Neurotensin and pain modulation

peptides 27 (2006) 2405–2414

available at www.sciencedirect.com

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Review

Neurotensin and pain modulation Paul R. Dobner * Department of Molecular Genetics and Microbiology, Program in Neuroscience, University of Massachusetts Medical School, 55 Lake Ave. North, Worcester, MA 01655, United States

article info

abstract

Article history:

Neurotensin (NT) can produce a profound analgesia or enhance pain responses, depending

Received 29 June 2005

on the circumstances. Recent evidence suggests that this may be due to a dose-dependent

Accepted 18 April 2006

recruitment of distinct populations of pain modulatory neurons. NT knockout mice display

Published on line 25 July 2006

defects in both basal nociceptive responses and stress-induced analgesia. Stress-induced antinociception is absent in these mice and instead stress induces a hyperalgesic response,

Keywords:

suggesting that NT plays a key role in the stress-induced suppression of pain. Cold water

Neurotensin

swim stress results in increased NT mRNA expression in hypothalamic regions known to

Antinociception

project to periaqueductal gray, a key region involved in pain modulation. Thus, stress-

Analgesia

induced increases in NT signaling in pain modulatory regions may be responsible for the transition from pain facilitation to analgesia. This review focuses on recent advances that

Abbreviations:

have provided insights into the role of NT in pain modulation. # 2006 Elsevier Inc. All rights reserved.

NT, neurotensin PAG, periaqueductal gray RVM, rostroventral medulla NRM, nucleus raphe magnus PVN, paraventricular nucleus of the hypothalamus VMR, visceromotor response CRD, colorectal distension NTR-1, neurotensin receptor-1 NTR-2, neurotensin receptor-2 CHO, Chinese hamster ovary SIAN, stress-induced antinociception mRNA, messenger ribonucleic acid CCK, cholecystokinin

Contents 1. 2.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . NT and NT receptor localization in the pain modulatory circuitry 2.1. Spinal column . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Periaqueductal gray and rostroventral medulla . . . . . . . . . .

* Tel.: +1 508 856 2410; fax: +1 508 856 5920. E-mail address: [email protected]. 0196-9781/$ – see front matter # 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2006.04.025

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1.

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Neurotensin modulates nociceptive responses. . . . . . . . . . . . . . . . . . . . . . . . . . . . NT modulates nociception through both NTR-1 and NTR-2 . . . . . . . . . . . . . . . . . . 4.1. Evidence that NTR-2 mediates the antinociceptive actions of NT . . . . . . . . 4.2. Evidence that NTR-1 is required for certain antinociceptive actions of NT . Endogenous NT facilitates nociception . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Antinociceptive effects of NT antagonists . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. NT and NTR-2 knockout mice display defects in nociceptive responses . . . Endogenous NT is required for stress-induced antinociception (SIAN) . . . . . . . . . Neurotransmitters involved in NT modulation of nociception. . . . . . . . . . . . . . . . 7.1. Monoamine transmitters involved in NT-induced antinociception . . . . . . . 7.2. Cholecystokinin (CCK) involvement in NT facilitation of nociception . . . . . Stress and chronic pain alter NT expression in the CNS . . . . . . . . . . . . . . . . . . . . Model for NT pain modulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Introduction

The discovery that electrical stimulation in certain regions of the rat central nervous system produces a profound analgesia [63,75] led to the identification of a pain modulatory circuit that includes the periaqueductal gray (PAG), the nucleus raphe magnus (NRM), and certain reticular structures surrounding these sites. Subsequent studies demonstrated that the analgesic effects of morphine and other opiates are likely to be mediated at least in part through this same circuitry [121]. This system is also activated in response to a variety of stressors, suggesting that the central modulation of nociception may be an important adaptive response to adverse environmental conditions [57]. The investigation of the possible contribution of endogenous opioids to stress-induced antinociception (SIAN) revealed that lower intensity stress results in a m opioid receptor-dependent antinociceptive response, while higher intensity stress is unaffected by m opioid receptor blockade [38,57,66,95]. Similar to intense stress, intracerbroventricular (i.c.v.) neurotensin (NT) administration produces a potent analgesia that is not blocked by m opioid receptor antagonists [21,22]. These effects are most likely the result of NT actions in the central pain modulatory circuitry; the subject of this review.

2. NT and NT receptor localization in the pain modulatory circuitry Immunohistochemical and in situ localization experiments have been used to map the distribution of NT-like peptides, NT receptors, and their corresponding mRNAs in the pain circuitry. NT is synthesized as part of a larger precursor protein along with the related companion peptide, neuromedin N [28,53], and the two peptides are generally co-expressed [26] and share the same functions. Three NT receptors have been described; however, only the two known G protein-coupled receptors, NTR-1 [94] and NTR-2 [64] will be considered here.

2.1.

Spinal column

NT-like immunoreactive (NT-IR) fibers and cell bodies have been detected in multiple brain regions involved in nocicep-

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tive transmission and modulation, including the PAG, the rostroventral medulla (RVM), and the dorsal horn of the spinal cord. The distribution pattern in the rat will be described in the most detail, since rats have been used extensively to investigate nociceptive modulation. NT-IR fibers and cell bodies are concentrated in laminae I and II of the dorsal horn of the spinal cord [37,46,48,88,102,103]. The greatest concentrations of NT-IR fibers and cell bodies are found within the inner portion of lamina II and the border region with lamina III, with the greatest numbers of cell bodies located at the outer border of lamina III, melding with regularly spaced groups of cells in the inner part of lamina II [86,88]. Similar results have been obtained in diverse mammalian species, including humans [62]. These NT-IR neurons have been reported to make direct synaptic contacts with both local intrinsic neurons located principally in the superficial laminae (I–III) and primary afferents [27,125]. These synapses are primarily asymmetric (presumably excitatory) [27,70,87], consistent with the co-localization of NT and glutamate, but not GABA in the rat spinal cord [96,97]. Collectively, these results suggest that NT may modulate both local excitatory and primary afferent neurotransmission in the spinal cord. Labeled NT binding studies indicate that NT receptors are also expressed predominantly in the superficial laminae of the dorsal horn of the spinal cord and spinal trigeminal nucleus [33,70,102,124,125]. These sites were particularly dense in the inner segment of lamina II in humans, but were also detected in deeper laminae [33]. A recent report indicates that NTR-2 is expressed at moderate levels in both cell bodies and fibers in the spinal trigeminal nucleus, although the distribution of this receptor in the spinal cord was not analyzed [83,84]. NTR-1 mRNA has been localized to a subpopulation of DRG neurons, but was not detected in the spinal cord of rats [125]. Clearly, further studies are necessary to complete the picture of NT receptor distribution in the spinal cord; however, the limited evidence available suggests that NTR-2 may play a more important role in modulating the activity of spinal neurons. NT can influence the activity of both DRG and spinal neurons. In isolated DRG preparations, iontophoretic application of NT inhibited a subpopulation of primary C-type neurons, but resulted in a rapid and prolonged stimulation of roughly half of the A-type neurons tested [125]. In the spinal cord, NT produced a dose-dependent excitation of neurons

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activated by both noxious and innocuous stimuli in both the anesthetized rat and spinalized cat [65,92]. These excitatory effects were mainly restricted to neurons located in laminae I– III in the cat [65,92]. Sciatic nerve stimulation results in Ca2+dependent NT release, suggesting that increased NT signaling could be involved in the processing of afferent stimulation [122].

2.2.

Periaqueductal gray and rostroventral medulla

The PAG is thought to play a key role in the descending modulation of nociceptive inputs via efferent output to the RVM, including a major NTergic projection to the NRM [12,13,118]. NT-IR cell bodies and fibers are heterogeneously distributed within the PAG in a variety of species [5,25,48,54,104], including humans [62]. In the rat, NT-IR fibers are most concentrated in the periaqueductal region of the PAG, particularly along the ventrolateral and ventral aspects of the cerebral aqueduct [12,89]. These include NT-IR fiber tracts that either enter or exit the PAG at the ventrolateral corners [89]. NT-IR cell bodies in colchicine-treated rats were most numerous in the ventromedial and ventrolateral PAG; however, numerous NT-IR neurons were also detected in the dorsal region [89,118]. Most NT-IR terminals in the PAG contacted dendrites of unlabeled neurons where they formed primarily symmetric synapses, although some contacted NTIR neurons and dendrites [118]. A small fraction of NT-IR terminals directly contacted PAG efferents to the NRM, but the bulk appeared to contact local circuit neurons within the PAG [118]. Thus, NT is likely to mediate, at least in part, neuronal transmission between the PAG and the NRM as well as influence neuronal activation in the PAG. NT binding studies first revealed the presence of NT receptor sites in the PAG, RVM, and the dorsal and medial raphe nuclei, which house numerous serotonin neurons. More recently, specific detection methods have been used to provide a clearer picture of the nature of these binding sites. NTR-1 was localized to dendrites and cell bodies both dorsal and ventral to the aqueduct in the PAG, and in the dorsal raphe nucleus using immunohistochemical methods [16]. A similar distribution of NTR-1 mRNA-positive neurons was observed using sensitive in situ hybridization methods, with the distribution shifting from the dorsolateral and ventromedial PAG rostrally to the ventrolateral region more caudally [4]. Recently, NTR-1 (but not NTR-2) has also been localized to spinally projecting serotonin neurons in the RVM [17]. NTR-2 has also been localized to neuronal cell bodies and processes within the PAG, the dorsal raphe nucleus, and the RVM, opening the possibility that NT could modulate neuronal activity in the PAG through both G protein-coupled NT receptors [80,84], consistent with functional studies reviewed below. NT has been shown to have mainly an excitatory effect on neurons in the PAG, including neurons that project to and activate neurons in the NRM [8,9,58,69]. These excitatory effects have been observed in PAG neurons in vivo [8], in slice preparations [9], and in dissociated cell culture [58], however, NT also occasionally inhibited these neurons [8]. Two different excitatory responses can be differentiated in vivo, one is rapid in onset and rapidly extinguished, while the other develops

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more slowly and persists for several minutes [8]. NT administration in the PAG results in neuronal excitation in the NRM [8], consistent with the observation that NT excited PAG neurons in dissociated cell culture that had previously been labeled in vivo following retrograde tracer microinjection in the RVM [58]. Opioids and inflammation produced at least shortterm increases in NT release in the PAG [93,120], suggesting that NT release from either PAG afferents or intrinsic PAG neurons may be involved in nociceptive responses. NT has also been shown to dose-dependently activate specific populations of neurons after focal infusion in the RVM [69]. Two classes of nociceptive modulatory neurons have been identified in the RVM and PAG, designated ‘‘on’’ and ‘‘off’’ cells [34,42]. These cells display opposite changes in activity immediately prior to and during the tail-flick response to noxious heat, with ‘‘off’’ cells decreasing and ‘‘on’’ cells increasing their firing rates. Interestingly, microinjection of low doses of NT into the RVM activates ‘‘on’’ cells, and this activation is correlated with enhanced nociceptive responding, but at higher doses, NT activates both classes of neuron, and produces an antinociceptive response [69]. These results suggest that NT is capable of activating substantial numbers of neurons in the RVM, and that differences in the intensity of NT signaling may result in the activation of different proportions of ‘‘on’’ and ‘‘off’’ cells, resulting in opposite modulatory effects on nociception.

3. Neurotensin modulates nociceptive responses Central NT administration was first reported to result in a m opioid receptor-independent antinociceptive response in the hot plate and acetic acid-induced writhing tests in rodents by Clineschmidt and co-workers [21–23]. This m opioid receptor independence has been confirmed in most [2,6,8,24,71], but not all studies [35,112,122]. Subsequent site-specific microinjection experiments revealed that NT most likely produces these antinociceptive effects through actions in the PAG, RVM, and various brain regions that provide afferent input to these structures, consistent with the anatomical localization studies described above [2,7,8,14,32,51,52,90,99,105,106,109,110]. NT microinjection experiments in the RVM also provided the first evidence that NT may also act to facilitate nociception under certain circumstances. NT was first shown to produce a long-lasting (up to 70 min) antinociceptive effect in the tailflick assay following intra-RVM administration [32], but subsequent dose response analyses revealed that NT produced a hyperflexive response at lower doses in addition to antinociception at higher doses [105], although this response proved to be somewhat variable [90]. Furthermore, NT was found to have similar biphasic effects on the visceromotor response (VMR) to colorectal distension (CRD), indicating that NT modulation extends to both visceral and somatic nociception [110]. These results indicate that NT can influence nociceptive transmission at several different points in the descending pain modulatory circuitry and that the intensity of NT signaling might dictate the direction of modulation.There is also limited evidence that NT may interfere with nociceptive transmission directly in the spinal cord, consistent with the

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anatomical data described above. Intrathecal NT administration increased both hot plate and aversive (hypertonic saline) response latencies [47,122]. These antinociceptive effects were at least partially blocked by naloxone in both assays, although naloxone administration alone (2 mg/kg) resulted in an apparent hyperalgesia in the chemical aversion test [47,122]. In contrast, intrathecal NT had no significant analgesic effect in the tail-flick assay [47,91]. These results suggest that NT action in the spinal cord can produce an analgesic response through a mechanism that may involve m opioid receptor signaling.

4. NT modulates nociception through both NTR-1 and NTR-2 4.1. Evidence that NTR-2 mediates the antinociceptive actions of NT There is now evidence that NT signaling through both NTR-1 and NTR-2 is required for different aspects of NT-induced antinociception, but much of the early evidence pointed toward an important role for NTR-2. Perhaps the earliest indication was that the antinociceptive effectiveness of a panel of NT analogs did not correlate with binding affinities for NTR-1. This indicated that a different NT receptor subtype was most likely involved, since where examined, the differences did not appear to be due to stability issues [2,35,56,98]. More recently, an excellent correlation has been shown between the EC50 values for various NT analogs in the acetic acid-induced writhing test and NTR-2 binding affinities [31], pointing toward the involvement of this receptor in certain antinociceptive responses. Chemical antagonist studies also provided an indication that NTR-1 was not involved in this response. Three NT antagonists have been used extensively to understand NT function, levocabastine, SR 48692 and SR 142948A. SR 142948A, but not SR 48692, pretreatment attenuated NT-induced antinociception in the phenyl-p-benzoquinone-induced writhing test in rats, although with bell-shaped dose–response characteristics [29,41]. Since SR 142948A blocks NT binding to both NTR-1 and NTR-2, while SR 48692 is relatively selective for NTR-1, these results provide a further indication that an NT receptor other than NTR-1 mediates the antinociceptive effects of NT in this test. Levocabastine, which selectively blocks NT binding to NTR-2 but is also a histamine H1 antagonist, partially blocked NT antinociception in both the acid-induced writhing and hot plate tests while highly selective H1 antagonists did not, consistent with the involvement of NTR-2 in these responses [30,99]. The antagonist experiments, although supporting the involvement of NTR-2, must be viewed with caution, since all of these compounds exhibit agonist activity under certain circumstances. Both SR 48692 and SR 142948A behave as agonists for both human and rat NTR-2 expressed in Chinese hamster ovary (CHO) cells, and stimulate several intracellular signaling pathways [36,113,123]. Initial observations that these compounds increase intracellular Ca2+ levels through either human or rat NTR-2 in both CHO cells [113,123] and rat cerebellar granule cells [82], have recently been challenged [36]. The selective NTR-2 antagonist levocabastine has also

been reported to act as an agonist or partial agonist at NTR-2 in some cases [30,36,64,82,123], but not others [76,99,113]. Fortunately, antisense inhibition experiments have provided an independent line of evidence for the involvement of NTR-2 in NT-induced antinociception. Central administration of phosphorothioate antisense oligonucleotides directed against NTR-2 twice daily for 3–4 days attenuated NT-induced antinociception in the acetic acid-induced writhing test, although this inhibition was incomplete and could be overcome at higher NT doses [31]. A likely explanation for this partial effect is that this antisense regimen did not completely inhibit NTR-2 expression, since 125 I-labeled NT binding to NTR-2 in brain membrane preparations was only reduced by about half [31]. In contrast, antisense inhibition of NTR-1 had no effect on NT-mediated antinociception, but did suppress turning behavior following unilateral NT microinjection in the striatum, a response thought to involve NTR-1 [40]. Taken together with the results from agonist and antagonist studies, these results provide fairly convincing evidence that NTR-2 is required for NT antinociception in the acetic acid-induced writhing test.

4.2. Evidence that NTR-1 is required for certain antinociceptive actions of NT In contrast to the results in the acetic acid-induced writhing test, both intra-PAG and systemic administration of an antisense peptide nucleic acid directed against the start of the coding region of NTR-1 significantly attenuated the antinociceptive response following NT administration in the PAG in the hot plate test [100,101]. The antisense injection regimen decreased NT receptor expression in the PAG and hypothalamus by 35–46%, and this level of inhibition was apparently sufficient to nearly completely block NT-induced analgesia [100,101]. Furthermore, the inhibition was specific, since antisense inhibition of NTR-1 expression had no effect on morphine analgesia, and conversely antisense inhibition of m opioid receptor expression had no effect on NT-induced antinociception, while it did attenuate the response to morphine [100,101]. Studies in two independently derived NTR-1 knockout mouse lines also point toward an important role for NTR-1 in NT-induced antinociception in the hot plate [73], but not the acetic acid-induced writhing test [74]. Thus, there appears to be a sharp distinction in the receptor requirements for NT-induced antinociception in the hot plate and writhing tests, perhaps due to a greater involvement of fear and anxiety circuits in the hot plate behavioral response [61] and a greater involvement of NTR-1 signaling in these circuits.

5.

Endogenous NT facilitates nociception

5.1.

Antinociceptive effects of NT antagonists

Several lines of evidence indicate that endogenous NT may predominantly facilitate nociception under basal conditions. First, central administration of an anti-NT antiserum had an antinociceptive effect that was dependent on the intensity of the noxious stimulus [15]. Second, disruption of NT signaling

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by several different means was found to augment morphine analgesia, suggesting that endogenous NT signaling may normally facilitate nociception [90,105]. Third, administration of SR 48692 alone produced an antinociceptive response as measured by the tail-flick test in rats, suggesting that endogenous NT signaling through an SR 48692-sensitive receptor, perhaps NTR-1, normally facilitates nociception [90]. Fourth, SR 48692 also principally decreased the VMR to CRD following either intra-RVM [110] or systemic [39] administration. These results echo observations that the administration of very low doses of NT into the RVM results in hyperreflexive responses in the tail-flick assay in rats [105], and support the hypothesis that NT mainly facilitates nociceptive responses under basal conditions, a view that is reinforced by more recent genetic experiments.

5.2. NT and NTR-2 knockout mice display defects in nociceptive responses The possibility that NT normally facilitates nociception is also supported by results from NT [39] and NTR-2 knockout mice [61]. NTR-2 knockout mice displayed an increased latency to jump in the hot plate test, although the latency to the first hind paw lick was similar to wild type [61]. The jump latency is thought to reflect tolerance to pain, while the jump is an escape response to the noxious thermal stimulus that most likely involves fear and anxiety brain circuits. The increased latency exhibited by NTR-2 knockout mice suggests that endogenous NT normally facilitates this response. However, reflex responses to pain were not altered in either NTR-2 or NTR-1 knockout mice, since no differences were observed in the latency to first hind paw lick in the hot plate test or the tailflick response under basal conditions [61,73]. In NT knockout mice, the VMR to CRD was reduced under basal conditions, adding further support to the hypothesis that endogenous NT signaling normally facilitates nociception [39].

6. Endogenous NT is required for stressinduced antinociception (SIAN) Several lines of evidence indicate that NT plays an important role in SIAN. Two forms of SIAN have been identified based on their sensitivity to m opioid receptor antagonists, with high intensity stress resulting in an antinociceptive response that is m opioid receptor-independent [57,66]. Central NT administration also produces an antinociceptive response that does not require m opioid receptor signaling, which led to the suggestion that NT is involved in SIAN. Furthermore, cold water swim stress increases NT mRNA expression in the lateral and medial preoptic hypothalamus [85], two regions that project to the PAG [1,67,77], suggesting that increased NT signaling could contribute to SIAN. Recent studies with NT knockout mice and NT antagonists in rats have revealed that NT plays an essential role in SIAN [39]. To investigate the possible role of NT in SIAN, Gui et al. examined the antinociceptive response to water avoidance stress in NT knockout mice and in rats pretreated with SR 48692 by measuring the VMR to CRD [39]. Water avoidance stress (30 min on a platform floating in 10 8C water into which

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the animals fell several times) resulted in a robust antinociceptive response in wild type mice and vehicle-treated rats, but instead resulted in a hyperalgesic response in both NT knockout mice and SR 48692 pretreated rats [39]. This reversal was not specific to water avoidance stress, since similar results were observed following prolonged restraint stress in rats [39]. Furthermore, the hyperalgesic response was significantly larger in female than in male rats [39], suggesting that this greater hyperalgesic response to stress may in part explain previous observations that female rats exhibit lower levels of SIAN than males [78,79]. This stress-induced hyperalgesia likely differs from the hyperalgesia caused by noxious irritants, since SR 48692 pretreatment blocked the hyperalgesia resulting from either mustard oil application to the hind leg or intracolonic acetic acid administration in rats [39,107]. These results strongly support the hypothesis that NT plays a key role in SIAN, and further indicate that stress normally activates counterbalancing antinociceptive and hyperalgesic mechanisms. The transition from NT facilitation to attenuation of nociception could involve stress-induced increases in NT signaling within the PAG or RVM [85], since a similar transition is observed in response to the microinjection of increasing doses of NT into the RVM [90,110].

7. Neurotransmitters involved in NT modulation of nociception 7.1. Monoamine transmitters involved in NT-induced antinociception Spinal monoaminergic pathways have been implicated in NTinduced antinociception in rats [68]. Depletion of spinal noradrenaline (by 70%) following i.c.v. administration of 6hydroxydopamine abolished the antinociceptive effect of intracisternal injection of NT without affecting basal responsiveness in the tail-flick test in mice. Similar results were obtained 1 week, but not 15 days after depletion of spinal serotonin (by 88%) through i.c.v. administration of 5,7dihydroxytryptamine. In contrast, repeated peripheral administration of p-chlorophenylalanine, which inhibits serotonin synthesis, decreased spinal serotonin levels by 50% without affecting NT-induced antinociception in mice [68] and similar depletion in rats (by 87% in whole brain) actually potentiated NT-induced antinociception [60]. There is also limited evidence that NT may influence antinociception through antagonism of dopamine signaling. Pretreatment with dopamine agonists antagonized NT-induced antinociception, while chlorpromazine, but not haloperidol, potentiated the response [44]. Thus, NT likely induces antinociception through the activation of noradrenaline neurons, possibly the A7 group, and perhaps serotonin neurons in the NRM. The electrophysiological effects of NT on serotonergic neurons in both the dorsal raphe and NRM have been examined in some detail [49,50,59]. In slice preparations, 64% of the serotonergic neurons in the ventral part of the dorsal raphe nucleus exhibited large excitatory responses to NT, which were blocked by SR 48692 indicating that NT activates these neurons through NTR-1 [49]. Immunohistochemical analyses indicate that both NTR-1 and NTR-2

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receptors are expressed in the dorsal raphe nucleus at low to moderate levels, and NT terminals contact serotonin neurons in the dorsal raphe nucleus [16,72,81]. NT activation was fairly short in duration due to rapid desensitization in the majority of responding neurons; however, the excitatory effects of NT persisted for >20 min in a significant number. NT also activated 64% of serotonergic neurons in dissociated cell cultures derived from the NRM; however, these effects were not blocked by either SR 48692 or SR 142948A, indicating that a novel NT receptor subtype could be involved [59].

7.2. Cholecystokinin (CCK) involvement in NT facilitation of nociception Several lines of evidence indicate that centrally administered NT facilitates nociception through a mechanism involving CCK signaling in the spinal cord. Intrathecal administration of a CCKB receptor antagonist blocked NT facilitation of the tailflick response [108]. Similarly, the ability of centrally administered NT to attenuate morphine analgesia (antianalgesia) was blocked by intrathecal administration of either CCKA or CCKB receptor antagonists in mice, although antianalgesia may not be strictly analogous to hyperalgesia [45]. Interestingly, morphine administration was found to increase NT release in the PAG [93]. Finally, both NT and CCK have been implicated in secondary hyperalgesia [108,111].

8. Stress and chronic pain alter NT expression in the CNS A variety of stressors increase NT mRNA expression in the hypothalamus and in some cases decrease expression in

certain other brain regions. A number of these increase NT mRNA levels in the paraventricular nucleus (PVN), including ether, dehydration, tail shock, immobilizaiton, and colchicine treatment [3,19,20,43,114,116]. This response is not universal since hypertonic saline and dehydration-induced anorexia increased NT mRNA expression in the lateral hypothalamus and decreased expression in the amygdala and arcuate [115,117]. Interestingly, cold water swim stress resulted in relatively rapid increases (within 1–3 h) in the medial preoptic and lateral hypothalamus in rats that had been pretreated with naloxone to block m opioid receptor-dependent SIAN [85], consistent with the involvement of NT in SIAN [39]. Three different models of chronic pain also resulted in increased NT and NT mRNA expression in brain regions related to pain modulation [119]. The development of chronic pain was most closely associated with increased NT mRNA expression in the cuneiform nucleus, lateral tegmentum, microcellular tegmental nucleus, the deep mesencephalic nuclei, the ventral and lateral PAG and the dorsal raphe nucleus, suggesting that increased NT signaling may either contribute to or oppose the development of chronic pain [119].

9.

Model for NT pain modulation

There is now fairly convincing evidence that endogenous NT both facilitates and attenuates somatic and visceral pain, and that stress may trigger increases in NT signaling that underlie the transition to antinociception (Fig. 1). Perhaps the most salient observation in this regard is that cold water swim stress results in a pronounced antinociceptive response, which is closely followed by increased NT mRNA expression in the medial preoptic and lateral hypothalamus [85].

Fig. 1 – Model for NT pain modulation. NT has been shown to facilitate nociception at lower doses, but to be antinociceptive at higher doses following microinjection into the RVM [90,109]. The available evidence suggests that this is due to the recruitment of ‘‘on’’ cells at low doses and the additional activation of ‘‘off’’ cells at higher doses [69]. Cold water swim stress increases NT expression in the lateral and medial preoptic hypothalamic areas that send afferent projections to the PAG [85], suggesting that stress may increase NT signaling in the PAG leading to the recruitment of ‘‘off’’ neurons. Recent evidence suggests that NT activation of NTR-1-positive spinally projecting serotonin neurons in the RVM mediate the antinociceptive effects of NT in the RVM [17]. Stress-induced NT signaling in the PAG is proposed to activate neurons controlling both descending noradrenergic and serotoninergic neurons leading to increased inhibition of dorsal horn neurons receiving nociceptive inputs and SIAN (right panel). In the absence of stress, NT likely selectively activates ‘‘on’’ cells facilitating pain transmission (left panel).

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Stimulation of these hypothalamic regions can induce an antinociceptive response most likely through projections to the PAG and RVM [1,18,55]. Furthermore, preliminary evidence suggests that NT mediates neuronal activation in the PAG following electrical stimulation of either the medial preoptic or lateral hypothalamus [10,11,55]. Increased afferent NT input could increase the overall level of NT signaling resulting in the activation of mechanisms required for antinociception, similar to what is observed upon microinjection of increasing doses of NT into the RVM [90,110]. The transition from pain facilitation to antinociception most likely involves an increasing recruitment of ‘‘off’’ cells, such as occurs in the RVM in response to increasing doses of NT [69]. Presumably, this activation results in increased noradrenergic and serotonergic signaling in the spinal cord and resulting suppression of nociceptive signaling, which may override CCK-dependent NT facilitation and also counteract stress-induced hyperalgesic mechanisms. Recent experiments in NT knockout mice have also provided direct evidence that NT facilitates basal nociception, and is required for the hyperalgesic response to intracolonic acetic acid administration and SIAN [39]. An important challenge for the future will be to pinpoint the defect(s) that underlie the observed phenotypic changes to provide insight into the mechanisms involved in nociceptive modulation. In the immediate future, it will be possible to examine NTdependent alterations in neuronal activity caused by stress and noxious stimulation using NT knockout mice. In addition, this model will be invaluable for examining NT’s role in somatic pain responses, hyperalgesia, chronic pain, and the antinociceptive actions of other analgesics, like morphine. Finally, targeted deletion of both NT and NT receptor genes using the Cre recombinase system can be used to identify neuroanatomical regions and neuronal populations where NT plays vital roles in these processes. These approaches and others will determine whether the model proposed above is ‘‘on’’ or ‘‘off’’.

Acknowledgements Work in the authors’ laboratory has been supported by grants from the NIH, the University of Massachusetts Medical School Diabetes and Endocrinology Research Center (DK032520), and the University of Massachusetts Medical School Program in Genetics.

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