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Proc. Natl. Acad. Sci. USA Vol. 96, pp. 7739-7743, July 1999 Colloquium Paper This paper was presented at the National Academy of Sciences colloquium "The Neurobiology of Pain," held December 11-13, 1998, at the Arnold and Mabel Beckman Center in Irvine, CA. Distinct neurochemical features of acute and persistent pain ALLAN I. BASBAUM* Departments of Anatomy and Physiology and W. M. Keck Foundation Center for Integrative Neuroscience, University of California, San Francisco, CA 94143 preprotachykinin-A gene, which encodes the peptides sub- stance P (SP) and neurokinin A (NKA). These studies have identified a specific behavioral phenotype in which the ani- mals do not detect a window of "pain" intensities; this window cuts across thermal, mechanical, and chemical modalities. The lowered thermal and mechanical withdrawal thresholds that are produced by tissue or nerve injury, however, were still present in the mutant mice. Thus, the behavioral manifesta- tions of threshold changes in nociceptive processing in the setting of injury do not appear to require SP or NKA. To identify relevant neurochemical factors downstream of the primary afferent, we are also studying the dorsal horn second messenger systems that underlie the development of tissue and nerve injury-induced persistent pain states. We have recently implicated the ~ isoform of protein kinase C (PKCy) in the development of nerve injury-induced neuropathic pain. Acute pain processing, by contrast, is intact in the PKCy-null mice. Taken together, these studies emphasize that there is a distinct neurochemistry of acute and persistent pain. Persistent pain should be considered a disease state of the nervous system, not merely a prolonged acute pain symptom of some other disease conditions. ABSTRACT To address the neurochemistry of the mech- inner part of lamina II. The same region can be stained for the anisms that underlie the development of acute and persistent P2X3 receptor and for FRAP. pain, our laboratory has been studying mice with deletions of Because these afferents are of small diameter, it has gen gene products that have been implicated in nociceptive pro- orally been assumed that they are nociceptors, even though ceasing. We have recently raised mice with a deletion of the some nonnociceptive, low-threshold mechanoreceptive, small diameter afferents have been identified (4~. Interestingly, the latter group did not express SP. Given that many neurons in the inner part of lamina II respond to nonnoxious stimuli (5, 6), it has been suggested that this region does not receive input from nociceptors. Despite these observations, we recently provided strong evidence in support of the hypothesis that the majority of the small-diameter afferents are nociceptors. Specifically, we showed that neurons in both populations express the vanilloid-1 receptor (VR1) to which capsaicin, the pungent ingredient in hot pepper, binds with high affinity (7~. The fact that these neurons express VR1 suggests that, at the minimum, these afferents are responsive to a noxious chemical stimulus. Furthermore, because the capsaicin receptor can be activated by noxious thermal stimuli (8), it is also likely that the predominantly nonpeptide population of C fiber includes heat nociceptors. Importantly, not all of the neurons were double labeled, and we identified a population of dorsal root ganglion (DRG) cells that was VR1 positive, but stained for neither SP nor IB4. Thus additional classes of primary afferent nociceptor remain to be characterized. The complexity of the DRG population is highlighted even further by the recent observa tion that there are at least two classes of heat nociceptors. Because only one of these responds to capsaicin (9) it will be of interest to determine the neurochemical phenotype of the non-VR1 heat nociceptor. These results establish that the two major classes of small diameter primary afferent include nociceptors, but that is just the first step in establishing their contribution to the genera tion of pain. For example, it is not at all clear to what extent they differ in the types of pain provoked by their activation. We also do not know whether coincident activity in the different classes of afferent affects the type/quality of pain that is provoked. Given that the afferents target very different pop ulations of neurons in the superficial dorsal horn, it is also of interest to address the contribution of these downstream neuronal populations. Importantly, many of the neurons tar geted by the peptide population are projection neurons, which transmit the nociceptive message to brainstem and/or thala mus. These are clustered in lamina I of the dorsal horn. Indeed, the large majority of neurons that express the neurokinin-1 (NK-1) receptor, which is targeted by SP-containing afferents, are projection neurons (10~. By contrast, the nonpeptide group of small-diameter afferents targets a region of the superficial dorsal horn that exclusively contains interneurons, namely the inner part of lamina II. Abbreviations: SP, substance P; VR1, vanilloid-1 receptor; NK-1, neurokinin-1; NKA, neurokinin A; PPT-A, preprotachykinin-A; PKC^y, "y isoform of protein kinase C. *To whom reprint requests should be addressed. e-mail: aib@phy. ucsf.edu. Recent studies have focused attention on the organization of two major small-diameter primary afferent systems that target dorsal horn neurons. Broadly speaking, they can be divided into a peptide-containing class of C fibers and one that is generally not associated with peptides (1~. The peptide- containing neurons can be demonstrated by immunostaining dorsal root ganglia with antisera directed against the prepro- tachykinin-A product substance P (SP). These neurons also coexpress calcitonin gene-related peptide (CGRP) and the neurotrophin receptor TrkA. The second group of afferents can be distinguished because they express a cell surface c~-galactosyl epitope, which can be stained with the lectin IB4. Most of the latter neurons also express a fluoride-resistant acid phosphatase (FRAP) (2) and the P2X3 subtype of purinergic receptor (3~. It has been suggested, however, that the failure of the peptide-containing group to immunostain for the P2X3 receptor reflects the inability of antisera to recognize the epitope in that class of neurons, because of coassembly with another subtype of purinergic receptor (E. W. McCleskey, personal communication). Perhaps of greater relevance to the functional differences of these two broad classes of afferent is that their patterns of axon termination in the spinal cord dorsal horn differ greatly. The terminals of the peptide-containing afferents are concentrated in laminae I and outer II of the superficial dorsal horn. The IB4-labeled afferents target the PNAS is available online at www.pnas.org. 7739

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7740 Colloquium Paper: Basbaum The problem is not a simple one to address, as it is presently not possible to selectively remove one or the other class of afferent or postsynaptic neuron. The only exception to this are the very recent studies of Mantyh and colleagues (11), who used a SP-saporin conjugate to selectively destroy superficial dorsal horn neurons that express the NK-1 receptor. Our approach to this problem has been to study the phenotype of mice in which the genes for critical neurotransmitters, neuro- transmitter receptors, or second messenger systems have been deleted. In the following discussion, I will highlight some of our progress in attributing different properties of the pain response to distinct gene products in afferents and second-order neu- rons. Preprotachykinin-A Products and Acute Pain Our first studies evaluated the phenotype of mice in which we deleted the preprotachykinin-A (PPT-A) gene, which encodes the two tachykinin peptides, SP and neurokinin A (NKA) (12~. These animals appeared normal in simple motor tests, includ- ing running on a rotating rod and when examined in an open field. As expected, we also found that plasma extravasation (PE) in response to injection of capsaicin was almost abolished in the mutant mice. That result was expected, as peripheral release of SP/NKA is generally required to evoke PE (13), a hallmark of neurogenic inflammation. In tests of acute pain, using mild to moderate noxious stimuli, the mutant mice also responded as the wild-type mice did. By contrast, when the stimulus intensity was increased we observed increased "pain" responses only in the wild type. For example, when the heat stimulus was increased from 52.5C to 55C, only the wild-type mice showed a decreased latency to respond. Decreased responsiveness in the setting of very intense stimuli was true for noxious chemical, thermal, and mechanical stimuli. These results indicate that SP/NKA contribute to the intensity coding of stimuli across modalities, but that the coding range is limited to one in which the stimulus is very intense. The fact that very intense stimuli are required to reveal the phenotype is consistent with our previous studies, which found that internalization of the NK-1 receptor (which provides a mea- sure of tachykinin release) occurs only when the stimulus is very intense (14~. This result is also consistent with neuro- chemical studies that found that higher intensities and higher frequencies of stimulation are required to evoked primary afferent release of SP, compared with glutamate (15, 16~. The latter is a major neurotransmitter of small-diameter primary afferents, and is found in the same terminals, albeit in a different population of synaptic vesicles (174. We presume that as the threshold for activating C-fiber nociceptors is crossed, glutamate is released at central synapses. Via an action at an a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor, the glutamate depolarizes postsynaptic projection neurons and interneurons of the dorsal horn, re- sulting in an "acute" pain message. What about the contribution of SP/NKA to persistent pain conditions?Allodynia refers to the production of pain (includ- ing reflex withdrawal) by nonnoxious stimuli. Hyperalgesia refers to an exaggerated pain response produced by noxious stimuli. Although most studies have reported that the devel- opment of allodynia and hyperalgesia results from injury- induced long-term changes/central sensitization of dorsal horn neurons secondary to activity at the N-methyl-D-aspartate (NMDA) receptor (18, 19), other studies have provided evi- dence for a contribution of tachykinins, notably SP (20-22~. It was thus surprising that we found no change in the develop- ment of mechanical or thermal allodynia after injury. Impor- tantly, the tests of allodynia involve stimuli that are at or near threshold. For example, within 24 hr of injecting complete Freund's adjuvant into the hindpaw, which produces a pro- found hindlimb inflammation, there is a dramatic drop in the Proc. Natl. Acad. Sci. USA 96 (1999) thermal and mechanical withdrawal threshold (allodynia) of the affected limb. We found that comparable allodynia was present in both wild-type and PPT-A-null mice. The thermal and mechanical allodynia produced after partial sciatic nerve section (23) was also intact, despite the loss of SP/NKA. Importantly, we did not specifically test for hyperalgesia, as this would have required using a noxious stimulus in the settin~ of . . 1nJury. ~ . . To determine whether the loss of the PPT-A gene products differentially affects the development of allodynia and hyper- algesia, we are using an electrophysiological approach to study the response of dorsal horn neurons under conditions of inflammation, and in response to noxious stimuli. In this setting the mice are anesthetized and thus it is possible to administer noxious stimuli, even to an inflamed hindpaw. Consistent with the behavioral analysis, our electrophysiolog- ical results to date suggest that SP/NKA are predominantly involved under acute conditions, when the stimulus is very intense. We do not have an explanation for the differences between our results and those of others that implicate SP and NKA in the development of allodynic states. A particularly interesting discrepancy concerns the observations of Neumann et al. (24), who reported that in the setting of persistent inflammation, SP is synthesized de novo in large-diameter afferents and that it contributes to the A ,l3 (i.e., large fiber- mediated) mechanical allodynia. Our results with the PPT-A-null mice also bear on the receptors that are targeted by SP and NKA. It is generally assumed that SP exerts its effects via the NK-1 receptor (25~. It is, thus, of great interest that the phenotypes of the PPT-A mice and of mice with a deletion of the NK-1 receptor (26) differ. Although we found that high-intensity stimuli are not processed normally in the PPT-A mutant mice, the response to acute noxious stimuli appears to be intact in the NK-1 receptor mutant animals. Furthermore, although the NK-1 receptor mutants showed reduced second-phase formalin test re- sponses, the PPT-A mutant mice had only reduced first-phase behavior. Although there is disagreement concerning the extent to which central sensitization contributes to second- phase pain in the formalin test (27, 28), the possibility that the time course and magnitude of central sensitization differ in the wild-type and mutant mice needs to be tested. We hypothesize that the differences between the PPT-A mutant and NK-1 receptor mutant mice are indicative of an action of SP and/or NKA at an additional tachykinin receptor. Although SP predominantly targets the NK-1 receptor, NKA has a higher affinity for the NK-2 receptor and has reasonable affinity for the NK-3 site (254. Importantly, there is evidence for an independent contribution of the NK-2 receptor (and thus of NKA) to nociceptive processing (29-31~. Of particular interest, iontophoresis of NK-2 antagonists reduces the hyper- excitability of dorsal horn neurons in the setting of inflamma- tion and blocks the central effects of NKA, but not of SP (32~. Fleetwood-Walker and colleagues (33) found that NK-2 an- tagonists were far more effective than NK-1 antagonists at reducing inflammation-induced up-regulation of dynorphin message in superficial dorsal horn. In preliminary studies, we found significant induction of Fos expression in laminae I and II after intrathecal injection of NKA in mice with a deletion of the NK-1 receptor. Finally, Duggan et al. (34) provided evidence that the central effects of SP and NKA differ, in part because of their differential susceptibility to the endopepti- dases that degrade them. Thus, if NKA targets a receptor to which SP has much lower affinity, its loss in the PPT-A-null mouse could have profound consequences independent of the loss of SP. Paradoxically, despite the pharmacological evidence for an independent NK-2 receptor contribution, neither in situ hy- bridization nor immunocytochemistry, with one recent excep- tion (35), has provided convincing evidence that the NK-2

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Colloquium Paper: Basbaum receptor is present in the dorsal horn. One possibility is that there is another receptor to which NKA binds, different from the cloned NK-2 receptor, but sensitive to NK-2 antagonists. A somewhat comparable situation exists for ~ opioid receptors (36), which are best distinguished pharmacologically. If we are to understand the mechanisms through which SP/NKA influ- ence short- and long-term nociceptive processing in the dorsal horn, it is essential that this paradox be addressed. To this end we are now generating mice that encode either SP or NKA, but not both. We will study these mice in both behavioral and electrophysiological tests of nociception and compare their phenotypes with those of the PPT-A-null and NK-1 receptor- null mice. Hopefully, this approach will address the differential contribution of SP and NKA to nociceptive processing. Protein Kinase C ~ and Persistent Pain The results described above provide important information on the contribution of the peptide-containing class of primary afferents to nociceptive processing. Much more difficult is the analysis of the contribution of the nonpeptide class of small- diameter afferents. Although these neurons express the P2X3 receptor, it is difficult to selectively activate this receptor in vivo so as to address its contribution to the generation of pain. Because of the difficulty in directly evaluating the afferents, we turned to a likely central target of the afferents, namely neurons in the inner part of the lamina II. These studies involved mice with a deletion of the gene that encodes the my isoform of protein kinase C (PKC^y) (37~. These mice were of interest for several reasons. Most importantly, there is considerable evidence that PKC contributes to the development of the long-term changes that underlie injury- associated allodynia and hyperalgesia (38-404. Unfortunately, there are no selective antagonists available for the many isoforms of PKC; thus it is impossible to determine their individual contribution. Furthermore, because the different isoforms are differentially distributed in the dorsal horn (see below), an understanding of the mechanisms through which they operate at the dorsal horn circuit level is very difficult to achieve. Thus, the opportunity to study mice with a deletion of one isoform is particularly useful. These mice were also of also interest because, unlike most PKC isoforms, PKC-y is not expressed until after birth; thus the likelihood that the deletion is associated with major developmental abnormalities, or for Proc. Natl. Acad. Sci. USA 96 (1999J 7741 compensatory responses to its loss during development, is significantly reduced. Any compensatory responses to the deletion would have to have occurred postnatally. The PKC^y- null mice are also of interest because studies in hippocampus revealed that the mice show a reduction of long-term poten- tiation (LTP) (41~. Although LTP and long-term changes in spinal cord nociceptive processing secondary to injury are not identical, it was reasonable to hypothesize that pain-related behaviors that have been associated with alterations in the excitability of dorsal horn nociresponsive neurons would be altered in these mice. Finally, and perhaps, most importantly, we found that the dorsal horn distribution of PKC^y differs greatly from that of the other isoforms. First, to our knowledge, PKC^y is the only isoform that is not found in dorsal root ganglia. Furthermore, PKC^y immunostaining in the spinal cord is restricted to a subpopulation of interneurons in the inner part of lamina II (Fig. 1~. This distribution greatly differs from that of other isoforms (e.g., PKCc~, ,131, and 132), which are found throughout the superficial dorsal horn as well as more ventrally. The presence of PKC^y in the inner part of lamina II raises the possibility that the phenotype of the deletion mutant is, at least in part, related to loss of the consequences of activation of the nonpeptide population of primary afferents that target this region. Importantly, the restriction of PKC^y to a single inter- neuron population significantly limits the circuits through which it can influence the development of persistent pain conditions. It also offers the hope that further neuroanatomi- cal studies at the light and electron microscopic level will be able to identify the major afferent inputs to and the connec- tions made by these neurons so that the circuits through which these neurons exert their effects can be understood. Our studies proved incredibly informative. In distinct con- trast to the mice with a deletion of the PPT-A gene, we found that in the PKC^y-null mice there was no defect in the processing of acute "pain" messages. For example, the PKC^y- null and wild-type mice behaved identically in tests of thermal pain. By contrast, when we tested the mice in models that involved persistent injury, for example, after partial sciatic nerve injury, we found a significant decrease in the magnitude of the mechanical and thermal allodynia that developed. This difference persisted for the duration of the experiment. We concluded that in the absence of PKC-y, partial nerve injury (Left) Micrograph from the lumbar spinal cord of the rat. It illustrates the restricted distribution of PKC-y immunoreactivity to interneurons in the inner part of lamina II of the superficial dorsal horn. (Right) Distribution of Fos-immunoreactive neurons in the L4 cord dorsal horn evoked by an injection of formalin into the ipsilateral hindpaw. The rat was killed 1 hr after the formalin injection. Note that there are many labeled neurons in the most superficial laminae (I and outer II), but that there is a distinct band (corresponding to the inner part of lamina II; arrows) in which there is very little Fos expression. Thus although the small-diameter afferents that innervate lamina II are chemonociceptors, their activation appears not to induce Fos in the neurons that they target. (x60.)

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7742 Colloquium Paper: Basbaum does not induce the hyperexcitability of dorsal horn neurons that is at the basis of the subsequent allodynia. Given that PKC^y is concentrated in interneurons of the inner part of lamina II, we presume that changes in the circuits that involve these neurons are critical to the failure of allodynia to develop after nerve injury. On the other hand, PKC^y is present throughout the neuroaxis. Thus, the defect in nerve injury-induced processing of nociceptive messages need not reflect the loss of enzyme function in the spinal cord. In preliminary studies using electrophysiological methods in the mouse spinal cord (42), however, we found that injury in the PKC^y-null mice does not lead to an enhancement of the response of nociceptive neurons to nonnoxious stimuli, as it does in the wild-type mice. This finding more directly points to the cord as the locus of the defect. Finally, we found that the spinal cord neuroanatomical consequences of nerve injury are also significantly reduced in the PKC^y-null mice. For example, numerous studies have demonstrated that there is a decrease in immunoreactivity for SP (43) and an increase in staining for the NK-1 receptor after sciatic nerve section (444. Although comparable changes were readily demonstrated in the wild-type mice, the changes were much reduced, and in some cases absent, in the mutant mice. Similarly, although we found that partial sciatic nerve section induces an up-regulation of neuropeptide Y immunoreactivity in the superficial dorsal horn, this was also significantly reduced in the PKC^y-null mice. Taken together, these results suggest either that the signal that is sent from the site of nerve injury to the dorsal root ganglia and spinal cord is not transmitted properly in the PKC^y-null mice, or that the response to the signal by dorsal horn neurons is altered. We favor the latter hypothesis and suggest that it is a signal in the nonpeptide population of small-diameter nociceptors that is not processed normally by the PKCy interneurons of the inner part of lamina II. How this comes about, and the extent to which such changes can be prevented or reversed, remains to be determined. The fact that the PKC^y-null mice behaved normally in tests of acute pain suggests that the contribution of these neurons is manifest only when noxious inputs persist, i.e., in the setting of injury. In fact, it is remarkably difficult to demonstrate that neurons in the inner part of lamina II, are activated by acute noxious stimuli. As noted above, electrophysiological studies have emphasized the responsiveness of many of the neurons in inner II to nonnoxious mechanical stimuli (5, 6~. Furthermore, even a very intense noxious stimulus rarely induces Fos expression in the interneurons of inner lamina II (45~. This is true even when formalin (Fig. 1) or capsaicin is used as the stimulus. Because the bulk of the small-diameter afferents that target inner lamina II express the capsaicin receptor, it is likely that this stimulus activates the interneurons of this region. We have tried repeatedly to find a stimulus that will induce Fos in the PKC-y population of neurons, but have not succeeded. To our knowledge, the only consistent stimulus that induces Fos in neurons of inner II is kainate injection in the rapine magnus of the medulla (46~. Studies in our laboratory, however, found that even this stimulus rarely induces Fos expression in the PKC^y population of interneurons. It is, of course, possible that the PKC^y neurons do not express Fos; that alone indicates that they are unusual, at least when compared with other dorsal horn neurons that receive inputs from primary afferent noci- 22. ceptors. 23. Given the nature of the afferents to the inner part of lamina 24. II (i.e., that they express VR1) we find it very difficult to accept the notion that this region exclusively, or even predominantly, subserves a nonnociceptive function. Rather, it appears that the conditions under which these neurons are activated re mains to be established. In part to address this question, we have established an in vitro spinal cord slice preparation in which translocation of PKC^y in these interneurons (which Proc. Natl. Acad. Sci. USA 96 (1999J occurs when the enzyme is activated) can be monitored. We hope that this will permit a more extensive analysis of the neurochemistry of the inputs that activate these neurons. That information will then be used to study the neurons in the intact animal. In summary, our studies using mice with deletions of specific genes have demonstrated that particular features of the re- sponses to acute and persistent injury conditions are differ- entially influenced by SP/NKA and PKC^y. 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