Molecular Brain Research 63 Ž1998. 180–183
Reduced nociceptive behavior in islet amyloid polypeptide žamylin/ knockout mice Samuel Gebre-Medhin
, Hindrik Mulder b,c , Yanzhen Zhang Christer Betsholtz a
, Frank Sundler b,
Department of Medical Biochemistry, Goteborg UniÕersity, Box 440 SE 405 30, Goteborg, Sweden ¨ ¨ Department of Physiology and Neuroscience, Section for Neuroendocrine Cell Biology, Lund UniÕersity, Lund, Sweden c Department of Cell and Molecular Biology, Lund UniÕersity, Lund, Sweden d HarÕard Institutes of Medicine, Boston, USA Accepted 22 September 1998
Abstract Islet amyloid polypeptide ŽIAPP or amylin. is predominantly expressed by insulin cells, but occurs also in primary sensory neurons in the rat. Here, using mice targeted for a null mutation in the IAPP gene, we establish murine expression of IAPP in sensory neurons; its distribution in a population of calcitonin gene-related peptide-containing neurons in the spinal cord and dorsal root ganglion is similar to that previously described in the rat. We also report the IAPP mutant mice display a reduced pain response in the paw formalin test. Adjuvant-induced joint inflammation was not altered in IAPP mutants, arguing against a peripheral inflammatory abnormality. These findings lead us to suggest that IAPP has a pro-nociceptive function in primary sensory neurons. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Islet amyloid polypeptide; Calcitonin gene-related peptide; Substance P; Gene knock out; Dorsal root ganglion ŽDRG. neurons; Paw formalin test; Adjuvant-induced inflammation
Islet amyloid polypeptide ŽIAPP., also termed amylin, is an insulin cell-derived glucoregulatory peptide, of which the human form has amyloidogenic properties w19x. There are structural Žapproximately 50%. and functional similarities between IAPP and the neuropeptide calcitonin gene-related peptide ŽCGRP., suggesting that these peptides may have overlapping or related functions w1,2,20x. Interestingly, IAPP is also expressed in a population of rat dorsal root ganglion ŽDRG. neurons containing CGRP andror substance P w13x; IAPP occurs in small- to medium-sized nerve cell bodies in DRG issuing fibers to the spinal cord dorsal horn and, to a lesser extent, peripheral tissues known to receive sensory innervation. Although the function of neuronal IAPP is unknown, its expression pattern in rat sensory neurons mimics that of CGRP and substance P following noxious stimulation Žup-regulation. w15x or axotomy Ždown-regulation. w14x; these are regulatory events
) Corresponding author. Fax: q46-31416108; E-mail: [email protected]
believed to reflect nociceptive adaptation w3,9,14–16x. Against this background, it may be conceived that IAPP is involved in the processing of somatosensory transmission. We recently generated IAPP gene knockout Ž IAPPyry . mice w7x. These mice display insulin- and glucoregulatory abnormalities due to the loss of IAPP in insulin cells, consistent with the proposed role for IAPP in glucose homeostasis. In the present study we confirm the expression of IAPP in primary sensory neurons in the mouse, and its loss in sensory neurons in IAPPyry mice. To address the function of IAPP in sensory neurons, we monitored nociceptive behavior and local inflammatory responses in IAPPyry mice and wild-type Ž IAPPqrq . controls using the paw formalin test, and a model of local adjuvant-induced inflammation. The animals used were 8–12 week-old male 129rC57BL6 hybrids and were F3-descendants from the same chimeric mouse Žimmunocytochemistry; paw formalin test., or 129rC57BL6rFVBrN and C57BL6 males Ž IAPPyry mice and IAPPqrq controls, respectively; adjuvant-induced inflammation test.. The experimental pro-
0169-328Xr98r$ - see front matter q 1998 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 9 - 3 2 8 X Ž 9 8 . 0 0 2 6 9 - 1
S. Gebre-Medhin et al.r Molecular Brain Research 63 (1998) 180–183
cedures were approved by The Animal Ethics Committee of the University of Goteborg. For immunocytochemistry, ¨ the spinal cord and DRG’s were excised from sacrificed mice, immersed overnight in 2% paraformaldehyde and 0.2% picric acid in phosphate buffer ŽpH 7.2., repeatedly rinsed in sucrose Ž10%.-enriched phosphate buffer ŽpH 7.2. for cryoprotection, and frozen on dry ice. Sections were cut Ž10 mm. in a cryostat and incubated with primary antibodies for single and double immunofluorescence, using antibodies to IAPP ŽF055-24, Amylin Pharmaceuticals, San Diego, CA, USA. and CGRP Ž8425; Euro-Diagnostica, Malmo, ¨ Sweden., as previously described w13x. Biphasic nociceptive behavioral responses were induced by injecting 0.02 ml of 2% paraformaldehyde in phosphate buffer ŽpH 7.2. subcutaneously under the dorsal surface of the right hind paw, as previously described w18x. Total time of licking of the injected paw during the early phase Ž0–5 min after injection. and the late phase Ž10–30 min after injection. was determined using a stop watch and defined as nociceptive responses as described w18x. Local adjuvant-induced joint inflammation was induced using Freund’s complete adjuvant ŽFCA., as previously described, w11,15x. Because a close correlation exists between the histopathological alterations in FCA-induced joint inflammation, and the circumference of the inflamed joint w4x, joint diameter was defined as the parameter of inflammation. Using a digital calliper, joint diameters were measured before Ž t s 0., and at 12 h, 24 h, 3 and 7 days after injection of adjuvant. The expression of IAPP and CGRP in DRG and in the dorsal horn of the spinal cord was investigated using immunofluorescence ŽFig. 1.. In IAPPqrq mice, IAPPimmunoreactivity occurred in the superficial layers of the dorsal horns of the spinal cord ŽFig. 1A., in a population of CGRP-immunoreactive nerve fibers Žnot shown.; these findings are similar to previous findings in the rat w13x. By contrast, IAPP-immunoreactive nerve fibers were lacking in the dorsal horn of the spinal cord in IAPPyry mice ŽFig. 1B., consistent with the disruption of the IAPP gene. In the same sections, double immunofluorescence demonstrated a dense plexus of CGRP-immunoreactive nerve fibers in the spinal dorsal horns of IAPPyry mice ŽFig. 1C., indistinguishable from that found in IAPPqrq mice Žnot shown.. Double immunofluorescence in DRG obtained from IAPPqrq mice revealed that IAPP occurred in a subpopulation of CGRP-immunoreactive small-sized nerve cell bodies, whereas IAPP-immunoreactive cell bodies were lacking in IAPPyry mice Žnot shown.. To study a possible role for neuronal IAPP in nociception, IAPPyry mice were examined in the paw formalin test, a test known to produce a biphasic nociceptive behavioral response in rodents w18x ŽFig. 2.. The duration of nociceptive behavior in the early phase did not differ significantly between IAPPyry mice and controls Ž53 " 10 s vs. 67 " 12 s for IAPPyry mice and IAPPqrq controls, respectively. ŽFig. 2.. In the late phase, however, IAPPyry
Fig. 1. Immunofluorescence of IAPP and CGRP in the murine spinal dorsal horn. IAPP-immunoreactivity in a wild-type spinal cord section ŽA.; superficial nerve fibre layers of the dorsal horn are stained, demonstrating that IAPP is expressed in murine sensory neurons. Double immunofluorescence in a spinal cord section obtained from a IAPPyry mouse ŽB, C.. Consistent with the IAPPyry genotype, IAPP-immunoreactivity is lacking in the dorsal horn ŽB., whereas CGRP-immunoreactive nerve fibers remain, forming a dense plexus ŽC.. Staining of blood vessels occurs due to the use of monoclonal antibodies Žsecondary antibody recognizes mouse immunoglobulin..
mice displayed a significant reduction in the duration of nociceptive behavior compared to controls Ž141 " 24 s vs. 215 " 33 s for IAPPyry mice and IAPPqrq controls, respectively; P s 0.027; two-tailed Mann–Whitney U– test. ŽFig. 2.. Because other sensory neuropeptides Že.g., substance P and CGRP. have been implicated in the control of local inflammation w8x, we reasoned that decreased nociceptive behavior in IAPPyry mice could reflect an altered inflammatory response to noxious stimulation. We therefore examined the impact and course of unilateral FCA-induced joint inflammation w11,15x ŽFig. 3.. The degree of inflammation, determined by measuring the ankle diameter, was monitored up to 1 week after injection of FCA ŽFig. 3.. The ipsilateral ankle diameter gradually increased during the test in both IAPPyry mice and IAPPqrq controls,
S. Gebre-Medhin et al.r Molecular Brain Research 63 (1998) 180–183
Fig. 2. Nociceptive behavior in the paw formalin test. Nociceptive responses in the early phase Ž0–5 min. and late phase response Ž10–30 min. following formalin injection is shown. In the late phase, IAPPyry mice Žblack bars; ns12. display a shorter duration of nociceptive behavior than wild-type controls Žwhite bars, ns10.. Duration of nociceptive behavior in the early phase did not differ between the two groups. Values are means"S.E.M. Statistical significance was accepted at P 0.05; ) P s 0.027; Mann–Whitney U-test.
indicating the onset of a local inflammation. However, no significant difference in ipsilateral ankle diameter was observed between the two groups at any time point studied ŽFig. 3., demonstrating that adjuvant-induced joint inflammation was not altered in the mutant mice. The contralateral ankle diameter remained unchanged during the test in both IAPPyry mice and IAPPqrq controls ŽFig. 3.. Reduced nociceptive behavior in IAPPyry mice implies that IAPP exerts a pro-nociceptive action in sensory neurons. A role for IAPP in nociception has previously been proposed based on its neuronal expression pattern in small-sized nerve cell bodies in rat DRG w13x; these neurons are known to give rise to C-type unmyelinated nerve fibers, which are involved in nociceptive transmission. Further support for a nociceptive role for IAPP comes from the notion that pro-nociceptive neuropeptides are generally believed to be excitatory transmitters promoting sensory transmission, e.g., pain, in the spinal cord. Thus, excitatory transmitters are down-regulated upon axotomy, a phenomenon believed to reflect anti-nociceptive adaptation w9x. Indeed, IAPP-expression is down-regulated in dorsal root ganglion neurons and in spinal cord nerve fibers following sciatic nerve transection w14x. The involvement of IAPP in nociception has also been inferred by its rapid up-regulation of in DRG after a noxious stimulus has been applied w15x. A similar regulation is also evident for other neuropeptides believed to be involved in nociception such as substance P and CGRP w3,16x. The importance of substance P in nociception has recently been demonstrated
by the finding that mice genetically devoid of substance P have no significant pain responses following formalin injection w21x. Release of sensory neuropeptides following noxious stimulation occurs both in peripheral tissues receiving sensory innervation, and in the dorsal horn of the spinal cord w6,8x. Neuropeptides are therefore proposed to modulate pain transmission by influencing local inflammation in peripheral tissues as well as regulating somatosensory processing in the spinal dorsal horn. However, normal local adjuvant-induced inflammation in peripheral tissues in IAPPyry mutants argues against a peripheral inflammatory mechanism as the underlying cause of reduced nociception in these mice. It is therefore possible that neuronal IAPP exerts its function in the dorsal horn of the spinal cord. In view of that a specific IAPP receptor has not yet been identified, we can only speculate on the precise mechanisms responsible for the sensory phenotype observed in IAPPyry mice. Cloning and functional characterization of CGRP-receptors has failed to demonstrate IAPP as a functional ligand w10,12x, but receptor specificity for this family of peptides has recently been shown to be complex involving receptor-activity-modifying proteins ŽRAMPs. w12x. Yet, the structural similarities between IAPP and CGRP w2,20x together with studies with CGRP-antagonists w5,17x suggest that the two peptides activate one or several common receptors. The sensory phenotype observed in IAPPyry mice is unexpected in
Fig. 3. Tarso-tibial joint diameters at different time points after unilateral injection of Freunds complete adjuvant. Ipsilateral Žleft. joint diameter increased similarly in IAPPyry mice Žblack symbols; ns8. and wildtype mice Žwhite symbols; ns8., demonstrating a similar onset and course of inflammation. The contralateral Žright. joint diameter remained constant during the test, ensuring that joint inflammation was not disseminated. Values are means"S.E.M.
S. Gebre-Medhin et al.r Molecular Brain Research 63 (1998) 180–183
view of that IAPP is expressed at a low level in a subpopulation of CGRP-containing sensory neurons w13x Žthis study.. There is, however, evidence of a dissociated regulation of IAPP and CGRP in neurons in innervating DRG after adjuvant-induced inflammation; up-regulation of IAPP expression is more rapid and transient than that of CGRP, which is slower and more sustained w15x. Hence, during situations of induced expression, IAPP may act at a time point that precedes the activity of CGRP. If this scenario is accepted, and common receptors are used, a time frame may exist in which functional compensation by CGRP for the lack of IAPP cannot occur. In summary, our studies have confirmed that IAPP is also a constituent of sensory neurons in mice. Genetic ablation of IAPP in sensory neurons produced a phenotype in which mice are more tolerant to a noxious stimulus. These findings suggest that IAPP, along with other sensory neuropeptides, modulate somatosensory transmission.
Acknowledgements We thank Amy Percy for providing FO55-24. This work was supported by the Swedish Medical Research Council, Swedish Diabetes Association, the Novo Nordisk, Society for Medical Research, Goteborg Medical Society, ¨ Crafoord’s, Konrad & Helfrid Johansson’s, Albert Pahls˚ son’s and Wiberg’s, Swedish Medical Society Research, Trygg-Hansa Research, and the Swedish Cancer Research Foundations, and by the Faculties of Medicine, Goteborg ¨ and Lund Universities.
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