Neurotensin activation of the NTR1 on spinally-projecting serotonergic neurons in the rostral ventromedial medulla is antinociceptive A.V. Buhler * , J. Choi, H.K. Proudfit, G.F. Gebhart Department of Pharmacology, Carver College of Medicine, The University of Iowa, Iowa City, IA 52242, USA Received 20 August 2004; received in revised form 7 December 2004; accepted 20 December 2004 Abstract Microinjection of neurotensin (NT) in the rostral ventromedial medulla (RVM) produces dose-dependent antinociception. The NTR1 (Neurotensin Receptor Subtype 1) may mediate part of this response, however definitive evidence is lacking, and the spinal mediators of NTR1-induced antinociception are unknown. In the present study, we used immunohistochemical techniques to show that the NTR1, but not the NTR2 is expressed by spinally projecting serotonergic neurons of the RVM. We also show that microinjection of NT or the NTR1- selective agonist PD149163 in the RVM both produce dose-dependent antinociception in the tail-flick test that is blocked by the NTR1- selective antagonist SR48692. The antinociception produced by NT or PD149163 is also blocked by intrathecal administration of the non- selective serotonergic receptor antagonist methysergide. The results of these experiments provide anatomical and behavioral evidence that activation of NTR1-expressing spinally projecting neurons in the RVM produces antinociception through release of serotonin in the spinal dorsal horn. These results support the conclusion that the NTR1 plays an important role in the central modulation of nociception. q 2005 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved. Keywords: Nucleus raphe magnus; Nociception; Immunohistochemistry; Serotonin; Dorsal horn; Neurotensin 1. Introduction The rostral ventromedial medulla (RVM), which includes the nucleus raphe magnus (NRM), is one of several brainstem nuclei that exert powerful modulatory influences on the spinal transmission of pain. Neurons in the RVM, including those that contain serotonin and GABA, send projections to the dorsal horn of the spinal cord directly (Antal et al., 1996; Bowker and Abbott, 1990) and indirectly through relays in the dorsolateral pons (Clark and Proudfit, 1991; Holden and Proudfit, 1998). Microinjection of neurotensin (NT) in the RVM has complex effects on nociception. Low doses of NT (0.03 pmol–0.03 nmol) facilitate nociception (Urban and Smith, 1993) whereas greater doses of NT (1.3–30 nmol) are antinociceptive (Behbehani and Pert, 1984; Fang et al., 1987; Holmes et al., 1999; Neubert et al., 2004; Sarhan et al., 1997; Urban and Smith, 1993; Urban et al., 1996b, 1999). These opposing effects of intra-RVM NT appear to be mediated by different neuronal pathways (Urban et al., 1996b) and have led to the suggestion that descending facilitation, or an imbalance of facilitation and inhibition, may underlie some chronic pain states (Porreca et al., 2002; Urban and Gebhart, 1999). The importance of the RVM neurotensinergic system is further supported by findings that morphine microinjection in the periaqueductal gray produces NT-mediated effects in the RVM (Smith et al., 1997; Urban and Smith, 1993, 1994). It is unclear which of three cloned NT binding proteins are responsible for the antinociceptive effects of NT; the NTR1 and NTR2 are G-protein coupled receptors (Chalon et al., 1996; Tanaka et al., 1990), while the functional activity of gp95/sortilin/NTR3 is undefined (Mazella et al., 1998; Nielsen et al., 1999). The NTR2 is expressed in the NRM (Sarret et al., 2003), but it is not known whether the NTR1 or sortilin/NTR3 are expressed by neurons in this region. However, reports that the antinociception produced by high doses of NT microinjected directly into the RVM 0304-3959/$20.00 q 2005 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.pain.2004.12.031 Pain 114 (2005) 285–294 www.elsevier.com/locate/pain * Corresponding author. Tel.:C1 319 335 7950; fax: C1 319 335 8930. E-mail address: amber-buhler@uiowa.edu (A.V. Buhler).