Department of Pharmacological and Physiological Sciences and the Committee on Neurobiology, The University of Chicago, Chicago, Illinois 60637
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Leung, Cynthia G. and Peggy Mason. Physiological survey of medullary raphe and magnocellular reticular neurons in the anesthetized rat. J. Neurophysiol. 80: 1630-1646, 1998. The present study was designed to provide a detailed and quantitative description of the physiological characteristics of neurons in the medullary raphe magnus (RM) and adjacent nucleus reticularis magnocellularis (NRMC) under anesthetized conditions. The background discharge and noxious stimulus-evoked responses of RM and NRMC neurons were recorded in rats lightly anesthetized with isoflurane. All cells that were isolated successfully were studied. After recording background discharge, the neuronal response to repeated noxious thermal and noxious mechanical stimulation of the tail was recorded. Most cells were identified as nonserotonergic by their irregular or rapid background discharge pattern. Because the spontaneous discharge of most RM nonserotonergic cells contained pauses and bursts, a comparison between the change in rate evoked by tail heat and the range of rate changes that occur spontaneously was used to classify cells. The mean responses of ON and OFF cells were more than four times the standard deviation of the changes in rate observed spontaneously. ON cells were excited in 86% of the tail heat trials tested. Similarly, OFF cells were inhibited in 97% of the noxious tail heat trials tested. The heat-evoked changes in ON and OFF cell discharge varied over more than two orders of magnitude and were greater in cells with greater rates of background discharge. The heat-evoked responses of ON and OFF cells had durations of tens of seconds to minutes and were always sustained beyond the visible motor response. Most ON and OFF cells responded to noxious tail clamp in a manner that was similar to their response to noxious heat. More than half of the NEUTRAL cells that were unresponsive to noxious heat were responsive to noxious tail clamp. A minority of ON, OFF, and NEUTRAL cells responded to innocuous brush stimulation with weak, transient responses. Although many cells discharged too infrequently to be classified, units with physiological properties that were different from those described above were rare. In conclusion, most RM and NRMC cells belong to three nonserotonergic physiological cell classes that can be distinguished from each other by the consistency, not the magnitude, of their responses to repeated noxious thermal stimulation. Because most of the heat-evoked change in ON and OFF cell discharge occurs after the conclusion of the initial motor withdrawal, ON and OFF cells are likely to principally modulate the response to subsequent noxious insults.
Neurons in the pontomedullary raphe magnus (RM) and adjacent nucleus reticularis magnocellularis (NRMC) project to the dorsal horn and are thought to participate in the modulation of spinal nociceptive transmission (Fields et al. 1991 Surgical preparation for acute recordings
Male Sprague-Dawley rats (240-400 g; Sasco, Madison, WI) were anesthetized initially with halothane. A tracheal catheter was inserted and connected to an open breathing circuit for administration of isoflurane. Inspired and endtidal isoflurane concentrations were monitored using a multichannel infrared analyzer (Datex Capnomac, Madison, WI). Throughout surgery, deep anesthesia was maintained with 1.8-2.0% isoflurane in oxygen. Core body temperature was maintained at 37-38°C by use of a water-perfused heating pad and a plastic cover over the rat. A catheter was inserted into the femoral artery for recording blood pressure, and electromyographic (EMG) electrodes were placed transcutaneously in the paraspinous muscles. A small midline craniotomy was made just posterior to the lamboid suture for the introduction of recording microelectrodes.
Surgical preparation for chronic recordings
A second group of rats (400-500 g) was prepared with miniature microdrives for chronic recording (unpublished data). Briefly, rats were anesthetized with pentobarbital sodium (Nembutal, 55 mg/kg) and placed in a stereotaxic apparatus. Bone screws were inserted through the skull for electroencephalographic recording, and stranded stainless steel wires were inserted through the biceps femoris and the deep muscles of the neck for EMG recording. A microdrive base and guide tube were affixed to the skull overlying the RM for extracellular unit recording. All electrodes were attached to a miniature connector, which was affixed to the skull with dental acrylic. Chronically prepared rats were used in the experimental protocol after Experimental protocol
Both acutely and chronically prepared rats were studied only in the anesthetized condition. Rats were anesthetized initially with halothane and then maintained on isoflurane administered via a nose cone. Chronically prepared rats were not restrained or held in a stereotaxic holder, whereas acutely prepared rats were placed in a standard stereotaxic apparatus. Rats were placed on top of a water-perfused heating pad, and core body temperature was maintained at 37-38°C. EMG electrodes were placed transcutaneously in the paraspinous muscles.
Extracellular recording and data acquisition
Extracellular unit recording was accomplished with stainless steel microelectrodes plated with platinum to a final resistance of 0.5-3.0 M Criteria for acceptable experiments
One experiment (4 cells) was rejected because the rat exhibited extreme variation in blood pressure (standard deviation >10 mmHg) within a period of steady-state anesthesia. Several other experiments were terminated when the rat displayed obvious respiratory difficulty. Cells (n = 6) were excluded from analysis if the recovered recording site was outside of the boundaries determined to encompass the RM and NRMC region. Because <10% of recording sites were found to be outside the RM and NRMC region, 15 cells for which recording sites were not found were included in the analysis. For each of these 15 cells, the recording depth was in the range (9.0-10.5 mm) that was reliably associated with RM or NRMC locations.
Analysis and cell classification
Most cells were assigned to one of four physiological cell classes according to their background and noxious-evoked discharge properties. First, cells were classified as p5HT (physiologically identified as serotonergic) or non-p5HT (physiologically identified as nonserotonergic) using a previously described algorithm based on the rate and variability of the background discharge (Fig. 1) (Mason 1997
Statistics
Each variable is expressed as a mean ± SE. Statistical tests were performed using Microsoft Excel 5.0 (Redmond, WA).
Histology
The recording site of the last cell recorded from each animal was lesioned by applying 20 µA of anodal current for 3-4 min. All other recording sites were calculated based on their stereotaxic distance from the marked recording site. Animals were perfused with a fixative containing 4% paraformaldehyde and 7% sucrose in 0.1 M phosphate buffer at pH 7.4. Transverse sections (80 µm) were cut on a freezing microtome, mounted on gelatin-coated slides, and stained with cresyl violet. Recording sites were examined microscopically and plotted onto standard sections.
Proportions of cell types
A total of 136 RM and NRMC neurons were recorded in the anesthetized rat. Because a smaller number of p5HT cells (n = 8; 6%) were recorded than would be expected by their observed proportion in anatomic studies (15-20%) (Potrebic et al. 1994 Background discharge patterns
Using a function that discriminates p5HT from non-p5HT cells (Mason 1997 Responses to noxious tail heat
ON CELLS.
The response thresholds (
OFF CELLS.
For OFF cells (n = 30), the response threshold ranged from 0.5 to 21.8 Hz and averaged 8.2 ± 1.1 Hz (Fig. 2). The mean heat-evoked change for OFF cells ranged over three orders of magnitude from NEUTRAL CELLS.
NEUTRAL cells (n = 20) had no consistent response to noxious tail heat (Fig. 2). NEUTRAL cells were excited (increased their discharge by P5HT CELLS.
Although eight p5HT cells were recorded, the discharge of only four p5HT cells was recorded in response to repeated tail heat trials. Two of these p5HT cells increased their discharge by 1 or 3 Hz in 100% of the trials tested. The discharge of the remaining two p5HT cells was not affected by noxious heat.
OTHER CELL TYPES.
Two cells exhibited increases in discharge in response to tail heat when their discharge was low before the trial onset and decreases in discharge when their discharge was high before the trial onset.2 The discharge of both of these cells varied in direct relation to the arterial blood pressure, which increased in trials that started when blood pressure was low and decreased in trials that started when blood pressure was high. In addition, one cell increased its discharge in response to noxious tail heat at a long latency (10-20 s). Because this neuron did not discharge before any of the tail heat trials, it may represent an OFF cell with a rebound burst discharge.
Responses to noxious and innocuous mechanical stimulation
No attempt was made to match the strength of the noxious mechanical and thermal stimuli, and the motor responses to these stimuli differed in their strength, muscle involvement, and timing. The movement evoked by noxious tail clamp was more vigorous than that evoked by heat and involved activation of hindlimb, trunk, and often forelimb musculature in addition to, and sometimes instead of, paraspinous muscles. The paraspinous muscle response, both visible and that revealed by the integrated EMG, to noxious tail clamp usually outlasted the stimulus and sometimes appeared to start after the stimulus offset (Figs. 4C, 5C, and 6).
Relationship between neuronal and motor responses to noxious heat
Because the discharge of ON and OFF cells has been proposed to gate noxious-evoked withdrawal movements (Fields et al. 1983
Relationship between neuronal and autonomic responses to noxious heat
The baseline blood pressure (OFF cells: 119 ± 4 mmHg; ON cells: 117 ± 2 mmHg) and heart rate (OFF cells: 345 ± 9 bpm; ON cells: 336 ± 6 bpm), measured before tail heat stimulation, were not different when recording ON or OFF cells. In response to noxious tail heat, heart rate nearly always increased (41/42 cells) by an average of 15 ± 1 bpm. The blood pressure response to noxious tail heat was more variable and included both pressor (n = 25 cells; mean: 11 ± 1 mmHg) and depressor (n = 17 cells; mean:
Recording locations
The recording sites for 121 cells were contained in RM or NRMC between the caudal pole of the facial nucleus and the nucleus of the trapezoid body, inclusive (Fig. 13). ON, OFF, and NEUTRAL cells were not preferentially distributed between these nuclei. p5HT cells were contained in either RM (n = 5) or in the ventral portion of NRMC, termed NRMC
Quantitative method for classifying RM and NRMC neurons
In the anesthetized rat, the spontaneous discharge of most RM cells is irregular and contains bursts and pauses that resemble noxious stimulus-evoked responses. A true "response" to noxious stimulation can only be distinguished from a naturally occurring change in discharge by a quantitative comparison between background and stimulus-evoked discharge patterns. Such a comparison reveals that the mean responses of ON and OFF cells are more than four times greater than the average change in rate that is observed spontaneously. Furthermore, as summarized in Fig. 3, these responses are elicited consistently by repeated trials of noxious stimulation.
Distribution and frequency of cell types in the RM and NRMC
In an attempt to completely survey all physiological cell types present in the RM and NRMC of the anesthetized rat, units were recorded solely based on isolation properties in the present study. Among nonserotonergic cells, the number of ON cells was greater than that of OFF cells, a finding that is in agreement with several previous studies in acutely anesthetized preparations (Chiang and Gao 1986 Classification of nonserotonergic regularly discharging neurons
In previous reports from this and other laboratories, cells with fast and regular discharge have been grouped together into a distinct cell class and termed "regular" cells (Gao et al. 1997 Limitations of this classification system
There are two limitations to the cell-classification system presented here. First, spontaneously inactive neurons were not studied. The second limitation is that this classification system is only directly applicable to the isoflurane-anesthetized condition. However, it is unlikely that the cell-class properties described here are dependent on the type or presence of anesthesia as similar physiological properties are observed in RM and NRMC cells recorded under halothane (Mason et al. 1990 Functional roles of ON and OFF cells
Based on their nocifensor reflex-related activity, OFF cells originally were hypothesized to gate spinal nociceptive transmission in a manner analogous to the gating of saccadic eye movements by omnipause neurons (Fields et al. 1983 Function of NEUTRAL cells
The lack of NEUTRAL cell responses to noxious thermal stimulation and opioid administration does not preclude a role for these cells in nociceptive modulation. It is possible that NEUTRAL cells are only affected by more robust stimuli or motor responses or by nonthermal modalities of noxious stimulation. In support of these ideas, 67% of the NEUTRAL cells, defined by their unresponsiveness to noxious heat, responded to noxious clamp of the tail with either an increase or a decrease in discharge. NEUTRAL cells then may represent ON or OFF cells that only respond to particular intensities and/or modalities of painful stimuli. Future experiments are required to examine this issue.
RM/NRMC and autonomic status
Previous reports (Leung and Mason 1996 Summary
The original cell classes described by Fields and colleagues
INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References
). Fields and colleagues introduced a system that divides neurons in the RM and NRMC into three physiological cell classes based on their activity before withdrawal from a noxious thermal stimulus in the lightly anesthetized rat (Fields et al. 1983
). According to this original description, OFF cells are inhibited and ON cells excited before the withdrawal reflex elicited by noxious stimulation. NEUTRAL cells were thought to be unaffected by such stimuli. Cells in these physiological classes also exhibit differential responses to opioids. OFF cells are excited, ON cells inhibited, and NEUTRAL cells unaffected by analgesic doses of opioids administered either systemically or by supraspinal microinjection (Barbaro et al. 1986
; Cheng et al. 1986
; Heinricher et al. 1992
). A number of such studies have led to the idea that ON and OFF cells, which project to the superficial dorsal horn, facilitate and inhibit, respectively, spinal nociceptive transmission (Fields et al. 1991
, 1995
).
; Hentall et al. 1993
; McGaraughty and Reinis 1993
; Morgan and Heinricher 1992
; Rosenfeld et al. 1990
; Thurston and Randich 1992
), there is no formal consensus on the criteria used. The few attempts to quantify the magnitude and variability of the noxious heat-evoked response across multiple tail heat trials have yielded data that differ from the original profile of ON, OFF, and NEUTRAL cell physiology. For example, one group of investigators reported that RM and NRMC neurons sometimes respond after the noxious heat-evoked reflex and that many RM neurons consistently respond to noxious heat even when no reflex is elicited (Thurston and Randich 1992
). Other investigators reported that the magnitude or presence of the neuronal response to various stimuli may depend on the level of background activity at the onset of the trial (Blair and Evans 1991
; Morgan and Heinricher 1992
).
METHODS
Abstract
Introduction
Methods
Results
Discussion
References
1 wk of recovery.
30 min, a metal microelectrode was introduced into the region of the RM and NRMC (P 0.8-2.6 mm from interaural zero, L 0.0-1.0 mm, V 8.5-10.5 mm from the cerebellar surface). In the case of chronically prepared rats, electrodes were introduced via a microdrive-microelectrode assembly (Biela Idea Development, Anaheim, CA) attached to the microdrive base. No search stimulus was used, and only units with background discharge were studied. To achieve as complete a sample as possible, every extracellular unit that was isolated successfully was studied. Each extracellular unit was discriminated with software that allowed for template-matching of waveforms sampled at 20 kHz (Spike2, CED, Cambridge, UK).
View larger version (37K):
[in a new window]
FIG. 4.
Peak ON cell discharge rate evoked by tail heat was similar to the peak discharge rate recorded during background conditions. This resulted in a greater evoked change in discharge when the prestimulus discharge rate was low. A: background discharge of an ON cell, in the absence of any stimulation, is shown below the heart rate (top; scale on right) and blood pressure (middle; scale on left) traces. B: tail heat-evoked responses recorded from the same ON cell as shown in A. Top to bottom: heart rate (scale on right), blood pressure (scale on left), neuronal discharge rate, the rectified electromyographic (EMG) recording from the paraspinous muscles, and stimulus temperature. C: tail clamp-evoked responses recorded from the same ON cell as shown in A and B. After 2 trials, this cell was lost. Top to bottom: heart rate (scale on right), blood pressure (scale on left), neuronal discharge rate, the rectified EMG recording from the paraspinous muscles, and stimulus application. Time calibration, shown in A, is applicable to all panels.
View larger version (52K):
[in a new window]
FIG. 5.
Neuronal discharge of an OFF cell in the absence of stimulation, in response to tail heat and clamp was recorded. A: background discharge in the absence of any stimulation. B: tail heat-evoked responses recorded from the same OFF cell as shown in A. Top to bottom: neuronal discharge rate, the rectified EMG recording from the paraspinous muscles, and stimulus temperature. C: tail clamp-evoked responses recorded from the same OFF cell as shown in A and B. Top to bottom: neuronal discharge rate, the rectified EMG recording from the paraspinous muscles, and stimulus application. Time calibration, shown in B, is applicable to all panels.
View larger version (26K):
[in a new window]
FIG. 6.
Tail clamp (A2-D2) evoked a similar neuronal response as tail heat (A1-D1) in ON (B, 1 and 2) and OFF (A, 1 and 2) cells but not in all NEUTRAL cells (C1-D2). For each of the 4 cells illustrated, poststimulus time histograms are shown for both tail heat (left) and tail clamp (right) trials. In each panel, the traces, from top to bottom, are the mean discharge rate (100 ms bins), the averaged rectified EMG, and the averaged stimulus (stimulus temperature in A1-D1 and tail clamp application in A2-D2). Tail clamp (A2 and B2) elicited a response that was similar to that evoked by tail heat (A1 and B1) in most ON (B) and OFF (A) cells. Although they did not respond to tail heat (C1 and D1), some NEUTRAL cells (C and D) responded to noxious tail clamp (C2 and D2). The number of trials averaged is 5 in all panels except B1 and D1 in which 6 trials are averaged. Time calibration, shown in A1, is applicable to all panels.
(FHC, New Brunswick, ME). Unit activity, concentration of expired isoflurane, paraspinous EMG, and arterial blood pressure (only in acute rat group) were acquired onto both a DAT tape recorder (collected at 10 kHz) and onto a Macintosh Quadra 950 equipped with a CED 1400 + A/D converter (20 kHz for the unit; all other measures collected at 250 Hz). Activity from single units was discriminated off-line (Spike2, CED).
). The probability of misclassification using this method is <10%. In the present study, the background discharge of isolated cells was recorded for 15 min and the mean (x) and standard deviation (SDISI) of the interspike intervals (ISI) were calculated from this recording. For each cell, the value of the function, y(x, SDISI) = 146
x + 0.98 * SDISI, was calculated. All cells with a negative function value were classified as p5HT and those with a positive function value were classified as non-p5HT.
View larger version (21K):
[in a new window]
FIG. 1.
Rate (abscissa) and regularity (ordinate) of the background discharge recorded from medullary raphe magnus (RM) and adjacent nucleus reticularis magnocellularis (NRMC) units. Two unclassified cells with mean interspike intervals (ISIs) of >10 s are not illustrated. A line representing the discriminant function [y(x, s) = 0] defines the optimal linear boundary between physiologically identified as serotonergic (p5HT) and physiologically identified as nonserotonergic (non-p5HT) cells and is illustrated on this graph.
).
2 SD10 s. This criterion then presented a problem in cases when a cell was not discharging at a rate
2 SD10 s before the tail heat stimulus. Thus for such cells, the change in the maximum ISI was calculated. This change in maximum ISI was considered to be an inhibitory response if it exceeded 2 SDISI (see preceding text). As with the previous method, this method allowed us to take into account the natural variability of RM cell discharge and to have confidence, at a P < 0.05 level, that an evoked response was unlikely to have occurred spontaneously.
), data from trials in which the cell did not respond and no withdrawal occurred were not included in these calculations. Cells that were excited by
50% of heat applications were considered ON cells. Cells that were inhibited by
50% of heat applications were considered OFF cells. Cells with <50% excitatory or inhibitory responses were classified as NEUTRAL cells. A minimum of three trials was required for classification. Therefore, cells that were not ON cells and did not discharge at rates of
1 Hz before at least three tail heat trials (that evoked withdrawals) were not classified.
).
RESULTS
Abstract
Introduction
Methods
Results
Discussion
References
), it is likely that serotonergic cells were sampled inadequately. Therefore the percentages of ON, OFF, NEUTRAL, and unclassified cells are reported as a proportion of the total number of non-p5HT cells (n = 128). Overall, cells were classified as ON (n = 49, 38%), OFF (n = 30, 23%), or NEUTRAL cells (n = 21, 16%). A proportion of the cells recorded could not be classified (n = 25, 20%) and three cells (2%) had physiological characteristics different from any of the established cell types.
), eight p5HT cells were identified in the present sample (Fig. 1). The mean rate of discharge of the p5HT cells was 2.0 ± 0.5 Hz. The CVISI of p5HT cells ranged from 0.29 to 0.55, reflecting the observation that p5HT cell discharge does not contain bursts of activity and is also not "clock-like" in its regularity (Mason 1997
).
; Leung and Mason 1995
), the discharge of most of these cells was highly irregular, consisting of alternating bursts of activity and periods of inactivity (see Figs. 4A and 5A). As expected from the irregularity in background discharge, the CVISI of most ON (39/49), OFF (24/30), and NEUTRAL (13/21) cells was greater than one (Wilbur and Rinzel 1983
) (Fig. 1).
2, and 17 of these cells had a CVISI > 5. The one cell in this group with a CVISI < 1 discharged only three action potentials during the entire 15-min period of background discharge recording.
2 SD10 s) of ON cells (n = 49) varied from 0.5 to 49.5 Hz and averaged 6.8 ± 1.3 Hz (Fig. 2). The mean ON cell discharge increase evoked by tail heat was 20.5 ± 4.0 Hz and ranged over two orders of magnitude from 0.8-132.8 Hz. Thus in the 10 s after the onset of tail heat stimulation, ON cells had a mean of 205 more action potentials than during the 10 s preceding the tail heat stimulus. ON cells with greater background discharge rates had larger heat-evoked responses (Fig. 3).
View larger version (26K):
[in a new window]
FIG. 2.
Tail heat-evoked responses for all OFF ( ), NEUTRAL (
), and ON (
) cells are illustrated in comparison with the response thresholds (
) for each cell. OFF cells were usually inhibited and and ON cells excited by tail heat stimulation. Heat-evoked changes in NEUTRAL cell discharge rarely exceeded the threshold for a significant response. All cells are arranged in classes and within the classes are ordered along the abscissa in ascending order of their response thresholds. Among NEUTRAL cells, units with mean tail heat-evoked decreases in discharge are grouped and followed by NEUTRAL cells with mean increases. Values >0.5 Hz are presented on logarithmic scales (top and bottom, respectively). Small changes in discharge (less than
0.5 Hz and <0.5 Hz) are represented on a linear scale (middle).
View larger version (22K):
[in a new window]
FIG. 3.
Mean heat-evoked responses of ON and OFF cells, but not of NEUTRAL cells, increased as background discharge rates increased. All points represent the means ± SE for cells that discharge at rates <1, 1-10, and >10 Hz. Error bars that are not visible are contained within the symbol.
).
2 s before the evoked burst (n = 93 trials), the mean latency was 3.06 ± 0.10 s after tail heat onset. For trials where an ON cell was active before the evoked burst (n = 116 trials), the latency to burst, estimated from the inflection point in the interspike intervals, averaged 2.94 ± 0.08 s after tail heat onset. The latencies to respond during trials when the cell was active or inactive before the trial onset were not different (t-test, P > 0.3).
0.5 to
62.4 Hz and averaged
17.1 ± 2.9 Hz, corresponding to an average decrease of 171 spikes in the 10 s after tail heat onset. OFF cells with greater background discharge rates had larger heat-evoked decreases in discharge (Fig. 3). OFF cells were inhibited in an average of 97 ± 1% of the tail heat trials (Fig. 2). No OFF cell was excited by tail heat.
). The remaining three cells continued discharging regularly and were inhibited for <1 s by noxious heat; in contrast, all other OFF cells (n = 27) were inhibited for >1 s by noxious heat. Although the inhibition of these cells was brief, it was highly significant (P < 0.0001) as it represented an increase of at least seven times SDISI over background discharge values. For instance, in one case where the mean ISI during background conditions was 19 ms with a standard deviation of 4 ms, the mean pause evoked by tail heat was 49 ms.
2 SD10 s; see METHODS) by 16 ± 4% of the trials and inhibited by 6 ± 2% of the trials. The mean change in discharge evoked by tail heat was unrelated to the cell's background discharge rate (Fig. 3).
View larger version (31K):
[in a new window]
FIG. 7.
Brush stimulation (A3 and B3) elicited a smaller response than that evoked by either tail heat (A1 and B1) or tail clamp (A2 and B2) in ON (B) and OFF (A) cells. Responses to tail heat and tail clamp (stimuli are shown bottom traces) are poststimulus time histograms (100-ms bins) averaged from 5 trials. Responses to brush are shown as histograms (100-ms bins). For brush stimulation, single trials were used and the stimulus site is listed below the stimulus trace. Time calibration, shown in A1, is applicable to all panels.
), ON and OFF cell responses to noxious tail heat were compared with evoked movements. The motor response to noxious tail heat was usually restricted to activation of paraspinous muscles that would lead to a flick of the tail (because the tail was taped down, no movement actually occurred). The mean duration of the early portion of the EMG response, which constituted the visible part of the motor response, was calculated from the averaged (rectified) EMG response recorded for each cell. The offset of this early EMG response occurred, on average, 8.7 ± 0.3 s after the stimulus onset (Fig. 8). The visible motor response outlasted the stimulus by >5 s in only 3 of 67 cases. In some cases, a small motor response, which was only apparent in the rectified EMG trace was recorded after the stimulus temperature returned to innocuous levels (Fig. 8D,
).
View larger version (28K):
[in a new window]
FIG. 8.
Neuronal response to noxious tail heat persisted beyond the early motor response in the case of ON (A and B) and OFF cells (C and D). This relationship held for all ON and OFF cells tested (E). Offset of a late motor response (D, ), which was only present in some cases, occurred close to the offset of the neuronal response (F). In A-D, the traces, from top to bottom, are the mean discharge rate (100-ms bins), the averaged rectified EMG, and the averaged stimulus temperature. Neuronal responses to tail heat are poststimulus time histograms averaged from 5 trials. Time calibration shown in C is applicable to A-D. E and F: latency to the offset of the neuronal response (measured from the poststimulus time histograms) is compared with the latency to the offset of the early (E) or late (F) motor response (measured from the mean rectified EMG) for each cell (symbols as in F). Offset latencies > 100 s were estimated at 100 s. Mean values for all ON and OFF cells are shown by
and
. The unity line (y = x) is presented on each graph.
1 Hz) before the stimulus application, noxious heat stimulation always evoked an inhibitory response in OFF cells (13/13 trials; Fig. 9A). The proportion of ON cell responses that occurred in trials that failed to elicit a motor withdrawal was significantly less than for OFF cells (
2 test, P = 0.002). To further examine the association or dissociation between the neuronal and motor responses, trials in which a motor response, but no neuronal response, occurred were examined. ON and OFF cells failed to respond in 27/227 and 3/106, respectively, of all trials that evoked a motor withdrawal. This difference was statistically significant (
2 test, P = 0.01).
View larger version (32K):
[in a new window]
FIG. 9.
ON (B and C) and OFF (A) cells responded to most, but not all, tail heat trials that failed to evoke a motor response. Top: poststimulus time histogram of neuronal discharge (100-ms bins). Bottom: averaged rectified paraspinous EMG. Tail heat stimulus is illustrated at the bottom of each column. A, 1 and 2: tail heat inhibits this OFF cell regardless of the motor response. A1: average response during 3 trials in which tail heat evoked a withdrawal response. A2: average response during 2 trials in which tail heat failed to evoke a withdrawal response. B, 1 and 2: tail heat evokes a burst in this ON cell regardless of the motor response. B1: average response during 2 trials in which tail heat evoked a withdrawal response. B2: average response during 5 trials in which tail heat failed to evoke a withdrawal response. C, 1 and 2: tail heat evokes a burst in this ON cell only in trials that also evoke a motor response. C1: average response during 2 trials in which tail heat evoked a withdrawal response. C2: average response during 5 trials in which tail heat failed to evoke a withdrawal response. Time calibration shown in C2 is applicable to all panels.
and
under the line). For six OFF cells and eight ON cells, the average response latency was greater than the average tail flick latency (Fig. 10D,
and
under the line).
View larger version (35K):
[in a new window]
FIG. 10.
Relationship between the latencies of ON and OFF cell responses and of the tail flick withdrawal. A: latency of the OFF cell pause is compared with the tail flick latency for all trials where the rat withdrew and the OFF cell responded. B: length of the OFF cell pause is compared with the tail flick latency for all trials where the rat withdrew and the OFF cell responded. C: latency to the ON cell burst is compared with the tail flick latency for all trials where the rat withdrew and the ON cell responded. D: mean latency of the tail flick withdrawal is compared with the mean latency of the cellular response for ON and OFF cells. Line y = x is presented in A, C, and D.
View larger version (22K):
[in a new window]
FIG. 11.
Poststimulus time histograms (100-ms bins) for ON (B, 1-3) and OFF (A, 1-3) cells were smoothed and aligned (at 0) to either the occurence of the tail flick ( ) or the onset of the heat stimulus (···). In different cells, the slope of the flick-aligned histogram was steeper (A1 and B1), similar (A2 and B2) or less steep (A3 and B3) than the slope of the heat-aligned histogram. Number in the box in the bottom right of A1-B3 is the ratio of the slope of the flick-aligned histogram to the slope of the heat-aligned histogram (Rmin in A, 1-3; Rmax in B, 1-3). C: frequency distribution of slope ratios for ON and OFF cells. Slope ratios were averaged within bins of 0.25.
10 ± 1 mmHg) responses.
View this table:
TABLE 1.
Number of trials in which ON and OFF cells responded to tail heat and there was a change in heart rate or blood pressure
View larger version (50K):
[in a new window]
FIG. 12.
Tail heat-evoked responses of ON and OFF cells were independent of the evoked changes in heart rate and blood pressure. Top to bottom: heart rate (scale on right), blood pressure (scale on left), neuronal discharge rate, the rectified EMG recording from the paraspinous muscles, and stimulus temperature. A: OFF cell that failed to respond to a tail heat trial (dashed arrow) that elicited a response in heart rate and blood pressure. B: ON cell that failed to respond to a tail heat trial (dashed arrow) that elicited a response in heart rate and blood pressure. In contrast, this ON cell responded to a tail heat trial that failed to elicit a response in blood pressure (small arrow).
(n = 2) (Newman 1985a
,b
). The locations of 15 cells, including 8 ON, 1 OFF, 1 NEUTRAL, 1 p5HT, 3 unclassified, and 1 "other" type cells could not be determined.
View larger version (20K):
[in a new window]
FIG. 13.
Distribution of recording sites for RM and NRMC cells. Locations of recorded cells are shown. Locations of 15 cells were not recovered (see text). VII n., facial nerve rootlets; n. VII, facial nucleus; NTB, nucleus of the trapezoid body; p, pyramid; tb, trapezoid body.
DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References
; Fields et al. 1983
; Guilbaud et al. 1980
; Morgan and Fields 1994
; Morgan and Heinricher 1992
; Rosenfeld et al. 1990
; Thurston and Randich 1992
). The proportions of OFF (23%) and NEUTRAL (16%) cells observed were likely to be slightly underestimated compared with other studies because ~20% of the RM/NRMC cells recorded could not be classified but likely belonged to one of these two cell classes. Intermediate cell types, such as cells with biphasic or long latency responses, were rarely observed. Thus it is likely that the original three cell classes described by Fields et al. (1983)
adequately describe the major nonserotonergic physiological cell types present in the RM and NRMC.
; Leung and Mason 1995
, 1996
; Oliveras et al. 1989
). This classification was based on initial findings that these cells had a distinctive discharge pattern, were opioid-insensitive, and were unaffected or weakly excited by noxious stimulation. The distinctive characteristics of these neurons suggested a physiological function different from that of irregularly firing neurons.
,b
; unpublished observations).
; Morgan and Heinricher 1992
), ketamine (McGaraughty and Reinis 1993
; McGaraughty et al. 1993
), and barbiturate (Fields et al. 1983
) anesthesia as well as under decerebrate conditions (Clarke et al. 1994
). Although it is possible that other types of noxious stimulus-evoked responses occur in the unanesthetized condition, none have been described that have not been observed in the anesthetized condition (Fornal et al. 1985
; McGaraughty et al. 1993
; Oliveras et al. 1989
, 1990
, 1991a
,b
; unpublished observations). Specifically, the four RM and NRMC cell types described above
ON, OFF, NEUTRAL, and p5HT
have been observed in the unanesthetized as well as anesthetized condition.
). That is, the inhibition of OFF cell discharge evoked by a noxious stimulus was thought to be necessary to disinhibit dorsal horn neurons, thereby permitting nociceptive transmission and the execution of nocifensor reflexes. The evidence for this idea included the findings that the OFF cell pause evoked by noxious tail heat occurred before the motor response and that the OFF cell inhibition was more rapid when aligned to the motor response than to the stimulus onset. In contrast, we observed that OFF cells often ceased discharging after the tail flick onset. Furthermore, the mean flick to heat slope ratio for OFF cells was distributed unimodally about a mean of 1.0. Our finding that OFF cells responded similarly to tail heat regardless of whether the stimulus elicited a tail flick response is also evidence that the OFF cell pause can be dissociated from the initiation of the motor response. Although differences in anesthetic agent, anesthetic depth, and the rise time of the tail heat stimulus may contribute to the differences between the present and previous findings, our results do not support a premotor function, analogous to that of the omnipause neuron, for OFF cells.
). Thus signals related to cardiovascular activity are one nonnociceptive source of RM input. Furthermore, there is evidence that RM ON and OFF cells respond to innocuous somatosensory and auditory stimulation in the unanesthetized condition (Oliveras et al. 1989
, 1990
; unpublished observations). Future experiments are needed to establish whether additional sources of nonsomatic input, such as vestibular- or respiratory-related signals, influence RM cell discharge. Even with our present knowledge, it is clear that nonnoxious inputs strongly influence RM ON and OFF cell discharge. Although the pain modulatory field has focused on pain's modulation of pain, these findings suggest that ON and OFF cells modulate nociceptive transmission in reaction to a number of physiological inputs, nonnociceptive as well as nociceptive.
). Furthermore, Ramirez and Vanegas (1989)
reported an example of phasic modulation in which tooth pulp stimulation shortened the withdrawal latency evoked by a subsequent tail heat stimulus. In summary, it is possible that ON and OFF cells play an important role in modulating the late responses to noxious stimulation as well as the responses to subsequent noxious insults.
; Thurston and Randich 1992
) have established that ON cells typically increase their discharge during periods of low blood pressure, whereas OFF cells burst during periods of high blood pressure. Because tail heat evokes changes in blood pressure and heart rate, it is possible that the changes in ON and OFF cell discharge evoked by tail heat are secondary to an autonomic response. This is unlikely, however, because the neuronal response to noxious heat occurred in trials that did not elicit a cardiovascular response. In addition, the responses of ON and OFF cells were similar regardless of whether tail heat produced a pressor or a depressor effect. Finally, the failure of ON and OFF cells to respond to noxious tail heat was never accompanied by the absence of a cardiovascular response.
ON, OFF, and NEUTRAL
are inclusive of the great majority of nonserotonergic cell types encountered in RM and NRMC. Serotonergic cells comprise the only necessary additional cell class. The assignation of RM and NRMC nonserotonergic cells to the ON, OFF, and NEUTRAL cell classes is best accomplished by a quantitative comparison of heat-evoked responses to the spontaneous variability present in discharge during periods without stimulation. The consistency, not the magnitude, of responses to repeated and standard noxious thermal stimulation distinguished ON and OFF from NEUTRAL cells.
![]() |
ACKNOWLEDGEMENTS |
---|
The authors thank A. Zhang, D. O. Chen, and C. Burgin for technical assistance, Drs. Donna Hammond, Bob McCrea, Phil Lloyd, and Jay Goldberg for helpful conversations during the course of the study, and J. Genzen and Dr. Keming Gao for comments on the manuscript. Isoflurane was generously provided by Anaquest Corporation.
This research was supported by the Brain Research Foundation and National Institutes of Health Grants NS-33984 and DA-07861. C. G. Leung was supported by National Institute of Drug Abuse Grant DA-05698 and by a grant from the Women's Council of the Brain Research Foundation.
![]() |
FOOTNOTES |
---|
Present address of C. G. Leung: Anesthesia Service (125), San Diego VA Medical Center, 3350 La Jolla Village Dr., San Diego, CA 92161.
1 This represented the first stimulus applied to the cell but not to the rat. Because of the protocol (see METHODS), a minimum of 15 min without stimulation preceded this first stimulus. However, the typical period without stimulation was closer to 1 h.
2
A similar observation was reported for the responses of RM and NRMC cells to sympathetic nerve stimulation (Blair and Evans 1991).
3 Because this cell did not discharge before any of the clamp trials, a possible inhibitory response cannot be ruled out.
4 A cutoff of 100 s was used in making these measurements.
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 30 December 1997; accepted in final form 24 June 1998.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|