons Respond to Noxious Colorectal Distension
Thaddeus S. Brink and Peggy Mason
Committee on Neurobiology and Department of Neurobiology, Pharmacology and Physiology, University of Chicago, MC 0926, Chicago,
Illinois 60637
Submitted 18 September 2002; accepted in final form 28 October 2002
Neurons in the medullary raphe magnus (RM) and the adjacent nucleus reticularis magnocellularis (NRMC) project to
the dorsal horn of the spinal cord (Basbaum and Fields 1978,
1979; Holstege and Kuypers 1982) where they modulate cutaneous nociceptive input (Fields et al. 1991; Mason 2001;
Sandkuhler 1996). Activation of RM and NRMC neurons can
both suppress and facilitate noxious heat-evoked motor and
cellular responses (Zhuo and Gebhart 1990, 1992a, 1997),
while inactivation or lesioning of RM and NRMC eliminates
both facilitation and suppression (Behbehani and Fields 1979;
Chung et al. 1987; Gebhart et al. 1983; Kaplan and Fields
1991; Sandkuhler and Gebhart 1984). This evidence indicates
that at least two cell classes exist within RM— one that facilitates and one that inhibits cutaneous nociception. Electrophysiological studies have characterized two classes of neurons that
are hypothesized to underlie the nociceptive inhibitory and
facilitatory effects of RM activation (reviewed in Fields et al.
1991; Mason 2001). ON cells are excited by noxious cutaneous
stimuli and inhibited by opiates and are thought to facilitate
nociceptive transmission. In contrast, OFF cells are inhibited by
noxious cutaneous stimuli, excited by opiates, and are thought
to inhibit nociceptive transmission. Both ON and OFF cells are
nonserotonergic (Gao and Mason 2000; Mason 1997; Potrebic
et al. 1994).
These results suggest that one physiological response—that
evoked by noxious tail heat— can predict the function of ON
and OFF cells. This is surprising because ON and OFF cell
responses to noxious tail heat are not perfectly correlated with
responses to noxious stimuli of other modalities (mechanical,
chemical) or applied to other cutaneous regions (Ellrich et al.
2001). Because there is limited information available regarding
the responses of RM and NRMC neurons to visceral stimulation (Chandler et al. 1994; Guilbaud et al. 1980; Lumb 1986;
Snowball et al. 1997), the present experiments were designed
to determine how ON and OFF cells respond to a physiological
visceral stimulus, distension of the colon and rectum (CRD).
A third type of nonserotonergic RM/NRMC neuron, the
NEUTRAL cell, does not respond to either noxious tail heat or
opioids. However, NEUTRAL cells typically respond to other
cutaneous somatosensory stimuli (Ellrich et al. 2000; Leung
and Mason 1998), leading to the idea that they modulate
responses to noxious stimulation of a different intensity or
modality than the tail heat stimulus used in most studies.
Finally, serotonergic neurons comprise a distinct cell class
distinguished by their unique neurochemistry and physiological discharge rate and pattern (Gao and Mason 2000; Mason
1997). Although most serotonin-containing RM and NRMC
neurons do not respond to either noxious cutaneous heat or to
opioids (Gao and Mason 2000; Gao et al. 1998), a majority
respond to mechanical stimulation within the retroperitoneum
(Gao and Mason 2001b). Together with the finding that serotonin receptor antagonists attenuate the heterotopic antinociception evoked by CRD (Zhuo and Gebhart 1992b), the sensitivity of serotonergic cells to retroperitoneal stimulation suggests that serotonergic cells may respond to CRD.
The studies reviewed in the preceding text provide indirect
evidence that cells in each of the physiological cell classes
contained in RM and NRMC may play a role in the modulation
of visceral nociception. The present investigation was designed
to address the physiological plausibility of such roles by examining whether cells in each cell class respond to CRD.
Therefore the responses of ON, OFF, NEUTRAL, and serotonergic
RM and NRMC cells to CRD were tested in lightly anesthetized rats.
Address for reprint requests: P. Mason, Dept. of Neurobiology, Pharmacology, and Physiology, University of Chicago, MC 0926, 947 E. 58th St.,
Chicago, IL 60637 (E-mail: [email protected]).
The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked ‘‘advertisement’’
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
INTRODUCTION
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0022-3077/03 $5.00 Copyright © 2003 The American Physiological Society
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Brink, Thaddeus S. and Peggy Mason. Raphe magnus neurons
respond to noxious colorectal distension. J Neurophysiol 89:
2506 –2515, 2003. First published November 13, 2002; 10.1152/
jn.00825.2002. Physiological studies of neurons in raphe magnus
(RM) and the adjacent nucleus reticularis magnocellularis (NRMC)
have demonstrated that the response to noxious cutaneous stimulation
predicts the response to opioid administration and therefore a cell’s
functional role in nociceptive modulation. Although visceral stimulation, like opioids, elicits antinociception, little is known about how
RM and NRMC cells respond to visceral stimulation. Therefore RM
and NRMC cells were tested for their responses to both colorectal
distension (CRD) and noxious cutaneous heat in halothane-anesthetized rats. Less than a third of serotonergic cells responded to CRD
with small increases or decreases in discharge rate. In contrast, almost
two-thirds of nonserotonergic cells responded to CRD stimulation
with either excitatory (35%) or inhibitory (30%) responses to CRD.
The response to heat did not predict the response to CRD with nearly
equal proportions of heat-excited, -inhibited, and -unaffected cells
being excited, inhibited, or unaffected by CRD. The dissociation
between the responses to cutaneous heat and CRD demonstrates that
cell classes based on the response to noxious heat are not homogeneous and may play multiple functional roles.
RAPHE MAGNUS NEURONS RESPOND TO COLORECTAL STIMULATION
METHODS
Surgery
Male Sprague-Dawley rats (n ⫽ 68, 250 –500 g; Charles River,
Portage, MI) were treated with atropine sulfate (40 ␮g in 0.1 ml sc)
and anesthetized with halothane. In early experiments, a Y tube was
inserted into the trachea, whereas in later experiments, halothane was
administered through a nose cone adapted for the stereotaxic. Halothane was maintained at 1.8 –2.0% in oxygen for the surgery. A
catheter was inserted into the femoral artery to record blood pressure.
Needle electrodes were placed bilaterally into the thorax to record the
electrocardiogram and into the paraspinous, quadriceps, and biceps
femoris muscles to record the electromyographic activity of the tail
and hindlimb muscles. A craniotomy was made over the cerebellum,
and the exposed dura was cut. Core temperature was maintained at
36 –38°C via a water-perfused heating pad. After the surgery, the
halothane concentration was lowered to 1% and the animal was
allowed to equilibrate for 30 – 60 min.
Both metal and glass microelectrodes were used. The glass micropipettes were filled with 2% Neurobiotin (Vector Labs, Burlingame,
CA) in a 0.1 M Tris (hydroxymethyl)aminomethane buffer (pH 7.4)
and 0.15 M KCl and were used with an impedance of 5–10 M⍀. Metal
electrodes were made of tungsten (A-M Systems, Pullman, WA).
Microelectrodes were lowered into the region of the RM and NRMC
(P 10.5–11.8 mm from bregma, L 0.0 –1.0 mm, V 8.5–10.5 from
cerebellar surface). Neurons were isolated by their spontaneous activity, and each extracellular unit that was successfully isolated was
studied. The unit waveform was acquired at 40 kHz by a CED
Micro1401 interface (CED, Cambridge, UK). Spike2 acquisition software (CED) stored the time of the spike and 30 digitized points from
the waveform. Individual waveforms were discriminated off-line using template-matching software.
Stimulation methods
Cells were tested for their responses to noxious tail or paw heat and
colorectal distension (CRD). Heat stimuli were applied using a peltier
device (Yale Instrumentation, New Haven, CT). For tail heat, the
peltier was placed on the ventrum of the tail at a point 6 –7 cm from
the distal tip, and for paw heat, the peltier was placed on the footpad
and toes of the hindpaw. Both the paw and the tail were affixed to the
peltier platform (2 cm square) so that they were exposed to the full
duration of the stimulus. Each heat stimulus consisted of a 2- to 3-s
ramp from 32 to 49 –56°C and a 4-s plateau at the peak temperature.
The peltier platform then ramped back down to 32°C over the course
of 3–7 s. Between thermal stimuli, the peltier platform was maintained
at 32°C.
To inflate the colon, the finger portion of a glove was secured onto
flexible tubing (Tygon R3603, 2 mm OD). This tubing was connected
to a 60-mm syringe with a side port connected to a manometer. The
glove finger was coated with petroleum jelly (Vaseline) and inserted
7–9 cm into the anus to a point just past the external sphincter muscle.
Unless otherwise stated, all distensions were applied for 20 s.
Experimental protocol
On isolation of a unit, repeated (2–5) trials of noxious cutaneous
heat (tail and/or paw heat) were interleaved with CRD trials. All
stimuli were separated by intervals of 3–5 min. In early experiments,
all cells were tested with heat intensities to 56°C and colorectal
distensions of 80 mmHg. In later experiments, the peak temperature of
the cutaneous stimulus was chosen to be the minimum that reliably
elicited a robust motor withdrawal and was typically 49 or 52°C and
initial CRD stimuli were applied at an intensity of 60 mmHg. If cells
J Neurophysiol • VOL
did not respond to the 60 mmHg CRD, the maximal intensity of 80
mmHg was applied. For each stimulus, three to five trials were
recorded.
The recording site for at least one cell per animal was labeled by
injection of current (5 min, 10-nA depolarizing for glass electrodes
and 4 min, 20-␮A hyperpolarizing for metal electrodes).
Histology
Animals were overdosed with 5% halothane and perfused with a
fixative containing 4% paraformaldehyde and 7% sucrose in 0.1 M
phosphate-buffered saline. The brain stem was removed, postfixed for
2–12 h, and then immersed in 30% sucrose in 0.1 M PBS. Coronal
sections (50 ␮m) were cut on a freezing microtome. Sections from
experiments with glass electrodes were processed for a DAB reaction
as previously described, and the site of Neurobiotin injection was
identified (Gao and Mason 1997). Lesion sites from experiments using
metal electrodes were recovered. Sections were mounted on gelatincoated slides and then stained with cresyl violet. Under ⫻50 magnification, the mediolateral and dorsoventral distances from midline and
the ventral midline edge of the section, respectively, were measured.
Sites were assigned an anterior-posterior location by comparison of
sections with a standard atlas (Paxinos and Watson 1986). Unlabeled
sites were located by their stereotaxic distance from marked recording
sites.
The nuclear boundaries used are modified from Newman (1985).
According to Newman, raphe pallidus (RP) extends rostrally to the
rostral pole of the inferior olive but does not continue into levels that
include the facial nucleus. It should be noted that this is different from
the nuclear boundaries illustrated in Paxinos’ atlas (Paxinos and
Watson 1986). Thus we considered RM to include a region 300 ␮m
wide centered on the midline and extending from the base of the brain
to a point 1,500 ␮m dorsal, at levels from –11.6 to ⫺10.4 relative to
bregma. A box representing this area is illustrated in Fig. 1, inset.
NRMC was considered to include a region that stretched laterally
from RM to the lateral edge of the pyramids and had a dorsal extent
of 1,000 ␮m. Cells dorsal to RM or NRMC were considered to be
located in nucleus reticularis gigantocellularis (NRGC).
Cellular analysis
Neurons were classified by their discharge pattern and their response to noxious heat as ON, OFF, NEUTRAL, or serotonergic cells
(Leung and Mason 1998). The mean (x) and SD (SDISI) of the
interspike intervals (ISI) were calculated from 15 min of discharge. In
addition, the coefficient of variation of the ISIs (CVISI) was calculated. Cells were classified as physiologically defined serotonergic
(p5HT) or nonserotonergic (non-p5HT) using a previously described
algorithm based on the rate and variability of discharge (Mason 1997).
For each cell, the value of the function, y(x, SDISI) ⫽ 146 ⫺x ⫹ 0.98
SDISI, was calculated, where x and SDISI are in ms. Cells were
classified as p5HT if the function value was less than zero and as
non-p5HT if greater than zero (Mason 1997). This algorithm has been
tested on nearly 100 cells and the probability of observed misclassification rate is ⬍10% (Gao et al. 1998; Gao and Mason 1997a,b, 2000,
2001a,b; Mason 1997, 2001).
As described previously, a quantitative method was used to determine whether responses to stimulation were different from spontaneously occurring changes in discharge (Leung and Mason 1998).
Briefly, the SD of the change across 10-s bins was calculated and
normalized to spikes/s (SD10s) as a measure of spontaneous variability
in discharge. Unit responses to stimulation were then calculated as the
difference in discharge before and after stimulus application (in
spikes/s). For both cutaneous heat and CRD stimulation, four sequential 10-s response periods were analyzed. Stimulus-evoked increases
in discharge that were ⬎2 SD10s were considered excitatory responses
and evoked decreases that were ⬎2 SD10s were considered inhibitory
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Electrophysiological methods
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RESULTS
Characterization of recorded cells: resting discharge and
response to noxious heat
responses. This method allows us to have confidence, at a P ⬍ 0.05
level, that a change evoked by a single stimulation trial represents a
response and is unlikely to have occurred spontaneously.
Any cell that responded to a majority of heat trials in the first
response period was considered responsive. Non-p5HT neurons that
were excited by tail or paw heat were considered ON cells, whereas
those that were inhibited were OFF cells. Non-p5HT cells that failed to
respond to cutaneous heat were NEUTRAL cells. Any cell that responded
to a majority of CRD trials in either of the first two response periods
(⫽stimulus duration) was considered responsive. By requiring a cell
to have a significant response to stimulation in most trials, the chance
of misidentifying a stimulation responsive cell is vanishingly small.
The duration of a cell’s response to either cutaneous heat or CRD
was defined by the number of sequential response periods in which the
cell was inhibited or excited (see Gao and Mason 2000). It is worth
noting that this analysis underestimates the time that a cell’s discharge
differs significantly from baseline values because it requires a change
that is sustained for 10 s and the threshold for significance is set very
stringently (see preceding text).
Statistics
Each variable is expressed as a mean ⫾ SE. Statistical tests were
performed using Microsoft Excel (Redmond, WA) or SigmaStat
(SPSS Science, Chicago, IL).
J Neurophysiol • VOL
Cellular response to CRD
Most of the p5HT cells (17/24; 71%) were unaffected by the
20-s CRD stimulus (Fig. 2A). Of the seven p5HT cells that
responded to CRD, three were excited and four inhibited (Fig.
2, B and C). Both the excitatory and inhibitory responses of
p5HT cells were small when present, averaging 13.6 ⫾ 6.6 and
–11.1 ⫾ 4.5 spikes/20 s, respectively. The ranges of excitatory
(3.8 –26.3 spikes/20 s) and inhibitory (⫺4.7 to ⫺24.3 spikes/20
s) responses were limited to less than one order of magnitude.
p5HT cell responses were also brief, being limited to the
duration of the stimulus presentation or less for five of the
seven p5HT cells responding (see Fig. 5).
Nearly two-thirds of non-p5HT cells (59/91; 65%) responded to CRD (Figs. 3 and 4). More than half of these
CRD-responsive non-p5HT cells that responded to CRD were
excited (n ⫽ 32) with a mean increase in cell discharge during
the 20-s CRD stimulus of 215.3 ⫾ 45.9 spikes (Fig. 4, A and
B). Excitatory responses ranged over more than two orders of
magnitude from 7.3 to 1,223.7 spikes (Fig. 5). When the
magnitudes of all non-p5HT cell responses were graphed in
ascending order, there was a discontinuity between responses
of ⱕ100 spikes and those that were 100 spikes/s (Fig. 4C).
Cells with the former responses were termed “small respond-
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FIG. 1. Distribution of recording sites for p5HT cells (■, Œ, ) and nonp5HT cells (䊐, ‚, ƒ) that were excited (Œ, ‚), inhibited (, ƒ), or unaffected (■,
䊐) by CRD. Inset: a line drawing of a section at –11.0 from bregma; the box
outlined (- - -) shows the area that is enlarged in each of the photomicrographs.
The labeled boxes show the regions considered to contain raphe magnus (RM)
and nucleus reticularis magnocellularis (NRMC) cells. In the interest of clarity,
all cell locations are shown on the right side of the brain, in some cases, at a
site exactly contralateral to their true location. Left to right: non-p5HT cells
that were unaffected, excited, and inhibited by colorectal distension (CRD) and
all p5HT cells (far right). The numbers indicate the distance from bregma for
each section within a row. VII, facial nucleus; NTB, nucleus of the trapezoid
body; p, pyramid.
A total of 115 cells were recorded from 68 rats. The recording sites were located within RM (n ⫽ 67), RP (n ⫽ 3), NRMC
(n ⫽ 25), or the ventral portion of NRGC (n ⫽ 7; Fig. 1).
Recording sites were concentrated at the level of the facial
nucleus but extended rostrally to the level of the trapezoid body
and caudally to the level of the inferior olive. The locations of
13 cells from eight animals could not be determined.
Using a quantitative analysis of the resting discharge (see
METHODS), 24 cells that fired slowly and steadily were classified
as p5HT. It should be noted that p5HT cells were located either
in RM (n ⫽ 14) or the ventral portion of NRMC (n ⫽ 8),
regions that contain many serotonergic cells (Fig. 1). The mean
discharge rate for p5HT cells was 2.0 ⫾ 0.2 spikes/s and the
mean CVISI was 0.41 ⫾ 0.03. A minority of the p5HT cells
(5/24) responded to noxious tail or paw heat with small
changes in discharge rate (⬍14 spikes in 10 s), consistent with
our previous report (Gao and Mason 2000). Of those that
responded, three were excited and two inhibited.
The remaining cells (n ⫽ 91) were non-p5HT and were
characterized as ON (n ⫽ 31; 34%), OFF (n ⫽ 15; 16.5%), or
NEUTRAL (n ⫽ 45; 49.5%) by their response to noxious tail (n ⫽
38) or paw (n ⫽ 53) heat (see Figs. 3 and 7). The background
discharge rates of ON, OFF, and NEUTRAL cells did not differ (P ⫽
0.9) and averaged 10.5 ⫾ 1.3 spikes/s. Although the majority
of these cells (50/91) discharged in a bursting pattern (CV ⬎1),
a minority (n ⫽ 28; 31%) had a regular discharge pattern
(CV ⬍ 0.5). Regularly discharging neurons were distributed
across each of the non-p5HT cell classes (13 NEUTRAL cells, 11
ON cells, 4 OFF cells) at the same proportion as nonregularly
discharging neurons (␹2, P ⫽ 0.99). All regularly discharging
non-p5HT cells had higher discharge rates (range: 6.4 – 80.4
spikes/s) than did p5HT neurons (range: 0.5–3.7 spikes/s).
RAPHE MAGNUS NEURONS RESPOND TO COLORECTAL STIMULATION
FIG. 3. Responses of a non-p5HT NEUTRAL cell to 3 trials each of noxious
tail heat and CRD. A: noxious tail heat (trace marked stimulus) elicited tail
withdrawal (not shown) and a pressor response (top) but did not consistently
change the discharge of this cell. B: CRD (80 mmHg; trace marked stimulus)
evoked a pressor response (top) and consistently excited this cell. In both
panels, cell discharge is shown as instantaneous discharge rate [1/interspike
interval (ISI)] and the time scale shown in A applies.
J Neurophysiol • VOL
FIG. 4. Mean excitatory and inhibitory responses of non-p5HT cells to
CRD (lines under traces in B and F). Only cells tested with 80 mmHg CRD are
included in the averages in A and B and D–F. In each panel, the gray region
shows the limits of the mean ⫾ SE of the response. A: the mean response for
cells that responded to CRD with an increase of ⱕ100 spikes in 20 s (n ⫽ 15).
B: the mean response for cells that responded to CRD with an increase of ⬎100
spikes in 20 s (n ⫽ 12). C: cells with excitatory responses to either 60 mmHg
(open symbols) or 80 mmHg (closed symbols) CRD were ranked and thereby
divided into small (below line) and large (above line) responders. D: the mean
response of cells that were inhibited by CRD and whose discharge remained
below baseline values after stimulus offset (n ⫽ 8). E: the mean response of
cells that were inhibited by CRD and whose discharge rebounded to a level
greater than baseline values after stimulus offset (n ⫽ 8). F: the mean response
of cells that were inhibited by CRD and whose discharge returned to baseline
values after stimulus offset (n ⫽ 5). The time scale in D applies to all panels
except C.
ers” (n ⫽ 18), and cells with the latter responses were termed
“large responders” (n ⫽ 14); the mean responses for each of
these groups are shown in Fig. 4, A and B. The majority (21/32)
of excitatory neuronal responses to CRD did not persist after
termination of the stimulus (Figs. 4, A and B, and 5). The
remaining cells, constituting a third of the total excited population, responded to CRD for ⱖ30 s (Fig. 5).
Just under half of the non-p5HT cells that responded to CRD
were inhibited (n ⫽ 27; Fig. 4, D–F). The mean CRD-evoked
decrease in cell discharge during the 20-s stimulus was
176.4 ⫾ 44.7 spikes and ranged over more than two orders of
magnitude from 7.7 to 1,147.8 spikes (Fig. 5). Most inhibitory
responses (19/27) did not persist after stimulus offset, either
returning to baseline values (n ⫽ 6; Fig. 4F) or rebounding to
a discharge rate above the baseline level (n ⫽ 10; Fig. 4E).
Because 40% of the inhibitory responses (n ⫽ 11) lasted ⱖ10
s longer than the stimulus, the mean total inhibitory response to
CRD (215.9 ⫾ 48.1 spikes) was much greater than the mean
response during the stimulus presentation alone (Figs. 4D
and 5).
A small proportion of the non-p5HT cells responded to CRD
in a complex pattern. For example in the cell illustrated in Fig.
6A, the cell’s discharge increased during the stimulus and then
went below baseline values after the stimulus offset. Such an
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FIG. 2. Responses of p5HT cells to CRD. A: this p5HT cell was unaffected
by CRD (80 mmHg; trace marked stimulus). The mean cell response (4 trials)
is seen in the solid histogram. The raster plots show responses to CRD during
4 individual trials. Top: average blood pressure and heart rate responses. B and
C: mean responses for p5HT cells that were excited (n ⫽ 3; B) or inhibited
(n ⫽ 4; C) by CRD. The gray region shows the limits of the mean ⫾ SE of the
response. The time scale in A applies to A only and that in C applies to B and
C. Bins are 0.5 s in all histograms.
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excitatory-inhibitory response was seen in three cells. Of the
10 cells that were inhibited by CRD and then rebounded to a
greater than baseline discharge rate, 6 increased their discharge
rate by ⬎2* SDISI in the 20- to 30-s response period after
stimulus offset (Fig. 6B). It is interesting to note that such
biphasic patterns, where the changes during and after the
stimulus are in opposite directions, were never observed in
response to noxious cutaneous heat (n ⫽ 46 in the present
study). Nine cells had no response to CRD during the stimulus
but consistently increased (n ⫽ 6; Fig. 6C) or decreased (n ⫽
3; Fig. 6D) their discharge after the stimulus offset. Similarly,
poststimulus excitatory (n ⫽ 9) and inhibitory (n ⫽ 1) responses to noxious cutaneous heat in NEUTRAL cells were observed (n ⫽ 10).
Relationship between responses to CRD and cutaneous heat
The majority of p5HT (13/24; 54%) cells did not respond to
either cutaneous heat or CRD. Most of the p5HT cells that
responded to CRD did not respond to heat stimulation (6/7),
and most of the cells that responded to heat stimulation did not
respond to CRD (4/5). One p5HT cell was inhibited by both
cutaneous heat and CRD.
J Neurophysiol • VOL
FIG. 6. Complex neuronal responses to CRD (80 mmHg). All histograms
are averages of a single cell’s response to 3–5 trials using a bin width of 0.25 s.
A: this cell’s discharge increased during the stimulus and then went below
baseline values after the stimulus offset. B: an example of a cell that was
inhibited during the CRD stimulus and then rebounded to a greater than
baseline discharge rate after stimulus offset. C: an example of a cell that had
no response to CRD during the stimulus but consistently increased its discharge after stimulus offset. D: an example of a cell that had no response to
CRD during the stimulus but consistently decreased its discharge after stimulus
offset. The mean stimulus application is shown below each histogram. The
scale in D applies to all panels.
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FIG. 5. The mean change in discharge evoked by CRD in all p5HT (Œ, })
and non-p5HT (‚, {) cells studied. For CRD-responsive cells (Œ, ‚), the
average magnitude and total duration of the response is shown. For CRDunresponsive cells (}, {), the average change in discharge during the 20-s
stimulus is shown for each cell. Increases and decreases in discharge are shown
on logarithmic scales. Response duration was measured in multiples of 10 s
(see METHODS) but symbols have been displaced to the left and right for
illustrative purposes.
For non-p5HT cells, the response to CRD was randomly
associated with the response to noxious heating of the paw or
tail (␹2, P ⫽ 0.7). Nearly equal numbers of ON cells were
excited (9/31), inhibited (n ⫽ 11), or unaffected (n ⫽ 11) by
CRD (Fig. 7A, top). Similarly, nearly equal numbers of NEUTRAL cells responded to CRD with an increase (16/45), decrease (n ⫽ 13), or no change (n ⫽ 16) in discharge (Fig. 7A,
middle). Finally, almost half of the OFF cells (7/15) were
excited by CRD and minorities were either inhibited (n ⫽ 3) or
unaffected (n ⫽ 5) (Fig. 7A, bottom). It is interesting to note
that the largest responses to CRD were observed in OFF cells
that were excited by CRD and ON cells inhibited by CRD
(Fig. 7A).
As illustrated in Fig. 7B, there was no obvious difference
between the response to noxious heat among cells with different responses to CRD. For example, the inhibitory responses of
OFF cells to noxious heat were similar among subpopulations
with dramatically different responses to CRD (compare Fig. 7,
A and B, bottom).
Excitatory responses to CRD were relatively nonadapting in
comparison with excitatory responses to heat that peaked immediately and then declined (Fig. 7). However, the inhibitory
responses to CRD and to heat were both nonadapting as they
were maintained throughout the stimulus. The inhibitory responses to CRD and to heat differed in their poststimulus form.
At stimulus offset, the discharge of most CRD-inhibited cells
returned quickly toward or beyond baseline values. In contrast,
among OFF cells, the return to baseline discharge rate after heat
offset was slow and gradual.
It is possible that responses to CRD and to heat are related
in some way that is obscured by the cell classification system
used. Therefore we compared the total change in spikes evoked
during the CRD and heat stimuli, regardless of whether that
change met our criteria for a response (Fig. 8). Figure 8 is a
double logarithmic plot that illustrates the mean change in
RAPHE MAGNUS NEURONS RESPOND TO COLORECTAL STIMULATION
2511
considered responsive to the stimulation because they do not
consistently change their discharge to a group of stimulation
trials.
Relationship between responses to CRD and cell location
There were no differences between the responses to CRD
among cells with different nuclear locations or cells located at
different mediolateral, dorsoventral or anterior-posterior sites.
DISCUSSION
CRD is a noxious visceral stimulus
In awake rats, CRD evokes a relaxation of the external anal
sphincter, a motor component of defecation, at intensities of
13–20 mmHg (Ness and Gebhart 1988b). At higher pressures,
FIG. 7. Mean cell responses to CRD (A) and noxious cutaneous heat (B) for
all non-p5HT cells. In each panel, the row denotes the response to noxious
cutaneous heat (ON, NEUTRAL, or OFF) and column denotes the response to CRD
(excited, unaffected, or inhibited). For instance, A, bottom left, shows the mean
response to CRD for OFF cells that were excited by CRD, whereas B, bottom
left, shows the mean response to noxious heat for the same population of cells.
All histograms are presented with the same vertical and time scales. The gray
region shows the limits of the mean ⫾ SE of the response. Bins are 0.25 s wide.
spikes evoked by CRD and heat for all p5HT and non-p5HT
cells. There are numerous non-p5HT cells in each quadrant of
the graph, supporting the idea that the response of non-p5HT
cells to heat cannot predict that cell’s response to CRD. It is
interesting to note that there are no p5HT cells that increase
their discharge in response to CRD but decrease their discharge
in response to heat.
Although no attempt was made to match the intensity of the
CRD and heat stimuli, the increases and decreases in nonp5HT cell discharge evoked by the two stimuli were similar in
magnitude (Fig. 8). Most cells, therefore fall close to the unity
lines illustrated in Fig. 8. Further, non-p5HT cells were equally
distributed on either side of the unity lines, demonstrating that
the mean population responses to CRD and to heat are approximately equal.
The changes in p5HT cell discharge evoked by CRD and
heat were consistently smaller than those observed in almost all
non-p5HT cells, even non-p5HT cells that were considered
unresponsive (Fig. 8). This reflects the finding that NEUTRAL
cells and cells unresponsive to CRD may change their discharge significantly in response to a minority of stimulation
trials (Leung and Mason 1998). However, these cells are not
J Neurophysiol • VOL
FIG. 8. A double logarithmic plot of the mean cell responses to CRD and
noxious cutaneous heat for all cells. For each cell, the average change in
discharge evoked during each stimulus, across all trials, is plotted. Thus for
both stimulus-responsive and -unresponsive cells, the evoked change is calculated as the change in spikes during the 10-s heat stimulus or the 20-s CRD
stimulus relative to the prestimulus baseline. In the case of the change evoked
by CRD stimulation, the number of spikes was divided by 2 and expressed in
units of spikes/10 s. Five cells that did not change their discharge rate in
response to one of the stimuli are plotted semi-logarithmically. Unity and unity
lines are illustrated. Both non-p5HT cells (E) and p5HT cells (ⴙ) are included.
Averages for non-p5HT cells (F) and p5HT cells (*) in each quadrant are also
illustrated.
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The present study demonstrates that most nonserotonergic
RM and NRMC cells respond to noxious intensities of CRD. In
contrast, few serotonergic neurons respond, and those that do
have weak responses. Although the response of nonserotonergic cells to noxious cutaneous heat predicts important pharmacological characteristics, it is clearly not predictive of the
response to CRD. The heterogeneity of responses to CRD
among ON, OFF, and NEUTRAL cells suggests that either important
functional subclasses of these cells exist or that the functional
importance of this classification system is limited.
2512
T. S. BRINK AND P. MASON
⬎22 mmHg, CRD evokes reactions that are consistent with its
being an aversive stimulus— hunching, tachycardia, and a
pressor response—in awake rats (Ness and Gebhart 1988b;
Ness et al. 1991). The latter two reactions are also observed in
lightly anesthetized rats. In the present study, all cells were
tested for their responses to either 60 or 80 mmHg distensions
for 20 s, stimuli that are clearly aversive to awake rats (Ness
and Gebhart 1988b; Ness et al. 1991).
Classification of serotonergic neurons
Serotonergic cells fail to respond to CRD
The paucity of serotonergic neurons that respond to CRD is
unexpected. Serotonin release from bulbospinal terminals has
been implicated in both the reaction to CRD and in the modulation of cutaneous nociception by CRD. Regarding the
former, an intracerebroventricularly administered 5HT3 receptor antagonist attenuates the blood pressure reaction elicited by
CRD in dogs (Miura et al. 1999). Together with the finding that
the pressor reaction evoked by CRD is greatly attenuated in the
spinalized rat (Ness and Gebhart 1988b), input from brain stem
serotonergic neurons may be critical to the production of the
behavioral reaction to CRD. Serotonin release has also been
implicated in the suppression of cutaneous heat-evoked withdrawals by CRD because serotonin receptor antagonists attenuate this suppression (Zhuo and Gebhart 1992b). However,
only 7 of 24 serotonergic neurons recorded in the present study
responded to CRD, and the observed responses were weak. The
most likely explanation for these findings is that serotonergic
neurons responsive to CRD are located outside of RM and
NRMC or tonic serotonergic discharge is sufficient for the
production of the full reaction to CRD and for CRD-evoked
antinociception.
Nonserotonergic cell responses to CRD
CRD-responsive RM and NRMC cells respond at short
latency and may therefore receive direct input from spinal
neurons. However, anatomical studies have revealed relatively
few spinal projections to RM and NRMC. Instead, ventral
medullary neurons are likely to receive somatosensory input
indirectly via projections from the PAG, hypothalamus, and
amygdala (Abols and Basbaum 1981; Hermann et al. 1997;
Holstege 1987; Murphy et al. 1999; Van Bockstaele et al.
J Neurophysiol • VOL
Somatic versus visceral responses of nonserotonergic RM
cells
Most ON (22/31), OFF (12/15), and NEUTRAL (29/45) cells
respond differently to noxious CRD than to noxious heat. For
instance, of 45 NEUTRAL cells, defined by their lack of a response to noxious heat, 16 were excited and 13 inhibited by
CRD. Our finding that cells do not respond in the same direction to visceral and cutaneous stimulation is consistent with
observations from a previous study of RM cell responses to
bladder stimulation in the cat. Chandler et al. (1994) showed
that nearly equal numbers of RM cells inhibited by noxious
pinch were excited, inhibited, or unaffected by bladder disten-
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Serotonergic neurons discharge in a slow and steady fashion
(Aghajanian and Vandermaelen 1982; Bayliss et al. 1997; Li
and Bayliss 1998a,b; Mason 1997; Wang et al. 2001). In the
present study, RM and NRMC neurons were classified as
p5HT or non-p5HT by an algorithm that utilizes their slow
discharge rate and steady pattern of firing. This algorithm has
been tested repeatedly by our laboratory (reviewed in Mason
2001). Notably, of 60 p5HT cells tested, 56 contained serotonin immunoreactivity, whereas none of the 56 cells classified
as non-p5HT contained serotonin immunoreactivity. This
would suggest that all of the non-p5HT cells are nonserotonergic, whereas about two of the p5HT cells recorded in the
present study may in fact be nonserotonergic. Such an error
would not change the basic findings of the present study—
namely, that most nonserotonergic RM and NRMC cells respond to CRD and that most serotonergic cells do not.
1991). The same projections may contribute to both cutaneous
and visceral responses in RM neurons.
Regardless of the route by which CRD information reaches
the ventral medulla, RM and NRMC responses to CRD are
ultimately dependent on input from dorsal horn cells. Dorsal
horn cells respond to CRD in at least six different ways, most
of which are relevant to the present study (Ness and Gebhart
1987, 1988a; Qin et al. 1999). Neurons with “abrupt” excitatory responses are excited by CRD at short latency and maintain discharge during the stimulus presentation but not afterward. Neurons with “sustained” excitatory responses differ
from abruptly responsive cells because they maintain an elevated level of discharge after termination of the CRD stimulus.
A third group of dorsal horn cells, the excitatory-inhibitory
cells, are excited by CRD during the stimulus and then show an
inhibition after stimulus termination. Among cells that are
inhibited by CRD, the inhibition is either confined to the
stimulus presentation, sustained beyond the stimulus presentation, or followed by an excitatory response after stimulus
termination (Qin et al. 1999). Nonserotonergic RM and NRMC
cells with excitatory responses to CRD stimulation that are
restricted to the stimulus presentation likely receive input primarily from the abrupt excitatory class of dorsal horn cells.
RM and NRMC cells with excitatory responses to CRD stimulation that persist beyond the stimulus presentation may receive input from both abrupt and sustained excitatory types of
dorsal horn cells. RM and NRMC cells with inhibitory responses to CRD may receive sign-inverting input from shortlatency abrupt and sustained cells as well as sign-conserving
input from one or more types of CRD-inhibited dorsal horn
cells. Finally, cells with biphasic response, either excitatoryinhibitory or inhibitory-excitatory, are found in both the dorsal
horn and in the medulla. Because some of the different classes
of dorsal horn cells can be distinguished by different thresholds
(Ness and Gebhart 1987, 1988a), an examination of the threshold for RM and NRMC cell responses to CRD would aid in
distinguishing between these possibilities. Such an examination is in progress.
Dorsal horn cells that respond to CRD receive convergent
cutaneous input from scrotal, perineal, and lower abdominal
regions (Ness and Gebhart 1987, 1988a). The present experiments demonstrate that many putative pain modulatory neurons in the brain stem also receive convergent cutaneous and
visceral input (30 of 91 non-p5HT cells; 1 of 24 p5HT cells).
This is certainly an underestimate of the true proportion of
convergent neurons as cells may respond to stimulus modalities or locations other than those used in this study.
RAPHE MAGNUS NEURONS RESPOND TO COLORECTAL STIMULATION
Functional implications
The response of spinal neurons to CRD increases after
spinalization and is unaffected by collicular decerebration,
providing evidence for a tonic inhibitory input from the brain
stem and possibly upper cervical cord (Ness and Gebhart 1987,
1989; Qin et al. 1999). In contrast, the cardiovascular reaction
to CRD is greatly attenuated by spinalization at either C1 or T6
while being unaffected by decerebration, suggesting that pronociceptive modulation descends from the brain stem (Ness
and Gebhart 1988b). In support of this lesion data, Zhuo and
Gebhart recently demonstrated that the behavioral and cellular
responses evoked by 80 mmHg CRD stimulation are either
attenuated or facilitated by RM stimulation depending on the
site and intensity of stimulation (Zhuo and Gebhart 2002; Zhuo
et al. 2002). Thus RM is at least one of the brain stem sites,
perhaps the primary site, that provides descending inhibitory
and facilitatory modulation to CRD-responsive circuits in the
spinal cord.
Nonserotonergic RM and NRMC cells that are classified by
their response to cutaneous noxious heat share additional classspecific physiological features. For instance, the response to
J Neurophysiol • VOL
noxious cutaneous heat also predicts a cell’s pattern of discharge across the sleep/wake cycle (Leung and Mason 1999).
Cells within a class also have consistent responses to other
manipulations such as nonnoxious thermoregulatory challenges (Young and Dawson 1987) and volume expansion
(Morgan and Fields 1993). Most importantly, the response to
noxious tail heat predicts the response to analgesic doses of
opioids in the anesthetized rat (Barbaro et al. 1986; Cheng et al.
1986; Heinricher and Drasner 1991; Heinricher et al. 1992).
Because the descending antinociceptive influence from RM/
NRMC is active (i.e., depends on activation of cells), RM and
NRMC cells that exert antinociceptive effects on spinal neurons must be activated by opioids (reviewed in Fields et al.
1991). Thus a cell’s response to opioids is critical to predicting
that cell’s function. On a cautionary note, opioids do not alter
the background discharge rate of ON cells in unanesthetized rats
(Martin et al. 1992). Further, the effects of opioids on OFF and
NEUTRAL cell discharge have never been tested in the unanesthetized rat. Thus the opioid response of RM and NRMC cells
in the natural, unanesthetized state may not be predicted accurately by the response to noxious cutaneous stimulation tested
in anesthetized animals.
Short of testing each cell’s response to opioid administration, the most reliable predictor, albeit not perfect, of a cell’s
response to opioids in the anesthetized rat is its response to
noxious thermal stimulation of the tail. The observation that
many ON, OFF, and NEUTRAL cells respond differently to tail heat
and to other cutaneous stimuli (Ellrich et al. 2000, 2001; Leung
and Mason 1998) is then puzzling as it suggests that the
response to tail heat, in particular, holds a special significance.
Our results and those of Chandler et al. (1994) further increase
the known physiological heterogeneity of cells in the ON, OFF,
and NEUTRAL cell categories. If the important functional classes
of RM and NRMC cells were uniform in their responses to
peripheral stimulation, the number of functional classes would
be quite large. Another possibility is that a smaller number of
moderately heterogeneous cell classes exist. For instance, a cell
inhibited by noxious thermal stimulation of most sites and
excited by CRD, but not one inhibited by CRD, may mediate
CRD-evoked suppression of cutaneous withdrawal reflexes.
Overall, the data suggest the existence of subgroups of ON, OFF,
and NEUTRAL cells, with varying functional roles in nociceptive
processing and modulation.
The authors thank Drs. Hayley Foo, Jonathan Genzen, Malcolm Nason, and
Madelyn Baez for comments on the manuscript.
T. Brink was supported by a National Institute of General Medicine Training
Grant (T32 GM-07839). This research was supported by the Brain Research
Foundation.
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