REVIEWS

STATE-DEPENDENT OPIOID CONTROL OF PAIN

Howard Fields Agonists for the µ-opioid receptor are powerful analgesics and are highly addictive; however, the contribution of the δ- and κ-opioid and opioid receptor-like receptors to motivational states is less clear. Agonists at each receptor modulate neurons in a circuit that selectively controls nociceptive transmission. This circuit can operate in both pain-inhibiting and pain-facilitating states, and the action of opioids contributes to and is determined by the state of the circuit. There is growing evidence that the state of the circuit is determined by aversive and appetitive motivational states, and that this contributes to adaptive behavioural choice.

Although the perception of pain is widely considered the action of the most potent analgesics2,3.Other to be the invariant consequence of the activation of members of the opioid receptor family regulate pain, peripheral nociceptors by potentially tissue-damaging but their contribution has been more difficult to study stimuli, such stimuli are just one of the many factors because their pain-relieving actions are neither as that are involved. There have been reports that, under robust nor as consistent as those of MOR ligands. So, conditions of great threat or strong emotion, people the functional significance of opioid receptor diversity with severe injuries (including open wounds and bone remains puzzling. However, experiments are beginning fractures) report little or no pain1.Drug actions can to shed light on the inconsistencies and paradoxes in also be highly variable; for example, opioid drugs that the field, and a deeper understanding is emerging are selective for a single receptor can either relieve or about how the members of this family of closely related worsen pain, depending on an animal’s behavioural receptors interact to provide a flexible choreography state. Such top–down variability of pain intensity and for pain control. Crucial to this improved understand- drug action highlights the importance of using a ing has been the systems neuroscience approach; systems neuroscience approach to study pain modula- the analysis of the firing properties and anatomical tion. This review focuses on the properties of an connectivity of neurons in defined opioid-sensitive opioid-sensitive pain-modulating circuit: how the pain-modulating circuits. synapses of its component neurons are affected by opioids, how this action modulates nociceptive Matching neural circuits to behaviour transmission, and how such modulation contributes Whether one’s goal is to explain the pharmacology of to behavioural choice. an exogenously applied opioid or to understand the Opioids such as morphine and heroin are not only physiology of endogenous opioid peptide function, the powerful analgesics; they also produce profound systems neuroscience approach has contributed to appetitive motivational actions. They can be addictive the discovery process by producing mechanistic models when used recreationally, and can enhance food of opioid function that are detailed and that have Box 0453, University of and alcohol consumption. For these reasons, opioid great predictive power. Because the biologically relevant California, 513 Parnassus receptors have received widespread interest from output of the nervous system is behaviour, circuits are Avenue, San Francisco, California 94143, USA. clinicians and basic scientists alike. Investigations into meaningfully defined in relation to a specific behaviour. e-mail: [email protected] pain have focused largely on the µ-opioid receptor Consequently, tracing a circuit requires the selection of doi:10.1038/nrn1431 (MOR/OPRM), because its activation is necessary for the behaviour of interest.

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The first step in determining how a drug affects Somatosensory Anterior cingulate cortex behaviour is to inject it directly into nuclei that are part cortex of the behaviourally-defined circuit. The effect of the drug is then explained by determining how it changes C the firing of neurons in that nucleus. Using innate nocifensive withdrawal reflexes as behavioural surro- Insular gates for pain, an opioid-sensitive circuit that selectively cortex controls nociceptive transmission has been defined4,5. Locally acting opioids robustly alter the activity of Thalamus neurons in several serially-connected relays within this circuit. These neurons modulate nociceptor-driven 6–8 behaviours and the synaptic mechanisms by which SMT opioids directly regulate their activity have been deter- mined9–11.Furthermore, through the use of selective opioid receptor antagonists, usefully constrained hypotheses of endogenous opioid function have been tested. Importantly, circuit models have been tested in human subjects through the use of functional imaging SRT Injury and opioid antagonists. Pain is a subjective experience II I and only humans can give ‘direct’ reports about what III IV Primary they feel, so the ability to conduct relevant experiments V afferent in humans provides a rare opportunity to validate nociceptors hypotheses about human subjective experience based on animal research. Therefore, the circuit analytical approach allows us to establish a chain of causality from Anterolateral molecular events at the synapse to human perception system and behaviour. Figure 1 | Schematic of afferent pathways underlying the sensation of pain. Injury activates the primary afferent Pain and pain-modulating circuits nociceptor (PAN), which transmits information to the dorsal horn The process that leads to pain perception is typically of the spinal cord. The terminals of the PAN contact neurons in specific laminae of the dorsal horn where they release glutamate initiated by the activation of peripheral receptors, which and peptides to activate the second order neurons. The axons of selectively detect intense, potentially tissue-damaging nociceptive dorsal horn neurons cross to the contralateral stimuli. These primary afferent nociceptors have been anterolateral quadrant to form an ascending tract, which studied extensively in animals and humans. We now terminates in the brainstem and several distinct areas of the know a great deal about the molecular mechanisms of thalamus, which contain higher order neurons that project to transduction and the relationship of the firing of these various cortical regions that mediate different aspects of the pain experience. These regions include somatosensory, anterior neurons to stimulus intensity and to the psychophysics cingulate and insular cortices. SMT, spinomesencephalic tract; 12–14 of perceived pain intensity .Furthermore, although SRT, spinoreticular tract. much remains to be learned about central processing, there is broad agreement on the general outlines of the afferent transmission pathways from primary affer- Defining the pain-modulation circuit. In the early 1970s, ent nociceptors through the dorsal horn and on to the the circumstances were in place for explosive growth thalamus and cortex15 (FIG. 1). in our understanding of pain-modulation circuits. At each of the identified nociceptive relay nuclei in Recording from single neurons in the CNS led to the rodents, cats and primates, neurons have been recorded conceptual breakthrough that these neurons are feature with activity increasing as a function of stimulus intensity detectors that are tuned to detect tissue-damaging stim- across the noxious range. Studies combining functional uli. Building on this discovery, scientists were able to show imaging and psychophysics have shown that activations that specific laminae (I, II and IV–VI) of the spinal cord in human thalamic and cortical nociceptive-receiving dorsal horn were a vital relay for nociceptive signals14.A areas correlate with perceived pain intensity13,16. key advance in the modulatory field was Wall’s discovery Furthermore, pain perception in humans is blocked by that nociceptive neurons in these laminae are subject to lesions in spinal cord and brain areas that are homolo- powerful control by supraspinal sites17. gous to those identified as pain pathways in animals. The Stimulation-produced analgesia was the next crucial consistent and lawful relationships between stimulus discovery. Working in rats and using simple withdrawal intensity, neuronal firing and human reports of pain reflexes as the pain measure, Reynolds18 and later intensity, and the striking anatomical homologies across Liebeskind and colleagues19,20 showed that stimulation of species have provided a simple, powerful and relatively a specific region of the midbrain — the periaqueductal complete explanation of the sensory processing that grey (PAG) — inhibited behavioural responses to nox- underlies pain perception. This robust and highly ious stimulation. In a dramatic extension of this finding, conserved afferent circuit provides a firm foundation for electrical stimulation of the midbrain PAG in humans the study of pain modulation. was reported by several neurosurgical groups to produce

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clinically significant pain relief (for reviews, see REFS 21,22). Importantly, stimulation of this midbrain site inhibited nociceptive dorsal horn neurons, indicating that ACC the behavioural changes were due to control of sensory transmission rather than motor responses23,24. Subsequent work using a combination of methods (brain mapping by electrical stimulation, anatomical T tract tracing, inhibition of withdrawal reflexes and dorsal H horn electrophysiology) rapidly led to detailed knowledge of the anatomy, physiology and pharmacology of this pathway7.The PAG receives direct inputs from the hypo- thalamus and from the LIMBIC forebrain, including several Amygdala regions of the frontal neocortex and the central nucleus of PAG the AMYGDALA (FIG. 2).The PAG controls nociceptive trans- mission indirectly by means of connections through neurons in the rostral ventromedial medulla (RVM) and the dorsolateral pontine tegmentum (DLPT). These two regions project through the spinal cord dorsolateral funiculus and selectively target the dorsal horn laminae RVM that house the nociceptive relay neurons. So, the selective control of pain by this circuit is explained by its anatomi- Dorsal cal selectivity for primary afferent nociceptor terminals horn and somata of dorsal horn neurons that respond to Pain noxious stimulation. transmission neuron Opioids in the pain-modulation circuit. Morphine is the prototypical MOR agonist. MOR agonists produce Figure 2 | Outline of opioid-sensitive pain-modulating analgesia through both pre- and postsynaptic mecha- circuit. This is a top–down pathway that can be activated by nisms at multiple CNS sites (FIG. 3). MOR agonists can both exteroceptive stimuli and certain motivational states. directly inhibit pain transmission at spinal levels Limbic forebrain areas, including the anterior cingulate cortex through actions on primary afferents25 and nociceptive (ACC), other frontal cortical areas, the hypothalamus (H) and relay neurons in the dorsal horn26,27,but this review will central nucleus of the amygdala project to the midbrain focus on supraspinal modulatory circuits. The MOR is periaqueductal grey (PAG), which can be thought of as a main output pathway of the limbic system. The PAG, in turn, present in all of the known supraspinal components of indirectly controls pain transmission in the dorsal horn through the pain-modulation circuit including the insular the rostral ventromedial medulla (RVM). This pathway can cortex,amygdala, hypothalamus, PAG, DLPT, RVM and exert both inhibitory (green) and facilitatory (red) control. A spinal cord dorsal horn2,28–30.Microinjection of MOR separate control channel through serotonergic neurons in the agonists into each of these sites inhibits behavioural RVM (yellow) can also modulate pain in a state-dependent responses to noxious stimulation4,5,31,32.Inactivating the manner. T, thalamus. RVM or cutting the axons of RVM neurons that project to the spinal cord dorsal horn reduces analgesia produced by morphine that has been given systemically Physiological activation of opioid circuits or microinjected into supraspinal sites. This shows So far, we have described the connectivity of the pain- that MOR-agonist analgesia depends on activation of modulation circuit and some of its pharmacological supraspinal neurons that project by way of the RVM to features. Clearly, the circuit can operate in a serial, the spinal cord dorsal horn (FIG. 2). opioid-linked fashion; but how is it normally activated? Beyond its distributed MOR agonist sensitivity, Noxious stimuli that are prolonged and inescapable are another distinctive feature of the pain-modulation circuit particularly effective for activation of the PAG–RVM is that the serial linkage of its component nuclei involves network. For example, forepaw shock in rats produces the release of endogenous opioids. The behavioural effect an acute anti-nociceptive effect that is blocked by the LIMBIC 37,38 A term that refers to a collection of activating the circuit at one site can be blocked by opioid antagonist naloxone and by RVM lesions . of cortical and subcortical microinjection of opioid antagonists at a downstream site There is evidence that similar mechanisms operate in structures that are important for in the pathway. For example, analgesia produced by MOR humans (see REF. 7 for a review). First, rodent and processing memory and agonists microinjected into the posterior hypothalamus human opioid receptors are almost pharmacologically emotional information. Prominent structures include or basolateral amygdala is reversed by a MOR antagonist identical. Second, endogenous opioid peptides and opi- 33 the hippocampus and amygdala. in the PAG , and PAG-elicited analgesia is blocked by oid receptors are present in human brain areas that are naloxone or selective MOR antagonists microinjected homologous to brainstem pain-modulating nuclei. AMYGDALA into the RVM34,35.Although the endogenous opioid that Third, analgesia has been produced in people by stimu- A small almond-shaped mediates these effects has not been identified, the fact that lating the PAG. Last, naloxone enhances experimental structure, comprising 13 nuclei, buried in the anterior medial an enkephalinase inhibitor injected into the RVM and clinical postoperative pain in human subjects who section of each temporal lobe. produces analgesia implicates the enkephalins36. have not received exogenous opioids39,40.

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a Presynaptic DAMGO It is now clear that this dual control results from the Control 70 DAMGO + 100 nM naloxone activity of two neuronal subpopulations. The two cell 5 60 classes, which are present in the PAG, DLPT and RVM, 8 2 exhibit phasic reciprocal changes in firing that precede 50 4,49,50 DAMGO 9 4 nociceptor-elicited withdrawal reflexes . One class, 40 termed ‘off cells’,shows a pause in firing that begins 10 nM 4 30 before the withdrawal reflex. The other class — ‘on cells’

Inhibition (%) — shows a burst of activity that begins before the reflex 10 20 4 (FIG. 4a) 30 nM 10 .Consistent with their role in pain modulation, 5 10 RVM on- and off-cell axons project directly and selectively to dorsal horn laminae that relay nociceptive 100 nM 0 signals51. –8 –7 –6 –5 –4 Log [DAMGO] (M) When MOR agonists are administered systemically, 300 nM either into the PAG or locally in RVM, off-cell firing Ca2+ accelerates and becomes continuous (FIG. 4c).With- GABA µ Somadendritic region drawal reflexes are inhibited and no off-cell pause 1 µM 15 mV terminal Cl– is seen. Selective blockade of off-cell activation 40 ms 52 GABAA prevents morphine’s anti-nociceptive effect .There- Primary cell fore, off-cell activation is necessary for the pain- inhibitory effects of MOR ligands given systemically b Postsynaptic or supraspinally53,54. DAMGO (300 nm)

15 mV On cells and pain facilitation. The correlation of on-cell discharge with withdrawal reflexes indicates that their 1 min AMPA-kainate action is to facilitate such responses and their dorsal ME (10 µM) 200 Potential (mV) NMDA horn projection target indicates that this effect is –120 –100 –80 –60 achieved through control of nociceptive transmission. 0 GIRK Several independent lines of evidence support this idea. µ Secondary Control Analgesic doses of MOR agonists silence on cells. On –200 cell cells in the RVM contribute to the enhanced nocicep- Control –400 tion that accompanies various manipulations, including Current (pA) Current nerve injury, inflammation, tonic activation of nocicep- –600 tors and systemic cytokine administration (see table 1 ME (10 µM) in REF. 42). Tonic activation of nociceptors results in –800 prolonged on-cell activity, which enhances certain with- drawal reflexes48,55.Furthermore, destruction of RVM Figure 3 | Synaptic actions of µ-opioid receptor (MOR) agonists in the rostral on cells by a MOR-selective neurotoxin blocks the  ventromedial medulla (RVM). Reproduced, with permission, from REF. 66 (1990) The hyperalgesic state elicited by nerve injury56,57.Finally, Physiological Society. a | MOR agonists reduce release of GABA (γ-aminobutyric acid) through selective activation of on cells enhances responses to a dose-dependent presynaptic action. Reduced GABA-mediated inhibition accounts for the 8 activation (disinhibition) of off cells by MOR agonists. b | By contrast, MOR agonists directly noxious stimulation . inhibit on cells (secondary cells in vitro) through activation of a potassium conductance. 2 4 AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid; DAMGO, [D-Ala , N-Me-Phe , Reciprocal and state-dependent neuronal activity. Gly-ol]enkephalin; GIRK, G-protein-coupled inwardly rectifying potassium channel; Recordings of pairs of neurons in the RVM of lightly 5 ME, [met ]enkephalin; NMDA, N-methyl-D-aspartate. anaesthetized rats demonstrate that on- and off-cell populations are active at different times6,58,59.In fact, these cells show reciprocal patterns of activity under vari- Bidirectional control of pain transmission ous conditions (FIG. 4a–c, TABLE 1). Despite the tendency One of the more striking and informative discoveries towards reciprocal firing, each population is capable of about the pain-modulating circuit is that it can facilitate independent action. For example, one can block the as well as inhibit nociceptive transmission41–43.In reflex-related on-cell burst without affecting the off-cell addition to the inhibition discussed above, stimulation pause60.Conversely, the activation of off cells by opioids of the RVM can enhance behavioural and dorsal horn can be blocked without affecting the on-cell burst. These neuronal responses to noxious stimulation44–46. findings indicate that the reciprocity of activity in the Furthermore, prolonged nociceptor inputs, including two RVM populations depends on shared upstream con- thermal and chemical stimulation, inflammation or nectivity rather than direct inhibitory connectivity nerve injury produce a state of generalized hyperalgesia within the RVM. that is reversed by lesions or reversible inactivation of the One of the more striking features of the pattern of RVM42,47,48.So, the activation of RVM neurons can gen- activity for both on- and off-cell populations is that it erate either facilitation or inhibition of pain transmission consistently varies with the animal’s state of arousal61. under different conditions. How are we to understand For example, in awake unrestrained rats, the off-cell this apparent paradox? population fires intermittently, only to accelerate and

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a Off cell

b Off A H

PAG TF 15 Hz R On cell On

RVM 15 Hz

TF 1 min

SC Off cell

c

µ-Opioid

On cell VII

RVM

TF TF TF TF TF TF 2 min µ-Opioid

d Bidirectional control of nociception

On Off

+ – PAN + T

Dorsal horn

Figure 4 | Two populations of neurons exert opposing modulatory actions. a | Recordings from neurons in the rostral ventromedial medulla (RVM) during application of noxious heat to the tail reveals one class that pauses (off cells) and one that bursts (on cells) just prior to the withdrawal tail flick (TF). A, amygdala; H, hypothalamus; PAG, periaqueductal grey; R, recording electrode; SC, spinal cord. b | In the absence of imposed stimuli, RVM cycles between periods of off- and on-cell activity. Reproduced, with permission, from REF. 59  (1989) Taylor and Francis. c | Reciprocal firing is also seen when µ-receptor agonists are microinjected directly into the RVM; off-cell discharge accelerates and on cells shut down with concomitant inhibition of the TF (open triangles). Reproduced, with permission, from REF. 70  (1994) Elsevier Science. d | On cells facilitate and off cells inhibit nociceptive transmission at the level of the dorsal horn. PAN, primary afferent nociceptor; T, pain transmission neuron.

become continuously active during slow-wave sleep62. The opioid receptor family and pain modulation Conversely, on cells show markedly reduced activity The known opioid receptors are members of the large during slow-wave sleep. Taken together, these findings G-PROTEIN-coupled receptor family. There are currently indicate that the modulatory circuit can operate in one four well-established members of the opioid receptor G PROTEIN of two opposing states: an on-cell state that enhances family — µ, δ (DOR/OPRD), κ (KOR/OPRK1) and A heterotrimeric GTP-binding nociceptive transmission and an off-cell state that opioid receptor-like (ORL1/OPRL) (for reviews, see and -hydrolysing protein that inhibits nociceptive transmission. Administration of REFS 63,64). The first three were defined on the basis of interacts with cell-surface receptors, often stimulating or MOR ligands changes the circuit into the off-cell state, ligand-binding studies, and subsequent cloning of their inhibiting the activity of a whereas the presence of a prolonged somatic noxious genes revealed high sequence homology. The ORL1 downstream enzyme. G proteins stimulus changes it into an on-cell state. The concept receptor was identified on the basis of its high sequence consist of three subunits: the that the modulatory circuit can operate in two distinct homology with the other three receptors. The anatomical α -subunit, which contains the modes is crucial for understanding how a given opioid distributions of DOR, KOR and ORL1 receptors parallel guanine-nucleotide-binding site; and the β- and γ-subunits, which ligand can have different behavioural effects when given that of the MOR; they are present in the component function as a heterodimer. at different times. nuclei of the pain-modulating circuit2,29,30,63,65.Ligands

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Table 1 | Rostral ventromedial medulla (RVM) neurons and behavioural state On cell Off cell Nociceptive Blocked by MOR Blocked by ORL response antagonist or KOR agonists MOR agonist – + – Y Y Acute MOR abstinence + – + N/A Y Tonic noxious stimulus + – + N/A N/A Low dose neurotensin (In RVM) + 0 + N/A N/A Threat or appetitive motivational state – + – Y Y +, increases; –, decreases; 0, no effect. KOR, κ-opioid receptor; MOR, µ-opioid receptor; N/A, not applicable; ORL, opioid receptor-like; Y, yes.

that are selective for each opioid receptor regulate various can block the analgesia that is produced by PAG activa- motivated behaviours including feeding, alcohol and tion78,79.Furthermore, in the RVM, DOR agonists psychostimulant consumption and pain. produce weak to moderate analgesia80 and changes in RVM on and off cells that are similar to but weaker Actions of MOR ligands on pain-modulating neurons. than those produced by MOR agonists81.There are no Morphine is the prototypical opioid ligand, and its published studies of DOR synaptic actions in the RVM. actions require the MOR3. In vitro studies of neurons in The modest cellular and behavioural effects of selec- pain-modulating nuclei have revealed several types tive agonists imply a relatively minor contribution of the of synaptic actions by MOR ligands (FIG. 3).Direct post- DOR to pain modulation. However, another possibility and presynaptic inhibition of GABA (γ-aminobutyric is that synthesis or cellular trafficking of the DOR is acid) release have been related to the nociception- variable, and more potent DOR effects could be modulating function of the PAG and RVM (FIG. 3).Direct demonstrated under the right conditions. For example, postsynaptic inhibition of subsets of PAG and RVM inflammation increases the targeting of the DOR to the neurons is produced by MOR agonists through activation plasma membrane in the spinal cord dorsal horn82. of an INWARDLY RECTIFYING POTASSIUM CHANNEL10,66. In vivo Furthermore, prolonged inflammation is associated with iontophoresis of morphine in the RVM selectively an enhanced anti-nociceptive effect for a DOR-selective inhibits on-cells, and blocks their excitation by applied ligand (deltorphin) in the RVM83.These results indicate glutamate. This shows that neurons in this region that are that DOR function might be more robust if the receptor postsynaptically inhibited by MOR agonists are on cells67. is studied under appropriate conditions. A subset of RVM neurons, which must include off cells, is not hyperpolarized by MOR agonists but does State-dependent effects of KOR and ORL1 ligands. KOR- have GABA-releasing inputs that are presynaptically selective agonists have at least two synaptic actions in the inhibited by MOR ligands67.Presynaptic inhibition of RVM. They directly hyperpolarize neurons that are not GABA-releasing inputs by MOR agonists has also been hyperpolarized by MOR agonists (off and/or neutral demonstrated in PAG neurons of rats and mice68,69.RVM cells), and they inhibit excitatory glutamatergic inputs to off cells are activated by local infusion of either MOR RVM neurons, including those that are hyperpolarized by

selective ligands or the GABAA receptor antagonist bi- MOR agonists (on cells). Nociceptin, a ligand that is selec- cuculline70,71, and this leads to an anti-nociceptive effect. tive for the ORL1 receptor, strongly hyperpolarizes all Because in vitro studies show no direct excitatory classes of neurons in the RVM and PAG through activa- effect of MOR ligands on any cell class, RVM off-cell tion of an inwardly rectifying potassium channel85,86.In excitation by local microinfusion of MOR agonists is at addition, nociceptin inhibits GABA release by a pre- least partly due to inhibition of GABA-releasing inputs. synaptic action84,86. In vivo,RVM microinjection of the Although presynaptic inhibition of glutamatergic trans- same ORL1 ligand strongly inhibits all on and off cells53. mission to on cells in both the RVM and PAG could In summary, KOR agonists act presynaptically and ORL1 contribute under some circumstances72,73, the analgesic agonists act postsynaptically to inhibit both on and effect of MOR agonists acting in both PAG and RVM is off cells in the RVM. FIGURE 5b summarizes the synaptic probably due to disinhibition of off cells. distribution of opioid receptors in the RVM on and off cell circuits. The cells of origin of the opioid-regulated Contribution of the δ-opioid receptor. The role of the afferent terminals of RVM on and off cells have not been DOR in pain modulation is puzzling (reviewed by REF.74). definitively identified. INWARDLY RECTIFYING DOR agonists microinjected into the PAG produce little In contrast to the robust cellular and synaptic actions POTASSIUM CHANNELS or no anti-nociceptive effect in the rat75,76.Consistent with of KOR and ORL1 in vitro, their behavioural effects on Potassium channels that allow long depolarizing responses, as this observation, in vitro experiments have failed to show pain transmission are highly variable.Various effects of they close during depolarizing either hyperpolarization of neurons or inhibition of supraspinal injection of ORL1 agonists have been pulses and open with steep transmitter release by DOR agonists in rat PAG10,73,77.By reported; no effect, enhancement of nociceptive respon- voltage dependence on contrast, in the C57B16/J mouse, DOR agonists hyperpo- siveness or enhancement followed by inhibition hyperpolarization. They are larize a small subset of PAG neurons (24% compared (reviewed by REF.87). Similarly, in comparison to MOR called inward rectifiers because 69,70 current flows through them more with 72% for MOR agonists) .In the RVM, DOR is agonists, KOR agonists produce analgesia that is weaker easily into than out of the cell. present on axon terminals, and DOR selective antagonists and more dependent on the type of noxious stimulus

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a These experiments illustrate the power (and the On Off necessity) of using neural circuit analysis to explain behavioural pharmacology. In addition to the descending control that is exerted by on and off cells, serotonergic Morphine + – neurons in the RVM that project to the dorsal horn On Off On Off provide a third, state-dependent element that controls nociceptive transmission (BOX 1). 0 – Naloxone + 0 b The biological imperative for pain modulation PAG Although the concept of state dependence is helpful for Activated by + + understanding the role of different opioid receptors in ? Activated by MOR action glut GABA Opioid glut pain control, it raises an intriguing question: what is the in PAG noxious stimulation biological meaning of a ‘behavioural state’? The available evidence indicates that the most promising framework –– for approaching this question is to conceptualize pain KOR RVM Off On MOR as primarily a motivational state that has a powerful ++ORL1 influence on decision making. Noxious stimuli produce distinctly unpleasant Spinal cord – + sensations and elicit various innate behaviours that are appropriate to a continuing physical threat such as Figure 5 | Shifts in nociceptive modulatory state during morphine analgesia and acute 93 naloxone-induced abstinence. a | Morphine activates off cells to inhibit pain (lower left), escape, defence and vocalization .Following injury, whereas when naloxone is used to precipitate acute abstinence, on cells are activated and nociceptive inputs elicit recuperative behaviours such as produce a hyperalgesic state (lower right). b | Synaptic distribution of opioid receptors within the quiescence, licking and guarding94.The nociceptive rostral ventromedial medulla (RVM). µ-Opioid receptor (MOR) is located on GABA (γ-aminobutyric input that activates and maintains these behaviours acid)-releasing terminals at off cells and the somadendritic region of on cells. Both cell classes can be conceptualized as inducing a drive state with κ have somadendritic opioid receptor-like (ORL1) receptors and both are excited by -opioid powerful motivational effects95.Because they induce a receptor (KOR)-bearing glutamatergic terminals (glut) that arise from different input neurons. motivational state, noxious stimuli serve as teaching Whereas MOR agonists produce anti-nociceptive effects by inhibiting on cells and disinhibiting off cells, ORL1 and KOR agonists acting in the RVM can block analgesia by inhibiting off cells or signals, allowing animals to avoid situations that have 96,97 block hyperalgesia by inhibiting on cells. PAG, periaqueductal grey. either caused or threatened tissue injury in the past . The powerful behavioural demand that is produced by noxious stimuli presents the animal with a biological that is used9,88,89.In addition, KOR agonists can problem. There will be circumstances — for instance, functionally antagonize MOR-mediated analgesia9,11,88–90. the presence of a predator — in which choosing to How can we resolve the discrepancy between the respond overtly to a noxious stimulus, such as with robust and consistent synaptic actions of KOR and vocalization or sudden movement, places the animal at ORL1 agonists and their weak and variable behavioural risk of even greater injury or death94.Exhibiting certain effects? The key is that the pain-modulating circuit has ‘pain’ behaviours in the presence of a competing domi- two opposing states, and that the behavioural effect nant male conspecific might significantly reduce repro- of an opioid depends on whether the circuit is in the on- ductive efficiency. So, when the motivational demand cell or off-cell state. These points are clearly illustrated for a behaviour that is typically elicited by a noxious by recent studies of the effects of KOR and ORL1 stimulus occurs in the presence of a biological cost for agonists on nociceptor-elicited behaviours, and on the its execution, a mechanism that can block the behaviour activity of RVM neurons during morphine analgesia confers a potential evolutionary advantage. (off-cell state) and during naloxone-precipitated Opioid-mediated inhibition of pain has been morphine abstinence (on-cell state). demonstrated in these situations. For example, naloxone- When morphine is administered (systemically or into reversible analgesia is induced in male rodents by the PAG), the on cells become silent and the off cells fire the presence of a predator98,or an aggressive male con- continuously (FIG. 4c and FIG. 5a). In the off-cell state, specific99.Through classical conditioning, initially neutral dorsal horn neurons and withdrawal reflexes are inhib- contextual cues can acquire the motivational power to ited. This inhibition is reversed by inactivation of elicit opioid-mediated analgesia. In the conditioned fear the RVM or selective inhibition of off-cell firing. In the model, after contingent pairing with an inescapable foot off-cell state, microinjection of either an ORL1 or KOR shock, an initially neutral light or tone can elicit an agonist will inhibit off cells and will have an anti- anti-nociceptive effect. This conditioned analgesia is analgesic (pain-promoting) action11,85.Conversely,if blocked by microinjection of MOR- but not KOR- or naloxone is given following systemic administration of DOR-selective antagonists into the basolateral amygdala, an analgesic dose of morphine, off-cell firing is shut PAG and RVM100–102.Similar to the analgesia that is down, on-cell firing increases and becomes continuous, elicited by PAG MOR agonists, the analgesia that accom- and withdrawal reflexes are enhanced91,92 (FIG. 5).In this panies conditioned fear is inhibited by the microinjection on-cell state, microinjection of an ORL1 or KOR agonist of a KOR agonist in the RVM103.Activation of the opioid- will inhibit on cells and will have an anti-hyperalgesic mediated anti-nociceptive network is part of the process (pain-reducing) action11,85. of deciding to respond to the anticipated threat rather

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Box 1 | Serotonergic neurons and pain modulation with either appetitive or aversive valence are associated with activation of opioid-mediated anti-nociceptive There is little doubt that serotonin (5-hydroxytryptamine, 5-HT) contributes to mechanisms. brainstem control of nociceptive transmission at spinal levels61,126,127.However,the weight of evidence indicates that rostral ventromedial medulla (RVM) serotonergic Reward expectancy, opioids and placebo neurons are part of a pathway that is anatomically coextensive and that interacts with, The concept that activation of opioid-mediated but is functionally distinct from, the opioid-mediated pain-modulatory circuit. In pain-modulatory circuits is driven primarily by adult rodents, about 20% of RVM neurons are serotonergic, and most project through motivational state provides a heuristic basis for inter- the dorsolateral funiculus to innervate the dorsal horn. However, cytochemical studies preting the literature on neural mechanisms of placebo of physiologically identified RVM neurons have clearly shown that serotonergic analgesia. Shortly after the discovery of endogenous neurons in the RVM are neither on nor off cells; the response of serotonergic RVM 128 opioids in the mid-1970s, placebo analgesia was neurons to noxious stimulation is weak and variable .Furthermore,in vivo studies in 111 adult animals indicate that serotonergic RVM neurons are not robustly affected by shown to be blocked by naloxone — a result that has 112,113 either PAG stimulation or morphine at doses that are sufficient to inhibit nociceptive been replicated several times .An important transmission61.By contrast, in vitro studies of spinally projecting serotonergic RVM mediating process that underlies placebo analgesia is 114 neurons in young rats have shown postsynaptic inhibition by µ-opioid receptor expectancy . (MOR)- and κ-opioid receptor (KOR)-selective agonists129, and MOR-mediated Expectancy can be induced verbally by telling an presynaptic inhibition of both glutamate and GABA (γ-aminobutyric acid) individual that they are about to receive a powerful transmission72.Serotonin has both excitatory and inhibitory synaptic actions on analgesic, or by conditioning115;for example, by giving nociceptive dorsal horn neurons130,131, and spinally administered 5-HT receptor an individual a treatment (pill, ointment or intravenous antagonists reduce both the pain-facilitating and inhibiting-effects of RVM infusion) that produces a powerful analgesic effect. stimulation132–135.The discharge patterns of serotonergic RVM neurons have been After conditioning, giving the subject a physiologically studied in anaesthetized cats. Their activity closely tracks periodic shifts in the inert treatment that closely resembles the appearance of sleep–wake cycle, being highest during waking, lower during slow-wave sleep and the actual analgesic produces a powerful, naloxone- lowest during paradoxical sleep. In summary, serotonergic RVM neurons provide a reversible analgesic effect116.By contrast, informing state-dependent and potentially bidirectional modulatory channel that is parallel to, the subject that they are getting an inert placebo will but operationally distinct from, the controls exerted by on and off cells. In vitro studies ‘reverse’ the effect of conditioning and prevent indicate that the serotonergic channel might be subject to opioid control under certain, the placebo-induced analgesic response117.From the as yet unspecified, conditions. The three channels converge at the level of the dorsal standpoint of motivational processes, an effective horn. The dynamic interplay between serotonin and the action of on and off cells placebo manipulation can be considered to be a represents an important field for future research. reward-predictive cue because pain relief is ‘rewarding’ (negative reinforcement). So, by virtue of leading than the ongoing tissue-damaging stimulus. The decision to the expectation of pain relief, placebo analgesics is the outcome of a computation of the relative cost and have appetitive motivational power. Just as the rodent probability of the threat compared with that of the anticipating a desired food reward engages its opioid- noxious stimulus94. mediated analgesia circuit, so might a person anticipating Consistent with a crucial role in decision making, pain relief engage a homologous opioid-mediated opioid-mediated pain-modulatory circuits can be circuit (see REF. 118). A sugar pill that resembles a pre- engaged during appetitive as well as aversive motiva- viously administered analgesic would have the advantage tional states. Feeding sucrose to animals104,105 or human of directly engaging opioid-mediated circuits through infants106 produces a naloxone-reversible analgesic its sweet taste as well as the cognitive expectation of effect. Sucrose-induced analgesia in rodents is blocked pain relief. by lesions of the ventromedial hypothalamus107,which Functional imaging studies support the idea that projects to both the PAG and RVM. Interestingly, the expectancy, and expectation of pain relief in particular, RVM on-cell burst and off-cell pause are reduced dur- can engage opioid-mediated pain-modulating circuitry. ing food or water consumption62.Furthermore, the Using positron emission tomography and an experi- anticipation of a food reward can have the same effect. mental pain model, Petrovic and colleagues119 studied For example, placing animals in an environment where brain areas that are activated by the powerful MOR they have previously received a desired food raises their agonist remifentanyl. The same subjects were then withdrawal threshold, and this effect is blocked by given a saline infusion with the instruction that it was a naloxone108.This demonstrates that food consumption powerful analgesic. Subjects who experienced significant or food predictive sensory cues activate an opioid- relief with the placebo infusion showed activation in mediated pain-modulatory circuit, increasing the areas that were largely coextensive with those activated probability that the animal will consume the food by the MOR agonist. These areas included the rostral despite conflicting drives. Consistent with a direct link anterior cingulate, and brainstem areas that overlap with BASAL GANGLIA A group of interconnected between opioid analgesia and appetitive choice, nuclei that have been implicated in pain modulation. subcortical nuclei in the microinjection of MOR (or MOR and DOR) agonists Wager and colleagues took the story further by showing forebrain and midbrain that into the nucleus accumbens (a region of the BASAL that activation of the anterior cingulate cortex (ACC) includes the striatum (putamen GANGLIA that is crucial for linking motivation to and midbrain PAG correlated with placebo analgesia in and caudate nucleus), globus action) induces both anti-nociception109 and con- human subjects120.Through its connection to the PAG, pallidus, subthalamic nucleus, 110 ventral tegmental area and sumption of sweet and rich foods and ethanol . the ACC is anatomically linked to the opioid-mediated substantia nigra. So, instinctive as well as learned motivational states pain-modulatory circuit121.

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It is noteworthy that the increase in activity in these Conclusions areas occurred prior to noxious stimulation. This As outlined above, our knowledge of the biological probably represents anticipation or expectation of pain significance of opioid receptors and endogenous opioid relief. Placebo administration activates areas of the ACC peptides has leaned heavily on the use of opioid antago- that are also activated by reward expectancy in nists. Although the pain inhibition that is associated with humans122 and primates123.The ACC also projects conditioned fear is primarily a MOR-mediated effect, to the nucleus accumbens124,which, as noted above, most published work has used non-selective opioid is crucial for linking motivation to action. It is note- antagonists such as naloxone and naltrexone, so the func- worthy that activity in the human nucleus accumbens tion of endogenous agonists for the other opioid receptor occurs rapidly following noxious stimulation and classes is currently obscure. Addressing their contribution precedes activation of cortical areas that have been impli- to motivational control will require the use of selective cated in pain perception125.This activity might reflect antagonists and some ingenuity to determine the behav- neural processing that underlies the ‘decision’ to either ioural conditions that are required to activate circuits that respond to or suppress ascending nociceptive pathways. release endogenous ligands for DOR, KOR and ORL1.

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124. Kunishio, K. & Haber, S. N. Primate cingulostriatal The authors use a marker injected into the spinal cord Acknowledgements projection: limbic striatal versus sensorimotor striatal input. and transported retrogradely to identify spinally The author would like to thank M. Heinricher for useful discus- J. Comp. Neurol. 350, 337–356 (1994). projecting RVM neurons. They show that a significant sions and for calling attention to several relevant references. The 125. Becerra, L., Breiter, H. C., Wise, R., Gonzalez, R. G. & number of spinally-projecting serotonergic neurons author is supported by PHS grants and the State of California Borsook, D. Reward circuitry activation by noxious thermal express functional µ- and κ-opioid receptors. Alcohol and Addiction Research Program at the University of stimuli. Neuron 32, 927–946 (2001). 130. Mason, P. & Gao, K. Raphe magnus serotonergic neurons California. 126. Suzuki, R., Morcuende, S., Webber, M., Hunt, S. P. & tonically modulate nociceptive transmission. Pain Forum 7, Dickenson, A. H. Superficial NK1-expressing neurons 143–150 (1998). Competing interests statement control spinal excitability through activation of descending 131. Huang, L.-Y. M. Tonic release of serotonin. Pain Forum 7, The author declares that he has no competing financial interests. pathways. Nature Neurosci. 5, 1319–1326 (2002). 151–154 (1998). 127. Le Bars, D. in Serotonin and Pain (eds Osborne, N. M. & 132. Yaksh, T. L. & Wilson, P. R. Spinal serotonin system Hamon, M.) 171–226 (John Wiley, New York, 1988). mediates antinociception. J. Pharmacol. Exp. Ther. 208, 128. Potrebic, S. B., Fields, H. L. & Mason, P. Serotonin 446–453 (1979). Online links immunoreactivity is contained in one physiological cell class 133. Zhuo, M. & Gebhart, G. F. Spinal serotonin receptors mediate in the rat rostral ventromedial medulla. J. Neurosci. 14, descending facilitation of a nociceptive reflex from the nuclei DATABASES 1655–1665 (1994). reticularis gigantocellularis and gigantocellularis pars alpha in The following terms in this article are linked online to: Using in vivo intracellular recording, on, off and the rat. Brain Res. 550, 35–48 (1991). Entrez Gene: neutral cells were identified physiologically and filled 134. Calejesan, A. A., Chang, M. H. & Zhuo, M. Spinal http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene with biocytin for later immunocytochemistry. serotonergic receptors meidate facilitation of a nociceptive OPRD | OPRK1 | OPRL | OPRM Serotonin was only found in neutral cells. reflex by subcutaneous formalin inejction into the hindpaw in 129. Marinelli, S., Vaughan, C. W., Schnell, S. A., Wessendorf, M. W. rats. Brain Res. 798, 46–54 (1998). FURTHER INFORMATION & Christie, M. J. Rostral ventromedial medulla neurons 135. Green, G. M., Scarth, J. & Dickenson, A. An excitatory role Encyclopedia of Life Sciences: http://www.els.net/ that project to the spinal cord express multiple opioid for 5-HT in spinal inflammatory nociceptive transmission; opiate receptors

receptor phenotypes. J. Neurosci. 22, 10847–10855 state-dependent actions via dorsal horn 5-HT3 receptors in Fields’ homepage: http://www.keck.ucsf.edu/labinfo/fields.htm (2002). the anaesthetized rat. Pain 89, 81–88 (2000). Access to this interactive links box is free online.

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