Chapter 16 Physiological functions of pontomedullary raphe and medial reticular neurons

G.Holstege, R. Bandler and C.B. Saper (Eds.)

Progress in Brain Research, Vol. 107 0 1996 Elsevier Science B.V. All rights resewed

CHAPTER 16

Physiological functions of pontomedullary raphe and medial reticular neurons Peggy Mason’ and Cynthia G. h u n g 2 ‘Department of Pharmacological and Physiological Sciences, The Universityof Chicago, MC 0926, 947 East 58th Street, Chicago, IL 60637, USA, and ’Committee on Neurobiology, The University of Chicago, MC 0926, 947 East 58th Street, Chicago, IL 60637, USA

Introduction In addition to evoking sensory perceptions and motor responses, painful stimuli produce changes in emotional state, affect and motivation. When confronted with a painful input, an organism alters its behavior to allow for a reflex withdrawal, escape andor quiescent behavior to progress. Although most noxious stimuli will produce changes in gross affect (Craig, 1989), only specific subsets of olfactory, auditory or visual stimuli evoke changes in behavior, emotion or affect. Thus, nociceptive transmission pathways appear to be more closely connected to affective and emotional circuitry than are other sensory systems. In the context of the emotional motor system, it is instructive to consider how nociceptive information is transmitted to higher neural centers which directly control emotional and affective behaviors. Ascending nociceptive transmission is strongly modulated such that the input stimulus is a poor predictor of any output measure. Cognitive, motivational, autonomic and sensory factors can alter the perceptions, affects, or motor response evoked by a standard stimulus. Such variability in pain responses is due, at least in part, to modulation of nociceptive transmission within the spinal and medullary dorsal horns. The best studied circuits for nociceptive modulation involve brainstem, spinal and primary afferent neurons. This chapter will focus on the no-

ciceptive modulatory role of neurons in the pontomedullary raphe magnus (RM) and the adjacent nucleus reticularis paragigantocellularis pars a (NRPGa) in the rat. This region is part of a system originally described by Oliveras (Oliveras et al., 1974, 1975) and then classically reported as the “endogenous analgesia system” by Fields and Basbaum (1978). In the classic formulation, neurons in the midbrain periaqueductal gray (PAG) were thought to suppress pain transmission via a monosynaptic relay with RM neurons, including serotonergic cells, that project to the spinal dorsal horn (see Fig. 1). Since the original description of descending pain modulation, our understanding of these pathways has grown in several ways. First, we now know that the PAG-RM-dorsal horn pathway is only one of several pathways which can affect spinal and trigeminal nociceptive transmission. Other brainstem areas, including the parabrachial nuclei (see Chapter 14, this volume), the lateral reticular nucleus (Janss and Gebhart, 1987; Janss and Gebhart, 1988), and the A5 and A7 catecholaminergic nuclei (Bumett and Gebhart, 1991; Yeomans et al., 1992), as well as forebrain areas such as the amygdala (see Chapter 14) and medial preoptic nucleus (Mokha et al., 1987) are now thought to play important roles in nociceptive modulation. Secondly, the pharmacology of pain modulation, once thought to center on endogenous opioids, serotonin and norepinephrine, has been compli-

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Fig. 1. The descending pain modulatory system involving the RhVNRPGa is illustrated. Putative pain modulatory centers are shaded. Descending projections between pain modulatory centers in the brainstem and the spinal dorsal horn are represented by arrows.

cated by the discovery of a multitude of peptides and amino acid transmitters contained in both pain modulatory and pain transmission neurons. The final major addition to the original “endogenous analgesia system” comes from recent evidence that pain is facilitated as well as inhibited (for review, see Fields, 1992). Bushnell et al. (1 985) trained monkeys and humans to detect increases in a thermal stimulus in one of two thermodes held at a baseline temperature. When the baseline was in the noxious range, the latency to detection was decreased when a visual cue correctly signaled which thermode would change in stimulus intensity. Since this decrease in detection latency does not occur when the baseline is in the innocuous range, nociceptive transmission appears to be specifically sensitive to attentional facilitation (Bushnell et al., 1985). In a similar paradigm, the neuronal discharge of multireceptive neurons in the medullary dorsal horn is directly correlated with detection speed (Dubner et al., 1989). These experiments suggest that attentional factors en-

hance pain perception and provide a physiological substrate for facilitation that, remarkably, acts at the first synapse in the ascending nociceptive pathway. Enhancement of nociceptive transmission has also been observed during naloxone-precipitated withdrawal from morphine in a variety of animals, including rats (Bederson et al., 1990; Kaplan and Fields, 1991). Nociceptive thresholds are lower following naloxone administration than during the baseline period prior to morphine. Withdrawal from noxious stimulation is also facilitated by internally aversive stimuli such as pyrogens or emetics (Mason, 1993; Watkins et al., 1994; Wiertelak et al., 1994). Enhanced nociception may be mediated by either an active facilitation or a release from inhibition. In the case of naloxone-precipitated withdrawal, Kaplan and Fields ( 1 99 1) demonstrated that lidocaine microinjection, which reversibly inactivates the RMNRPGa, reverses the observed decrease in nociceptive threshold. Thus, cells in RMMRPGa actively facilitate nociceptive transmission during naloxone-precipitated opioid withdrawal (Kaplan and Fields, 1991). Consistent with this idea, some RMINRPGa neurons are activated during opioid withdrawal (see below). Active facilitation is also likely during pyrogen-induced nociceptive enhancement since electrolytic lesions of RM block the increased nociceptive responsiveness produced by systemic pyrogen (Watkins et al., 1994). In support of RM cells having a role in pyrogen-evoked pain facilitation, more RM neurons express c-fos immunoreactivity after pyrogen than after vehicle administration (Watkins et al., 1994). Since little data are available on the mechanisms of pain facilitation, this chapter focuses primarily on the inhibition of nociceptive transmission. The mechanisms by which RM/NRPGa neurons contribute to the antinociception evoked by brainstem stimulation or systemic morphine, is examined. In addition, evidence that RM/NRPGa neurons modulate autonomic, motivational or behavioral responses as well as somatomotor responses to painful stimuli is reviewed.

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Connectivity of RM/NRPGa cells RMNRPGa neurons have anatomical connections which support a role for these cells in nociceptive modulation. Efferent projections from RM/ NRPGa cells to the spinal dorsal horn are strongly suggestive of a primary role for RWNRPGa neurons in sensory modulation (Basbaum and Fields, 1979; Skagerberg and Bjorklund, 1985; Basbaum et al., 1986). Within the dorsal horn, the terminals of RWNRPGa cells are concentrated in laminae I, I1 and V, regions where primary afferent nociceptors terminate and nociceptive projection neurons are present. This projection to the nociceptive laminae of the dorsal horn suggests that RMINRPGa cells are primarily involved in modulating nociceptive sensory inputs. RMINRPGa cells also project to the intermediolateral cell column and the ventral horn, albeit less strongly than to the dorsal horn (Basbaum and Fields, 1979; Skagerberg and Bjorklund, 1985; Basbaum et al., 1986; Zagon and Smith, 1993). Interneurons, including ones with direct projections to preganglionic sympathetic neurons (see Chapter 3, this volume), are located in laminae II and V of the dorsal horn. Therefore, disynaptic and oligosynaptic connections from RWNRPGa cells to autonomic and motor neurons in the spinal cord are also possible. Physiological studies have confirmed a monosynaptic projection from RM/NRPGa neurons to nociceptive dorsal horn cells but have not resolved whether mono-, di- and/or oligo-synaptic connections from RM/NRPGa to the dorsal horn are critical to antinociception. Only a few studies have intracellularly recorded the responses of dorsal horn cells evoked by RMINRPGa stimulation in anesthetized animals (Giesler et al., 1981; Light et al., 1986; Mokha and Iggo, 1987). In both cat and monkey, the predominant short-latency effect of RM stimulation on dorsal horn cells is an inhibitory post-synaptic potential (IPSP). The IPSP evoked by RM stimulation has a duration of more than 300 ms and can last over 1.5 s (Giesler et al., 1981; Light et al., 1986; Mokha and Iggo, 1987). This long lasting IPSP is likely due to the activa-

tion of numerous RM cells with different conduction velocities (Light et al., 1986). Excitatory responses to Rh4 stimulation are also observed, usually occurring at shorter latencies than the inhibitory responses, and are predominant in nonnociceptive and multireceptive neurons (Light et al., 1986). These results can be interpreted as evidence for RM excitation of inhibitory interneurons. Consistent with this idea, intracellular labeling of two cells that were excited by RM stimulation revealed that these neurons had axonal collaterals in laminae I, 11, IV and V (Light et al., 1986). Such local circuit neurons may contribute secondarily to the long-lasting IPSP elicited by RM stimulation. Future studies are needed to clarify the synaptic route by which RM/NRPGa cells affect nociceptive transmission. Afferents to RMNRPGa arise from neurons in the pontine parabrachial nuclei, the mesencephalic reticular formation, PAG, the medial preoptic area, and the amygdala (Holstege et al., 1985; Holstege, 1987, 1988; Van Bockstaele et al., 1991). While each of these areas putatively plays a part in nociceptive modulation, they are also likely to participate in the modulation of the cardiovascular and respiratory systems, as well as thermoregulatory, sexual and species-specific behaviors (see Chapters 17 and 18, this volume). For instance, PAG activation affects blood pressure, heart rate, vasomotor tone, lordosis, vocalization and defense behaviors as well as nociceptive reflexes and the responses of nociceptive dorsal horn cells (Depaulis and Bandler, 1991). Thus, while the afferent input to RMINRPGa cells is consistent with these cells contributing to pain modulation, it is also consistent with their having a role in autonomic modulation and in the coordination of the affective component of behavior.

Role of RM/NRPGa in nociceptive modulation A considerable body of evidence suggests that the RM/NRPGa can modulate nociceptive transmission under experimental conditions. Electrical activation of RMINRPGa neurons results in either the suppression or facilitation of nociceptive re-

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flexes and the responses of nociceptive dorsal horn cells (Fields and Anderson, 1978; Basbaum and Fields, 1984; Fields et al., 1991; Zhuo and Gebhart, 1992). The effect produced is dependent on the stimulus intensity used and the particular site stimulated. Similarly, microinjection of Lglutamate into RM/NRPGa sites can either inhibit or facilitate nociceptive transmission depending on the dose administered (Zhuo and Gebhart, 1992). A variety of other chemicals, including morphine, opioid peptides, selected peptidase inhibitors, the GABAA receptor antagonist bicuculline, and a2 adrenoceptor agonists, also produce nociceptive inhibition when injected into the RWNRPGa (Fields et al., 1991). To date, al adrenoceptor agonists are the only compounds that facilitate nociceptive responsiveness when injected into RMINRPGa (Haws et al., 1990). These studies demonstrate that nociceptive inhibition is elicited by most manipulations of the RM/NRPGa, although RMINRPGa activation can produce either antinociception or nociceptive facilitation. Lesion studies demonstrate that the RM/ NRPGa may also be necessary for the production of antinociception under specific conditions. Electrolytic lesion or chemical inactivation of the RM/NRPGa produces a decrease in the maximal antinociceptive effect of PAG activation (Behbehani and Fields, 1979; Gebhart et al., 1983; Prieto et al., 1983; Sandkuhler and Gebhart, 1984). The antinociception produced by systemic morphine is also attenuated by either electrolytic lesions (Chance et al., 1978; Proudfit, 1980a,b; Azami et al., 1982; Young et al., 1984) or microinjection of naloxone into the RM/NRPGa (Dickenson et al., 1979; Azami et al., 1982). These studies provide evidence that neurons in RWNRPGa contribute to the antinociception evoked by either PAG stimulation or systemic morphine.

Role of serotonergic RM/NRPGa cells in nociceptive modulation Serotonin has long been considered important in both descending pain modulation and in the generation of opioid anaigesia (Vasko et al., 1984)

(for reviews, see LeBars, 1988; Sawynok, 1989). Consequently, a number of investigations have sought to distinguish the relative contributions of serotonergic and non-serotonergic mechanisms in the production of antinociception. Unfortunately, the attention afforded to the role of serotonin in nociceptive modulation has not resulted in consistent experimental results or a clear conceptual framework. Antinociception evoked by RMINRPGa activation or systemic opioids is partially attenuated by either systemic depletions of serotonin (Akil and Liebeskind, 1975; Deakin and Dostrovsky, 1978; Besson and Oliveras, 1980; Rivot et al., 1980) or serotonin antagonists administered intrathecally (Hammond and Yaksh, 1984; Barbaro et al., 1985; Jensen and Yaksh, 1986), suggesting that serotonergic bulbo-spinal neurons contribute to RMNRPGa mediated antinociception. Consistent with this idea, opioid antinociception is potentiated by serotonin uptake inhibitors (Larsen and Amt, 1984). In addition, morphine administration increases serotonin release or serotonin metabolites in the spinal cord (Shiomi et al., 1978; Vasko and Vogt, 1982) while exogenous application of serotonin to the spinal dorsal horn consistently inhibits both nociceptive reflexes and the activity of nociceptive cells (Belcher et al., 1978; Yaksh and Wilson, 1979). The above findings suggest that serotonin plays an important role in the nociceptive modulation mediated by RM/NRPGa cells. However, there is also evidence that serotonin is not necessary for RM/NRPGa evoked antinociception. Although systemic depletion of serotonin is effective in attenuating antinociception evoked by RM stimulation or morphine, selective chemical lesions of RM/NRPGa serotonergic neurons, effective in reducing spinal serotonin content to 55% of control levels, do not attenuate the antinociception evoked by systemic morphine in the nociceptive tail flick test (Mohrland and Gebhart, 1980). In addition, recent microdialysis studies in the anesthetized cat and the awake rat suggest that RM activation has inconsistent effects on serotonin release in the dorsal horn. Although some in-

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creases in spinal serotonin release were evoked by RM stimulation or supraspinal opioid administration in these studies, the increased serotonin levels were not correlated with either RM-evoked inhibition of nociceptive neurons (Sorkin et al., 1993) or behavioral antinociception (Matos et al., 1992). Furthermore, the earliest effects of RM stimulation on dorsal horn neurons are mediated by neurons with conduction velocities of 5 m / s or greater (Giesler et al., 1981; Light et al., 1986). These cells are unlikely to include serotonergic bulbospinal neurons that have unmyelinated axons (Basbaum et al., 1988) and likely conduct with velocities of under 1 m / s . Finally, the physiological profile of serotonergic cells makes it unlikely that these cells are necessary for RMMRPGa mediated antinociception (see below).

RM/NRPGa is one of several sites that synergistically contribute to nociceptive modulation Since RM/NRPGa lesions rarely block antinociception completely, RIWNRPGa may interact with other neural regions to produce maximal antinociception. Pharmacological studies indicate that opioids act at both spinal and multiple supraspinal sites to synergistically evoke antinociception. Doses of morphine, which are without effect when administered only intrathecally or only supraspinally, interact in a supra-additive fashion to produce antinociception when delivered to both sites (Yeung and Rudy, 1980; Siuciak and Advokat, 1989). Likewise, opioid agonism within the PAG and RM/NRPGa interact synergistically (Rossi et al., 1993). The nature of the interactions between opioid agonism in RMINRPGa and the spinal cord or between PAG, RMMRPGa and the spinal cord have not been tested. The neural circuits which support synergistic interactions have not been described and the mechanism of the proposed opioid synergism remains unclear. Studies on the interaction of terminals within the dorsal horn, that are derived from supra- and proprio-spinal sources, are required for a complete understanding of opioid synergism.

If the synergistic interactions make a substantially larger contribution to antinociception than does the activation of any one site, the design of current physiological experiments which examine the activity of only one nociceptive modulatory site, may be fundamentally flawed. It is possible that future experiments will need to simultaneously examine neurons at multiple levels of the neuraxis.

Electrophysiologicalstudies of RM/NRPGa neurons To determine which physiological populations of RMINRPGa neurons contribute to antinociception and under what circumstances, it is necessary to first understand what is known of the physiological characteristics of RMMRPGa cells. We will first describe the properties of RIWNRPGa neurons in the anesthetized rat, the paradigm under which these cells have been most thoroughly studied. In a later section, differences between the characteristics of RMMRPGa cells recorded in anesthetized and unanesthetized rats are discussed. In the anesthetized rat, RMINRPGa cells can be classified according to their responses to various types of stimuli, including somatic, thermal or autonomic stimuli. One classification scheme which has been of great heuristic value divides RIWNRPGa cells based on their responses to noxious stimulation and opioid administration (Fields et al., 1983a; Barbaro et al., 1986; Cheng et al., 1986; Chiang and Gao, 1986) (see Fig. 2). OFF cells are inhibited by noxious stimulation that evokes a withdrawal reflex and are activated by analgesic doses of opioids. ON cells exhibit increased firing rates in response to noxious stimuli and are inhibited by morphine administration. NEUTRALcells are weakly andor inconsistently excited by noxious stimulation and unaffected by opioid administration. At least 30% of the neurons in each cell group project to the spinal cord (Vanegas et al., 1984).

ON and OFF cells Experiments designed to elucidate the function

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Fig. 2. RMINRPGa neuronal responses to noxious thermal stimulation and systemic morphine. (A) Withdrawal from a noxious thermal stimulus is indicated by increased activity of the paraspinous EMG (top trace). The remaining traces demonstrate neuronal responses to the noxious stimulus. Each line represents a single action potential. All traces are 9 s long. (B) Rate meter records of RM/NRPGa neuronal activity before and after administration of morphine are shown. Morphine sulfate (5 m a g , i.p.) was injected at the arrow. Unit activity is expressed in spikesh (Hz). Each record is 16 min long.

of each cell class have focused primarily on the activity of ON and OFF cells. Manipulations that inhibit nociceptive reflexes consistently activate OFF cells. Thus, OFF cells are excited by opioids administered systemically or by microinjection into the PAG or RM/NRPGa (Fields et al., 1983b; Cheng et al., 1986; Morgan et al., 1992; cf. Thurston and Randich, 1992). Microinjection of the GABA, receptor antagonist, bicuculline, into the RM/NRPGa excites OFF cells and increases the nociceptive threshold (Heinricher and Tortorici, 1992). Iontophoretic application of opioids, which does not alter nociceptive responsiveness, has no affect on OFFcell activity (Heinricher et al., 1992). ON cells are inhibited by opioid administration and activated by manipulations which are associated with facilitated nociceptive responsiveness. The latter manipulations include naloxone-precipitated withdrawal from systemic morphine, conditioning with heterosegmental noxious stimulation,

and volume expansion (Ramirez and Vanegas, 1989; Bederson et al., 1990; Morgan and Fields, 1993). In the case of ON cell activation during opioid withdrawal, the level of activation is correlated with the decrease in nociceptive threshold (Bederson et al., 1990). Studies which demonstrate OFF cell activation during antinociception and ON cell activation during enhanced nociception have led to the hypothesis that OFF cells inhibit and ON cells facilitate nociceptive transmission. Paired recordings in the anesthetized rat reveal that ON and OFF cells do not fire simultaneously but tend to discharge in phase with other neurons of the same physiological cell class (Barbaro et al., 1989). Since nociceptive responsiveness varies temporally in the anesthetized rat, with a time constant in the minute range (Hentall et al., 1991), the nociceptive modulatory effect of ON and OFF cells can be studied by testing the response to a standard noxious stimulus during

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spontaneous periods of ON or OFF cell activity. Fields and colleagues performed this experiment and demonstrated that the latency of the withdrawal from a noxious thermal stimulus to the tail was shorter, by approximately 0.5 s, during ON cell activity than during OFF cell activity (Heinricher et al., 1989). Since the difference in the tail flick latency recorded during ON and OFF cell activity is less than one second (Heinricher et al., 1989), the physiological activation of OFF cells likely has only a small influence on nociceptive transmission in the anesthetized rat. This interpretation is consistent with the finding that OFF cells are activated by morphine administered into the lumbar intrathecal space, a manipulation which reduces the response to noxious stimulation of the hind body but not, at least grossly, to noxious stimulation of the forelimbs and head (Yaksh and Wilson, 1979; Heinricher and Drasner, 1991). While there may be some heretofore unmeasured increase in the cervical and trigeminal nociceptive threshold, it is clear that OFF cell activation alone does not produce a profound antinociception. There are several possible interpretations of this data: (1) the antinociceptive effect of RM/NRPGa OFF cell activation is small; (2) concurrent activation or inhibition of a second RMINRPGa cell class is required for the full antinociceptive effect of RM/NRPGa OFF cell activation; or (3) activation of another brainstem or spinal region, acting in synergy with RWNRPGa, is required for OFF cells to exert their full antinociceptive effects. Future experiments will need to discriminate between these possibilities.

REGULAR NEUTRAL cells resemble ON and OFF cells in that they cycle through periods of activity and periods of inactivity. In contrast, REGULAR NEUTRAL cells exhibit low variability in their discharge rate (see Fig. 2). Since REGULAR NEUTRAL cells appear to have no phasic inputs or properties, it is possible that these cells function to regulate nociceptive transmission in a tonic manner. A subpopulation of NEUTRAL cells contains serotonin (Pan et al., 1993; Potrebic et al., 1994). Of 25 RM/NRPGa cells that were physiologically characterized and intracellularly labeled, none of the 17 ON and OFF cells contained serotonin immunoreactivity while half of the 8 NEUTRAL cells labeled were immunoreactive for serotonin (Potrebic et al., 1994). Consistent with this finding, previous studies have reported that physiologically identified serotonergic neurons’ are unaffected by opioid administration (Auerbach et al., 1985; Chiang and Pan, 1985; Chiang and Gao, 1986; Fornal et al., 1990). In light of previous reports that systemic morphine evokes spinal serotonin release (Shiomi et al., 1978; Vasko and Vogt, 1982), it is paradoxical that opioid antinociception occurs in the absence of an increase in serotonergic cell firing. In this regard, Matos et al. (1992) did not consistently observe increased serotonin release in the dorsal horn following supraspinal morphine. It is currently unknown whether serotonergic neutral cells belong to the REGULAR andor the IRREGULAR physiological subclasses. Intracellularly labeled serotonergic neurons in the rat dorsal raphe have a steady and slow (<5 Hz) discharge rate (Aghajanian and Vandermaelen, 1982). If

Neutral cells

At present, an unequivocal physiological marker for mammalian serotonergic neurons has only been directly demonstrated for cells located in the dorsal raphe and recorded in the anesthetized rat (Aghajanian and Vandennaelen, 1982). Most investigators have used criteria such as ( 1 ) long-duration action potentials; (2) slow, steady discharge rate; (3) inhibition by serotonin-1A agonists; (4) slow conduction velocity; (5) decreased activity during REM sleep; (6) presence after serotonin neurotoxin treatment; or some combination of the above factors (Jacobs and Azmitia, 1992), to “identify” serotonergic neurons. However, it is possible that (1) some neurons that are physiologically identified as serotonergic are not; andor (2) some serotonergic neurons, with different physiological characteristics from those described above, are currently omitted from study.

NEUTRAL cells are thus named because they do not respond to opioids administered systemically, supraspinally or iontophoretically (Fields et al., 1983b; Cheng et al., 1986; Heinricher et al., 1992). They are weakly and inconsistently excited by noxious pinch or heat (Leung and Mason, 1995). NEUTRAL cells can be divided into subpopulations of REGULAR and IRREGULAR cells, according to their spontaneous activity patterns. Most IR-

’

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serotonergic neurons within the RM/NRPGa resemble those in the dorsal raphe, then one would expect that REGULAR NEUTRAL cells are serotonergic. Consistent with this idea, both REGULAR NEUTRAL cells and physiologically identified serotonergic neurons are weakly affected or unaffected by changing the depth of general anesthesia (Heym et al., 1984; Leung and Mason, 1995). However, unlike the findings for dorsal raphe serotonergic neurons and for physiologically identified serotonergic neurons in other regions, REGULAR NEUTRAL cells discharge at rates up to 20Hz (Leung and Mason, 1995). Thus, future studies will need to directly establish the unequivocal physiology of serotonergic and nonserotonergic neurons in the RMINRPGa. RM/NRPGa neurons in awake animals

Little evidence exists regarding the roles of ON and OFF cells in the unanesthetized, awake animal. Oliveras observed only ON-like cells2 and NEUTRAL cells in the RM/NRPGa of unanesthetized rats (Oliveras et al., 1990, 1991a,b). Neurons inhibited by noxious stimulation were only recorded after induction of barbiturate anesthesia. These results have been interpreted to imply that OFF cells are either an artifact of barbiturate anesthesia or are not active in the unanesthetized rat. However, OFF cells have been found in rats anesthetized with ketamine (McGaraughty and Reinis, 1993), halothane (Mason et al., 1990) or isoflurane (Leung and Mason, 1995), suggesting that OFF cell activity is not simply a consequence of barbiturate anesthesia. Furthermore, McGaraughty and Reinis have recorded spontaneously active RMINRPGa units that are excited by morphine in unanesthetized rats (McGaraughty et al., 1993). The observation of OFF-like cells by McGaraughty but not by Oliveras may be the result of differences in experimental protocol. McGaraughty recorded OFFlike cells in rats that did not receive any somatic On-like cells, recorded by Oliveras et al. (1990, 1991a.b). are excited by noxious and innocuous stimulation. Morphine administration inhibits only the responses evoked by noxious stimulation but does not inhibit the spontaneous activity of these units.

stimulation. Since OFF cells are inhibited by noxious stimuli, it is not surprising that spontaneously active OFF cells are not observed in rats which receive repeated and/or intense noxious stimulation. Alternatively, because the om-like units observed by McGaraughty were not tested for their responses to noxious somatic stimuli, it is possible that morphine excites RM/NRPGa cell types other than OFF cells in the unanesthetized rat. Finally, the excitation of om-like cells by barbiturate anesthesia observed by Oliveras may reflect a transient excitatory response to increasing anesthetic concentration, an effect which has recently been described in the isoflurane-anesthetized rat (Leung and Mason, 1995). Further studies of individual RM/NRPGa neurons recorded during both steady state anesthesia and waking will be required to elucidate the role of ON, OFF and NEUTRAL cells in the unanesthetized animal.

Non-nociceptiveroles for RM/NRPGa cells Although the anatomical and physiological data above suggest that RMINRPGa cells function to modulate nociceptive transmission, existing data cannot exclude the possibility that these cells serve other functions. Indeed, electrophysiological experiments have demonstrated correlations between the activity of RMINRPGa neurons and cardiovascular, thermoregulatory, behavioral state and motor measures (Dickenson, 1977; Young and Dawson, 1987; Jacobs and Azmitia, 1992; Thurston and Randich, 1992). Cardiovascular modulation

In cats, electrical or chemical stimulation of the RM/NRPGa evokes an increase in arterial blood pressure with little or no change in heart rate (Adair et al., 1977; Yen et al., 1983; McCall, 1984). Activation of RMINRPGa cells may evoke cardiovascular responses via direct projections to thoracic preganglionic sympathetic neurons (Bacon et al., 1990). RM/NRPGa ON cells may also affect cardiovascular function via specific projections to the rostra1 and caudal ventrolateral me-

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dulla, a region that contains autonomic modulatory neurons (Mason and Fields, 1989). In addition, some RMINRPGa cells respond to stimulation of the vagus nerve (Blair and Evans, 1991; Thurston and Randich, 1995), suggesting that afferent autonomic input may influence RMINRPGa neurons. Furthermore, some RM/NRPGa cells respond to the peripheral administration of phenylephrine or norepinephrine, which produces a pressor response (Yen and Blum, 1984; Thurston and Randich, 1992). Cardiovascular inputs to RM/NRPGa cells and cardiovascular modulation evoked by RWNRPGa stimulation may underlie interactions between the cardiovascular and somatomotor systems (also see Chapters 17 and 18, this volume). Thermoregulatory transmission

RMINRPGa cells receive ascending thermal input, in the non-noxious range, from large areas of the skin and scrotum (Dickenson, 1977; Hellon and Taylor, 1982). RM lesions block the responses of hypothalamic neurons to changes in skin temperature and modify thermoregulatoty behavior, evidence that RM relays thermal cutaneous input from the spinal cord to thermosensitive neurons in the medial hypothalamus (Hellon and Taylor, 1982; Taylor, 1982). Neuronal responses to thermal and nociceptive inputs are associated such that ON cells are excited by cool cutaneous inputs while OFF cells are excited by warm cutaneous inputs (Young and Dawson, 1987). This association may serve to coordinate thermoregulatory and somatomotor responses to environmental and internal stimuli. Sexual reflexes

Sexual reflexes in the female and male rat are under tonic inhibition from bulbar neurons, an inhibition that is mimicked by serotonergic agonists (Marson and McKenna, 1992). In the male rat, lesions of the lateral portions of NRPGa release the sexual coitus reflex from tonic inhibition, suggesting that this bulbar region is involved in regulating sexual function (Marson et al., 1992; Mar-

son and McKenna, 1992). Consistent with this idea, injections of pseudorabies virus into the penile bulbocavernous muscle is transneuronally transported to medullary sites including RM, NRPGa and raphe pallidus (Marson et al., 1993). The participation of RWNRPGa neurons in the modulation of sexual reflexes may be initiated by dense projections from the medial preoptic nucleus, a region with a well established role in sexual behavior (see Chapter 22, this volume). Behavioral state

The spontaneous activity of physiologically identified serotonergic (see above) cat raphe cells is strongly correlated with behayioral state, a correlation that is independent of many other factors such as temperature or nociceptive responsiveness (Jacobs and Azmitia, 1992). In cats, physiologically presumed serotonergic neurons are most active during alert waking and least active during REM sleep with intermediate discharge rates observed during quiet waking and slow wave sleep. Environmental manipulations, including opioid administration, alter the activity of physiologically identified serotonergic neurons only inasmuch as they change the behavioral state. The above findings have led Jacobs to hypothesize that serotonergic RMINRPGa cells modulate dorsal horn transmission in accordance with behavioral state (Jacobs and Azmitia, 1992). While it is possible that the primary function of RWNRPGa is nociceptive modulation, it is perhaps more likely that RMINRPGa cells modulate a variety of physiological and behavioral processes. As has been described in invertebrates, it is possible for an ensemble of neurons to form several different circuits, each with a distinct behavioral outcome (Marder, 199I), Thus, different physiological conditions or challenges may prompt RM/NRPGa neurons to form several distinct circuits capable of a variety of modulatory effects.

Future directions Our understanding of RMINRPGa function has

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grown tremendously since the original hypothesis of an “endogeneous analgesia system” was introduced. Important issues still remain to be addressed in future studies. The mechanism of the RWNRPGa evoked inhibition of nociceptive transmission is poorly understood. It will be crucial to delineate the synaptic circuits within the dorsal horn, by which RWNRPGa cells affect nociceptive transmission. Secondly, the role of synergistic interactions between brainstem nuclei and the spinal cord, under physiological conditions, has not been adequately explored. It is unclear whether RWNRPGa neurons can act alone to modulate nociception or if they must act in concert with other regions of the CNS. Thirdly, while current evidence is suggestive that Ow cells inhibit and ON cells facilitate nociceptive transmission, further studies are needed to define the maximal effect that each of these cell classes can have on nociception. The role of each subtype of NEUTRAL cell in nociceptive modulation and the unequivocal physiological definition of serotonergic cells are also important issues for future experiments. Finally, the physiology of RWNRPGa cells must be studied using an integrative approach in awake animals in order to fully understand the role of this region in nociception as well as in numerous other physiological processes and emotional behaviors.

Acknowledgements This research was supported by the Louis Block Foundation, Brain Research Foundation, and NIDA grant R01 DA07861 (PM). CL was supported by NIGMS training grant 5T32GM07151.

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