The NK1 receptor mediates both the hyperalgesia and the resistance to morphine in mice lacking noradrenaline Luc Jasmin*†, Duc Tien*, David Weinshenker‡, Richard D. Palmiter‡, Paul G. Green§, Gabriella Janni¶, and Peter T. Ohara¶ Departments of *Neurological Surgery, §Oral Surgery, and ¶Anatomy, University of California, San Francisco, CA 94143-0112; and ‡Department of Biochemistry and Howard Hughes Medical Institute, University of Washington, Seattle, WA 98195 Contributed by Richard D. Palmiter, November 8, 2001 Noradrenaline (NA), a key neurotransmitter of the endogenous difficult interpretative scenarios because of the many non- pain inhibitory system, acutely inhibits nociceptive transmission noradrenergic neurons and͞or projections that are removed (including that mediated by substance P), potentiates opioid an- simultaneously. algesia, and underlies part of the antinociceptive effects of the To circumvent the problem of incomplete and variable nor- widely prescribed tricyclic antidepressants. Lesions of noradrener- adrenergic denervation, we chose to take a genetic approach to gic neurons, however, result in either normal or reduced pain studying the role of noradrenergic transmission in nociception behavior and variable changes in morphine antinociception, un- and morphine analgesia. Specifically, we studied pain behavior dermining the proposed association between noradrenaline (NA) and morphine analgesia in mice missing the gene coding for deficiency and chronic pain (hyperalgesia). We used mice lacking dopamine ␤-hydroxylase (DBH). A unique advantage of these the gene coding for dopamine ␤-hydroxylase, the enzyme respon- knockout mice is that NA can be restored, which allowed us to sible for synthesis of NA from dopamine, to reexamine the conse- determine whether the absence of NA, rather than some unrec- quences of a lack of NA on pain behavior. Here, we show that ognized developmental defect, is responsible for the findings. absence of NA in the central nervous system results in a substance NA is restored by administering a synthetic amino acid precursor P-mediated chronic hyperalgesia (decreased nociceptive threshold) of NA, L-threo-3,4-dihydroxyphenylserine (DOPS), which is to thermal, but not mechanical, stimuli and decreased efficacy of converted to NA by aromatic amino acid decarboxylase (AADC; morphine. Contrary to studies that show substance P-mediated ref. 20). When carbidopa, an inhibitor of AADC that does not hyperalgesia requires intense stimuli, we found that even a mild cross the blood–brain barrier, is coadministered with DOPS, NA stimulus is sufficient to evoke substance P-dependent hyperalgesia is produced essentially only in the CNS. in the NA-deficient mice. Restoring central NA normalized both the nociceptive threshold and morphine efficacy, which is consistent Materials and Methods with a tonic inhibitory effect of NA on nociceptive transmission. The DbhϪ/Ϫ mice used in this study were adult males, 20th Unexpectedly, however, antagonists to the substance P receptor generation 129͞SvEv and C57BL͞6J mixed genetic background. (the NK1 receptor) could achieve the same effect as NA replace- DbhϪ/Ϫ mice were phenotyped by identifying animals with ptosis ment. We conclude that when unopposed by NA, substance P and delayed growth and were genotyped by PCR. Controls were acting at the NK1 receptor causes chronic thermal hyperalgesia, Dbhϩ͞Ϫ littermates with normal levels of NA and adrenaline and that the reduced opioid efficacy associated with a lack of NA (21); they are normal for all phenotypes tested (20). Procedures is due to increased NK1-receptor stimulation. for the maintenance and use of the experimental animals were approved by the Animal Care and Use Advisory Committees at oradrenaline (NA) is an essential neurotransmitter of the the University of Washington and the University of California Nendogenous pain inhibitory system (1, 2) that tonically and San Francisco and were carried out in accordance with National phasically inhibits spinal nociceptive transmission, including that Institutes of Health regulations on animal use. mediated by substance P (3, 4). By stimulating central nervous system (CNS) ␣ adrenoreceptors, NA increases threshold and Behavioral Testing. For each individual experiment, the same 2 Ϫ/Ϫ latency to noxious stimuli without affecting responses to innoc- number of animals (between 8 and 12) was used for Dbh and uous stimuli, and potentiates the antinociceptive effects of control groups. Thermal nociceptive responses were assessed by opiates (5–10). using the hot-plate, tail-flick, and cold-plate tests. Individual Based on the above understanding, it has been suggested that mice were placed on the hot plate at 50°C (Stoelting), and the noradrenergic dysfunction is a key component of certain chronic delay for the first hindpaw lift was timed. Cut-off was at 30 s, NEUROBIOLOGY intractable pain disorders (11). A major caveat is that this except for experiments with morphine in which 50 s were used. hypothesis is based primarily on experimental studies that use The threshold for heat withdrawal was determined by setting the acute rather than chronic pain. Although acute lesions or hot plate at incrementing temperatures (43–47°C) and by count- inhibition of noradrenergic spinal afferents produce a state of ing the number of hindpaw lifts during a period of 4 min for each hyperalgesia and reduced antinociceptive effects of opiates (2, temperature. A delay of 1 h was allowed before each mouse was 10, 12), the increased pain behavior abates over time. In fact, exposed to a higher temperature. The tail-flick test was carried long-term effects of noradrenergic denervation have been re- ported to result in decreased pain behavior, whereas the antino- Abbreviations: NA, noradrenaline; CNS, central nervous system; DBH, dopamine ␤-hydrox- ciceptive effect of morphine is either reduced, unchanged, or ylase; DOPS, L-threo-3,4-dihydroxyphenylserine; AADC, aromatic amino acid decarboxyl- increased (13–17). These conflicting results can be caused in part ase; MPE, maximum possible effect; CFA, complete Freund’s adjuvant. by the variability in the extent of the removal of noradrenergic See commentary on page 549. neurons or terminals which, when using a neurotoxin, is both †To whom reprint requests should be addressed. E-mail: [email protected]. incomplete and nonselective. Moreover, because noradrenergic ʈPresent address: Department of History, Stanford University, Stanford, CA 94305. terminals contain more than one neurotransmitter (18, 19), the The publication costs of this article were defrayed in part by page charge payment. This effects of their elimination are difficult to interpret. Surgical article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. lesions of noradrenergic nuclei or pathways result in even more §1734 solely to indicate this fact. www.pnas.org͞cgi͞doi͞10.1073͞pnas.012598599 PNAS ͉ January 22, 2002 ͉ vol. 99 ͉ no. 2 ͉ 1029–1034 Downloaded by guest on October 2, 2021 out by placing the ventral surface of the middle third of the tail over a radiant heat source (Ugo Basile, Varese, Italy). With- drawal of the tail stopped the heat source, and the time was recorded automatically. Cut-off was set at 15 s. The cold-plate test (22) was performed by placing each mouse on a 4°C surface for 5 min. The number of hindpaw lifts during 4 min was recorded (22). Mechanical responses were assessed by using the tail-pressure and von Frey filament tests. For the tail-pressure test, mice were placed in a Plexiglas cylinder, and an increasing pressure was applied on the junction of the proximal and middle third of their tails with a Randall-Selitto apparatus (1-mm tip; Stoelting) until the tail was withdrawn. The test was repeated three times every 5 min. The threshold for withdrawal from mechanical stimulation to the plantar aspect of the hindpaws was determined with von Frey monofilaments (Stoelting). Filaments with a bending force from 0.01 g to 2.0 g were applied to the plantar aspect of each hindpaw until a paw withdrawal occurred in more than half of the trials. Progressively finer filaments were applied until the mechanical threshold was found. Motor func- tion was assessed by using a RotaRod (3.2-cm diameter rod, Ugo Basile) which was set at a constant low speed of 50 cm͞min. Once all animals were walking, the RotaRod was switched to an accelerating mode, reaching a cut-off speed of 400 cm͞min at 5 min. The maximal time in seconds each mouse was able to remain on the RotaRod was recorded. Animals were trained for 1 week before data collections. Exploratory behavior was as- sessed with the Hole board test (Stoelting). Mice were placed in the middle of a square surface (40 ϫ 40 cm) perforated with 16 equally spaced 2.9-cm holes and allowed to explore freely for 4 min. The number of holes visited during the test period was recorded. Motor function and exploratory behavior were as- sessed for the highest dose of each drug resulting in an antino- ciceptive effect to ensure that the increase in withdrawal latency was due to sensory change rather than a psychomotor effect. Peripheral Vascular Perfusion. Hindpaw cutaneous blood perfusion was assessed with a 2D Laser Doppler Imager (Moor Instru- ments, Axminster, U.K.) equipped with a near-infrared (780 nm) laser. This system measures blood flow in deep-lying subcuta- neous vessels (23) for both hindpaws without any physical Fig. 1. Brainstem noradrenergic cell groups and their projections. (a, aЈ) contact. Mice were lightly anesthetized with 1% isoflurane Ϫ/Ϫ mixed with 100% oxygen, which was delivered by means of a Immunolabeling for DBH was absent from Dbh brainstem noradrenergic cell groups such as the locus coeruleus compared with normal labeling in vaporizer connected to a face mask. Scanning was done twice ͞ control mice (arrow). (b) Neurons in noradrenergic cells groups contain equiv- over 60 s at a resolution of 0.2 mm pixel for each animal. alent amounts of AADC in DbhϪ/Ϫ (arrow pointing to locus coeruleus) and control mice (see arrow, Fig.