Download Modulation of Sympathetic and Somatomotor Function by the

J Neurophysiol 92: 510 –522, 2004.
First published February 18, 2004; 10.1152/jn.00089.2004.
Modulation of Sympathetic and Somatomotor Function by the Ventromedial
Medulla
Malcolm W. Nason, Jr. and Peggy Mason
Committee on Neurobiology and Department of Neurobiology, Pharmacology and Physiology, University of Chicago,
Chicago, Illinois 60637
Submitted 29 January 2004; accepted in final form 13 February 2004
The ventromedial medulla (VMM), which includes raphe
magnus (RM), raphe pallidus (RP), and the adjacent nucleus
reticularis magnocellularis (NRMC), has been implicated in
nociceptive and cardiorespiratory modulation and in the premotor control of cold defense (Mason 2001; Morrison 2001a).
However, studies directed at understanding VMM’s role in
each of these functions have focused on pharmacological or
electrical activation of different regions within the ventromedial medulla. For instance, nociceptive modulatory studies
have focused on the effect of microstimulation of and microinjection into RM and NRMC (Drower and Hammond 1988;
Zhuo and Gebhart 1990, 1992a, 1997), whereas the role of
VMM in cold defense has been tested using microinjection into
RP (Morrison et al. 1999). Since neurons throughout VMM
have large dendritic arbors that cross cytoarchitectonic boundaries (Edwards et al. 1987; Fox et al. 1976; Gao and Mason
1997; Leontovich and Zhukova 1963; Maciewicz et al. 1984;
Mason et al. 1990; Newman 1985; Potrebic and Mason 1993;
Ramon-Moliner and Nauta 1966; Valverde 1961), it is unlikely
that a microinjection into any single nucleus in VMM will
affect neurons confined to that nucleus. Furthermore, with few
exceptions, experiments examining VMM’s role in nociceptive
modulation have not recorded thermoregulatory measures and
experiments examining VMM control of cold defense have not
recorded dependent variables related to nociceptive sensitivity.
Thus it is not clear whether microinjections into individual
ventromedial medullary nuclei elicit changes in autonomic
control or nociceptive modulation or simultaneously affect
both functions. Therefore in these experiments, the effects of
microinjections into midline and lateral regions of VMM on
somatomotor, cardiorespiratory, and thermoregulatory function were studied.
A wealth of data implicates the VMM in nociceptive modulation. Electrical or chemical stimulation in the VMM modulates the cellular and motor reactions to noxious stimulation
(Dostrovsky et al. 1982; Jensen and Yaksh 1986; Satoh et al.
1983; Zhuo and Gebhart 1990, 1992a, 1997; Zorman et al.
1981). RM and NRMC cells receive afferents from the periaqueductal gray matter (PAG), a site that evokes descending
opiate analgesia, and project to the superficial dorsal horn of
the spinal cord, where nociceptive sensory afferents synapse
(Abols and Basbaum 1981; Fields et al. 1995; Urban and Smith
1994; Vanegas et al. 1984). Lesioning or inactivating the
VMM greatly attenuates the antinociceptive effects of systemic
opioids, opiate injection into PAG, or PAG stimulation (Behbehani and Fields 1979; Chung et al. 1987; Proudfit 1980;
Proudfit and Anderson 1975; Sandkuhler and Gebhart 1984;
Urban and Smith 1994). These findings suggest that, when
activated by rostral regions, RM and NRMC cells modulate
nociceptive dorsal horn cells. While activation of RM and
NRMC cells typically suppresses the nociceptive responses of
dorsal horn cells and the withdrawal movements evoked by
noxious stimulation, facilitation may also result (Zhuo and
Gebhart 1990, 1992a, 1997). Both the suppression and the
facilitation of spinal nociceptive transmission are due to excitation, and not disinhibition, of medullary neurons (Behbehani
and Fields 1979; Kaplan and Fields 1991).
Address for reprint requests and other correspondence: P. Mason, Dept. of
Neurobiology, Pharmacology and Physiology, Univ. 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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0022-3077/04 $5.00 Copyright © 2004 The American Physiological Society
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Nason Jr., Malcolm W. and Peggy Mason. Modulation of sympathetic and somatomotor function by the ventromedial medulla. J
Neurophysiol 92: 510 –522, 2004. First published February 18, 2004;
10.1152/jn.00089.2004. The ventromedial medulla is implicated in a
variety of functions including nociceptive and cardiovascular modulation and the control of thermoregulation. To determine whether
single microinjections into the ventromedial medulla elicit changes in
one or multiple functional systems, the GABAA receptor antagonist
bicuculline was microinjected (70 nl, 5–50 ng) into the ventromedial
medulla of lightly anesthetized rats, and cardiovascular, respiratory,
and nociceptive measures were recorded. Bicuculline microinjection
into either the midline raphe or the laterally adjacent reticular nucleus
simultaneously increased interscapular brown adipose tissue temperature, heart rate, blood pressure, expired [CO2], and respiration rate
and elicited shivering. Bicuculline microinjection also decreased the
noxious stimulus-evoked changes in heart rate and blood pressure,
decreased the frequency of heat-evoked sighs, and suppressed the
cortical desynchronization evoked by noxious stimulation. Although
bicuculline suppressed the motor withdrawal evoked by noxious tail
heat, it enhanced the motor withdrawal evoked by noxious paw heat,
evidence for specifically patterned nociceptive modulation. Saline
microinjections into midline or lateral sites had no effect on any
measured variable. All bicuculline microinjections, midline or lateral,
evoked the same set of physiological effects, consistent with the lack
of a topographical organization within the ventromedial medulla.
Furthermore, as predicted by the isodendritic morphology of cells in
the ventromedial medulla, midline bicuculline microinjection increased the number of c-fos immunoreactive cells in both midline
raphe and lateral reticular nuclei. In summary, 70-nl microinjections
into ventromedial medulla activate cells in multiple nuclei and elicit
increases in sympathetic and somatomotor tone and a novel pattern of
nociceptive modulation.
MULTIPLE MODULATORY ROLES FOR THE VENTROMEDIAL MEDULLA
J Neurophysiol • VOL
METHODS
Surgical preparation
The methods for all experiments were approved by the University
of Chicago Institutional Animal Care and Use Committee and conformed to the guidelines of National Institutes of Health and the
International Association for the Study of Pain.
Male Sprague-Dawley rats (250 – 450 g; n ⫽ 110; Charles River,
Portage, MI) were pretreated with atropine sulfate (40 ␮g in 0.1 ml ip)
and anesthetized with halothane. Rats were placed on a heated (36°C)
water blanket and covered with a plastic blanket for the duration of the
experiment. A Y-tube was inserted into the trachea through which
humidified halothane (2%) in oxygen was administered. Expired
[CO2] was measured through a 23-gauge, blunted needle placed at the
end of the Y tube. A catheter filled with heparinized (10 IU/ml) saline
was inserted into the right femoral artery to record blood pressure. To
record the EEG, two stainless steel screws with attached wire leads
were affixed to the lateral skull and coated with dental cement.
Stainless steel electrodes (Grass, Warwick, RI) were inserted bilaterally into the thorax to record the EKG and into the paraspinous
muscles to record the EMG during tail withdrawal movements. Needle electrodes were also placed through the skin of the upper thigh
into multiple muscles, including the quadriceps and hamstring, to
record the EMG from paw withdrawal movements. BAT is the major
nonshivering thermogenic organ in the rat and the largest single
deposit of BAT is found interscapularly. Therefore a thermistor (Thermometrics, Edison, NJ) with a time constant of 90 ms was inserted
through a 1-cm cut in the skin into a pocket 3–5 mm deep in the BAT.
The overlying skin incision was closed with wound clips. Core temperature was monitored with a rectal thermistor. A craniotomy allowed for reflection of the dura over the cerebellum and insertion of
a micropipette. Animals were allowed to equilibrate for ⱖ1 h at 1%
halothane after surgery and exhibited no visible signs of discomfort or
distress.
Peltier thermodes (2 ⫻ 2 cm) were placed on the ventrum of the
tail, 5– 8 cm from the distal tip, and on the ventrum of the left hind
foot, just proximal to the distal joint of the toes, and extending toward
the heel.
Data collection
Data were collected onto a Pentium III PC using a Power 1401 A/D
converter (CED, Cambridge, UK) and analyzed using Spike 4.0
(CED). Halothane concentration and expired [CO2] were monitored
by a Datex Capnomac (Tewksbury, MA). EEG, blood pressure, and
EKG were collected at 250 Hz. EMG signals were rectified, integrated, and collected at 1.2 kHz, while temperature (stimulus, core,
and BAT) and [CO2] were collected at 100 Hz.
Protocol
Peltier thermodes were held at a constant temperature of 32°C by
computer-driven Peltier controllers (Yale Instrumentation Laboratory,
New Haven, CT). Noxious thermal stimulation was applied to the
hindpaw and the tail alternately with 5-min intervals between stimulus
site trials. Each stimulus consisted of a 3-s ramp from 32°C to a
plateau of 56°C for 3 (hind paw) or 4.5 s (tail), followed by a 2- to 6-s
return to 32°C. The animal’s tail or paw was strapped on to the
thermode throughout baseline and stimulus conditions. There were
three to five trials at each stimulus site prior to microinjection.
After baseline recording, a borosilicate glass micropipette (⬃25 ␮m
tip diam) was introduced into the midline (L 0.0 mm) or lateral (L 1.0
mm) portions of the VMM (P 2.0 –2.6 mm from interaural 0, V 9.5
mm from the cerebellar surface). There were no changes in heart rate,
blood pressure, EMG, or EEG on introduction of the injection pipette
into either locus. A picospritzer (WPI, Sarasota, FL) was used to inject
saline or 5, 15, or 50 ng of bicuculline methiodide (Sigma, St. Louis,
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Strong lines of evidence also implicate RP in the descending
control of thermoregulatory cold defense. Bicuculline microinjection into RP elicits nonshivering thermogenesis in the
interscapular brown adipose tissue (BAT) (Morrison et al.
1999), whereas muscimol microinjection into RP blocks the
BAT activation evoked by cooling the hypothalamus or forebrain injection of prostaglandin E2 (Morrison 2001b, 2003;
Nakamura et al. 2002). Pseudorabies virus injections into most
sympathetic nervous system targets tested, including BAT,
consistently reveal retrograde trans-synaptic labeling of RM,
NRMC, and RP neurons (Bamshad et al. 1999; Cano et al.
2003; Strack et al. 1989a,b). VMM neurons, both serotonergic
and nonserotonergic, respond to nonnoxious changes in skin
temperature (Dickenson 1977; Rathner et al. 2001; Young and
Dawson 1987), and c-fos labeling in the VMM has been
reported after cold challenge (Cano et al. 2003; Martinez et al.
2001; Morrison et al. 1999). VMM neurons project to the
sympathetic intermediolateral cell column, where targeted cells
include preganglionic sympathetic neurons (Bacon et al. 1990;
Basbaum et al. 1978; Henry and Calaresu 1974; Holstege and
Kuypers 1982), further evidence that VMM cells could control
BAT activation. However, neurons throughout VMM also
project to the superficial dorsal horn, where thermoreceptors as
well as nociceptors terminate. Moreover, electrical stimulation
in RM modulates the responses of thermoreceptive neurons in
the dorsal horn (Sato 1993), raising the possibility that VMM
modulates thermoregulation via effects on thermoreceptive
pathways that either ascend to the hypothalamus or serve as
afferent input to local preganglionic sympathetic neurons.
The role of the VMM in cardiorespiratory function is not
well understood, but there are strong hints that such a role
exists. Both depressor and pressor responses to electrical stimulation of the medullary raphe, sometimes accompanied by
bradycardia or tachycardia, have been reported (Adair et al.
1977; Blessing and Nalivaiko 2000; Henry and Calaresu 1974;
McCall 1984; McCall and Harris 1987; Yen et al. 1983).
Bicuculline microinjections into the midline raphe result in
sympathetically mediated increases in heart rate and blood
pressure (Cao and Morrison 2003). Inactivation of the medullary raphe by muscimol microdialysis greatly reduces the respiratory response to increased CO2 (Messier et al. 2002). Both
serotonergic and nonserotonergic neurons in the medullary
raphe contribute to the respiratory reflex evoked by elevated
CO2 (Messier et al. 2004; Nattie et al. 2004). Finally, microinjection of a substance P antagonist causes respiratory arrest
and death (G. F. Gebhart, personal communication; Aimone
and Gebhart 1986).
To determine whether microinjection into single sites of the
VMM elicits single or multiple modulatory changes, the
GABAA receptor antagonist bicuculline was microinjected into
the midline raphe and into the lateral portion of NRMC, and
the effects on nociception, BAT temperature, and cardiorespiratory physiology were simultaneously examined. By using
c-fos immunoreactivity as a gross marker for neuronal activity
(Morgan and Curran 1991; Wang and Redgrave 1997), the
anatomical extent of the neurons excited by microinjections
was estimated. The results demonstrate that small (70 nl)
microinjections activate neurons in a broad area of the medullary raphe and adjacent reticular formation and elicit sympathoexcitatory and somatomotor effects, including a novel pattern of nociceptive modulation.
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M. W. NASON AND P. MASON
stimulus. Noxious stimulation elicited an enhanced expiration that we
have termed a sigh (see below). Sighs were stereotyped, and therefore
the incidence of sighs was compared before and after drug or saline
microinjection. Animals without sighs (n ⫽ 4) and animals whose
sighs occurred outside the time scope of our analysis (n ⫽ 6; within
30 min of injection) were omitted from analysis.
All EMG signals were full-wave rectified. A 30-s integral of the
EMG prior to each noxious stimulus (ending at the beginning of the
stimulus ramp) was used as a measure of resting motor tone. The
motor response to noxious stimulation was calculated as the difference between the integrated EMG activity for 30 s before and 30 s
beginning at the start of the plateau phase of the stimulus and including the 3- (paw) or 4.5-s (tail) plateau. All responses were normalized
to the mean response prior to injection. In experiments exhibiting
significantly increased EMG baseline activity (see below), the maximum EMG activity following noxious stimulation was used as the
assessment of the response before and after injection. Normally, when
EMG signals exceeded 5 V, the amplifier gain was reduced. Unfortunately, in several experiments (such as that shown in Fig. 5), this
was overlooked, resulting in the truncation of some EMG values and
causing some EMG data to be underestimated. Resting EMG values
from experiments exhibiting increased EMG baseline activity were
removed from the general population and compared separately. Withdrawal latencies were determined, off-line, by visual inspection of the
5- (foot) and 10-s (tail) records following the noxious stimulus. In
cases where no tail withdrawal occurred, a latency of 10 s was
assigned to the trial (Carstens and Wilson 1993). All heat stimuli to
the paw evoked a motor withdrawal response that was observed by
eye as well as recorded electromyographically.
c-fos experiments
Analysis
Systolic blood pressure was computed from the peaks in the recorded blood pressure from each heartbeat and converted by linear
interpolation to an array of 10 samples/s. Similarly, heart rate was
calculated from the sampled EKG as the reciprocal of the interval
between sequential heartbeats and converted by linear interpolation
into an array of 10 samples/s. Resting blood pressure and heart rate
values were calculated as the average measure during the 30 s before
each noxious thermal stimulus. Blood pressure and heart rate values
are expressed in millimeters of mercury (mmHg) and beats per minute
(bpm), respectively.
Noxious stimulation typically elicited changes in blood pressure
and heart rate. For these measures, stimulus-evoked changes were
calculated as the difference of the maximum value during the poststimulus period (⫹30 s) minus the resting value. The tachycardia
evoked by paw heat was somewhat variable and was observed in
most, but not all (66/79), animals. Heart rate was labile, fluctuating in
seemingly random fashion around a mean. Because of this variation,
changes in heart rate ⬍2 SD from resting levels were considered
insignificant. In 13 animals, heart rate changed by less than this
criterion and was considered insignificant; these animals were not
used in further analysis of the heat-evoked tachycardia.
EEG power spectra with 1,024 points were created by summing the
power in the frequency range of 0 – 4 Hz for 30-s epochs before and
after the stimulus. This low frequency power typically decreases in
response to noxious stimulation (Grahn and Heller 1989), and the
percentage decrease was used as a measure of cortical “desynchronization.” In some animals (n ⫽ 6), the EEG was never sufficiently
synchronized for a noxious heat-evoked desynchronization to occur;
EEG recordings from these animals were not further analyzed.
Since [CO2] was obtained at some distance from the trachea, all
values were considered as relative, rather than absolute, measures and
were normalized to the average preinjection resting [CO2] for each
experiment. Resting respiratory rate was calculated as the reciprocal
of the interval between the [CO2] peaks for the 30 s before the noxious
J Neurophysiol • VOL
To estimate the number and distribution of cells that were activated
by microinjection, a group of rats (n ⫽ 25) received microinjections
as above and were processed for c-fos immunoreactivity (Wang and
Redgrave 1997). Animals were anesthetized with halothane and
placed in a nose cone delivering oxygenated halothane. A craniotomy
was completed as described above. To estimate the physiological
effects of the microinjections, EKG and core temperature were recorded. However, additional physiological measures were not recorded to reduce the activation of c-fos due to surgical procedures.
Anesthetic was lowered to 1% and animals were allowed to equilibrate for 1 h. Microinjections (70 nl) of 5, 15, or 50 ng bicuculline or
saline were made as described above. Either fluorescent beads (Molecular Probes, Eugene, OR) or Fast green was added to the injectate.
After a 1-h rest period, animals were killed, perfused, and postfixed
for 4 –12 h.
The medulla was cut transversely into 40-␮m sections. Sections
containing the microinjection site were identified by the presence of
fluorescent beads or Fast green. Typically 8 –10 sections distributed
about the central site were processed for c-fos immunocytochemistry.
In brief, sheep anti-c-fos antibody (Cambridge Research Biochemicals, Cheshire, UK) was preabsorbed with rat liver powder at 4°C for
24 h in 0.9% phosphate buffered saline containing 1% normal goat
serum (NGS) and 0.3% Triton X-100 (termed “buffer” below). Tissue
was incubated for 30 min in 50% ethanol and washed with buffer
three times at room temperature. Sections were incubated in 3% NGS
in buffer for 30 min. Sections were transferred directly into sheep
anti-c-fos antibody (1:2000) at 4°C for 48 h. After incubation in the
primary antibody, sections were washed in buffer and placed in
biotinylated rabbit anti-sheep antibody (Vector Labs, Burlingame,
CA) at 1:250 dilution for 2 h at room temperature. Tissue was washed
and transferred to 1:1,000 ExtraAvidin HRP (Sigma) for 2 h. Finally,
sections were washed and processed with di-aminobenzidine as previously described (Mason and Fields 1989).
All c-fos immunoreactive cells in the VMM, medial to the facial
nucleus and ventral to the medial longitudinal fasciculus, were plotted
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MO) in 70 nl of phosphate buffered saline or Dubelco’s NES, (both
pH 7.4). The dose of 15 ng used by Morrison et al. (1999) formed the
starting point for our study. Semi-log doses were chosen on either side
of that starting point. The injection lasted 30 –100 s and was monitored using a calibrated reticule. Trials resumed 60 –100 s after drug
injection. In the case of some injections, both saline and bicuculline,
changes in blood pressure or respiration rate occurred during the
injection itself; these injections were not analyzed further. Additional
injections were made after a minimum of 1.5 h, including a 20- to
30-min rest interval (i.e., no stimulus trials), and after all baseline
measures had returned to preinjection levels or had remained constant
for ⱖ30 min. No animal received ⬎4 injections. Animals were
occasionally supplemented with 5 ml of 10% buffered dextrose sc at
the beginning of the rest period, ⱖ30 – 45 min before the time of the
next microinjection. At the end of recording, 35–70 nl of 1% Fast
green (Sigma) was injected at the stereotaxic coordinates of the last
microinjection.
Animals were overdosed with halothane and perfused transcardially
with phosphate buffered saline, followed by 10% formalin, and immersed in 30% sucrose in 0.1 M PBS until equilibration. Brains were
sectioned (50 ␮m) in the coronal plane, and marked injection sites
were visualized and plotted on standard sections. The raphe nuclei
were considered to include a region 400 ␮m wide centered on the
midline. A small region between the pyramids with densely packed,
small cells was considered to be RP. RM was considered to be the
raphe region stretching from the dorsal boundary of RP to a point 1.5
mm dorsal to the ventral surface of the brain. In the case of animals
that received multiple injections, the locations of unmarked injections
were determined by their distances from labeled sites with known
stereotaxic coordinates.
MULTIPLE MODULATORY ROLES FOR THE VENTROMEDIAL MEDULLA
(Neurolucida, Colchester, VT) using a 20⫻ objective (Nikon). In
addition, c-fos immunoreactive cells in the dorsal cochlear nucleus
that were consistently labeled in each animal, as has been noted before
(Erickson and Millhorn 1994), were also plotted. The median number
of neurons labeled per section in each animal and at each site was
compared across experimental groups.
Statistical analyses were completed using Excel 9.0 (Microsoft
Corp., Redmond, WA) and SigmaStat 2.0 (SPSS Corp, Chicago, IL).
Drug effects were compared using two-way repeated measures
ANOVAs and the Student-Newman-Keuls post hoc test. A P value ⱕ
0.05 was considered significant.
RESULTS
Drug-evoked changes in resting variables
BAT temperature, blood pressure, heart rate, respiration rate,
EMG magnitude, and EEG delta frequency power were analyzed for 30 s prior to each noxious stimulus trial. These
measures comprise the resting variables. Prior to microinjection, none of the resting variables analyzed differed between
the experimental and control groups. Furthermore, none of the
resting variables changed after injection of saline into either
RM or NRMC (Fig. 1).
BAT temperature began to increase within 5 min of bicuculline microinjection (Fig. 2) and had increased significantly
by an average of 1.3 ⫾ 0.2°C 20 min after bicuculline microinjection. BAT temperature remained elevated for another 20
min before slowly returning to baseline values. A significant
increase of 34.3 ⫾ 0.3% in expired [CO2] occurred during the
rise in BAT temperature (n ⫽ 32). Bicuculline elevated core
body temperature significantly, but in contrast to its effects on
BAT temperature, evoked a small increase of only 0.3 ⫾ 0.1°C
after a long delay (n ⫽ 40).
Injection of bicuculline significantly elevated blood pressure
by 23.7 ⫾ 2.0 mmHg (n ⫽ 56) and increased heart rate by
92.4 ⫾ 6.5 bpm (n ⫽ 53), and respiration rate by 0.4 ⫾ 0.1
breaths/s (n ⫽ 32) for 20 –30 min regardless of injection site
(Fig. 2). Increases in heart rate were sometimes accompanied
by alternating strong and weak heartbeats within the same
cardiac rhythm, a phenomenon known as pulsus alternans (Fig.
3; Lab and Seed 1993). Pulsus alternans occurred in 20 –30%
of the animals that received bicuculline but was never observed
in saline treated animals (0/26).
As shown in Fig. 2, medullary microinjections of all three
doses of bicuculline elicited tonic increases in resting EMG
activity (5/12 low , 4/24 mid-, and 12/20 high dose), averaging
7.0 ⫾ 2.9 times baseline levels. The increases in the baseline
EMG activity of paraspinous muscles and limb muscles were
not significantly different. Such increases in EMG activity
were not observed in any of the 26 saline-treated animals (Figs.
1 and 4). This increase in resting EMG activity was observed
in all muscles recorded and may represent a shivering response
(Fuller et al. 1975). Although the animal often visibly shivered,
a rigorous distinction between shivering and high muscle tone
could not be made as hardware rectification of the EMG signal
prevented testing of the spectral content of the EMG signal.
Microinjections that evoked an increase in resting EMG activity were more likely to also evoke pulsus alternans (10/22) than
those that did not (7/57; ␹2, P ⫽ 0.001).
Resting EEG delta activity did not change after injection of
bicuculline into either RM or NRMC.
Somatomotor responses to noxious heat
Paw heat evoked significant withdrawal movements that
were unaffected by saline microinjection (Fig. 4). Injections of
bicuculline into either RM or NRMC significantly increased
FIG. 1. Microinjection of saline (dashed line marked by # in
top trace) into nucleus reticularis magnocellularis (NRMC) does
not affect baseline physiological measures or alter the response to
noxious stimulation. Core and brown adipose tissue (BAT) temperatures, heart rate, blood pressure, respiration rate, and [CO2]
magnitude remain unchanged after injection. ⽧ over the [CO2]
magnitude trace indicates occurrences of sighs. Noxious stimulation of the tail (‚) or paw (Œ) was alternated with 5-min intervals
between successive trials. Saline had no effect on the motor
response to paw or tail heat. Expanded time views of paw heats
marked “Fig. 4A” and “Fig. 4B” are found in those figures.
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None of the recorded physiological measures changed after
saline microinjection into either RM or NRMC. In contrast,
microinjections of bicuculline, at every dose tested and at both
sites, produced similar multiple physiological effects. Since
there were virtually no dose- or site-dependent effects, all data
from animals injected with bicuculline were combined, and
will be described. Drug-evoked changes in resting physiological measures are considered first followed by a description of
drug-evoked changes in the motor and autonomic responses to
noxious heat stimulation.
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the magnitude of noxious paw heat-evoked withdrawals by an
average of 2.3- to 2.9-fold (Figs. 4 and 5A). No change in paw
withdrawal latency (3.2 ⫾ 0.2 s before, 3.5 ⫾ 0.3 s after)
accompanied this change in withdrawal strength (Fig. 6).
Our observation that bicuculline microinjection elicited an
enhancement of the noxious heat-evoked paw withdrawal appeared to be at odds with the previously established suppression of tail flick by bicuculline microinjection into RM
(Drower and Hammond 1988). Therefore in most experiments,
tail heat and paw heat stimulation trials were presented alternately. In contrast to its effects on paw heat-evoked withdraw-
als and in agreement with published reports, bicuculline, into
either RM or NRMC, significantly attenuated the tail withdrawal from noxious heat (Fig. 5). Bicuculline significantly
decreased the magnitude of tail withdrawal movements to 28 ⫾
10% of baseline values and increased the withdrawal latency
from 6.8 ⫾ 0.4 to 9.4 ⫾ 0.4 s (Fig. 5).
Cardiorespiratory and cortical responses to noxious heat
Both paw and tail heat elicited transient changes in blood
pressure, heart rate, respiration, and EEG delta frequency
FIG. 3. Bicuculline microinjection often resulted in pulsus
alternans and muscle shivering. A: 3-min recording of heart beat
interval (top), blood pressure (middle), and flexor EMG (bottom)
starting 20 s after bicuculline microinjection. B–D: expanded
traces of the time points indicated in A. B: 30 s after microinjection, heart rate, blood pressure, and EMG were at nearbaseline values. C: 65 s after microinjection, shivering has
begun and the 1st indications of pulsus alternans were present.
D: 4 min after microinjection, both pulsus alternans and shivering were present. Time scale in B applies to B–D.
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FIG. 2. Microinjection of 50 ng bicuculline (dashed line
marked by * in top trace) into NRMC changes both baseline
physiological measures and the response to noxious stimulation. BAT temperature, heart rate, blood pressure, respiration
rate, and [CO2] magnitude increase after injection. ⽧ above
the [CO2] trace indicates occurrences of sighs. Trials of noxious stimulation of the tail (‚) and paw (Œ) were alternated
with 5-min intervals. Paw heat-evoked EMG responses were
increased, whereas tail heat-evoked EMG responses were
reduced after bicuculline injection. Expanded time views of
paw heats marked “Fig. 5A” and “Fig. 5B” are found in those
figures.
MULTIPLE MODULATORY ROLES FOR THE VENTROMEDIAL MEDULLA
515
FIG. 4. Noxious paw heat (bottom trace marked stimulus)
evokes a tachycardia (HR), a pressor response (BP), changes in
respiration including a sigh (expired [CO2]), a cortical desynchronization (EEG), and a motor withdrawal (Paw EMG) both
before (A) and after (B) saline microinjection. Time scale in A
applies to A and B. Traces are found at a more compressed time
scale in Fig. 1.
heat-evoked tachycardia and pressor reactions is at least partially a depression and is not entirely attributable to a “ceiling
effect.”
Paw heat elicited a consistent and stereotyped respiratory
response (Figs. 4 and 5A). This consisted of a long (⬃0.5 s, 2
times baseline duration) inspiration that preceded an enhanced
expiration, of normal duration, that peaked at two times baseline [CO2] levels. This expiration was followed by 2–3 s of
shallow, rapid breaths, sometimes interspersed with pauses.
This sequence was termed a “sigh” (Issa and Porostocky 1993)
and was marked by the maximum [CO2] level recorded during
the enhanced expiration. Sighs appeared in all but four animals,
but never occurred in response to every noxious heat within an
experiment. Sighs were typically most pronounced for the first
noxious stimulation of the experiment and remained consistent
in size (⬃90% of maximum) and latency thereafter. Sigh
incidence did not change in saline-treated rats (n ⫽ 13), averaging 0.58/stimulus before injection and 0.48/stimulus afterward. In contrast, bicuculline injected at either site significantly
decreased the incidence of sighs (n ⫽ 28) from 0.64/stimulus
to 0.04/stimulus (t-test for proportions, P ⬍ 0.001).
Paw heat significantly decreased EEG delta activity by
60.2 ⫾ 0.2% (n ⫽ 53), a phenomenon that we refer to as
cortical “desynchronization” (Figs. 4 and 5A). After injection
FIG. 5. Tachycardia, pressor response, changes in respiration, and cortical desynchronization evoked by noxious paw heat (A) are entirely blocked (the former 3) or
attenuated (the latter reaction) by bicuculline microinjection (B). Motor withdrawal evoked by noxious paw heat
(A) is much larger after bicuculline microinjection (B).
Also note that there are changes in the resting blood
pressure and respiration rate after bicuculline microinjection. Time scale in A applies to A and B. Traces are found
at a more compressed time scale in Fig. 2.
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power (Figs. 4 and 5A). However, noxious paw heat evoked
larger and more consistent responses than did tail heat. Therefore only the responses to paw heat were analyzed in detail and
are reported below.
As seen in Figs. 4 and 5A, noxious paw heat elicited a
pressor response, tachycardia, change in respiratory pattern
(see following text), and decrease in EEG delta power. These
reactions were transient with all physiological measures returning to baseline within 30 s. None of the analyzed responses to
paw heat changed after injection of saline into either RM or
NRMC (Figs. 1 and 4).
Bicuculline microinjection suppressed cardiorespiratory and
cortical reactions to noxious paw heat (Figs. 2 and 5). Paw heat
evoked a mean pressor response of 22.5 ⫾ 0.5 mmHg (n ⫽
56), which decreased by 50 –100% (mean: 72.3 ⫾ 5.5%) after
bicuculline injection. Noxious paw heat evoked a mean tachycardia of 28.1 ⫾ 2.2 bpm (n ⫽ 53) that was significantly
reduced by 28 –100% (mean, 44.4 ⫾ 7.8%) after bicuculline
injection. The reduction in pressor and tachycardic reactions to
paw heat appeared within 90 s after injection and remained
constant until ⱖ25 min after injection. While cardiovascular
reactions evoked by noxious paw heat were suppressed, they
were still evident in most animals after bicuculline microinjection (Figs. 2 and 7). Therefore the suppression of noxious
516
M. W. NASON AND P. MASON
FIG. 6. A: magnitude of motor withdrawal from noxious
paw heat increased after bicuculline microinjection, but
withdrawal from noxious tail heat decreased. B: latency to
withdrawal from noxious paw heat did not change after
bicuculline microinjection, whereas the latency to tail withdrawal increased. Since there were no dose- or site-dependent effects, data from all bicuculline microinjections were
grouped together. *Significant differences (P ⬍ 0.01).
Injection sites
As shown in Fig. 8, midline microinjection sites were concentrated within RM, on the midline, and dorsal to RP. Lateral
microinjection sites were located in NRMC, just dorsal to the
lateral edge of the pyramid. Injection sites were labeled by the
co-injectate, Fast green, which stained an ovoid area with the
longer axis oriented dorso-ventrally. The stained area was
centered on the midline and reached approximately to each
medial edge of the pyramids and was readily apparent for
⬃300 –350 um in the rostro-caudal plane.
Distribution of neurons affected by injections
To delineate the area within which neurons were influenced
by drug microinjections, rats that were minimally prepared (see
METHODS) received microinjections of 5, 15, or 50 ng of bicuculline or saline into the midline. Fast green or fluorescent
beads labeled a small region on the midline, presumably the
center of the injection. The area containing c-fos immunoreactive cells always included this region and neither had any
effect on staining patterns. An average of 15.6 ⫾ 4.3 c-fos
positive cells were observed after saline microinjections (n ⫽
5; Fig. 9A). In contrast, after midline microinjections of bicuculline, at every dose, a significantly greater number of c-fos
labeled cells was observed (n ⫽ 20; average for all 3 doses:
88.2 ⫾ 9.3; Fig. 9, B–E). There were no differences between
the numbers of cells labeled after each dose of bicuculline. It
should be noted that the area delineated by the c-fos positive
stained cells always exceeded the area demarcated by the Fast
green or bead co-injectate. In anesthetized rats, neurons in the
dorsal cochlear nucleus (DCN) contain c-fos immunoreactivity
constitutively (Erickson and Millhorn 1994). Therefore the
number of c-fos labeled DCN cells was used as a control for
any variation in histological processing. The number of c-fos
labeled cells in the DCN was not different in rats receiving
saline or bicuculline (Fig. 9F). There were significantly fewer
labeled DCN neurons in rats that received 50 ng bicuculline
than in rats that received 5 ng bicuculline. It is therefore
possible that the number of VMM neurons labeled with c-fos
immunoreactivity in the former group is underestimated.
In animals that received bicuculline microinjections, c-fos
immunoreactive neurons were concentrated on the midline,
labeling neurons in both RP and RM (Fig. 9, B–D). However,
c-fos immunoreactive neurons were present, at a lower density,
bilaterally throughout the NRMC. Scattered labeling was
FIG. 7. Noxious stimulation of the paw
(Œ) evoked a tachycardia, pressor reaction,
and sometimes a sigh (⽧ above [CO2] trace).
After 15 ng bicuculline (dashed line marked
by @ in top trace), heat-evoked reactions
were reduced but not abolished. The noxious-evoked tachycardia observed after 15
ng bicuculline peaked below the maximum
baseline achieved after microinjection of 50
ng bicuculline (dashed line marked by * in
top trace).
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of bicuculline (n ⫽ 36) into RM or NRMC, the evoked desynchronization was significantly attenuated such that paw heat
evoked only a 35.8 ⫾ 2.9% reduction in EEG delta activity
(Fig. 5B).
MULTIPLE MODULATORY ROLES FOR THE VENTROMEDIAL MEDULLA
517
A
E
Saline
Mean Cells in Ventromedial Medulla
120
500 µm
60
0
0
F
B
5
15
50
Mean Cells in Dorsal Cochlear N.
170
Bicuculline (5 ng)
85
0
0
FIG.
8. Microinjection sites were concentrated within RM or NRMC, just
dorsal to the lateral edge of the pyramid. F, saline microinjection sites; Œ, 50
ng bicuculline microinjection sites. Photomicrographs of the ventromedial
medulla. These sites are representative of all microinjection sites. Interaural
level of each photomicrograph is listed to the right.
sometimes observed in nucleus reticularis paragigantocellularis pars lateralis and the parapyramidal region lateral to the
pyramids (data not shown).
In rats that were processed for c-fos immunoreactivity, heart
rate was monitored using transcutaneous needle electrodes to
measure the physiological effect of each microinjection. The
greater number of c-fos immunoreactive cells in bicucullinetreated rats was associated with a significant increase in heart
rate (Fig. 9G; average for all 3 doses: 85.3 ⫾ 7.5 bpm, n ⫽ 17)
that was similar to that observed in rats that were fully instrumented (82.9 ⫾ 5.6 bpm). Heart rate did not change after saline
injections (n ⫽ 5) in either minimally (Fig. 9G) or fully (Fig.
1) instrumented rats.
DISCUSSION
This study demonstrates that microinjection of the GABAA
receptor antagonist, bicuculline, into either the medullary midline raphe or adjacent reticular region, simultaneously elicits
thermoregulatory, cardiorespiratory, somatomotor, and nociceptive modulatory effects. Bicuculline microinjection deJ Neurophysiol • VOL
G
5
15
50
Mean Change in Heart Rate
100
C
Bicuculline (15 ng)
50
0
0
5
15
50
Bicuculline Dose (ng)
D
Bicuculline (50 ng)
FIG. 9. c-fos–labeled cells throughout ventromedial medulla (VMM) are
observed after microinjection of bicuculline (B–D), but not saline (A) into the
midline raphe. A–D: c-fos immunoreactive cells from animals that received
saline or bicuculline. Each montage includes the cells present on 1 section for
each of 5 animals randomly selected from every treatment group. Within each
montage, the cells from different animals are shown in different colors.
Contours show the outline of the medullary section and are shown in the same
color as the cells contained within. E: average number of c-fos–labeled cells in
VMM per section for each treatment group. F: average number of c-fos–
labeled cells in the dorsal cochlear nucleus per section for each treatment
group. G: average change in heart rate observed after microinjection for each
treatment group.
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pressed most reactions to cutaneous noxious heat, including the
somatomotor withdrawal to tail heat as well as cardiorespiratory changes and cortical desynchronization evoked by paw
heat. However, contrary to these suppressive effects, bicuculline microinjection markedly enhanced the motor withdrawal
evoked by noxious paw heat. This finding is strong evidence
that RM/NRMC differentially modulates multiple circuits engaged by noxious stimulation, an idea supported by previous
reports (Jasmin et al. 1997; Mason et al. 2001). Moreover, the
simultaneously observed opposing effects of bicuculline microinjection on tail flick and paw withdrawal are unequivocal
evidence for a form of nociceptive modulation that is somatotopic, rather than diffuse.
After bicuculline microinjection into either RM or NRMC,
the autonomic and somatomotor state was consistent with the
physiology of an “excited” animal. Heart rate, blood pressure,
and BAT temperature were all elevated. Respiration rate and
expired [CO2] increased and shivering was elicited in many
cases. However, this excited physiology was not accompanied
by changes in resting cortical synchrony. Thus there is no
evidence that bicuculline’s effects on the body’s physiology
were secondary to an evoked cortical arousal. Instead, bicu-
518
M. W. NASON AND P. MASON
culline microinjection may increase sympathetic and somatomotor outflow more directly via descending projections from
VMM to the spinal cord.
Methods: bicuculline microinjection
Anatomical locus
As mentioned in the Introduction, studies examining nociceptive responsiveness have focused on injections into RM,
whereas studies that measure thermoregulatory variables have
employed injections into the most ventral region of the raphe,
the RP. However, this distinction is likely spurious as the
dendrites of cells located on the midline at the level of the
facial nucleus extend both dorsally and laterally (Edwards et al.
1987; Fox et al. 1976; Gao and Mason 1997; Leontovich and
Zhukova 1963; Maciewicz et al. 1984; Mason et al. 1990;
Newman 1985; Potrebic and Mason 1993; Ramon-Moliner and
Nauta 1966; Valverde 1961). Furthermore, cells in the adjacent
reticular nuclei extend dendrites medially into all parts of the
midline raphe and often reach the contralateral reticular nucleus. The spread of radioactive bicuculline is approximated
well by the spread of Fast green (Yoshida et al. 1991). Since
the latter was limited to a small sphere in this study, it is
unlikely that diffusion can account for the distribution of c-fos
immunoreactive cells. Therefore even the small volumes (ⱕ70
nl) and concentrations injected by us and others (Morrison et
al. 1999; Samuels et al. 2002) would be expected to affect cells
in the ventral and dorsal portions of the midline as well as cells
J Neurophysiol • VOL
Sympathetic mediation of autonomic effects
Bicuculline microinjections caused an increase in BAT temperature at short latency that was almost an order of magnitude
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Bicuculline is a competitive GABAA receptor antagonist and
as such blocks transmission through that receptor, thereby
disinhibiting neurons. Like opioid agonists, bicuculline is
thought to produce antinociception when microinjected into
RM or NRMC through the selective disinhibition of one class
of RM neurons, the nociceptive-inhibitory OFF cells (Heinricher
et al. 1991). However, when ⱖ50 ng bicuculline is injected
into RM, other RM cell types, including nociceptive-facilitatory ON cells, are also excited (Heinricher and Tortorici 1994).
This may occur because bicuculline nonselectively activates
neurons through a block of small-conductance calcium-activated potassium channels (SK channels) (Grunnet et al. 2001;
Khawaled et al. 1999), resulting in depolarization and an
increase in impedance. Since SK channels are present within
the medullary raphe (Stocker and Pedarzani 2000), the bicuculline microinjections in this study likely increased the discharge of multiple RM neuronal types, including serotonergic
cells (Bagdy et al. 2000; Levine and Jacobs 1992).
Microinjection of an excitatory (or disinhibitory) drug is a
method that has been used extensively to determine the function of a group of activated cells in the complex mammalian
CNS. However, there is no direct evidence that microinjections
mimic physiological events. The heterogeneous group of cells
activated by microinjections may never be collectively engaged by normal physiological events. Furthermore, microinjections cannot tell us the effect of patterned activity in affected
neurons or whether individual neurons contribute to one or
more of the observed effects. Nonetheless, microinjections
remain the best available technique to elucidate the full range
of outputs evoked by the mass activation of the neurons affected.
located more laterally, not because of drug diffusion, but
because of the dendritic morphology of the cells in the region.
Our observations that midline microinjections of bicuculline,
but not saline, stimulate c-fos immunoreactivity in neurons
throughout raphe and NRMC bilaterally are in agreement with
this prediction.
Our original intention was to use small microinjections to
locally excite cells and thereby test whether single ventromedial medullary nuclei were uni- or multi-functional. However,
we observed that even using very small injections with a
limited diffusion of ⬍300 ␮m, cells in a large area, including
cells belonging to three to four nuclei, were “activated,” as
assessed by c-fos immunoreactivity. These cells represent the
bicuculline-responsive neurons with either somata or dendrites
located within the immediate diffusion zone of the microinjection site. Since our methods closely mirrored those of Morrison
and utilized smaller volumes than those used by others, the
microinjections made by these other groups are likely to have
also activated cells within multiple cytoarchitechtonically
bounded VMM nuclei. This is likely but not tested, because no
previous study attempted to assess the distribution of activated
cells.
Since we were ultimately unable to activate neurons restricted to a single VMM nucleus, an exclusive and unique
correspondence between a single nucleus and a single “function” remains a formal possibility. However, leaving aside the
conceptual ambiguity in what constitutes or defines a single
“function,” the extreme heterogeneity of neurons contained
within each of the VMM nuclei with respect to neurochemistry, electrophysiology, connectivity, and morphology makes
this idea unlikely. The best evidence that at least a subgroup of
neurons within a single VMM nucleus share a common function is the finding that a cold (4°C) challenge elicits c-fos
immunoreactivity in cells in RP, suggesting that neurons in RP,
and not those in NRMC and RM, produce thermoregulatory
effects (Cano et al. 2003; Martinez et al. 2001; Morrison et al.
1999). However, even in the limited fields of view that are
illustrated in these papers, scattered c-fos immunoreactive cells
are evident in RM and NRMC as well as RP. Further, exposure
to a milder cold temperature (12°C) sufficient to evoke an
increase in BAT temperature (Sakurada and Shido 1993) does
not result in the induction of c-fos immunoreactivity anywhere
in the brain (C. B. Saper and K. Kanosue, personal communication). Thus the activation of VMM neurons in response to a
4°C challenge may represent these cells’ involvement in something other than, or in addition to, cold defense, such as the
reaction to stress (Zaretsky et al. 2003). Finally, we observed in
the present experiments that injections into the lateral portions
of NRMC, where very few RP cells extend dendrites, elicited
the same increases in BAT temperature as did midline injections. Thus it is unlikely that the neurons involved in cold
defense are restricted to RP. Instead it is likely that the heterogeneous neurons distributed throughout the cytoarchitechtonic structures within VMM have multiple functions including
the modulation of cardiorespiratory, thermoregulatory, and nociceptive function as demonstrated here.
MULTIPLE MODULATORY ROLES FOR THE VENTROMEDIAL MEDULLA
Somatomotor effects
Bicuculline, but not saline, microinjections evoked tachypnea and increased [CO2] levels, and often caused shivering.
Direct projections from the medulla to phrenic motoneurons
and to the premotor neurons in the ventral respiratory group
may contribute to these bicuculline-induced changes in respiration (Gao and Mason 1997, 2001; Hosogai et al. 1998;
Mason and Fields 1989). The observed tachypnea could also be
secondary to the increase in metabolic rate produced by both
elevated brown adipose tissue activity and shivering. Since
J Neurophysiol • VOL
virus injection into somatic muscle labels VMM neurons at
short survival times (Kerman et al. 2003), VMM projections to
the ventral horn may be important in eliciting shivering.
Nociceptive modulatory effects
Consistent with previous results that activation of the VMM
by either bicuculline or excitatory amino acid microinjection
reliably blocks tail flick (Drower and Hammond 1988; Heinricher and Tortorici 1994; Heinricher et al. 1991; Jensen and
Yaksh 1984; Satoh et al. 1983; Zhuo and Gebhart 1992b), we
observed that the tail flick was suppressed by bicuculline
administration into either the medullary raphe or NRMC. Our
findings that the cardiovascular, respiratory, and cortical responses to noxious paw heat were also attenuated after bicuculline microinjection are novel evidence that multiple reactions to noxious stimulation are suppressed by medullary raphe
and reticular neurons. It is unlikely that the sympathoexcitatory
effects of bicuculline microinjection into VMM underlie the
decreases in noxious-evoked cardiorespiratory responses because paw heat still evoked increases in blood pressure and
heart rate, even when baseline blood pressure and heart rate
were significantly elevated (Fig. 7). Instead our results strongly
suggest that VMM exerts its nociceptive modulatory effects on
an early stage of central sensory processing, perhaps even at
the first synapse between nociceptors and secondary sensory
neurons in the dorsal horn. The attenuation of the cortical
desynchronization evoked by paw heat may also be due to an
inhibition of nociceptive neurons in the dorsal horn, particularly those that project to the forebrain and are involved in
evoking arousal in response to noxious inputs.
Although inhibition of most reactions to noxious stimulation can be explained by an inhibitory effect early in the
nociceptive transmission pathway, the enhancement of the
motor withdrawal from paw heat cannot be explained by this
mechanism. Our finding that bicuculline microinjection into
VMM facilitated the motor response elicited by noxious
paw heat was unexpected, because brain stem mediated
antinociception has previously been described as affecting
the entire body (Fardin et al. 1984a,b; Oliveras et al. 1974,
1978). However, there is some precedence for tail reactions
being suppressed and hindpaw reactions facilitated by the
same or similar manipulations. Bicuculline microinjection
into RM in awake rats suppresses the tail flick while decreasing the latency to a paw paddle reaction evoked by heat
(Drower and Hammond 1988). Furthermore, hot water immersion of the hindpaw blocks the noxious heat-evoked tail
flick while tail immersion decreases paw withdrawal latency
(Morgan et al. 1994). The full expression of both of these
effects—tail flick inhibition and paw withdrawal facilitation—is attenuated after transection of the spinal cord, indicating supraspinal, possibly medullary, involvement in
such differential modulation.
The opposing effects of VMM bicuculline on paw and tail
withdrawals could be explained if nociceptive-inhibiting
VMM cells act earlier in the dorsal horn than do nociceptive-facilitating VMM cells. In essence, this allows VMM’s
descending inhibitory influence to act as a gate and VMM’s
descending facilitation to control the gain of spinal nociceptive circuits. As discussed above, bicuculline microinjection is likely to have increased the discharge of both
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larger than the evoked increase in core temperature. The observed elevation in BAT temperature is therefore unlikely to be
secondary to a change in core temperature or hormonal status.
More likely, this temperature elevation represents an increase
in sympathetic outflow since the BAT is only innervated by the
sympathetic system (Bamshad et al. 1999). Such an increase
has been demonstrated after bicuculline microinjection into
either RM or RP (Morrison et al. 1999).
It has been proposed that medullary raphe activation elicits
an increase in BAT temperature through the direct activation of
preganglionic sympathetic neurons (Morrison 2001a; Morrison
et al. 1999). The primary evidence that VMM neurons are
premotoneurons is that the labeling of VMM cells after
pseudo-rabies injections into BAT tissue occurs at a time that
suggests a retrograde trisynaptic connection (BAT to postganglionic, postganglionic to preganglionic, preganglionic to raphe). However, this data may be misleading because most
spinal neurons infected with PRV at short survival times
(thought to suggest a disynaptic connection) are in fact interneurons rather than preganglionic motoneurons (Nadelhaft and
Vera 1995, 1996; Vera and Nadelhaft 2000). Thus the VMM
neurons labeled shortly thereafter would be presynaptic to
interneurons rather than to motoneurons. Furthermore, the variety of VMM’s visceral and sympathetic targets suggests a
general modulatory rather than a specific driving effect on
autonomic function (Marson 1997; Nadelhaft and Vera 1995,
1996, 2001; Vizzard et al. 1995). A simple alternative to the
premotoneuron model is that VMM activation elicits an increase in BAT temperature by modulating the excitability of
preganglionic sympathetic neurons. This could occur through
connections to premotoneurons in the intermediate gray or via
projections from VMM to thermoreceptive cells in the superficial dorsal horn (Sato 1993) that are themselves presynaptic
to preganglionic sympathetic neurons (Cabot 1996).
Increases in blood pressure and heart rate were evoked by
bicuculline microinjection into the midline raphe or the more
laterally located NRMC, a finding consistent with others (Morrison 1999; Morrison et al. 1999). Increases in heart rate and
blood pressure elicited by midline raphe injections of bicuculline are independent of hormonal release and are sympathetically mediated (Cao and Morrison 2003). Pseudorabies virus
injections into a large number of sympathetic nervous system
targets including the stellate ganglion (which provides sympathetic innervation of the heart and BAT) consistently reveal
labeling in RM, RP, and NRMC (Jansen et al. 1995a,b; Smith
et al. 1998; Strack et al. 1989a). Thus anatomical and physiological evidence strongly supports sympathetic mediation of
cardiovascular changes evoked by bicuculline into RM/RP or
NRMC.
519
520
M. W. NASON AND P. MASON
ACKNOWLEDGMENTS
The authors thank Dr. Keming Gao for help with the initial experiments, J.
Streets for programming assistance, and Drs. Patrice Guyenet, Ron Harper, and
Hayley Foo for comments on the manuscript.
GRANTS
M. W. Nason was supported by National Institute of General Medical
Sciences Grant T32 GM-07839.
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nociceptive-inhibiting and facilitating neurons in VMM. If
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sensory neurons, incoming nociceptive information would
be largely blocked when they are active. The simultaneous
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supported by qualitatively different spinal circuits. The tail
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incomplete inhibition, leaving a substantial paw nociceptive
signal for descending facilitation to act on. In summary, the
proposed mechanism may explain how VMM can specifically pattern nociceptive transmission, simultaneously inhibiting some reactions and facilitating others.
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