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. Author manuscript; available in PMC: 2022 Sep 15.
Published in final edited form as: Brain Res. 2021 May 28;1767:147539. doi: 10.1016/j.brainres.2021.147539

Responses of neurons in rostral ventromedial medulla to nociceptive stimulation of craniofacial region and tail in rats

Jing-Shi Tang 1, Chen Yu Chiang 2,4, Jonathan O Dostrovsky 2, Dongyuan Yao 3, Barry J Sessle 2,4,*
PMCID: PMC8349861  NIHMSID: NIHMS1712595  PMID: 34052258

Abstract

The rostral ventromedial medulla (RVM) plays a key role in the endogenous modulation of nociceptive transmission in the central nervous system (CNS). The primary aim of this study was to examine whether the activities of RVM neurons were related to craniofacial nociceptive behaviour (jaw-motor response, JMR) as well as the tail-flick response (TF). The activities of RVM neurons and TF and JMR evoked by noxious heating of the tail or perioral skin were recorded simultaneously in lightly anaesthetized rats. Tail or perioral heating evoked the TF and JMR, and the latency of the JMR was significantly shorter (P<0.001) than that of the TF. Of 89 neurons recorded in RVM, 40 were classified as ON-cells, 27 as OFF-cells, and 22 as NEUTRAL-cells based on their responsiveness to heating of the tail. Heating at either site caused an increase in ON-cell and decrease in OFF-cell activity before the occurrence of the TF and JMR, but did not alter the activity of NEUTRAL cells. Likewise, noxious stimulation of the temporomandibular joint had similar effects on RVM neurons. These findings reveal that the JMR is a measure of the excitability of trigeminal and spinal nociceptive circuits in the CNS, and that the JMR as well as TF can be used for studying processes related to descending modulation of pain. The findings also support the view that RVM ON- and OFF-cells play an important role in the elaboration of diverse nociceptive behaviours evoked by noxious stimulation of widely separated regions of the body.

Keywords: rostral ventromedial medulla, neurons, craniofacial, descending modulation, pain

1. Introduction

The rostral ventromedial medulla (RVM) plays a major role in the endogenous modulation of nociceptive transmission in the spinal and trigeminal nociceptive pathways in the central nervous system (CNS). The RVM region in the rat includes nuclei raphe magnus, reticularis gigantocellularis pars alpha and reticularis paragigantocellularis lateralis. It receives spinal and trigeminal somatosensory afferent inputs that include nociceptive inputs relayed through the spinal dorsal horn (SDH), medullary dorsal horn (MDH; also known as trigeminal subnucleus caudalis), parabrachial nucleus (PBN), locus coeruleus (LC) and subnucleus reticularis dorsalis (SRD), and receives descending projections from several higher brain centres such as the midbrain periaqueductal grey (PAG) and hypothalamus (for review, see Fields and Basbaum, 1994; Mason, 2001; Dubner et al., 2014; Ossipov et al., 2014; Chichorro et a., 2017; Martins and Tavares 2017; Chen and Heinricher, 2019). The major projection sites of the RVM include the SDH and MDH (eg, Basbaum et al., 1978; Lovick and Wolstencroft, 1983; Vanegas et al., 1984a,b; Mason and Fields, 1989; Fields et al., 1995; Salas et al., 2016), and local electrical stimulation of the RVM or microinjection of neurochemical mediators (eg opioids, GABA, excitatory amino acids) into the RVM modulates nociceptive behaviour and the activity of SDH and MDH nociceptive neurons (eg, Oliveras et al., 1975; Basbaum et al., 1976; Willis et al., 1977; Sessle et al., 1981; Chiang et al., 1994; Meng et al., 2000; Lambert and Zagami, 2009; Chebbi et al., 2014; Salas et al., 2016; for review, see Fields and Basbaum, 1994; Dubner et al., 2014; Ossipov et al., 2014; Chichorro et al., 2017; Martins and Tavares, 2017). Through these processes and its receipt of nociceptive afferent inputs as well as descending inputs from brainstem and higher levels of the CNS, the RVM is viewed as an integral CNS site by which descending controls may exert their modulatory influences on pain and also may make a contribution to CNS mechanisms by which nociceptive afferent inputs elicit diffuse noxious inhibitory controls (DNIC) (eg, Le Bars et al., 1979; Fields and Basbaum, 1994; Mason, 2001; Dubner et al., 2014; Ossipov et al., 2014; Chichorro et al., 2017; Martins and Tavares, 2017; Chen and Heinricher, 2019).

The neurons in the RVM have commonly been functionally classified as ON-cells, OFF-cells and NEUTRAL-cells in relation to the occurrence of the tail-flick response (TF), with ON-cells facilitating and OFF-cells inhibiting this spinally mediated nociceptive reflex : ON- and OFF-cells increase and decrease, respectively, their discharges just prior to and during the TF elicited by noxious thermal stimulation of the tail, and NEUTRAL-cells are generally reported to show no consistent changes in activity related to the TF (eg, Fields et al., 1983, 1995; Brink et al., 2012; Salas et al., 2016; Follansbee et al., 2018; Chen and Heinricher, 2019). The activity of the ON-cells and OFF-cells can also be modulated by noxious mechanical stimuli and by inflammatory irritants such as capsaicin, histamine and Complete Freund’s Adjuvant applied to other spinally innervated tissues, and their modulation involves several endogenous chemical mediators (eg, glutamate, opioid, gamma-amino butyric acid [GABA]) as well as cytokines and glial cells acting within the RVM (eg, Brink et al., 2012; Hellman and Mason, 2012; Dubner et al., 2014; Salas et al., 2016; Follansbee et al., 2018; Chen and Heinricher, 2019).

There is considerable evidence from these various studies that supports the view that ON-cells and OFF-cells in the RVM respectively facilitate and inhibit the activity of SDH and MDH neurons and thereby underpin the crucial role played by the RVM in endogenous pain control, but the exact mechanisms by which these influences are accomplished are still unclear (see Zhang et al.,2015; Chichorro et al., 2017; Francois et al., 2017; Martins and Tavares, 2017; Chen and Heinricher, 2019). There is also limited information available of the effects on RVM neurons of noxious craniofacial stimulation. Except for reports that the activity of NEUTRAL- cells as well as ON-cells and OFF-cells may be modulated by noxious mechanical and algesic chemical stimulation of some craniofacial tissues (eg, Schnell et al., 2002; Khasabov et al., 2015), the extent to which ON-cell, OFF-cell and NEUTRAL-cell activity patterns operate in relation to craniofacial nociceptive behaviours is virtually unexplored, but is likely a key feature underlying CNS mechanisms modulating sensory and motor functions in acute and chronic craniofacial pain states. Therefore, the primary aim of the present study was to examine whether the activities of RVM neurons were related to craniofacial nociceptive behaviour (jaw-motor response, JMR) as well as the TF evoked by noxious thermal stimulation of the tail or perioral skin

2. Results

2.1. Nocifensive responses evoked by noxious stimulation

In all 39 rats tested, hot water (55°C) immersion of the tail evoked a TF. The TF latency measurements were made by applying a total of 55 trials of the tail heating in 19 rats (ie, ~3 trials/rat); (each trial consisted of a sequence of 3–8 consecutive heat stimuli. The mean (±SEM) latency of the TF in these 19 rats was 6.0±0.3s(eg, Figs. 1, 2, 3). Furthermore, such noxious stimulation of the tail usually could also evoke a jaw-motor response (JMR; represented by anterior digastric [DIG] and masseter [MASS] muscle electromyographic [EMG] responses); a JMR was concomitantly evoked in 84% (46/55) of the tail heating trials (eg, Figs, 1, 2, 3) with a mean latency of 5.1±0.3 s. The mean latency of the JMR evoked by tail heating was significantly shorter than that of the TF (P<0.001, paired t-test, Fig.3A and B).

Fig. 1.

Fig. 1.

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An ON-cell excited by noxious thermal stimulation of the tail. The neuronal ongoing activity was increased prior to the occurrence of the tail-flick response (TF, force scaled in g) and jaw-motor response (JMR; represented by anterior digastric [DIG] and masseter [MASS] muscle EMG responses). The force of the tail flick movement was monitored during heating of the tail produced by immersing the distal portion of the tail into a hot water bath at 55°C. The changes in neuronal activity were judged as a meaningful increase only if the firing rate increased to 150% or more of the baseline value that was an average of the baseline firing rate during the 3–5 min period before the sequence of heating stimuli. Responses to eight consecutive heating stimuli are superimposed. The dotted line indicates the onset of the TF.

Fig. 2.

Fig. 2.

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An OFF-cell inhibited by noxious thermal stimulation of the tail. The neuronal ongoing activity abruptly stopped prior to the occurrence of the tail-flick response (TF, force scaled in g) and jaw-motor response (JMR; represented by anterior digastric [DIG] and masseter [MASS] muscle EMG responses). The force of the tail flick movement was monitored during heating of the tail by immersing the distal portion of the animal’s tail into a hot water bath at 55°. The changes in neuronal activity were judged as a meaningful decrease only if the firing rate decreased below 50% of the baseline value that was an average of the baseline firing rate during the 3-5 min period before heating. Responses to three consecutive heating stimuli are superimposed. The dotted line indicates the onset of the TF.

Fig. 3.

Fig. 3.

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A, Comparison between mean latency of tail-flick response (TF) and mean latency of jaw-motor response (JMR). The TF (black bar) and JMR (shaded bar) were evoked during alternate noxious heating of the tail and perioral skin (upper lip). The data are presented as mean±SEM. The difference in TF and JMR latencies is statistically significant (***P<0.001); B, Individual JMR latency values plotted against their TF latency values, either during heating of the lip (●) or the tail (○). Note that in both heating conditions, most points are located to the left of the diagonal line.

Radiant heat was applied to the perioral skin in 19 of the 39 rats (which had been previously applied with the TF test) and evoked a JMR in all 47 trials, and it also evoked a TF in 96% (45/47) of the trials (eg, Figs. 4, 5) at a mean perioral skin threshold temperature of 46.7±0.9°C. The latency of the JMR (3.9±0.3 s) evoked by perioral skin heating was significantly shorter than that of the TF (4.7±0.3 s; P<0.001, paired t-test, also see Fig. 3A and B).

Fig. 4.

Fig. 4.

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An ON-cell excited by noxious thermal stimulation of the perioral skin (upper lip). The neuronal ongoing activity was increased prior to the occurrence of the tail-flick response (TF, force scaled in g) and jaw-motor response (JMR; represented by anterior digastric [DIG] and masseter [MASS] muscle EMG responses). The TF and ON-cell activity were assessed as in Fig. 1. Responses to eight consecutive heating stimuli are superimposed. The dotted line indicates the onset of the JMR.

Fig. 5.

Fig. 5.

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An OFF-cell inhibited by noxious thermal stimulation of the perioral skin (upper lip). The neuronal ongoing activity abruptly stopped prior to the occurrence of the tail-flick response (TF, force scaled in g) and jaw-motor response (JMR; represented by anterior digastric [DIG] and masseter [MASS] muscle EMG responses). The TF and OFF-cell activity were assessed as in Fig. 2. Responses to three consecutive stimuli are superimposed. The dotted line indicates the onset of the JMR.

Radiant heat that was also applied to the left hindpaw in 7 other rats to test the effects of this additional noxious stimulus on RVM neuronal activity (see below). Hindpaw heating was found to evoke a hindpaw withdrawal reflex (HWR) that was monitored in the 7 animals by recording the heat-evoked EMG activity in the biceps femoris muscle (eg, Fig. 6). Heating of the hindpaw evoked not only a HWR but also elicited concomitantly a TF and JMR in all these animals at a mean threshold temperature of 45.2±0.3°C; the mean latencies were 4.7±0.3 s, 4.8±0.3 s and 4.5±0.3 s, respectively, and there was no significant difference between the latencies.

Fig. 6.

Fig. 6.

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An OFF-cell inhibited by noxious thermal stimulation of the left hindpaw (LHP). The neuronal ongoing activity abruptly stopped prior to the occurrence of the tail-flick response (TF), hindpaw withdrawal response (HWR) as well as jaw-motor response (JMR). The HWR is represented by biceps femoris (BF) muscle EMG responses, and the JMR is represented by anterior digastric (DIG) muscle EMG responses). The TF and OFF-cell activity were assessed as in Fig. 2. Responses to three consecutive heating stimuli are superimposed. Note that there were no clear differences in the time intervals between the changes of neuronal activity and the onsets of the HWR, JMR and TF. The dotted line indicates the onset of the HWR.

2.2. Activity of RVM neurons during nocifensive responses evoked by noxious stimulation.

A total of 89 neurons in the RVM and the adjacent region were recorded in 39 rats. Of these neurons, 40 were classified as ON-cells, 27 as OFF-cells, and 22 as NEUTRAL-cells on the basis of their activity in relation to the TF evoked by noxious heating of the tail. All of the ON-cells were activated not only in relation to the heat-evoked TF but could also be excited by noxious pinch stimulation delivered to all limbs, trunk, ears and facial skin. The OFF-cells were inhibited by these noxious mechanical stimuli as well as before and during the TF. The NEUTRAL-cells, which showed no change in activity in relation to the TF, also could not be activated or inhibited by any of these other mechanical stimuli.

Of the 89 neurons, 55 neurons that were recorded in 19 rats and that had histologically confirmed locations in RVM (eg, Fig. 7) were analyzed in detail for their activity patterns in relation to the TF and JMR evoked by tail and perioral heat stimulations. In the 27 ON-cells analyzed, their increase in firing produced by noxious heating of the tail began at a mean time interval of 964±180 ms before the onset of the TF (eg, Fig. 1). The activity of the 10 OFF-cells analyzed was relatively stable prior to the TF, but was markedly reduced well before and during the TF (eg, Fig.2); the onset of reduced firing began at a mean time interval of 1370±237 ms before the onset of the TF. As noted above, the JMR could also be evoked by noxious heating of the tail. The 27 ON-cells and 10 OFF-cells defined by their activity patterns in relation to the TF also showed changes in activity in relation to the JMR evoked by the tail stimulus (eg, Figs.1, 2). The mean time interval between the onset of changes in neuronal firing and the onset of the JMR evoked by tail heating was 189±166 ms for the ON-cells, and 165±355 ms for the OFF-cells. These time intervals for ON-cell and OFF-cell activity changes in relation to the JMR evoked by tail heating were significantly shorter than those relative to the TF (eg, Figs. 1, 2; and summary in Fig. 8A (for ON-cells, P< 0.001; for OFF-cells, P<0.01 paired t-test). The activities of the 18 NEUTRAL-cells analyzed were not significantly changed by noxious heating of the tail.

Fig. 7.

Fig. 7.

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Reconstruction of the location of each single unit recording site (for the group of 19 rats) in the rostral ventromedial medulla (RVM). The recording sites are shown at two anterior-posterior levels. The sites of neurons classified as ON-cells, OFF-cells and NEUTRAL-cells according to their response to noxious thermal stimulation applied to the tail are indicated by the symbols shown. The number indicated at the lower right part of each diagram refers to the anterior-posterior level in relation to the bregma (Paxinos and Watson, 1998). Abbreviations: 7, Facial nucleus; 7n, facial nerve root; Sp5, trigeminal spinal tract nucleus.

Fig. 8.

Fig. 8.

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Bar graphs comparing the time intervals between the onsets of inhibition of OFF-cells and excitation of ON-cells and the tail-flick response (TF) or the jaw-motor response (JMR). The TF (black bar) and JMR (shaded bar) were evoked by heating of either the tail (A) or the perioral skin (upper lip; B). The time intervals between the onset of changes of neuronal activity and the onset of the JMR were significantly shorter than those for the TF. Data are presented as mean±SEM (**P<0.01; *** P<0.001).

The activity of RVM neurons in relation to the JMR and TF evoked by noxious heat stimulation of the perioral skin was examined in 45 of the 55 neurons that were analyzed in detail and had been tested and classified as ON-cells, OFF-cells, or NEUTRAL-cells based on their activities relative to the TF elicited by heating of the tail. A total of 21 of the 27 ON-cells excited by the noxious heating of the tail were also tested and shown to be excited by noxious heating of the perioral skin (eg, Fig.4, Fig. 8B); their increase in firing began at a mean interval of 267±161 ms prior to the onset of the JMR. The activity of the 10 OFF-cells that was attenuated by the tail stimulation was also reduced by the perioral noxious heating (eg, Fig.5, Fig. 8B); their reduced firing started at a mean interval of 470±153 ms prior to the onset of the JMR, In the case of the TF evoked by noxious heating of the perioral skin, the mean time interval between the onset of changes in neuronal firing and the onset of the TF evoked by perioral heating was 1118±168 ms for the ON-cells, and 1307±158 ms for the OFF-cells. For both ON- and OFF-cells, these time intervals between the onset of the changes in neuronal activity and the onset of the JMR were significantly shorter than those between the onset of the changes in neuronal activity and the onset of the TF (Figs.4 and 5 and. 8B (for ON-cells, P<0.001, n=21; for OFF-cells, P<0.01, n=10, paired t-test). The 14 NEUTRAL-cells tested did not change their activity during the perioral noxious thermal stimulation.

As noted above, noxious heating of the left hindpaw elicited the TF and JMR as well as the HWR, and was used as an additional test of effects of noxious stimulation of spinally innervated tissues on RVM neuronal activity. No ON-cells or NEUTRAL-cells were tested but all 7 OFF-cells tested in 7 rats with noxious heating of the hindpaw showed reduced activity (eg, Fig. 6) that started prior to all three motor responses (HWR, TF and JMR); the mean time intervals between the onset of the decrease in activity and the onset of the HWR, TF and JMR during hindpaw stimulation were 401±79 ms, 394±69 ms and 346±60 ms, respectively. There were no significant differences between these time intervals.

In addition, as an added test of effects of noxious stimulation of craniofacial tissues, the effects of mustard oil injection into the left TMJ on the activity of RVM neurons were examined in 13 rats. In the 13 ON-cells tested, mustard oil injection (but not its vehicle mineral oil) produced a marked increase (1947±667% of the baseline level of activity; P<0.001, paired t-test) at a mean latency of 5.9±1.6 s; by 10 min, the neuronal activity had returned to baseline level. Injection of mustard oil (but not vehicle) into the TMJ markedly reduced the activity of the 10 OFF-cells tested to 18.6±8.6% of the baseline level (P<0.001, paired t-test) at a mean latency of 4.5±1.7s; the attenuation of neuronal activity lasted as long as 2 min. mustard oil injection into the TMJ did not alter the activity of all four NEUTRAL-cells tested.

3. Discussion

The present study has provided novel findings that noxious heating of the tail in lightly anaesthetized rats evokes not only a TF but also a JMR, a sensorimotor response that was monitored by recordings of evoked EMG activity in the DIG and MASS muscles. Likewise, noxious heating of the perioral skin could elicit these nociceptive reflex behavioural responses, and the mean latency of the JMR was significantly shorter than that of the TF regardless of whether the stimulation site was the tail or perioral skin. Moreover, the activities of ON-cells and OFF-cells in the RVM were respectively increased or decreased by the noxious stimulation of either site, and the time intervals between the onset of the change in the ON- and OFF-cell activities and the onset of the JMR also were significantly shorter than that of the TF. In addition noxious stimulation of the hindpaw or TMJ produced similar changes in the activities of ON-cells and OFF-cells of the RVM. Taken together, these findings suggest that craniofacial nociceptive sensorimotor responses such as the JMR can be used as a measure of the excitability of trigeminal and spinal nociceptive circuits in the CNS for studying processes related to descending modulation of pain. Furthermore, the findings also reveal that the ON-cells and OFF-cells in the RVM are intimately involved in the integration of the excitatory and inhibitory effects on behavioural responses manifested in widely separated regions of the body that can be reflexly evoked by the same noxious stimulus irrespective of whether it is applied to the craniofacial region near the tip of the nose or to the tip of the tail.

This study included the TF as a measure of nociceptive reflex behaviour in relation to RVM neuronal activities since it has been extensively used as a parameter of nociceptive reflex behaviour in numerous earlier studies examining the functional role played by the RVM and related CNS sites in the modulation of nociceptive processes and nociception (for review, see Fields and Basbaum, 1994; Mason, 2001; Ossipov et al., 2014; Dubner et al., 2014; Chichorro et al., 2017; Martins and Tavares, 2017; Chen and Heinricher, 2019). The latency of the TF evoked by noxious heating of the tail in the present study is comparable to that reported in earlier studies in anaesthetized rats (eg, Berge et al., 1988; Morgan et al., 1994; Bannon and Malmberg, 2007). Hindpaw heating that evoked the HWR (and also evoked a TF and JMR) was also used in some rats of the present study as an added measure of effects on RVM neurons of a noxious stimulus of tissues supplied by spinal nerves. The latency of the HWR evoked by noxious heat applied to the hindpaw is comparable to that reported in earlier studies in anaesthetized animals (eg, Carstens and Campell, 1992; Morgan et al., 1994; Morgan, 1998; Kincaid et al., 2006; Devonshire et al., 2015).

In the case of the JMR evoked by noxious heating of the perioral skin near the tip of the nose, this study has provided novel findings of the occurrence of this response as manifested by the increased EMG activity concomitantly occurring in both the DIG and MASS muscles, at a mean latency of 3.9 ± 0.3 s and a mean threshold of 46.7± 0.9°C. The heat-evoked EMG activity in both these jaw muscles, one a jaw-opening muscle (DIG) and the other a jaw-closing muscle (MASS), contrasts with the jaw-opening reflex that can be evoked in the DIG by noxious stimulation (eg, see Dubner et al., 1978; Ellrich et al, 2001; Sessle, 2006), but appears similar to the sustained increase that has been previously documented in these muscles when other types of noxious stimuli (eg, mustard oil, capsaicin, glutamate) are applied to orofacial tissues such as TMJ, muscle, and tooth pulp (Yu et al., 1995; Cairns et al., 1998; Sunakawa et al., 1999; Ro et al., 2002; Lam et al., 2009; Narita et al., 2012; Filippini et al., 2020). Indeed, the present study also used mustard oil application to the TMJ in some rats as an added noxious stimulation of craniofacial tissues and showed for the first time that, like perioral heating, it was effective in inducing increased activity of ON-cells and decreased activity of OFF-cells. Mustard oil is a well-known inflammatory irritant, and when applied to the TMJ or other craniofacial tissues it elicits increased EMG activity of DIG and MASS muscles in lightly anaesthetized animals (Yu et al., 1995; Sunakawa et al., 1999; Narita et al., 2012; Filippini et al., 2020; Yao et al., 2020) that has been implicated as a “splinting” action in these jaw-opening (i.e. DIG) and jaw-closing (i.e. MASS) muscles to minimize jaw movement in the presence of pain (Sessle, 2000, 2006; Murray and Lavigne, 2014). The increased jaw muscle activity has also been viewed as a sensorimotor reflection of trigeminal central sensitization that has been documented by the occurrence of hyperexcitability of nociceptive neurons of the MDH following application of mustard oil to the TMJ or mustard oil and other noxious stimuli (eg, heat) to other craniofacial sites (eg, Hu et al., 1992; Yu et al., 1993; Chiang et al., 1998, 2007; Imbe et al., 2001; Lam et al., 2009; Nakaya et al., 2016; Tashiro and Bereiter, 2020). Thus, like the mustard oil-evoked responses, the JMR evoked by perioral heating and reflected in increased EMG activity in the jaw muscles can be viewed as a “surrogate” measure of the excitability of trigeminal nociceptive circuits in the CNS such as the MDH and thereby a useful metric for studies of CNS modulatory mechanisms.

The MDH is considered the trigeminal analogue of the SDH and like the SDH receives nociceptive inputs from A-delta and C-fibre afferents activated by noxious mechanical, thermal or algesic chemical stimuli of the types used in the present study (for review, see Dubner et al., 1978; Sessle, 2000; Ringkamp et al., 2013; Cairns et al., 2001; Chichorro et al., 2017). Both the MDH and SDH relay the nociceptive afferent information that they receive to the thalamus and other higher centres in the CNS as well as to spinal cord and brainstem sites such as the SRD, PBN and RVM; some of these sites also include the motoneuron pools in the spinal ventral horn and trigeminal motor nucleus subserving respectively the TF, HWR, and JMR (see Dubner et al., 1978; Sessle, 2000, 2006; Dostrovsky and Craig, 2013; Chichorro et al., 2017; Chen and Heinricher, 2019; Todd, 2010; Koch et al., 2018). The nociceptive afferent inputs to the MDH and SDH and their projections via the brainstem, including the RVM, to the motoneuron pools may explain the novel findings of the present study that widely separated nociceptive reflex behavioural responses (eg, TF and JMR) can be concomitantly evoked by noxious stimulation of any one of the craniofacial, hindpaw and tail sites tested. For example in the case of the JMR, nociceptive afferent inputs into the brainstem from sites in the craniofacial region such as the perioral skin and TMJ have been demonstrated to be relayed via the MDH directly or indirectly to the trigeminal motor nucleus which contains the brainstem motoneurons supplying the DIG and MASS muscles: there are neuroanatomically and electrophysiologically delineated projections from MDH to the trigeminal motor nucleus and disruption of MDH synaptic transmission blocks the JMR that is expressed as increased EMG activity in the DIG and MASS muscles evoked by noxious facial and TMJ stimuli (Dubner et al., 1978; Yu et al., 1995; Tsai et al., 1999; Cairns et al., 2001). In addition, it is noteworthy that the MDH is also the source of some of the descending projections from the brainstem that directly or indirectly access the lower levels of the spinal cord (see Dubner et al., 1978; Sessle, 2000, 2006; Chichorro et al., 2017) which are the sites of the ventral horn motoneurons supplying the muscles involved in the HWR and TF. Likewise, there are CNS pathways explaining our novel finding that noxious stimulation of the tail or hindpaw can evoke all three nociceptive reflex behavioural responses: there are spinal internuncial pathways connecting higher and lower levels of the spinal cord that could provide the CNS neural circuitry underlying the HWR and TF, and there are some ascending spinal pathways projecting to the brainstem where they could evoke excitatory or inhibitory influences on regions (eg MDH, reticular formation, PBN) acting as reflex interneuronal relay sites to trigeminal and spinal motoneurons (see Mandadi et al., 2013; Dostrovsky and Craig, 2013).

A key role in the elicitation or inhibition of these nociceptive reflex behavioural responses is played by the RVM. This brainstem structure has previously been shown to project to the MDH and SDH, and to receive nociceptive afferent inputs from widespread parts of the body (for review, see Chen and Heinricher, 2019; Khasabov et al., 2015; Vanegas et al., 1984a,b; Fields et al., 1995; Dostrovsky and Craig, 2013), consistent with the present findings of the respective excitatory or inhibitory effects of noxious craniofacial, hindpaw and tail stimulation on the activity of ON-cells or OFF-cells in the RVM. The RVM receives some of these inputs from the SDH and MDH and other caudal brainstem sites such as SRD, LC and PBN, and as well receives projections from higher CNS regions such as the PAG and hypothalamus. Disruption of RVM function can interfere with the influences exerted by these regions on nociceptive processes, and a variety of cellular mechanisms including glutamatergic, opioid, GABAergic and cannabinoid processes and glia regulate its activity and influences (for review, see Dubner et al., 2014; Ossipov et al., 2014; Chichorro et al., 2017; Chen and Heinricher, 2019). Thereby, by way of its inputs, outputs and intrinsic modulatory processes, the RVM acts as an integral CNS site by which descending controls exert their modulatory influences on spinal and trigeminal nociceptive transmission and pain behaviour and also may contribute to CNS mechanisms by which nociceptive afferent inputs induce DNIC in laboratory animals and conditioned pain modulation in humans (eg, Le Bars et al., 1979; Oliveras et al., 1975; Hu, 1990; Bouhassira et al., 1993; Chiang et al., 1994; Meng et al., 2000; Lambert and Zagami, 2009; Wang et al., 2010; Chebbi et al., 2014; Follansbee et al., 2018; for review, see Fields and Basbaum, 1994; Mason, 2001; Dubner et al., 2014; Ossipov et al., 2014; Chichorro et al., 2017; Chen and Heinricher, 2019; Yarnitsky et al., 2019).

In the present study, the RVM neurons were functionally classified in accordance with the well-documented scheme of earlier studies whereby their activity can be linked to the onset of the TF elicited by noxious heating of the tail (eg, Fields et al., 1983, 1995; Ellrich et al., 2001; Schnell et al., 2002; Brink et al., 2012; Salas et al., 2016; Follansbee et al., 2018; Chen and Heinricher, 2019; for review, see Fields and Basbaum, 1994; Mason, 2001; Ossipov et al., 2014; Chichorro et al., 2017). The activity patterns and the timings of the onset or offset of activity of the ON-cells and OFF-cells in relation to this nociceptive reflex behaviour were generally similar to those documented in the earlier studies, with ON-cells showing increased activity and OFF-cells manifesting decreased activity. Our finding that noxious stimulation of craniofacial tissues (perioral skin and TMJ) also modulates these two types of RVM neurons is also consistent with earlier findings that activation of afferent inputs from various craniofacial tissues can modify ON-cell and OFF-cell activities (Ellrich et al., 2001; Schnell et al., 2002; Edelmayer et al., 2009; Khasabov et al., 2015) although a differential response pattern reported for some RVM neurons to stimulation of tissues supplied by trigeminal vs spinal nerves (Ellrich et al., 2001; Snell et al., 2002) was not observed in the present study. Schnell et al. (2002) and Khasabov et al. (2015) have reported that some NEUTRAL-cells can also be modulated by nociceptive inputs, including those activated by craniofacial stimulation. However, our finding that the activity of NEUTRAL-cells could not be modulated by any of the noxious stimuli used in the present study is consistent with most of the earlier studies that have characterized these RVM neurons.

The findings of the present study clearly show that the activity patterns of the ON- cells and OFF-cells in relation to the TF evoked by noxious stimulation of the tail were also apparent when the noxious stimulation was instead applied to craniofacial or hindpaw tissues. Furthermore, these ON-cell and OFF-cell patterns of activity evoked by these noxious stimuli were also closely linked to the elicitation of the JMR and HWR as well as the TF, and the times of onset of ON-cell activity and offset of OFF-cell activity in relation to the onset of the TF and JMR were significantly shorter for the JMR than the TF, regardless of whether the stimulus was to the tail or the lip. Given these findings and current knowledge of the input and output features of these RVM neurons and concepts of their functional role in endogenous pain modulation (for review, see Fields and Basbaum, 1994; Mason, 2001; Ossipov et al., 2014; Dubner et al., 2014; Chichorro et al., 2017; Martins and Tavares, 2017; Chen and Heinricher 2019), the present study has provided further insights into the integral role that is played by the RVM in elaborating diverse nociceptive behaviours that can be evoked from widely separated parts of the body.

In conclusion, the findings of this study reveal that the JMR can be used as a measure of the excitability of trigeminal and spinal nociceptive circuits in the CNS, and that the JMR as well as the TF and HWR can also be used for studying processes related to descending modulation of pain. The findings also support the view that RVM ON-cells and OFF-cells may play a key role in the elaboration of diverse nociceptive behaviours evoked by noxious stimulation of widely separated regions of the body.

4. Experimental Procedure

4.1. Animal preparation

The experiments were performed on male Sprague-Dawley rats, weighing between 220 and 400 g. The animals were anaesthetized initially with sodium pentobarbital (55mgkg−1, ip) for the surgical procedures. After cannulation of the trachea and left jugular vein, a pair of Teflon-coated stainless-steel wires (diameter 0.1 mm, exposed tip 1.0 mm) was inserted into the left anterior digastric muscle (DIG) and another pair was inserted into the left masseter muscle (MASS) for recording jaw EMG activity in order to monitor the JMR. A pair of EMG electrodes was also placed in the biceps femoris muscle to monitor the HWR in some animals. The animal’s head was positioned in a stereotaxic frame, and a small craniotomy was performed over the cerebellum to allow for subsequent insertion of a glass microelectrode into the RVM for recording of single unit activity (see below). During the experiment, the animal was maintained in a state of light anaesthesia by intravenous infusion of methohexital sodium at a constant rate (Brietal 30–40 mg kg−1h−1) to allow the TF, HWR and JMR to be elicited at a stable latency (see below). Heart rate, expired percentage CO2 and rectal temperature were continuously monitored and maintained at 330–430 beats/min, 4–5% and 37–38°C, respectively.

4.2. Stimulation and Recording procedures

In 39 rats, a TF was evoked by immersing the rat’s tail into 55°C hot water at a depth of about 3–4 cm from the tip of the tail. The TF was considered to be evoked when there was a flick of the tail, the force of which was monitored with a mechanical force transducer (Transbridge TBM4M, World Precision Instruments, USA) attached to the tail with a thread. For evoking the JMR, radiant heat from a distance of 4.0–4.5 cm was applied to the left perioral skin (upper lip, near the tip of the nose) in 19 of the 39 rats via a projector lamp (Osram, 8V, 50W) focused on the tip of a thermocouple (Physitemp Bat-12 model digital thermocouple thermometer, IT-2 probe, 0.15 s response time constant) fixed to the perioral skin with cyanoacrylate glue. The skin temperature was monitored by the thermocouple’s voltage output. An analogous heat stimulus and thermocouple were also applied to the ventral surface of the left hindpaw in 7 of the rats in order to evoke the HWR. The JMR or HWR was considered to be evoked when an increase occurred in the EMG activities of both the DIG- and MASS muscles or the biceps femoris muscle. An increase in EMG activity associated with the JMR or HWR was considered to occur when it was ≥150% of the baseline EMG activity recorded in the 30 s preceding the stimulus; the latencies of the JMR and HWR were measured as the interval between the onset of the heat stimulus and the onset of the EMG activity changes. Each noxious thermal stimulus, in a sequence of 3–8 consecutive heat stimuli (termed a “trial”), was terminated at 6 s for perioral skin and 10 s for tail or hindpaw, to avoid thermal damage to the tissues. A stable TF, JMR, and HWR could be elicited repeatedly when a time interval of 5 min was allowed between each noxious thermal stimulus in a sequence. The skin threshold temperature for eliciting the JMR or HWR in each animal was taken as the average skin temperature for 3 consecutive heat stimuli at the time the reflex occurred.

In all the 39 rats, single unit extracellular recordings were made in the RVM (AP −9.5 to −11.5 mm relative to bregma; DV −8.0 to −9.2 mm from the cerebellar surface at the midline) with a glass micropipette filled with 0.5M sodium acetate containing 2% Pontamine Sky Blue (impedance 10–20 MΩ at 1000 Hz). The micropipette was advanced into the medulla with an auto-microdrive while mechanical stimuli (brush, pinch) were applied to different parts of the body surface. The activity of single units was amplified and filtered (Krohn-Hite model 3700 filter, typically 100–10000 Hz), and the signal was fed to an audio monitor (Grass AM8, Grass Instrument Co, USA) and oscilloscopes (Iwatsu DS-6411, Tektronix 5113, USA) and analyzed as noted below. When a single unit was clearly differentiated from background activity, the neuron was tested for responses to innocuous and noxious mechanical stimuli applied to all limbs as well as the trunk, face, and ears in order to determine its mechanoreceptive field. Then the spontaneous activity of the RVM neuron was continuously recorded, and examined specifically before, during and after the TF evoked by dipping the tail in hot water (see above) and before, during and after the JMR evoked by the noxious heat stimulation of the perioral skin in 19 of the 39 rats. In the 7 other rats, the activity of OFF cells was also examined before, during and after the HWR evoked by the noxious heat stimulation of the hindpaw. The effects of mustard oil 20% 20μl) or its vehicle (mineral oil, 20 μl) injected into the left temporomandibular joint (TMJ) were also examined on the activity of RVM neurons in a separate group of 13 rats. First the vehicle was injected, and then after a 15–20 min period, the mustard oil was injected during the neuronal recording, and the neuronal activity was recorded for another 15 min or more.

RVM neurons were functionally classified into three classes according to the criteria proposed previously (Fields et al., 1983, 1995): ON-cells which were excited just prior to and during the TF elicited by noxious heating of the tail; OFF-cells which were inhibited just prior to the TF; and NEUTRAL-cells which did not change their firing rate prior to and during the TF. Briefly, the baseline level of activity of the neuron was determined by averaging the neuronal firing rate over the 3–5 min period before commencement of each heating trial. The beginning of the evoked activity of ON-cells was considered to occur when the neuronal activity increased to 150% or more of the baseline level. The beginning of the reduced firing of OFF-cells was judged to occur when the neuronal firing rate was reduced below 50% of the baseline level of activity.

4.3. Histology

Histological verification and reconstruction of neuronal recording sites was made as previously described (eg, Hu et al., 1992; Tsuboi et al., 2011; Wang et al., 2013). Briefly, at the end of each recording experiment, the recording site of the last neuron studied was marked by ejecting pontamine sky blue from the micropipette with a cathodal current through the electrode tip (10 μA for 10–20 min s) and 20 min later an overdose of barbiturate (100 mgkg−1) was administered intravenously. The animal was then perfused transcardially with 0.9% saline followed by 10% formalin. The brain was then removed and fixed in fresh formalin for 3–7 days. The brainstem was removed and 100 micron sections were cut with a freezing microtome and mounted and stained with Cresyl violet. The recording sites were reconstructed and plotted on coronal sections (see Fig. 7) modified from the atlas of Paxinos and Watson (1998). The locations of unmarked recording sites were determined by their distances from the marked one on the basis of the microdrive readings. Evans Blue dye (5 mg kg−1) was injected through the catheter in the left external jugular vein. In those animals receiving mustard oil injected into the TMJ during the experiment, the injection site was visually confirmed to be localized in TMJ tissues by the appearance of extravasated dye in the tissues, as previously described (Haas et al. 1992; Hu et al. 1993).

4.4. Data analysis

Neuronal activity, EMG activity, skin temperature, and TF mechanical force were simultaneously recorded, collected and processed with a digital data acquisition system (CED 1401 and Spike2 program, Cambridge, UK). The EMG signals were rectified and integrated, and all the recorded data were analyzed off-line with the Spike2 program for the time period starting at 10 s before and continuing for 20 s after the onset of either the TF, JMR or HWR which was referred as the “0” time-point on the time axis. Peri-stimulus time histograms (sum of 3–8 heat stimuli for OFF-cells and ON-cells, respectively, bin=250 ms) of the neuronal responses evoked by the noxious stimuli were displayed in association with the EMG signals, heat stimulus temperature and TF curves.

The latencies of the TF, JMR and HWR as well as the time interval between changes in the firing rate of RVM neurons and the onset of the TF, JMR or HWR were measured. Evoked EMG activity related to the JMR and HWR was expressed as percentage change from baseline control values, with the data points representing the integrated area for each 30s segment (10s before and 20s after the onset of the TF, JMR or HWR) in association with the firing rate of RVM neurons. All data are indicated as the mean±SEM and analyzed for statistical significance (P<0.05) by paired t-test.

HIGHLIGHTS.

  1. Noxious stimulation of either perioral skin or tail evokes a jaw motor response (JMR) and tail flick.

  2. Each of these noxious stimuli modulates rostral ventromedial medulla (RVM) neuronal activity.

  3. The JMR can be used for studying processes related to descending modulation of pain.

  4. RVM ON- and OFF-cells play an important role in modulating diverse nociceptive behaviours.

5. Acknowledgments

The authors thank K. MacLeod and S. Carter for their technical assistance and Dr. J. W. Hu for his guidance. This study was funded by the U.S, National Institute of Dental and Craniofacial Research grant DE-04786 to B. J. Sessle. The funding source played no role in study design, in collection, analysis or interpretation of the data, and in the writing and submission of this article.

Abbreviations:

CNS

central nervous system

DIG

anterior digastric muscle

DNIC

diffuse noxious inhibitory controls

EMG

electromyographic

GABA

gamma-amino butyric acid

HWR

hindpaw withdrawal reflex

JMR

jaw-motor response

LC

locus coeruleus

MASS

masseter muscle

MDH

medullary dorsal horn

PAG

midbrain periaqueductal grey

PBN

parabrachial nucleus

RVM

rostral ventromedial medulla

SDH

spinal dorsal horn

SRD

subnucleus reticularis dorsalis

TF

tail-flick response

TMJ

temporomandibular joint

Footnotes

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