Physiological Identification of Pontomedullary Serotonergic Neurons
in the Rat
PEGGY MASON
Department of Pharmacological and Physiological Sciences and the Committee on Neurobiology, University of
Chicago, Chicago, Illinois 60637
INTRODUCTION
Serotonin within the spinal cord arises from cells in the
caudal brain stem, largely from neurons in the pontomedullary raphe and reticular nuclei (Dahlstrom and Fuxe 1964;
Fuxe 1965; Jacobs and Azmitia 1992). Serotonin-containing
axonal terminations in the dorsal horn are concentrated in
the superficial laminae, where nociceptors terminate, and
arise primarily from neurons in the pontomedullary raphe
magnus (RM) and adjacent nucleus reticularis magnocellularis (NRMC) (Jones et al. 1991; Kwiat and Basbaum 1992;
Oliveras et al. 1977). Although a large body of evidence
suggests that SEROTONERGIC neurons in the RM and NRMC
are important for descending pain modulation (LeBars 1988;
Potrebic et al. 1995; Sawynok 1989), little is known about
the physiological characteristics of these cells. The lack of
an established method for distinguishing pontomedullary SEROTONERGIC cells from their nonserotonergic neighbors
solely on the basis of electrophysiological properties is a
major obstacle to understanding the functional role of these
cells. The present study was designed to establish electrophysiological criteria that identify pontomedullary SEROTONERGIC cells.
Early extracellular studies revealed that most cells located
in pontomesencephalic noradrenergic or serotonergic nuclei
discharge with a slow and regular pattern, prompting many
to speculate that a slow, regular discharge is a marker for
monoaminergic neurons (Aghajanian et al. 1968; AstonJones and Bloom 1981; Heym et al. 1982b; Moore and
Bloom 1979). In a landmark study, Aghajanian and Vandermaelen (1982) demonstrated, in the anesthetized rat, that all
intracellularly labeled ‘‘slowly, rhythmically firing’’ midbrain dorsal raphe (RD) and locus coeruleus neurons contained serotonin and norepinephrine histofluorescence, respectively. Aghajanian’s study provided convincing evidence that all slow, rhythmic cells in RD and locus coeruleus
are monoaminergic. It did not, however, address whether
some cells that are not slow and regular are also monoaminergic. To determine whether the discharge of monoaminergic cells is uniformly slow and rhythmic, it is essential to
study all neurons that are encountered, regardless of the
similarity of their discharge pattern to that described by Aghajanian and Vandermaelen for RD and locus coeruleus cells.
The ‘‘slow and regular’’ standard for monoaminergic cells
has been extended to cells in many monoaminergic nuclei,
including SEROTONERGIC cells in the pontomedullary raphe
(Auerbach et al. 1985; Chiang and Gao 1986; Fornal et al.
1990; McCall and Clement 1989; Wessendorf and Anderson
1983; Wessendorf et al. 1981). However, as discussed by
Aghajanian and Vandermaelen, it is unclear whether the
discharge of SEROTONERGIC and noradrenergic cells, located
in less homogeneous monoaminergic nuclei than the RD and
the locus coeruleus, conforms to the slow and regular pattern.
0022-3077/97 $5.00 Copyright q 1997 The American Physiological Society
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Mason, Peggy. Physiological identification of pontomedullary serotonergic neurons in the rat. J. Neurophysiol. 77: 1087–1098,
1997. Spinal serotonin is derived entirely from bulbar sources and
plays an important role in spinal modulatory processes, including
pain modulation. Establishing the electrophysiological properties
of SEROTONERGIC bulbospinal neurons in the pontomedullary raphe
and reticular formation is critical to understanding the physiological role of serotonin in the spinal cord. Neurons were characterized
by their responses to noxious stimulation and their background
discharge pattern in the lightly anesthetized rat. Characterized cells
were intracellularly labeled with Neurobiotin, which was visualized
with a Texas Red fluorophore. Sections containing the labeled cells
were processed for serotonin immunocytochemistry with the use
of a Bodipy fluorophore. Forty-seven intracellularly labeled cells
were tested for serotonin immunoreactivity. The labeled neurons
were located in raphe magnus, the nucleus reticularis magnocellularis, and the adjacent reticular and raphe nuclei at levels from the
inferior olivary complex to the superior olivary complex. SEROTONERGIC cells were located in the raphe nuclei, in nucleus reticularis
magnocellularis pars alpha, and in nucleus reticularis paragigantocellularis lateralis, but not in nucleus reticularis magnocellularis
pars beta or nucleus reticularis gigantocellularis. Thirteen intracellularly labeled cells contained serotonin immunoreactivity. The
background discharge rate of SEROTONERGIC cells averaged 1.8 Hz
(range: 0.5–3.1 Hz). Discharge was steady and without sustained
pauses or bursts in firing. Most serotonin-immunoreactive cells
were unaffected or slightly excited by pinch and were unaffected
by noxious heat. Three SEROTONERGIC cells were weakly excited
by both noxious pinch and heat, whereas two SEROTONERGIC cells
were briefly inhibited by these stimuli. Cells that lacked serotonin
immunoreactivity were heterogeneous and included ON, OFF, and
NEUTRAL cells. Nonserotonergic cells differed from SEROTONERGIC
cells in having an irregular discharge pattern and/or a high mean
discharge rate. A linear discriminant function, employing background discharge characteristics as independent variables, was calculated that successfully classified 13 of 13 SEROTONERGIC and 32
of 33 nonserotonergic neurons. The probability of misclassification
with the use of this discriminant function was estimated to be
õ10%. Employing the discriminant function on a test group of
cells whose immunochemical content was unknown revealed a
population of SEROTONERGIC-LIKE cells that resembled the labeled
SEROTONERGIC cells in background discharge pattern, response to
noxious stimulation, and nuclear location. The discharge of pontomedullary SEROTONERGIC neurons is slow and steady, suggesting
that these neurons may have a role in the tonic, rather than phasic,
modulation of spinal processes.
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METHODS
Experimental protocol
Male Sprague Dawley rats (250–500 g; Sasco, Madison, WI,
n Å 95) were used. Rats were pretreated with atropine sulfate (40
mg in 0.1 ml sc) 10 min before anesthetic induction with halothane.
A Y tube was inserted into the trachea and anesthesia was maintained with 2% halothane in oxygen during surgery. A posterior
craniotomy was made overlying the cerebellum and the exposed
dura was cut. Electrodes were inserted bilaterally into the thorax
to record the electrocardiogram and into the parispinous muscles
to record the electromyographic activity during tail withdrawal
(see below). Core body temperature was maintained at 36–387C.
After surgical preparation, the halothane concentration was reduced
to 1% and the animal was allowed to equilibrate at this concentration for 30 min before recording.
Glass micropipettes (resistance 30–80 MV ) were filled with a
solution of 2% Neurobiotin (Vector Laboratories, Burlingame,
CA) in 0.1 M tris(hydroxymethyl)aminomethane buffer (pH 7.4)
and 0.15 M KCl. Microelectrodes were lowered through the cerebellum and fourth ventricle to reach the brain stem. The dorsal
edge of the RM was encountered 8.7–9.0 mm below the cerebellar
surface. At this depth, neurons were isolated by their spontaneous
activity. When cells were isolated, their waveforms had an initial
positive potential of 2–10 mV and resembled those described by
Furshpan and Furakawa (1962) as originating from recording sites
juxtaposed to the soma of the Mauthner cell. These extracellular
recordings never showed any evidence of injury discharge and
were stable for up to 20 min. After background cell discharge was
recorded for 300 s, each cell was characterized by its responses to
noxious stimulation (see next paragraph). After the cell characterization was performed extracellularly, depolarizing current was
used to impale the cell. Successful impalement was marked by a
large increase in spike height and a membrane potential of 030 to
040 mV. Neurons were intracellularly labeled with Neurobiotin
by injecting constant depolarizing current (0.3–3.0 nA) for 30 s
to 15 min.
The cell’s responses to noxious heat of the tail (2–5 trials) and
noxious pinch of both hind paws and tail were recorded. The noxious heat stimulus consisted of a step to 567C generated by a radiant
heat source focused on the ventrum of the tail. Tail temperature
was not monitored; the thermal stimulator was calibrated to heat
to 567C in separate experiments. Noxious pinch was accomplished
by squeezing either a digit, in the case of the hindpaws, or the tail
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with untoothed forceps. The same pinch was judged to be painful
when applied to a fold of the investigator’s skin.
Animals were killed with a pentobarbital overdose 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 30–60 min in fixative, and cryoprotected in 30%
sucrose in phosphate-buffered saline. Coronal serial 40- to 50-mm
sections were cut on a freezing microtome.
Histological processing
Sections were incubated in 0.5% Triton X-100 in phosphatebuffered saline for 30 min at room temperature and then transferred
to 0.4% Avidin D conjugated to Texas Red (in 0.5% Triton X100 in phosphate-buffered saline) (Vector Laboratories, Burlingame, CA) for 4 h at room temperature. Sections containing labeled
neurons were identified with the use of a fluorescent microscope
and processed for serotonin immunocytochemistry as described
previously (Potrebic et al. 1994). Briefly, the primary antibody
solution consisted of rabbit anti-5HT antibodies (INCStar, Stillwater, MN) that were preabsorbed with 15–20 mg of acetone-extracted rat liver powder and used at a final dilution of 1:5,000 in
buffer. The secondary antibody solution consisted of 10 mg/ml of
Bodipy goat anti-rabbit antisera (Molecular Probes, Eugene, OR)
and was applied for 6–8 h. Mounted sections were coverslipped
with DPX mountant (BDH Laboratory Supplies, Poole, UK) and
stored at 0407C. When the primary antibody was omitted from
the staining protocol, the Bodipy conjugated secondary antibody
produced no specific staining.
For each section with a labeled cell, a control section, taken
from the caudal midbrain, was processed for serotonin immunocytochemistry in the same solutions. The control section was viewed
before the sections containing the labeled cells. If the control section was judged to be poorly stained for serotonin, compared with
previously established standards, the cell was not considered as
part of the histological component of this experiment. On the few
occasions when sections were poorly stained (6 cells in 4 rats),
this could be explained by procedural errors such as accidentally
overfixing the tissue.
Anatomic analysis
For intracellularly labeled neurons, somata were examined on a
Zeiss Axiophot 50 equipped with 120 and 163 Plan-NEOFLUAR
objectives. The Texas Red label was viewed with the Zeiss filter
set 00 and the Bodipy label with an altered filter set 10. Because
the Texas Red label was sometimes visible when viewed through
filter set 10, this filter set was modified by replacing the transmission filter with a D540/40 filter (Chroma Technology, Brattleboro,
VT) which blocks transmission of wavelengths ú540 nm. All cells
were examined with the use of the 163 objective, with a numerical
aperture of 1.25.
The locations of labeled cells were reconstructed at low magnification by the use of a computer reconstruction system (Neurolucida, MicroBrightField, Chestertown, VT). The locations of all
serotonin-immunoreactive cells visible with a 120 objective were
marked on this reconstruction. The rostrocaudal coordinate of each
labeled cell was then determined by comparing the cell’s location
with the plates in the atlas of Paxinos and Watson (1986). The
other two coordinates were determined by measuring the mediolateral distance to the midline and the dorsoventral distance to the
top of the basilar artery.
For each intracellularly labeled cell, the cell group containing
its soma was identified. Because the distribution of SEROTONERGIC
somata is restricted, the recorded sites of all SEROTONERGIC cells
must be within the confines of nuclei that contain SEROTONERGIC
cells. Therefore the parcellation of pontomedullary raphe and reticular nuclei described by Newman (1985a,b) was adapted (see also
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This issue is particularly important in the rat RM and NRMC
(RM/NRMC), a region where no more than 25% of the
cells contain serotonin (Moore 1981; Potrebic et al. 1994).
In a study in which physiologically characterized and intracellularly labeled RM neurons were directly tested for serotonin immunoreactivity, SEROTONERGIC cells were insensitive to noxious stimulation (Potrebic et al. 1994). However,
because other neurons that were insensitive to noxious stimulation did not contain serotonin immunoreactivity (Potrebic
et al. 1994), this physiological characteristic is insufficient
to identify pontomedullary SEROTONERGIC cells.
The goals of the present study were 1) to characterize the
responses of pontomedullary serotonergic cells to noxious
stimulation and 2) to develop an electrophysiological
method by which pontomedullary serotonergic neurons
could be distinguished from their nonserotonergic neighbors.
The results presented demonstrate that pontomedullary SEROTONERGIC cells have distinct physiological characteristics
and can be distinguished from their nonserotonergic neighbors by their background discharge pattern.
IDENTIFICATION OF PONTOMEDULLARY SEROTONERGIC NEURONS
Electrophysiological analysis
Unit activity and other physiological measures were continuously acquired onto both a VHS tape recorder (Vetter, Rebersburg,
PA) and onto a Macintosh Centris 650 (2 kHz) equipped with a
12-bit A-D converter (NB-MIO-16L), a direct memory access
board (NB-DMA 2800), and LabView software (National Instruments, Austin TX). Physiological analysis was performed with
LabView software (National Instruments, Austin TX). Statistical
analyses were performed with SAS (Cary, NC). Illustrations were
created with Adobe Photoshop (Adobe Systems, Mountain View,
CA) and Igor (WaveMetrics, Lake Oswego, OR).
The background discharge was recorded extracellularly before
intracellular impalement. From this record, the mean interspike
interval, its SD, and its coefficient of variation (CVISI ) were calculated. All illustrations show the instantaneous discharge, which is
defined as the reciprocal of the interspike interval preceding the
occurrence of each action potential. This illustration style obviates
the need for arbitrarily binning the data. In graphs of instantaneous
rate, a point at 50 Hz reflects an action potential that occurred
20 ms after the preceding action potential: it does not reflect the
occurrence of 50 action potentials within a bin. Adjacent points
are joined by lines and the graph is filled to the zero line.
Responses to noxious stimulation were analyzed off-line and used
to classify nonserotonergic cells as ON, OFF, or NEUTRAL (Fields et
al. 1983). Cells that were consistently excited by noxious tail heat,
before the tail flick withdrawal, were classified as ON cells. Similarly,
OFF cells responded to noxious tail heat with large and consistent
decreases in discharge rate. Cells that were neither ON nor OFF were
classified as NEUTRAL. NEUTRAL cells were either unaffected or weakly
affected by noxious stimulation. The responses of SEROTONERGIC cells
to noxious pinch and heat were also analyzed off-line, after the immunochemical content of the cells was already known. Although SEROTONERGIC cells resembled NEUTRAL cells in their responses to noxious
stimulation (see below), their serotonin content is hypothesized to be
more critical to their function than is their response to noxious stimulation (see INTRODUCTION ). Therefore all intracellularly labeled cells
that contained immunoreactive serotonin are referred to simply as
SEROTONERGIC cells.
RESULTS
Intracellular labeling of SEROTONERGIC and
nonserotonergic cells
Of 119 cells that were physiologically characterized, 47
were successfully labeled and tested for serotonin immuno-
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reactivity. Of these 47, 13 contained serotonin immunoreactivity (see Fig. 2), whereas 34 did not contain serotonin
immunoreactivity (see Fig. 3).
As noted in METHODS, a brain was included in the histological sample only if there was adequate staining of SEROTONERGIC cells in a section taken through the midbrain. As a
further control for the immunohistochemical process, the
number of serotonin-immunoreactive cells was counted in
each pontomedullary section that contained an intracellularly
labeled cell. Similar numbers of serotonin-immunoreactive
cells were seen in sections containing intracellularly labeled
cells that were or were not SEROTONERGIC, evidence that the
lack of serotonin immunoreactivity in intracellularly labeled
nonserotonergic cells was not simply the result of inadequate
immunochemical processing.
Nuclear location of labeled cells
The locations of intracellularly labeled cells are shown in
Fig. 1, A and B ( ● ). Separate panels are used to depict the
locations of serotonergic (Fig. 1A) and nonserotonergic
(Fig. 1B) cells and the nuclear boundaries in the region
(Fig. 1C). The locations of serotonin-immunoreactive cells,
plotted from one representative section at each rostrocaudal
level, are depicted by small dots in Fig. 1, A–C. Labeled
SEROTONERGIC cells were located in RM (n Å 4), NRMCa
(n Å 4), RP (n Å 2), NRPGl (n Å 2), and raphe obscurus
(n Å 1). Labeled nonserotonergic cells were located within
regions where serotonergic cells are present, including RM
(n Å 9), NRMCa (n Å 2), NRPGl (n Å 1), and raphe
obscurus (n Å 1). For example, the intracellularly labeled
nonserotonergic cell illustrated in Fig. 3A1 is in very close
proximity to the serotonin-immunoreactive neuron shown in
Fig. 3A2.
Nonserotonergic cells were also located in regions lacking
SEROTONERGIC cells, such as NRMCb (n Å 13) and nucleus
reticularis gigantocellularis (n Å 8). Although serotoninimmunoreactive cells were observed in the arcuate nucleus,
no SEROTONERGIC or nonserotonergic cell was intracellularly
labeled in this area.
Responses to noxious stimulation of
nonserotonergic cells
SEROTONERGIC
and
Previous work suggests that SEROTONERGIC cells do not
have inhibitory or excitatory responses to noxious tail heat
(Potrebic et al. 1994). In line with that report, most serotonin-immunoreactive cells were either unresponsive to noxious pinch and heat (n Å 4) or slightly excited by pinch and
unaffected by noxious heat (n Å 4) (see Fig. 4, A and C).
Figure 4 illustrates the range of SEROTONERGIC cell responses
to noxious heat (left) and pinch (right). In this figure, the
calculated instantaneous discharge rate (see METHODS ) is
shown in a graph below a raster plot of the same activity.
A SEROTONERGIC cell that was insensitive to both noxious
heat and pinch is shown in Fig. 4A. An example of a SEROTONERGIC cell that was insensitive to noxious heat but slightly
excited by noxious pinch is shown in Fig. 4C. Three SEROTONERGIC cells were weakly excited by both noxious pinch and
heat (see Fig. 4B), whereas two were inhibited by both
noxious pinch and heat (see Fig. 4D).
Nonserotonergic cells responded to noxious stimulation
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Kruger et al. 1995). This parcellation resembles that of Kruger et
al. (1995) for the rat and that of Taber et al. (1960) for the cat.
Newman’s anatomic system represents a departure from previous
work in the field, including work by this author. However, it clearly
distinguishes nuclei that contain SEROTONERGIC cells from nuclei
that do not contain SEROTONERGIC cells, an important advantage in
the present study.
The cytoarchitechtonic boundaries of Newman are illustrated in
Fig. 1C. The most important change in this parcellation from that
of Paxinos and Watson (1986) and Jones (1995) is that the nucleus
located lateral to the RM is termed NRMC (see Fig. 1, C2–C5)
instead of nucleus reticularis paragigantocellularis (Basbaum and
Fields 1984) or nucleus reticularis gigantocellularis pars alpha
(Paxinos and Watson 1986). As seen in Fig. 1, C2–C5, NRMC
consists of two subnuclei, a ventrally located pars alpha (NRMCa )
and a dorsally located pars beta (NRMCb ). NRMCb is distinguishable from NRMCa by its larger, more densely packed cells.
Neurons containing serotonin are found in RM, raphe pallidus
(RP), raphe obscurus, NRMCa, nucleus reticularis paragigantocellularis lateralis (NRPGl) (see Fig. 1C, dots), and the arcuate nucleus (see Fig. 1, C1–C4, dots within pyramids) (Bowker et al.
1983; Steinbusch et al. 1981). It is important to note that SEROTONERGIC cells are absent from NRMCb (see Fig. 1, C2–C5).
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FIG . 1. Location of labeled cells and serotonin-immunoreactive cells within the pontomedullary raphe and reticular cell
groups studied. In all columns, the ventromedial brain stem has been enlarged and the locations of serotonin-immunoreactive
cells (dots) are illustrated. A1–A5: locations of all intracellularly labeled SEROTONERGIC cells ( ● ) and SEROTONERGIC-LIKE
cells ( s ) are shown in the left column. SEROTONERGIC-LIKE cells are cells that have the physiological characteristics of
SEROTONERGIC cells (as judged by the discriminant function; see text) but that were not directly tested for serotonin
immunoreactivity. In A and B, some serotonin-immunoreactive cells near the labeled cells are covered by black ( ● ) or opaque
( s ) filling. B1–B5: locations of intracellularly labeled nonserotonergic cells ( ● ) and nonserotonergic-like cells ( s ) are
shown. Nonserotonergic-like cells are cells that have the physiological characteristics of nonserotonergic cells (as judged by
the discriminant function; see text) but that were not directly tested for serotonin immunoreactivity. C1–C5: nuclear
boundaries of the pontomedullary raphe and reticular cell groups studied are shown with the locations of serotonin-immunoreactive cells (dots) overlaid. The anteroposterior level for each trio of sections is as follows (mm caudal to interaural 0):
A1–C1: 03.4 to 02.65; A2–C2: 02.6 to 02.45; AC–C3: 02.4 to 02.2; A4–C4: 02.1 to 01.7; A5–C5: 01.6 to 01.1.
ION, inferior olivary nuclei; N. VII, facial nucleus; NRGC, nucleus reticularis gigantocellularis; NRMCa, nucleus reticularis
magnocellularis pars alpha; NRMCb, nucleus reticularis magnocellularis pars beta; NRPGl, nucleus reticularis paragigantocellularis lateralis; p, pyramid; RD, raphe dorsalis; RM, raphe magnus; RO, raphe obscurus; RP, raphe pallidus.
in patterns that were consistent with previous descriptions
of ON, OFF, or NEUTRAL cells (Fields et al. 1983; Leung and
Mason 1995). The 34 intracellularly labeled cells that did
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not contain serotonin immunoreactivity included 4 ON, 6
OFF, and 24 NEUTRAL cells. To determine whether SEROTONERGIC cells physiologically resemble ON and OFF cells, the
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IDENTIFICATION OF PONTOMEDULLARY SEROTONERGIC NEURONS
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FIG . 2.
GIC cells.
Photomicrographs of 3 SEROTONERFor each cell, the top row (1) shows
the intracellular label visualized with Texas
Red and the bottom row (2) shows the serotonin immunoreactivity visualized with Bodipy.
A: SEROTONERGIC cell in NRMCa. B: SEROTONERGIC cell in RM. C: SEROTONERGIC cell in
NRMCa. Calibration bar in C2 represents 25
mm for all images.
FIG . 3. Photomicrographs of 2 nonserotonergic cells located close to
unlabeled SEROTONERGIC cells. For both cells, the left column (1) shows
the intracellular label visualized with Texas Red and the right column (2)
shows the serotonin immunoreactivity visualized with Bodipy. A: nonserotonergic NEUTRAL cell in RM. B: nonserotonergic NEUTRAL cell in RM.
Calibration bar in B2 represents 25 mm for all images.
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tail heat evoked a stronger inhibition in a typical OFF cell
(see Fig. 5E) that outlasted the stimulus. This OFF cell did
not resume discharging for ¢3–4 s after tail heat termination
and firing remained below baseline rates for another 10–15
s after stimulus termination.
As typified by the examples above, the responses of nonserotonergic ON and OFF cells to noxious stimulation are stronger
and more sustained than those of SEROTONERGIC cells. Thus,
judging from their responses to noxious stimulation alone, all
SEROTONERGIC cells would be classified as NEUTRAL cells because they did not respond at all or responded only weakly to
noxious tail heat. However, in the present paper, all intracellularly labeled and serotonin-immunoreactive cells are grouped
separately from nonserotonergic NEUTRAL cells, and are called
SEROTONERGIC cells (see METHODS ).
Discharge pattern of
SEROTONERGIC
cells
Previous studies of SEROTONERGIC and other monoaminergic cells have emphasized these cells’ slow and regular discharge patterns (see INTRODUCTION ). To test whether the
discharge patterns of SEROTONERGIC and nonserotonergic
cells were different, 5 min of background discharge was
recorded and analyzed. SEROTONERGIC cells discharged
slowly and without prolonged bursts or pauses in firing (see
Fig. 6A), a pattern that resembles that previously described
for SEROTONERGIC RD cells.
The mean discharge rate for the 13 SEROTONERGIC cells
was 1.8 Hz, with a range of 0.5–3.1 Hz (see Table 1, Figs.
6A and 7A). Although SEROTONERGIC cells did not alternate
between inactivity and bursts of activity, they were not
‘‘clocklike’’ either (see Fig. 6A). To measure regularity,
the SD of the interspike intervals and the CVISI were calculated for each cell.1 The CVISI for SEROTONERGIC cells ranged
from 0.23 to 0.74 and averaged 0.40 (see Table 1, Fig. 7B).
To determine whether there was a systematic relationship
between physiological properties and anatomic location, measures of rate (mean interspike interval) and regularity (CVISI )
for each cell were plotted against every coordinate value of
1
Readers may interpret the CVISI values with the following benchmarks
in mind. As discharge approaches perfect regularity, the CVISI approaches
0. As a cell’s discharge approaches a Poisson distribution of intervals, the
CVISI approaches unity. A cell with bursting discharge has a CVISI that is
greater than unity (Stein 1967; Wilbur and Rinzel 1983).
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noxious heat-evoked responses of SEROTONERGIC cells were
compared with those characteristic of nonserotonergic ON
and OFF cells (see Fig. 5). As stated above, noxious heat
did not evoke a response in most SEROTONERGIC neurons
(n Å 8) (Fig. 5, A and B). Among SEROTONERGIC cells that
responded to noxious heat (n Å 5), responses were typically
small and transient. The typical inhibitory response of a
SEROTONERGIC cell (Fig. 5C) is much weaker and shorter in
duration that that of a typical OFF cell (Fig. 5E). Similarly,
the typical excitatory response of a SEROTONERGIC cell (Fig.
5D) is also weaker and shorter in duration that that of a
typical ON cell (Fig. 5E).
In the SEROTONERGIC cell with the largest excitatory response to heat (see Fig. 4B), the mean discharge rate increased from 3 to 6 Hz. Within 4 s of stimulus termination,
this cell’s discharge returned to baseline values. In contrast,
in the ON cell illustrated in Fig. 5F, the discharge rate increased from 13 to 26 Hz during the tail heat stimulus and
remained elevated for 11 s after stimulus termination. In the
SEROTONERGIC cell with the greatest inhibitory response to
heat (see Fig. 4D), the cell was briefly inhibited by tail heat
but resumed firing during the stimulus application. Noxious
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somatic location (rostrocaudal, mediolateral, and dorsoventral;
see METHODS ). A least-squares linear regression analysis revealed that no significant correlations existed between background discharge properties and cellular location.
Discharge characteristics of nonserotonergic cells
The background discharge of nonserotonergic cells was
also recorded and analyzed quantitatively. ON and OFF cells
had low discharge rates averaging 5.8 and 4.5 Hz, respectively (see Table 1). The discharge of both ON and OFF cells
contained pauses and bursts in activity that measured tens
of seconds to minutes in duration. This discharge irregularity
was evident in both groups having a mean CVISI of ú1 (see
Table 1). As illustrated in Fig. 6B, the intraburst discharge
of both ON and OFF cells was also irregular. Thus ON and
OFF cells can be distinguished from SEROTONERGIC cells by
the irregularity of their discharge.
The background discharge of NEUTRAL cells was heterogeneous with 9 cells having a regular discharge pattern and 15
NEUTRAL cells having an irregular discharge pattern (Leung
and Mason 1995). Regularly discharging NEUTRAL cells discharged at a mean rate of 20.5 Hz and had a mean CVISI of 0.24
(see Table 1). A typical example of such a cell is illustrated in
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Fig. 6B. Because the regularly discharging NEUTRAL cell with
the lowest rate of discharge fired at a rate of 10.0 Hz, these
cells were easily distinguished from SEROTONERGIC cells by
their relatively high discharge rate. Irregularly discharging NEUTRAL cells (n Å 15) fired at a mean rate of 7.6 Hz and had a
mean CVISI of greater than unity (see Table 1). These NEUTRAL
cells could be distinguished from SEROTONERGIC cells by their
irregular discharge pattern and in some cases by their high
mean discharge rate (see Figs. 6B and 7A).
Both the mean interspike interval and the CVISI of SEROTONERGIC cells overlapped with those of nonserotonergic cells.
However, when the mean interval and CVISI are plotted on a
two-dimensional graph (see Fig. 7B), there is no overlap between SEROTONERGIC and nonserotonergic cells. A two-way
analysis of variance showed that the mean interspike interval
and CVISI for SEROTONERGIC and nonserotonergic cells were
significantly different [F(2,43) Å 7.885, P Å 0.001]. Thus
nonserotonergic cells discharged more rapidly and/or more
irregularly than SEROTONERGIC cells (see below).
Discriminant analysis of SEROTONERGIC and
nonserotonergic discharge patterns
The nonoverlapping distributions of background discharge
properties for known SEROTONERGIC and nonserotonergic
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FIG . 4. Response of 4 SEROTONERGIC cells to noxious heat (left) and noxious pinch (right). Line under each trace: timing
of the stimuli. Arrow: timing of the tail withdrawal from noxious heat. In this and the following figures, the instantaneous
discharge rate is plotted on the ordinate and the time at which the action potential occurred is plotted on the abscissa.
Although the instantaneous discharge rate cannot be equal to 0, any instantaneous rate õ1 represents a pause of ú1 s. A:
RP cell does not respond to either noxious heat or noxious pinch. B: RM cell is slightly excited by both pinch and heat. C:
RP cell is excited by noxious pinch but unaffected by noxious heat. D: RM cell is inhibited by both pinch and heat. Time
calibration in C, left trace applies to the left traces in A and B as well. Time calibration in C, right trace applies to the right
traces in A, B, and D as well. Time calibration in D, left trace applies only to that trace.
IDENTIFICATION OF PONTOMEDULLARY SEROTONERGIC NEURONS
1093
cells suggested that a function could be described that would
separate the two cell groups. The method used for the development of this function was a linear discriminant analysis
(Morrison 1990). This analysis was performed on the discharge data from 13 SEROTONERGIC and 33 nonserotonergic
cells. This data set, which was used to develop the discriminant function, is termed the ‘‘training’’ data set. The discharge pattern of one nonserotonergic cell was not recorded
on tape before impalement and was therefore not included
in this analysis.
As independent variables, the mean interspike interval
( 1U , in ms) and the SD of the intervals (s, in ms) were
chosen. The SD was used in place of the CVISI because its
use led to a better discrimination. The discriminant analysis
characterizes each unit by the linear sum, yi Å b0 / b11U i /
b2si . The mean values of yi for the SEROTONERGIC and nonserotonergic cells are called yp and yn , respectively. The coefficients, b1 and b2 , are then chosen to maximize the difference between the values of yp and yn . The constant, b0 , is
set to (yp / yn )/2. For the training data set containing 46
/ 9k0e$$mr08 J641-6
known SEROTONERGIC and nonserotonergic cells, the resulting discriminant function is
y( 1U , s) Å 146 0 1U / 0.98s
(1)
The straight line representing y( 1U , s) Å 0 defines the
optimal linear boundary between the two groups and is illustrated in Fig. 7A. When plotted in log-log coordinates, a
curved line results. If the value of this function is õ0
[y( 1U , s) õ 0, points below the line in Fig. 7A], the cell is
likely to be SEROTONERGIC, and if the value of the function
is ú0 [y( 1U , s) ú 0, points above the line in Fig. 7A], the
cell is likely to be nonserotonergic. The discriminant function defines two conditions that must be met for a cell to be
classified as SEROTONERGIC: 1) because s must be ú0, then
1U ú 146 ms or, equivalently, the discharge rate must be
õ7 Hz; and 2) the discharge must be sufficiently regular
such that s õ 1U by ¢146 ms.
All 13 SEROTONERGIC neurons fell below the line (all ●
are below the function line), whereas 32 of 33 nonserotonergic cells are located above the line. One nonserotonergic
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FIG . 5. Responses of 4 SEROTONERGIC cells to noxious tail heat are compared with those of an ON cell and an OFF cell.
Line under each trace: timing of the tail heat stimulus. Arrow: timing of the withdrawal. A: RP cell does not respond to
noxious tail heat. B: NRMCa cell is unaffected by noxious tail heat. C: NRMCa cell is weakly inhibited by noxious tail
heat. D: NRMCa cell is weakly excited by noxious tail heat. E: NRMCa OFF cell is strongly inhibited by noxious tail heat.
F: NRPGl ON cell is strongly excited by noxious tail heat. Each trace is 50 s in duration. Calibration bar in B: 10 s.
1094
P. MASON
cell was misclassified as a SEROTONERGIC cell (see s below
the line in Fig. 7A). This cell had a mean interspike interval
of 5,640 ms ( õ0.2 Hz) and an SD of 4,650 ms, suggesting
that the discriminant function may not be appropriate for
cells with extremely low discharge rates. The value of the
function for several other cells was very close to 0. However,
for each of these cells the value of the function (and the
center of the symbol in Fig. 7) was consistent with the
serotonin immunoreactivity of the cell. The discharge rate
and regularity for nonserotonergic ON ( n ), OFF ( , ), and
NEUTRAL ( s ) cells overlapped (see Fig. 7A).
Only 1 of 46 cells independently tested for serotonin immunoreactivity was misclassified, suggesting a 2% probability of misclassification when using the training data set. To
employ this discriminant function on cells that will be recorded in the future, it is important to estimate the probability
of misclassification for cells whose serotonin content is not
known. One would expect that the misclassification rate
would increase because future cells are not part of the training data set that was used to calculate the discriminant function. To estimate this error rate, a cross validation procedure
was performed (Lachenbruch and Mickey 1968). In this
procedure, 1 of the 46 identified cells was removed from
the sample and a new discriminant function was calculated
TABLE
from the remaining 45 cells. The new discriminant function
was then used to classify the removed cell. This process was
repeated for each cell in the sample. When this procedure
was performed on the present data set, 2 of 33 (6.1%) nonserotonergic cells were misclassified as SEROTONERGIC cells
and 1 of 13 (7.7%) SEROTONERGIC cells was misclassified
as nonserotonergic cells. This result suggests a misclassification rate of 5–10%.
Application of the discriminant function to an unknown
sample
One method for assessing the discriminant function’s validity is to apply the function to a ‘‘test’’ sample and compare
the resulting cell classes with those in the original, training
sample. Therefore the discriminant function was used to
classify 72 cells that were subjected to the same physiological protocol as above but were not immunochemically tested
for serotonin because they were not intracellularly labeled
(n Å 43), because the immunocytochemistry was inadequate
(n Å 6), or because they were processed with diaminobenzidine and their serotonin content was not determined (n Å
23). These cells with unknown immunochemical content
constitute a sample of test cells. The discriminant scores
1.
Cell Type
N
SEROTONERGIC
ON
OFF
NEUTRAL-regular
NEUTRAL-irregular
All nonserotonergic
13
4
6
9
14
33*
Mean Discharge Rate
Range of
Discharge Rate
{
{
{
{
{
{
0.5–3.1 Hz
0.6–12.1
0.2–13.9
10.0–45.5
0.2–22.2
0.2–45.5
1.8
5.8
4.5
20.5
7.6
10.3
0.2
2.4
2.5
3.7
1.7
1.8
Values are means { SE. * The data from one nonserotonergic
was not recorded on tape.
/ 9k0e$$mr08 J641-6
NEUTRAL
Mean CVISI
Range of CVISI
{
{
{
{
{
{
0.23–0.74
0.43–1.65
0.76–2.63
0.12–0.52
0.36–2.22
0.12–2.63
0.40
1.08
1.35
0.24
1.05
0.88
0.04
0.25
0.30
0.04
0.18
0.12
(irregular type) cell is not included here because its background discharge
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FIG . 6. Background discharge of SEROTONERGIC and nonserotonergic cells. Each trace is 1 min in length and is taken
from a 5-min recording, from which the mean discharge rate and the coefficient of variation printed above each trace was
calculated. A: discharge of 4 SEROTONERGIC cells. B: discharge of 4 nonserotonergic cells.
IDENTIFICATION OF PONTOMEDULLARY SEROTONERGIC NEURONS
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FIG .
7.
Comparison of the rate and regularity of background discharge from SEROTONERGIC cells ( ● ) and nonserotonergic
( s ), ON ( n ) and OFF ( , ) cells. A: log-log plot of the mean interspike interval vs. the SD of the interspike interval
for all labeled cells. B: log-log plot of the mean interspike interval vs. the coefficient of variation for immunochemically
tested cells and for SEROTONERGIC-LIKE ( ) and nonserotonergic-like units (same symbols as in A, with center dots).
NEUTRAL
were calculated, from Eq. 1, for the cells in the test sample.
To determine the accuracy of the classification procedure,
the responses to noxious stimulation and nuclear locations
of test cells with a negative value were then compared with
those of known SEROTONERGIC neurons. Similarly, test cells
with a positive value were compared with known nonserotonergic neurons.
Of the 72 test cells, 18 had negative scores and were
/ 9k0e$$mr08 J641-6
classified as SEROTONERGIC-LIKE neurons. The scores for all
other test cells were positive and these cells are referred to
as nonserotonergic-like neurons. The proportion of SEROTONERGIC-LIKE neurons in the test sample (18 of 72, 25%) was
similar to the proportion of SEROTONERGIC neurons in the
training sample (13 of 47, 28%).
The test sample of SEROTONERGIC-LIKE and nonserotonergic-like units resembled their training counterparts in their
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1096
P. MASON
DISCUSSION
In the present study, the physiological characteristics of
pontomedullary SEROTONERGIC cells in five cytoarchitechtonically distinct nuclei were found to be qualitatively and
quantitatively distinct from those of their nonserotonergic
neighbors. SEROTONERGIC cells were distinguishable by their
slow, nonbursting pattern of discharge. Because all pontomedullary cells with slow, rhythmic discharge contained
serotonin, previous studies that classified slow and regularly
firing cells as SEROTONERGIC were probably correct in this
assignation. However, the discharge of several intracellularly
labeled SEROTONERGIC cells was neither ‘‘rhythmic’’ nor
‘‘regular.’’ The discharge of all SEROTONERGIC cells is best
described as steady and without sustained pauses or bursts
in firing. It is therefore possible that SEROTONERGIC cells
with a slow and steady, but not regular, discharge pattern
have been misidentified as nonserotonergic in previous studies.
A quantitative analysis of SEROTONERGIC and nonserotonergic neurons in the pontomedullary raphe and reticular
nuclei of the anesthetized rat revealed that SEROTONERGIC
neurons have a mean interspike interval that is greater than
/ 9k0e$$mr08 J641-6
the SD of the interspike interval by Ç150 ms or more. This
quantitative physiological method for identifying SEROTONERGIC and nonserotonergic neurons is simple and easily
employed and has an estimated misclassification rate of
õ10%. To employ this algorithm, 5 min of background
discharge must be recorded and the interspike intervals collected. After the mean and SD of the interspike interval are
calculated, these numbers are used to calculate the value of
the discriminant function; SEROTONERGIC-LIKE cells will have
a negative value and nonserotonergic-like cells will have a
positive value. Because units located outside of the circumscribed regions containing serotonin-immunoreactive neurons cannot contain serotonin, it is important to localize the
recording site for each potential SEROTONERGIC cell in a
section stained for serotonin immunoreactivity. In this regard
it should be noted that inferior olivary cells discharge similarly to SEROTONERGIC cells, and have a negative value in
the discriminant function described above, but are located
in a region that does not contain any SEROTONERGIC cells 2
(unpublished observations).
Although the background discharge was recorded extracellularly for 5 min in the present study, shorter recordings
also reveal the same patterns (P. Mason and D. L. Chen,
unpublished observations). The steady discharge of a regularly discharging nonserotonergic NEUTRAL cell is immediately distinguishable from that of a SEROTONERGIC cell by
its high discharge rate. Discharge during the burst of an ON,
OFF, or irregularly discharging NEUTRAL cell is less regular,
and often has a higher mean rate, than the steady firing
pattern of a SEROTONERGIC cell. These differences can be
observed in records of °1 min (see Fig. 6).
Limitations of the current study
There are three limitations to the present study. First, it
is possible that some nonserotonergic cells contained low
concentrations of serotonin that were below the detection
threshold of the immunochemical procedure. Second, no
cells, SEROTONERGIC or not, located within the pyramids (the
arcuate nucleus) were successfully labeled. It is therefore
unclear whether the arcuate SEROTONERGIC cells have physiological characteristics similar to those of SEROTONERGIC
cells located in other pontomedullary raphe and reticular
nuclei.
The most serious limitation to the present study is the
limited context, i.e., the halothane-anesthetized rat, for which
the present findings provide direct evidence. Future studies
are needed to compare the discharge pattern of SEROTONERGIC-LIKE cells in anesthetized and unanesthetized conditions.
Despite this need, there is some evidence that SEROTONERGIC
cells discharge with a similar pattern when recorded under a
variety of anesthetics as well as in the absence of anesthesia.
SEROTONERGIC cells recorded in the halothane-anesthetized
rat in the present study resemble pontomedullary raphe cells
in awake cat (Auerbach et al. 1985; Fornal et al. 1985; Heym
et al. 1982a; Jacobs and Azmitia 1992) and in isofluraneanesthetized rats (P. Mason and C. G. Leung, unpublished
observations). Furthermore, cells with a slow and regular
discharge in the awake cat RD, a sample that likely included
2
Inferior olivary cells can also be distinguished electrophysiologically by
their complex extracellular waveform (Crill 1970).
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responses to noxious stimulation. As was the case for positively identified SEROTONERGIC cells, most SEROTONERGICLIKE cells did not respond to either pinch or heat (n Å 9)
or were slightly excited by both pinch and heat ( n Å 5). The
remaining SEROTONERGIC-LIKE units were slightly excited by
pinch but unaffected by heat (n Å 2) or were inhibited by
both pinch and heat (n Å 2). Nonserotonergic-like cells
included 7 ON, 11 OFF, and 36 NEUTRAL cells; these three
cell classes were present in similar proportions in the training
and test samples. As with cells in the training sample, the
responses of nonserotonergic-like ON and OFF cells were
typically larger in magnitude and longer in duration than
were the responses of SEROTONERGIC-LIKE neurons.
Only 8 of the 18 SEROTONERGIC-LIKE units were labeled.
Like the SEROTONERGIC cells, these cells were located in RM
(n Å 5), NRMCa (n Å 1), NRPGl (n Å 1), and RP (n Å
1) (see Fig. 1A, s ). No SEROTONERGIC-LIKE units were in
nucleus reticularis gigantocellularis or NRMCb, two nuclei
that do not contain serotonin-immunoreactive cells. Eighteen
nonserotonergic-like units were labeled. These cells were
located in RM (n Å 8), NRMCb (n Å 5), NRMCa (n Å
2), nucleus reticularis gigantocellularis (n Å 2), and NRPGl
(n Å 1) (see Fig. 1B, s ).
As dictated by the discriminant analysis, the background
discharge patterns of SEROTONERGIC-LIKE units resembled
those of SEROTONERGIC cells. Figure 7B shows the distribution of 1U and CVISI for both the training and test cells. The
line in Fig. 7B represents the line given by 0 Å (146/ 1U ) 0
1/ 0.98 CVISI , which is the discriminant function (Eq. 1)
in terms of 1V and CVISI . The mean interval and CVISI of
the SEROTONERGIC-LIKE cells overlapped with those of SERO). In addition, the
TONERGIC cells (see Fig. 7B, ● and
mean interval and CVISI of nonserotonergic-like neurons
overlapped those of nonserotonergic cells (see Fig. 7B, open
symbols with and without center dot), evidence that the
intracellularly labeled and immunochemically tested sample
contains cell types that are representative of the pontomedullary population.
IDENTIFICATION OF PONTOMEDULLARY SEROTONERGIC NEURONS
at least some SEROTONERGIC cells, did not alter their discharge by ú20% after administration of a sedative bolus of
the anesthetic chloral hydrate, evidence that the physiological characteristics of candidate SEROTONERGIC cells in the
anesthetized state resemble those during the unanesthetized
waking state (Heym et al. 1984).
SEROTONERGIC
neurons are not
ON
or
OFF
cells
neurons are unlikely to be necessary for
RM antinociception
SEROTONERGIC
Several lines of evidence, accumulated over the last 25
years, have led to the hypothesis that SEROTONERGIC bulbospinal neurons are important mediators of RM-mediated inhibition of spinal nociceptive transmission (LeBars 1988;
Potrebic et al. 1994; Sawynok and Reid 1987). Because OFF
cells are the hypothesized pain inhibitory cells of the RM,
there was an expectation that OFF cells would contain serotonin (Fields et al. 1991). However, the present study confirms
a previous report that OFF cells do not contain serotonin
(Potrebic et al. 1994). In support of the idea that SEROTONERGIC cells do not mediate the descending inhibition of nociceptive transmission, the optimal RM stimulation parameters
for evoking serotonin release differ significantly from those
for eliciting antinociception. This was most clearly demonstrated by Sorkin et al. (1993), who showed that high-frequency stimulation of RM at intensities that suppress nociceptive neuronal responses does not evoke serotonin release.
In contrast, stimulation of the RM at slower rates evokes
serotonin release in the dorsal horn but does not inhibit
spinal nociceptive transmission (Bowker and Abhold 1990;
Hammond et al. 1985; Sorkin et al. 1993).
ing somatosensory (both nociceptive and nonnociceptive),
autonomic, and motor processes, are modulated in accordance with behavioral state (Kleitman 1963). Given that
serotonin has a net inhibitory effect on pain transmission
(Belcher et al. 1978; Duggan 1992; Yaksh 1985; Yaksh and
Wilson 1979) and that at least some SEROTONERGIC cells
are tonically more active during waking than sleeping (see
above), then one can hypothesize that nociceptive sensitivity
will be greater during sleeping than during waking. Indeed,
rats withdraw from noxious paw heat more rapidly during
slow-wave sleep than during waking (Escobedo and Mason
1995). In further support of a waking ‘‘analgesia’’ mediated
by monoamines, systemic methysergide, a serotonin antagonist, causes dorsal horn neurons that only responded to innocuous stimulation to become responsive to noxious pinch
and heat in awake cats (Saito et al. 1990). Future experiments are required to determine whether the state-dependent
discharge of SEROTONERGIC cells is sufficient to explain the
state-dependent changes in nociceptive sensitivity.
Conclusions
In the anesthetized preparation, SEROTONERGIC cells discharge slowly and steadily and are not strongly affected by
noxious stimulation. The slow tonic discharge of pontomedullary SEROTONERGIC cells resembles that of SEROTONERGIC
cells in pontomesencephalic nuclei as well as that reported
for other monoaminergic cells (Jacobs and Azmitia 1992;
Monti 1993; Moore and Bloom 1979). The finding that SEROTONERGIC cells have a tonic discharge pattern has important implications for the functional role of these cells.
Like other monoaminergic cells, SEROTONERGIC cells are
likely to function in the modulation of spinal processes during tonic behavioral or social states.
The author thanks I. Escobedo and D. L. Chen for invaluable technical
assistance, Drs. D. A. Hanck and T. Karrison for assistance with the discriminant analysis, and Drs. McCrea and J. M. Goldberg for helpful conversations throughout the study. The author also thanks Drs. McCrea and H. L.
Fields for comments on the manuscript. Special thanks are due to Dr.
Goldberg for invaluable advice and support.
This research was supported by the Brain Research Foundation and National Institute of Neurological Disorders and Stroke Grant NS-33984.
Address for reprint requests: P. Mason, Dept. of Pharmacological and
Physiological Sciences, University of Chicago, MC 0926, 947 East 58th
St., Chicago, IL 60637.
Received 13 August 1996; accepted in final form 29 October 1996.
REFERENCES
Functional implications
Phasic pain modulation, such as that which occurs after
noxious stimulation, has been the focus of intensive investigation (Basbaum and Fields 1984). SEROTONERGIC cells are
unlikely to participate in such phasic modulation because
they are not strongly inhibited or excited by noxious stimulation. The phasic responses of nonserotonergic ON and OFF
cells are better suited to participation in the pain modulation
that follows noxious stimulation. In support of this idea,
brief noxious stimulation evokes an inhibition of OFF cells,
an excitation of ON cells, and an increase in subsequent
nociceptive sensitivity (Ramirez and Vanegas 1989).
SEROTONERGIC cells that discharge slowly and steadily are
likely to serve a tonic function. All spinal functions, includ-
/ 9k0e$$mr08 J641-6
AGHAJANIAN, G. K., FOOTE, W. E., AND SHEARD, M. H. Lysergic acid diethylamide: sensitive neuronal units in the midbrain raphe. Science Wash.
DC 161: 706–708, 1968.
AGHAJANIAN, G. K. AND VANDERMAELEN, C. P. Intracellular identification
of central noradrenergic and serotonergic neurons by a new double labeling procedure. J. Neurosci. 2: 1786–1792, 1982.
ASTON-JONES, G. AND BLOOM, F. E. Activity of norepinephrine-containing
locus coeruleus neurons in behaving rats anticipates fluctuations in the
sleep-waking cycle. J. Neurosci. 1: 876–886, 1981.
AUERBACH, S., FORNAL, C., AND JACOBS, B. L. Response of serotonincontaining neurons in nucleus raphe magnus to morphine, noxious stimuli, and periaqueductal gray stimulation in freely moving cats. Exp. Neurol. 88: 609–628, 1985.
BASBAUM, A. I. AND FIELDS, H. L. Endogenous pain control systems: brainstem spinal pathways and endorphin circuitry. Annu. Rev. Neurosci. 7:
309–338, 1984.
BELCHER, G., RYALL, R. W., AND SCHAFFNER, R. The differential effects of
09-02-97 13:43:57
neupal
LP-Neurophys
Downloaded from http://jn.physiology.org/ by 10.220.32.246 on June 18, 2017
Although the classification of ON and OFF cells is admittedly crude, the responses of SEROTONERGIC cells to noxious
pinch and/or heat appear different from those of ON and OFF
cells and are similar to those of NEUTRAL cells. First, the
discharge of most SEROTONERGIC neurons was unaffected by
noxious tail heat. Second, the SEROTONERGIC cells that did
respond to noxious tail heat had weak responses that did not
outlast the stimulus. The present results are consistent with
the previous findings that RM/NRMC ON and OFF cells are
nonserotonergic (Potrebic et al. 1994) and that physiologically presumed SEROTONERGIC cells are weakly sensitive or
insensitive to noxious stimulation (Auerbach et al. 1985;
Chiang and Gao 1986; Wessendorf and Anderson 1983).
1097
1098
P. MASON
/ 9k0e$$mr08 J641-6
LEBARS, D. Serotonin and pain. In: Neuronal Serotonin, edited by N. N.
Osborne and M. Hamon, New York: Wiley, 1988, p. 171–226.
LEUNG, C. G. AND MASON, P. Effects of anesthetic depth on the activity of
pontomedullary raphe and medial reticular neurons in the rat. Brain Res.
699: 71–82, 1995.
MCCALL, R. B. AND CLEMENT, M. E. Identification of serotonergic and
sympathetic neurons in medullary raphe nuclei. Brain Res. 477: 172–
182, 1989.
MONTI, J. M. Involvement of histamine in the control of the waking state.
Life Sci. 53: 1331–1338, 1993.
MOORE, R. Y. The anatomy of central serotonin neuron systems in the rat
brain. In: Serotonin Neurotransmission and Behavior, edited by B. L.
Jacobs and A. Gelperin. Boston, MA: MIT Press, 1981, p. 35–71.
MOORE, R. Y. AND BLOOM, F. E. Central catecholamine neuron systems:
anatomy and physiology of the norepinephrine and epinephrine systems.
Annu. Rev. Neurosci. 2: 113–168, 1979.
MORRISON, D. F. Multivariate Statistical Methods. New York: McGrawHill, 1990.
NEWMAN, D. B. Distinguishing rat brainstem reticulospinal nuclei by their
neuronal morphology. I. Medullary nuclei. J. Hirnforsch. 26: 187–226,
1985a.
NEWMAN, D. B. Distinguishing rat brainstem reticulospinal nuclei by their
neuronal morphology. II. Pontine and mesencephalic nuclei. J. Hirnforsch. 26: 385–418, 1985b.
OLIVERAS, J. L., BOURGOIN, S., HERY, F., BESSON, J. M., AND HAMON, M.
The topographical distribution of serotoninergic terminals in the spinal
cord of the cat: biochemical mapping by the combined use of microdissection and microassay procedures. Brain Res. 138: 393–406, 1977.
PAXINOS, G. AND WATSON, C. The Rat Brain In Stereotaxic Coordinates.
San Diego, CA: Academic, 1986.
POTREBIC, S. B., FIELDS, H. L., AND MASON, P. Serotonin immunoreactivity
is contained in one physiological cell class in the rat rostral ventromedial
medulla. J. Neurosci. 14: 1655–1665, 1994.
POTREBIC, S. B., MASON, P., AND FIELDS, H. L. The density and distribution
of serotonergic appositions onto identified neurons in the rostral ventromedial medulla. J. Neurosci. 15: 3273–3283, 1995.
RAMIREZ, F. AND VANEGAS, H. Tooth pulp stimulation advances both medullary off-cell pause and tail flick. Neurosci. Lett. 100: 153–156, 1989.
SAITO, Y., COLLINS, J. G., AND IWASAKI, H. Tonic 5-HT modulation of
spinal dorsal horn neuron activity evoked by both noxious and nonnoxious stimuli: a source of neuronal plasticity. Pain 40: 205–219, 1990.
SAWYNOK, J. The 1988 Merck Frosst Award. The role of ascending and
descending noradrenergic and serotonergic pathways in opioid and nonopioid antinociception as revealed by lesion studies. Can. J. Physiol.
Pharmacol. 67: 975–988, 1989.
SAWYNOK, J. AND REID, A. Effect of 6-hydroxydopamine-induced lesions
to ascending and descending noradrenergic pathways on morphine analgesia. Brain Res. 419: 156–165, 1987.
SORKIN, L. S., MC ADOO, D. J., AND WILLIS, W. D. Raphe magnus stimulation-induced antinociception in the cat is associated with release of amino
acids as well as serotonin in the lumbar dorsal horn. Brain Res. 618: 95–
108, 1993.
STEIN, R. B. Some models of neuronal variability. Biophys. J. 7: 37–68,
1967.
STEINBUSCH, H. W., NIEUWENHUYS, R., VERHOFSTAD, A. A., AND VAN DER
KOOY, D. The nucleus raphe dorsalis of the rat and its projection upon
the caudatoputamen. A combined cytoarchitectonic, immunohistochemical and retrograde transport study. J. Physiol. Lond. 77: 157–174, 1981.
TABER, E., BRODAL, A., AND WALBERG, F. The raphe nuclei of the brain
stem of the cat. I. Normal topography and cytoarchitechture and general
discussion. J. Comp. Neurol. 114: 161–187, 1960.
WESSENDORF, M. W. AND ANDERSON, E. G. Single unit studies of identified
bulbospinal serotonergic units. Brain Res. 279: 93–103, 1983.
WESSENDORF, M. W., PROUDFIT, H. K., AND ANDERSON, E. G. The identification of serotonergic neurons in the nucleus raphe magnus by conduction
velocity. Brain Res. 214: 168–173, 1981.
WILBUR, W. J. AND RINZEL, J. A theoretical basis for large coefficient of
variation and bimodality in neuronal interspike interval distributions. J.
Theor. Biol. 105: 345–368, 1983.
YAKSH, T. L. Pharmacology of spinal adrenergic systems which modulate
spinal nociceptive processing. Pharmacol. Biochem. Behav. 22: 845–
858, 1985.
YAKSH, T. L. AND WILSON, P. R. Spinal serotonin terminal system mediates
antinociception. J. Pharmacol. Exp. Ther. 208: 446–453, 1979.
09-02-97 13:43:57
neupal
LP-Neurophys
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5-hydroxytryptamine, noradrenaline and raphe stimulation on nociceptive
and non-nociceptive dorsal horn interneurones in the cat. Brain Res. 151:
307–321, 1978.
BOWKER, R. M. AND ABHOLD, R. H. Evoked changes in 5-hydroxytryptamine and norepinephrine release: in vivo dialysis of the rat dorsal horn.
Eur. J. Pharmacol. 175: 101–106, 1990.
BOWKER, R. M., WESTLUND, K. N., SULLIVAN, M. C., WILBER, J. F., AND
COULTER, J. D. Descending serotonergic, peptidergic and cholinergic
pathways from the raphe nuclei: a multiple transmitter complex. Brain
Res. 288: 33–48, 1983.
CHIANG, C. Y. AND GAO, B. The modification by systemic morphine of the
responses of serotonergic and non-serotonergic neurons in nucleus raphe
magnus to heating the tail. Pain 26: 245–257, 1986.
CRILL, W. E. Unitary multiple-spiked responses in cat inferior olive nucleus.
J. Neurophysiol. 33: 199–209, 1970.
DAHLSTROM, A. AND FUXE, K. Evidence for the existence of monoaminecontaining neurons in the central nervous system. I. Demonstration of
monoamine in the cell bodies of brain stem neurons. Acta Physiol. Scand.
Suppl. 232: 1–36, 1964.
DUGGAN, A. W. Neuropharmacology of pain. Curr. Opin. Neurol. Neurosurg. 5: 503–507, 1992.
ESCOBEDO, I. AND MASON, P. Nociceptive responsiveness is greater during
slow wave sleep than during waking. Soc. Neurosci. Abstr. 21: 387, 1995.
FIELDS, H. L., BRY, J., HENTALL, I., AND ZORMAN, G. The activity of neurons in the rostral medulla of the rat during withdrawal from noxious
heat. J. Neurosci. 3: 2545–2552, 1983.
FIELDS, H. L., HEINRICHER, M. M., AND MASON, P. Neurotransmitters in
nociceptive modulatory circuits. Annu. Rev. Neurosci. 14: 219–245,
1991.
FORNAL, C., AUERBACH, S., AND JACOBS, B. L. Activity of serotonin-containing neurons in nucleus raphe magnus in freely moving cats. Exp.
Neurol. 88: 590–608, 1985.
FORNAL, C. A., LITTO, W. J., MORILAK, D. A., AND JACOBS, B. L. Singleunit responses of serotonergic neurons to vasoactive drug administration
in behaving cats. Am. J. Physiol. 259 (Regulatory Integrative Comp.
Physiol. 28): R963–R972, 1990.
FURSHPAN, E. J. AND FURAK AWA, T. Intracellular and extracellular responses in the several regions of the mauthner cell of the goldfish. J.
Physiol. Lond. 732–771, 1962.
FUXE, K. Evidence for the existence of monoamine-containing neurons in
the central nervous system. IV. Distribution of monoamine nerve terminals in the CNS. Acta Physiol. Scand. Suppl. 247: 37–80, 1965.
HAMMOND, D. L., TYCE, G. M., AND YAKSH, T. L. Efflux of 5-hydroxytryptamine and noradrenaline into spinal cord superfusates during stimulation
of the rat medulla. J. Physiol. Lond. 359: 151–162, 1985.
HEYM, J., STEINFELS, G. F., AND JACOBS, B. L. Medullary serotonergic neurons are insensitive to 5-MeoDMT and LSD. Eur. J. Pharmacol. 81:
677–680, 1982a.
HEYM, J., STEINFELS, G. F., AND JACOBS, B. L. Chloral hydrate anesthesia
alters the responsiveness of central serotonergic neurons in the cat. Brain
Res. 291: 63–72, 1984.
HEYM, J., TRULSON, M. E., AND JACOBS, B. L. Raphe unit activity in freely
moving cats: effects of phasic auditory and visual stimuli. Brain Res.
232: 29–39, 1982b.
JACOBS, B. L. AND AZMITIA, E. C. Structure and function of the brain serotonin system. Physiol. Rev. 72: 165–229, 1992.
JONES, B. E. Reticular formation: cytoarchitecture, transmitters, and projections. In: The Rat Nervous System, edited by G. Paxinos. San Diego, CA:
Academic, 1995, p. 155–171.
JONES, B. E., HOLMES, C. J., RODRIGUEZ, V. E., AND MAINVILLE, L. GABAsynthesizing neurons in the medulla: their relationship to serotonin-containing and spinally projecting neurons in the rat. J. Comp. Neurol. 313:
349–367, 1991.
KLEITMAN, N. Sleep and Wakefulness. Chicago, II: Univ. of Chicago Press,
1963.
KRUGER, L., SAPORTA, S., AND SWANSON, L. W. Photographic Atlas of the
Rat Brain. New York: Cambridge Univ. Press, 1995.
KWIAT, G. C. AND BASBAUM, A. I. The origin of brainstem noradrenergic
and serotonergic projections to the spinal cord dorsal horn in the rat.
Somatosens. Mot. Res. 9: 157–173, 1992.
LACHENBRUCH, P. A. AND MICKEY, M. A. Estimation of error rates in discriminant analysis. Technometrics 10: 1–10, 1968.