Distinct pathways for norepinephrine- and opioid-triggered antinociception from the amygdala.

BACKGROUND: The amygdala has an important role in pain and pain modulation. We showed previously in animal studies that α2 -adrenoreceptor activation in the central nucleus of the amygdala (CeA) mediates hypoalgesia produced by restraint stress, and that direct application of an α2 -agonist in this region produces analgesia. AIMS: In the present animal experiments, we investigated the pathways through which α2 -sensitive systems in the CeA produce behavioural analgesia. The CeA has dense connections to a descending pain modulatory network, centred in the midbrain periaqueductal grey (PAG) and the rostral ventromedial medulla (RVM), which is implicated in various forms of stress-related hypoalgesia and which mediates the antinociceptive effect of morphine applied in the basolateral amygdala. We investigated whether this circuit mediates the hypoalgesic effects of α2 -adrenergic agonist administration into the CeA as well as the contribution of endogenous opioids and cannabinoids. We also tested the possibility that activation of α2 -receptors in the CeA produces antinociception by recruitment of noradrenergic pathways projecting to the spinal cord. RESULTS: Hypoalgesia resulting from bilateral application of the α2 -adrenergic agonist clonidine in the CeA was not reversed by chemical inactivation of the RVM or by systemic injections of naloxone (µ-opioid antagonist) or rimonabant (CB1 antagonist). By contrast, spinal α2 -receptor blockade (intrathecal idazoxan) completely prevented the hypoalgesic effect of clonidine in the CeA, and unmasked a small but significant hyperalgesia. CONCLUSION: In rats, adrenergic actions in the CeA mediating hypoalgesia require spinal adrenergic neurotransmission but not the PAG-RVM pain modulatory network, or opiate or cannabinoid systems.

Neuropeptide Y in the rostral ventromedial medulla reverses inflammatory and nerve injury hyperalgesia in rats via non-selective excitation of local neurons.

Chronic pain reflects not only sensitization of the ascending nociceptive pathways, but also changes in descending modulation. The rostral ventromedial medulla (RVM) is a key structure in a well-studied descending pathway, and contains two classes of modulatory neurons, the ON-cells and the OFF-cells. Disinhibition of OFF-cells depresses nociception; increased ON-cell activity facilitates nociception. Multiple lines of evidence show that sensitization of ON-cells contributes to chronic pain, and reversing or blocking this sensitization is of interest as a treatment of persistent pain. Neuropeptide Y (NPY) acting via the Y1 receptor has been shown to attenuate hypersensitivity in nerve-injured animals without affecting normal nociception when microinjected into the RVM, but the neural basis for this effect was unknown. We hypothesized that behavioral anti-hyperalgesia was due to selective inhibition of ON-cells by NPY at the Y1 receptor. To explore the possibility of Y1 selectivity on ON-cells, we stained for the NPY-Y1 receptor in the RVM, and found it broadly expressed on both serotonergic and non-serotonergic neurons. In subsequent behavioral experiments, NPY microinjected into the RVM in lightly anesthetized animals reversed signs of mechanical hyperalgesia following either nerve injury or chronic hindpaw inflammation. Unexpectedly, rather than decreasing ON-cell activity, NPY increased spontaneous activity of both ON- and OFF-cells without altering noxious-evoked changes in firing. Based on these results, we conclude that the anti-hyperalgesic effects of NPY in the RVM are not explained by selective inhibition of ON-cells, but rather by increased spontaneous activity of OFF-cells. Although ON-cells undoubtedly facilitate nociception and contribute to hypersensitivity, the present results highlight the importance of parallel OFF-cell-mediated descending inhibition in limiting the expression of chronic pain.

The dorsomedial hypothalamus mediates stress-induced hyperalgesia and is the source of the pronociceptive peptide cholecystokinin in the rostral ventromedial medulla.

While intense or highly arousing stressors have long been known to suppress pain, relatively mild or chronic stress can enhance pain. The mechanisms underlying stress-induced hyperalgesia (SIH) are only now being defined. The physiological and neuroendocrine effects of mild stress are mediated by the dorsomedial hypothalamus (DMH), which has documented connections with the rostral ventromedial medulla (RVM), a brainstem region capable of facilitating nociception. We hypothesized that stress engages both the DMH and the RVM to produce hyperalgesia. Direct pharmacological activation of the DMH increased sensitivity to mechanical stimulation in awake animals, confirming that the DMH can mediate behavioral hyperalgesia. A behavioral model of mild stress also produced mechanical hyperalgesia, which was blocked by inactivation of either the DMH or the RVM. The neuropeptide cholecystokinin (CCK) acts in the RVM to enhance nociception and is abundant in the DMH. Using a retrograde tracer and immunohistochemical labeling, we determined that CCK-expressing neurons in the DMH are the only significant supraspinal source of CCK in the RVM. However, not all neurons projecting from the DMH to the RVM contained CCK, and microinjection of the CCK2 receptor antagonist YM022 in the RVM did not interfere with SIH, suggesting that transmitters in addition to CCK play a significant role in this connection during acute stress. While the RVM has a well-established role in facilitation of nociception, the DMH, with its well-documented role in stress, may also be engaged in a number of chronic or abnormal pain states. Taken as a whole, these findings establish an anatomical and functional connection between the DMH and RVM by which stress can facilitate pain.

Neural basis for improgan antinociception.

Improgan, the prototype compound of a novel class of non-opioid analgesic drugs derived from histamine antagonists, attenuates thermal and mechanical nociception in rodents following intracerebroventricular (i.c.v.) administration. Improgan does not bind to known opioid, histamine or cannabinoid receptors, and its molecular target has not been identified. It is known however, that improgan acts directly in the periaqueductal gray and the rostral ventromedial medulla to produce its antinociceptive effects, and that inactivation of the rostral ventromedial medulla prevents the antinociceptive effect of improgan given i.c.v. Here we used in vivo single-cell recording in lightly anesthetized rats to show that improgan engages pain-modulating neurons in the medulla to produce antinociception. Following improgan administration, OFF-cells, which inhibit nociception, became continuously active and no longer paused during noxious stimulation. The increase in OFF-cell firing does not represent a non-specific neuroexcitant effect of this drug, since ON-cell discharge, associated with net nociceptive facilitation, was depressed. NEUTRAL-cell firing was unaffected by improgan. The net response of rostral ventromedial medulla (RVM) neurons to improgan is thus comparable to that evoked by mu-opioids and cannabinoids, well known RVM-active analgesic drugs. This common basis for improgan, opioid, and cannabinoid antinociception in the RVM supports the idea that improgan functions as a specific analgesic agent.

Brainstem control of cerebral blood flow and application to acute vasospasm following experimental subarachnoid hemorrhage.

Symptomatic ischemia following aneurysmal subarachnoid hemorrhage (SAH) is common but poorly understood and inadequately treated. Severe constriction of the major arteries at the base of the brain, termed vasospasm, traditionally has been thought to be a proximal event underlying these ischemias, although microvascular changes also have been described. The vast majority of studies aimed at understanding the pathogenesis of ischemic deficits, and vasospasm have focused on the interaction of the "spasmogen" of the extravasated blood with the smooth muscle and endothelium of the arteries. This has led to a comparative neglect of the contribution of the CNS to the maintenance of cerebral perfusion. In the present study, we focused on the role of the rostral ventromedial medulla (RVM) in modulating cerebral perfusion at rest and following an experimental SAH in the rat. Changes in cerebral blood flow (CBF) were measured using laser-Doppler flowmetry and three-dimensional optical microangiography. Focal application of a GABA(A) receptor agonist and antagonist was used to respectively inactivate and activate the RVM. We show here that the RVM modulates cerebral blood flow under resting conditions, and further, contributes to restoration of cerebral perfusion following a high-grade SAH. Failure of this brainstem compensatory mechanism could be significant for acute perfusion deficits seen in patients following subarachnoid hemorrhage.

Descending control of nociception: Specificity, recruitment and plasticity.

The dorsal horn of the spinal cord is the location of the first synapse in pain pathways, and as such, offers a very powerful target for regulation of nociceptive transmission by both local segmental and supraspinal mechanisms. Descending control of spinal nociception originates from many brain regions and plays a critical role in determining the experience of both acute and chronic pain. The earlier concept of descending control as an "analgesia system" is now being replaced with a more nuanced model in which pain input is prioritized relative to other competing behavioral needs and homeostatic demands. Descending control arises from a number of supraspinal sites, including the midline periaqueductal gray-rostral ventromedial medulla (PAG-RVM) system, and the more lateral and caudal dorsal reticular nucleus (DRt) and ventrolateral medulla (VLM). Inhibitory control from the PAG-RVM system preferentially suppresses nociceptive inputs mediated by C-fibers, preserving sensory-discriminative information conveyed by more rapidly conducting A-fibers. Analysis of the circuitry within the RVM reveals that the neural basis for bidirectional control from the midline system is two populations of neurons, ON-cells and OFF-cells, that are differentially recruited by higher structures important in fear, illness and psychological stress to enhance or inhibit pain. Dynamic shifts in the balance between pain inhibiting and facilitating outflows from the brainstem play a role in setting the gain of nociceptive processing as dictated by behavioral priorities, but are also likely to contribute to pathological pain states.

Purinergic receptor immunoreactivity in the rostral ventromedial medulla.

The rostral ventromedial medulla (RVM) has long been recognized to play a pivotal role in nociceptive modulation. Pro-nociception within the RVM is associated with a distinct functional class of neurons, ON-cells that begin to discharge immediately before nocifensive reflexes. Anti-nociceptive function within the RVM, including the analgesic response to opiates, is associated with another distinct class, OFF-cells, which pause immediately prior to nocifensive reflexes. A third class of RVM neurons, NEUTRAL-cells, does not alter firing in association with nocifensive reflexes. ON-, OFF- and NEUTRAL-cells show differential responsiveness to various behaviorally relevant neuromodulators, including purinergic ligands. Iontophoresis of semi-selective P2X ligands, which are associated with nociceptive transmission in the spinal cord and dorsal root ganglia, preferentially activate ON-cells. By contrast, P2Y ligands activate OFF-cells and P1 ligands suppress the firing of NEUTRAL cells. The current study investigates the distribution of P2X, P2Y and P1 receptor immunoreactivity in RVM neurons of Sprague-Dawley rats. Co-localization with tryptophan hydroxylase (TPH), a well-established marker for serotonergic neurons was also studied. Immunoreactivity for the four purinergic receptor subtypes examined was abundant in all anatomical subdivisions of the RVM. By contrast, TPH-immunoreactivity was restricted to a relatively small subset of RVM neurons concentrated in the nucleus raphe magnus and pallidus, as expected. There was a significant degree of co-localization of each purinergic receptor subtype with TPH-immunoreactivity. This co-localization was most pronounced for P2Y1 receptor immunoreactivity, although this was the least abundant among the different purinergic receptor subtypes examined. Immunoreactivity for multiple purinergic receptor subtypes was often co-localized in single neurons. These results confirm the physiological finding that purinergic receptors are widely expressed in the RVM. Purinergic neurotransmission in this region may play an important role in nociception and/or nociceptive modulation, as at other levels of the neuraxis.

Alpha(2)-noradrenergic antagonist administration into the central nucleus of the amygdala blocks stress-induced hypoalgesia in awake behaving rats.

Stress-induced hypoalgesia (SIH) is an adaptive behavioral phenomenon mediated in part by the amygdala. Acute stress increases amygdalar noradrenaline levels and focal application of alpha(2)-adrenoceptor agonists in the central nucleus of the amygdala (CeA) is antinociceptive. We hypothesized that alpha(2)-adrenoceptor antagonist administration into the CeA may block SIH. Bilateral microinjections of drug or saline via chronically implanted CeA cannulae were followed by either a period of restraint stress or rest. The nocifensive paw-withdrawal latency (PWL) to a focused beam of light was measured. PWLs were longer in restrained rats, constituting SIH. Microinjection of the alpha(2)-adrenoceptor antagonist idazoxan into the CeA prior to restraint blocked SIH. Idazoxan administration in unrestrained rats had no effect. Microinjection of the alpha(2)-adrenoceptor agonist clonidine in unrestrained rats caused dose dependent hypoalgesia, mimicking the effects of environmental stress. alpha(2)-Adrenoceptor function in the CeA is necessary for restraint-induced SIH.

Are opioid-sensitive neurons in the rostral ventromedial medulla inhibitory interneurons?

mu-Opioid agonists frequently activate output neurons in the brain via disinhibition, that is, by inhibiting "secondary cells," which results in disinhibition of "primary cells," considered to be output neurons. Secondary cells are generally presumed to be inhibitory interneurons that serve only to regulate the activity of the output neurons. However, studies of the opioid-sensitive neurons in the rostral ventromedial medulla, a region with a well-documented role in nociceptive modulation, indicate that the opioid-inhibited neurons in this region (termed "on-cells" when recorded in vivo) have a distinct functional role that parallels and opposes the output of the subset of RVM neurons that are activated following opioid administration, the "off-cells." The aim of the present study was to analyze the relative timing of on- and off-cell reflex-related firing in the rostral ventromedial medulla to help determine whether on-cells are likely to function as inhibitory interneurons in this region. On- and off-cells display complementary firing patterns during noxious-evoked withdrawal: off-cells stop firing and on-cells show a burst of activity. If on-cells are inhibitory interneurons mediating the off-cell pause, the on-cells would be expected to begin their reflex-related discharge before the off-cells cease firing. To examine this we recorded activity of on- and off-cell pairs during heat-evoked paw or tail withdrawal in lightly anesthetized rats. For each cell pair, we measured the onsets of the off-cell pause and the on-cell burst. Contrary to what would be expected if on-cells were inhibitory interneurons, off-cells typically ceased firing before on-cells began reflex-related firing, with a mean 481 (+/-69) ms lag between the final off-cell spike and the first on-cell spike. This suggests that on-cells do not mediate the off-cell pause, and points instead to presynaptic mechanisms in opioid-mediated disinhibition of medullary output neurons. These data also support an independent role for on-cells in pain modulation.

Noradrenergic agonist administration into the central nucleus of the amygdala increases the tail-flick latency in lightly anesthetized rats.

The amygdala is a medial forebrain structure with an established role in nociceptive modulation, including the expression of stress-induced hypoalgesia (SIH). Projections from the locus coeruleus increase levels of noradrenaline in the amygdala during acute stress. alpha(2)-Noradrenergic receptor agonists have significant clinical utility as analgesic agents. We therefore hypothesized that alpha(2)-noradrenergic activation of the amygdala may result in behaviorally measurable hypoalgesia. Lightly anesthetized rats underwent microinjection of the alpha(2)-noradrenergic agonist clonidine into the amygdala and intermittent measurement of thermal nociception using the tail-flick latency (TFL). Bilateral microinjection of clonidine into the central nucleus of the amygdala (CeA) resulted in a significant, dose-dependent increase in TFL. This effect was blocked by systemic pre-treatment with the alpha(2)-antagonist yohimbine or by local pre-injection of the alpha(2)-antagonist idazoxan but not by local pre-injection of the alpha(1)-antagonist WB-4101. When injected alone, no antagonist resulted in a significant change in TFL compared with baseline. Clonidine injection into the amygdala but outside the CeA, including the basolateral nucleus of the amygdala, did not significantly alter TFL. These results demonstrate that anatomically and pharmacologically specific activation of alpha(2)-receptors in the CeA in lightly anesthetized rats results in behaviorally measurable antinociception.

Purinergic actions on neurons that modulate nociception in the rostral ventromedial medulla.

The rostral ventromedial medulla (RVM) serves as a critical link in bulbo-spinal nociceptive modulation. Within the RVM, 'off-cells' pause and 'on-cells' discharge immediately prior to a nocifensive reflex. These neurons are also activated and inactivated, respectively, by local or systemic application of opioids. Off-cell activation leads to behavioral anti-nociception and on-cell activation to hyperalgesia. Thus, on- and off-cell populations allow bi-directional modulation of nociception by the RVM. A third neuronal population, neutral cells, shows no reflex-related change in discharge. The role of neutral cells in nociception, if any, is unknown. We investigated the responses of on-, off- and neutral cells to the iontophoretic application of purinergic ligands in lightly anesthetized rats. On-cell firing increased rapidly in response to application of ATP and to the P2X-receptor agonist, alpha,beta-methylene ATP. Off-cell firing increased gradually in response to ATP and to the P2Y-receptor agonist, 2-methylthio-ATP. All of these responses were attenuated or reversed by the non-specific P2-receptor antagonists, suramin and pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS). Activation of off-cells was preferentially antagonized by the relatively selective P2Y antagonist, MRS2179. By contrast with activation of on- and off-cells by ATP, neutral cell firing was depressed by ATP, adenosine and the P1-receptor agonist, 5'-(N-ethylcarboxamido) adenosine (NECA). Neutral cell responses to these agonists were at least partially reversed by the adenosine-receptor antagonist, 8-phenyltheophylline (8PT). These data imply that on-cells preferentially express P2X-receptors, off-cells P2Y-receptors and neutral cells P1-receptors. Immunohistochemical localization of purinergic receptors confirms the presence of some subtypes of P2X, P2Y and A1 receptors on neuronal cell bodies and fibers within the RVM. The differential responses of on-, off- and neutral-cells to purinergic ligands highlight the value of pharmacological signatures in further delineation of the anatomy, connectivity and function of this therapeutically important system.

Dissociation of hyperalgesia from fever following intracerebroventricular administration of interleukin-1beta in the rat.

Interleukin-1beta (IL-1beta) is a cytokine that contributes to the hyperalgesia, inactivity, and fever associated with illness. These three components of the illness response occur simultaneously following peripheral administration of IL-1beta. The objective of the present study was to determine whether hyperalgesia, inactivity, and fever correspond following central administration. Rats were injected with IL-1beta (0.05 pg-50 ng/10 microl) into the lateral ventricle and core body temperature and activity were assessed for 5.5 h using radio telemetry while rats remained in their home cage. Rats were removed from the cage periodically to assess nociception by measuring the latency for hindpaw withdrawal to radiant heat. The two highest doses of IL-1beta (5 and 50 ng) caused an increase in core body temperature and a decrease in activity beginning 105 min following administration. No change in nociception was evident at any time after administration of IL-1beta regardless of dose. These data indicate that the hyperalgesia associated with fever is triggered by a peripheral, not a central action of IL-1beta, presumably by activation of vagal afferents.

Prostaglandin E2 in the medial preoptic area produces hyperalgesia and activates pain-modulating circuitry in the rostral ventromedial medulla.

Prostaglandin E2 (PGE2) produced in the medial preoptic region (MPO) in response to immune signals is generally accepted to play a major role in triggering the illness response, a complex of physiological and behavioral changes induced by infection or injury. Hyperalgesia is now thought to be an important component of the illness response, yet the specific mechanisms through which the MPO acts to facilitate nociception have not been established. However, the MPO does project to the rostral ventromedial medulla (RVM), a region with a well-documented role in pain modulation, both directly and indirectly via the periaqueductal gray. To test whether PGE2 in the MPO produces thermal hyperalgesia by recruiting nociceptive modulating neurons in the RVM, we recorded the effects of focal application of PGE2 in the MPO on paw withdrawal latency and activity of identified nociceptive modulating neurons in the RVM of lightly anesthetized rats. Microinjection of a sub-pyrogenic dose of PGE2 (50 fg in 200 nl) into the MPO produced thermal hyperalgesia, as measured by a significant decrease in paw withdrawal latency. In animals displaying behavioral hyperalgesia, the PGE2 microinjection activated on-cells, RVM neurons thought to facilitate nociception, and suppressed the firing of off-cells, RVM neurons believed to have an inhibitory effect on nociception. A large body of evidence has implicated prostaglandins in the MPO in generation of the illness response, especially fever. The present study indicates that the MPO also contributes to the hyperalgesic component of the illness response, most likely by recruiting the nociceptive modulating circuitry of the RVM.

Activation of brainstem N-methyl-D-aspartate receptors is required for the analgesic actions of morphine given systemically.

The analgesic actions of opioids are in large part mediated by activation of brainstem pain modulating neurons that depress nociceptive transmission at the level of the dorsal horn. The present study was designed to characterize the contribution of N-methyl-D-aspartate (NMDA)- and non-NMDA-mediated excitatory transmission within the rostral ventromedial medulla (RVM) to the activation of brainstem inhibitory output neurons and analgesia produced by systemic morphine administration. The NMDA receptor antagonist D-2-amino-5-phosophonopentanoic acid (AP5), the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione disodium (CNQX) or saline was infused into the RVM of lightly anesthetized rats while recording the activity of identified pain modulating neurons: 'off-cells', thought to inhibit nociceptive transmission, and 'on-cells', thought to facilitate nociception. Nociceptive responsiveness (tail flick latency) was not affected by either antagonist. AP5, but not CNQX, attenuated or blocked activation and disinhibition of off-cells and the antinociception produced by systemically administered morphine. Reflex-related discharge of on-cells was unaffected by AP5, but significantly attenuated by CNQX. The present results highlight two important aspects of RVM pain modulatory circuits. First, morphine given systemically produces its analgesic effect at least in part by recruiting an NMDA-mediated excitatory process to activate off-cells within the RVM. This excitatory process may play a role in the analgesic synergy produced by simultaneous mu-opioid activation at different levels of the neuraxis. Second, reflex-related activation of on-cells is mediated by a non-NMDA receptor, and this activation does not appear to play a significant role in regulating reflex responses to acute noxious stimuli. Excitatory amino acid-mediated excitation thus has at least two distinct roles within the RVM, activating off-cells and on-cells under different conditions.

Circuitry underlying antiopioid actions of cholecystokinin within the rostral ventromedial medulla.

It is now well established that the analgesic actions of opioids can be modified by "anti-analgesic" or "antiopioid" peptides, among them cholecystokinin (CCK). Although the focus of much recent work concerned with CCK-opioid interactions has been at the level of the spinal cord, CCK also acts within the brain to modify opioid analgesia. The aim of the present study was to characterize the actions of CCK in a brain region in which the circuitry mediating the analgesic actions of opioids is relatively well understood, the rostral ventromedial medulla (RVM). Single-cell recording was combined with local infusion of CCK in the RVM and systemic administration of morphine in lightly anesthetized rats. The tail-flick reflex was used as a behavioral index of nociceptive responsiveness. Two classes of RVM neurons with distinct responses to opioids have been identified. OFF cells are activated, indirectly, by morphine and mu-opioid agonists, and there is strong evidence that this activation is crucial to opioid antinociception. ON cells, thought to facilitate nociception, are directly inhibited by opioids. Cells of a third class, NEUTRAL cells, do not respond to opioids, and whether they have any role in nociceptive modulation is unknown. CCK microinjected into the RVM by itself had no effect on tail flick latency or the firing of any cell class but significantly attenuated opioid activation of OFF cells and inhibition of the tail flick. Opioid suppression of ON-cell firing was not significantly altered by CCK. Thus CCK acting within the RVM attenuates the analgesic effect of systemically administered morphine by preventing activation of the putative pain inhibiting output neurons of the RVM, the OFF cells. CCK thus differs from another antiopioid peptide, orphanin FQ/nociceptin, which interferes with opioid analgesia by potently suppressing all OFF-cell firing.

The role of excitatory amino acid transmission within the rostral ventromedial medulla in the antinociceptive actions of systemically administered morphine.

Two classes of neurons with distinct responses to opioids have been identified in the rostral ventromedial medulla (RVM), a region with a well-documented role in nociceptive modulation. 'Off-cells' are activated, indirectly, by opioids, and are likely to exert a net inhibitory effect on nociceptive processing. 'On-cells' are directly inhibited by opioids, and there is evidence that these neurons can, under various conditions, facilitate nociception. We showed previously that excitatory amino acid (EAA) neurotransmission is crucial to the nocifensor reflex-related on-cell burst, but plays little role in maintaining the ongoing activity of off-cells. The aim of the present study was to determine whether EAA transmission contributes to the activation of off-cells and the concomitant behavioral antinociception that follow systemic opioid administration. The non-selective EAA receptor antagonist kynurenate was infused into the RVM (1 nmol/200 nl) of lightly anesthetized rats prior to administration of morphine (1.5 mg/kg i.v). Off-cell, on-cell and neutral cell firing, as well as, tail flick response (TF) latencies were recorded. Kynurenate, significantly attenuated the characteristic opioid activation of off-cells. As a group, off-cells in kynurenate-treated animals did not become continuously active, and continued to exhibit tail-flick related pauses in firing. On-cell and neutral cell responses to opioid administration were unchanged. Opioid inhibition of the TF was also reduced, although baseline TF latency was unaffected, by RVM kynurenate. EAA-mediated activation of off-cells, thus has an important role in opioid analgesia. The present observations underscore the importance of excitatory interactions among opioid-sensitive nociceptive modulatory circuits for systemic morphine analgesia, suggesting that such interactions are a critical factor in the synergistic relationships which have been demonstrated among these sites.

Orphanin FQ/nociceptin: a role in pain and analgesia, but so much more.

The publication of the delta opioid receptor sequence led to the cloning of three homologous receptors: the mu and kappa opioid receptors, and a novel opioid-like orphan receptor. The orphan receptor's endogenous ligand, a 17-amino-acid peptide that resembles dynorphin, was named 'orphanin FQ' and 'nociceptin' (OFQ/N1-17). The OFQ/N1-17 receptor is expressed widely in the nervous system, and it is becoming clear that the peptide is likely to participate in a broad range of physiological and behavioral functions. At the cellular level, OFQ/N1-17 has much in common with the classical opioids; however, functional studies are now revealing distinct actions of this peptide. Identified only two years ago, OFQ/N1-17 has already attracted a great deal of attention. The number and diversity of papers focused on OFQ/N1-17 at the recent meeting of the Society for Neuroscience augur an exciting future for this new peptide.

Analysis of excitatory amino acid transmission within the rostral ventromedial medulla: implications for circuitry.

Two classes of neurons with distinct responses to opioids have been identified in the rostral ventromedial medulla (RVM), a region with a well-documented role in nociceptive modulation. 'On-cells' are directly inhibited by opioids, and opioids can thus gain access to the modulatory circuitry of the RVM by an action on these neurons. 'Off-cells' are likely to exert a net inhibitory effect on nociceptive processing, and are activated by opioids. Because the opioid activation of off-cells is indirect, it has been proposed that on-cells function as inhibitory interneurons, and that opioid-induced suppression of on-cell firing in turn activates off-cells via disinhibition. The aim of the present study was to test this possibility. We had previously shown that excitatory amino acid (EAA) neurotransmission is crucial to the nocifensor reflex-related on-cell burst. We therefore infused the non-selective EAA receptor antagonist kynurenate (0.5-2 nmol, 200-500 nl) into the RVM while recording activity of on-, off- and neutral cells in lightly anesthetized rats. Kynurenate infusions produced a significant decrease in on-cell firing, with suppression of the on-cell burst. Off-cells nonetheless continued to display a tail flick-related pause in firing. Tail flick latency was used as an index of nociceptive responsiveness, and was unaffected by kynurenate infusions. These results demonstrate that a burst of on-cell firing is not required in order for the off-cell to exhibit a reflex-related pause in discharge, and do not support the proposed crucial role for on-cells as inhibitory interneurons within the RVM. In addition, preferential suppression of on-cell tiring was not associated with an increase in tail flick latency. This suggests that, under the conditions of these experiments, on-cell discharge is not a potent regulator of moment-to-moment variations in nociceptive responsiveness.

Reflex-related activation of putative pain facilitating neurons in rostral ventromedial medulla requires excitatory amino acid transmission.

Although the importance of the rostral ventromedial medulla in pain modulation is generally accepted, the recognition that it can exert both pain facilitating and pain inhibiting influences, and that its constituent neuronal population is physiologically and pharmacologically heterogeneous, is relatively recent. A class of neuron which may be a source of facilitating influences from the rostral ventromedial medulla has been identified in electrophysiological experiments. These neurons, termed "on-cells," are characterized by a sudden burst of activity beginning just before nocifensive reflexes. This burst of firing is thought to be a significant factor in brainstem control of nociceptive transmission under physiological conditions. The aim of the present study was to determine whether an excitatory amino acid is involved in generation of the reflex-related burst that defines on-cells, and more generally, to examine the role of excitatory amino acid neurotransmitters within the rostral ventromedial medulla of the rat. Iontophoretic application of the broad-spectrum excitatory amino acid receptor antagonist kynurenate significantly reduced the reflex-related on-cell burst, whereas ongoing firing was unaffected. Spontaneous activity of other medullary neurons was unchanged. These data demonstrate that release of an endogenous excitatory amino acid neurotransmitter is necessary for the activation of on-cells that is associated with nocifensive reflexes. In contrast, these receptors evidently play a much less significant role in maintaining the ongoing activity of any cell class in the rostral ventromedial medulla in lightly anaesthetized rats.

Effects of iontophoretically applied serotonin on three classes of physiologically characterized putative pain modulating neurons in the rostral ventromedial medulla of lightly anesthetized rats.

The importance of the rostral ventromedial medulla (RVM) in nociceptive modulation is well documented, and several lines of evidence point to a role for serotonin (5HT) in regulating the activity of pain modulating neurons in this region. The aim of the present study was to examine the effect of iontophoretically applied 5HT upon the firing of three physiologically defined classes of RVM neurons with distinct roles in pain modulation. The predominant effect across all classes was a facilitation of ongoing or evoked activity. A minority of cells within each class were inhibited by 5HT itself, but agonists at 5HT1 receptor types inhibited the majority of cells tested. The results thus indicate that the behavioral effects of manipulating 5HT within the RVM cannot be attributed to a selective influence on a particular cell class.