Nocistatin and nociceptin exert opposite effects on the excitability of central amygdala nucleus-periaqueductal gray projection neurons

Abstract

Central amygdala nucleus (CeA)-periaqueductal gray (PAG) pathway is the component of descending antinociceptive circuitry. Nociceptin/orphanin FQ (N/OFQ) and nocistatin (NST) produce supraspinal pronociceptive and antinociceptive effects, respectively. We hypothesized that opposite effects of N/OFQ and NST on supraspinal pain modulation result from their opposing effects on the excitability of CeA-PAG projection neurons. This hypothesis was tested by investigating electrophysiological effects of N/OFQ and NST on medial CeA neurons that project to PAG (CeA_M-PAG). N/OFQ hyperpolarized CeA_M-PAG projection neurons by enhancing inwardly rectifying potassium conductance. In contrast, NST depolarized CeA_M-PAG neurons by causing the opening of TRPC cation channels via G_αq/11-PLC-PKC pathway. CeA_M-PAG neurons hyperpolarized by N/OFQ express CRF or neurotensin mRNA. NST-responsive CeA_M-PAG neurons contain CRF or substance P mRNA. Our study provides the evidence that the molecular and cellular basis for opposite effects of N/OFQ and NST on supraspinal pain regulation is their opposing effects on the excitability of peptidergic CeA_M-PAG neurons.

Introduction

Nociceptin/orphanin FQ (N/OFQ) was originally identified as the endogenous ligand for ORL1 (opioid receptor-like 1 receptor), which exhibits a high degree of sequence homology to that of μ-, δ- or κ-opioid receptor (Meunier et al., 1995, Reinscheid et al., 1995). In contrast with the analgesia produced by opioid peptides, intracerebroventricular injection of N/OFQ induced supraspinal hyperalgesia (Meunier et al., 1995, Reinscheid et al., 1995, Mogil and Pasternak, 2001) and reversed supraspinal analgesia induced by exogenously applied μ-, δ- and κ-opioid receptor agonists (Grisel et al., 1996, Mogil et al., 1996, Calo et al., 1998). N/OFQ is derived from a larger precursor named prepronociceptin/orphanin FQ (ppN/OFQ) that contains several biologically active peptides (Mollereau et al., 1996, Nothacker et al., 1996). Nocistatin (NST) is also an active neuropeptide cleaved from ppN/OFQ (Okuda-Ashitaka et al., 1998, Okuda-Ashitaka and Ito, 2000). In spite of being generated from the same precursor ppN/OFQ, NST is unable to bind to N/OFQ receptor (NOP receptor; formerly ORL1), and specific binding sites for NST are expressed in the brain and spinal cord (Okuda-Ashitaka et al., 1998). Furthermore, NST and N/OFQ produce opposite biological effects in the CNS, and NST has been shown to antagonize several N/OFQ-mediated effects in the brain (Nicol et al., 1998, Hiramatsu and Inoue, 1999, Okuda-Ashitaka and Ito, 2000, Olszewski et al., 2000; Gavioli et al., 2002). In contrast with N/OFQ-mediated pronociceptive effect at the supraspinal level, NST has been shown to induce analgesic effect following intracerebroventricular administration (Okuda-Ashitaka et al., 1998, Nakagawa et al., 1999). NST also antagonizes N/OFQ-induced hyperalgesic effects at the supraspinal level (Okuda-Ashitaka et al., 1998, Zhao et al., 1999, Okuda-Ashitaka and Ito, 2000, Liu et al., 2006). Thus, NST acts as a functional antagonist of N/OFQ in modulating nociceptive transmission. The cellular and molecular basis for opposite effects of N/OFQ and NST on supraspinal pain modulation remains unknown.

Nociceptive transmission in the dorsal horn of spinal cord is modulated by brain descending antinociceptive circuitry (Fields, 2000, Millan, 2002). Initial studies identified midbrain periaqueductal gray (PAG) and rostral ventromedial medulla (RVM) as key components of brainstem descending pathway that inhibits pain transmission in the spinal dorsal horn (Fields et al., 1991, Bandler and Shipley, 1994). Analgesic effect produced by PAG excitation is mediated by an excitatory innervation from PAG to RVM, which projects to the dorsal horn of spinal cord and inhibits nociceptive transmission (Fields et al. 1991). Multiple lines of evidence indicate that the amygdaloid complex is also a major component of brain descending pain-modulating pathway and plays an important role in regulating spinal nociceptive transmission (Manning, 1998, Millan, 2002). Electrical or chemical stimulation of central amygdala nucleus (CeA), which is one of output nuclei of amygdala and predominant source of amygdaloid projection to the brainstem (Sah et al., 2003), inhibits spinal pain transmission and produces an antinociceptive effect (Oliveira and Prado, 2001, Manning et al., 2003, Ortiz et al., 2007). CeA sends a dense ipsilateral projection to lateral and ventrolateral regions of PAG (Rizvi et al., 1991). Inactivation of ventrolateral PAG neurons significantly inhibits antinociceptive effect induced by electrical stimulation of CeA (Oliveira and Prado, 2001). Therefore, CeA activation-induced analgesia depends on CeA-PAG connection and results from the activation of CeA-PAG-RVM-spinal cord descending antinociceptive circuitry. The amygdaloid complex plays an essential role in processing emotional information and coordinating appropriate behavioral and autonomic responses including fear-induced antinociception (LeDoux, 2000, Sah et al., 2003). Previous studies demonstrated that stress-induced analgesia requires an intact amygdala and is mediated by CeA-PAG neuronal circuitry (Bellgowan and Helmstetter, 1996, Emery et al., 2001). Bilateral lesion of CeA significantly reduces the analgesic effect of systemically administered morphine or cannabinoid receptor agonist WIN55,212-2 (Manning and Mayer, 1995, Manning, 1998, Manning et al., 2003). Thus, CeA-PAG-RVM-spinal cord pain modulatory circuitry contributes to antinociceptive effects produced by psychological state of fear and numerous neurotransmitters including opioid peptides.

A large number of peptidergic neurons in the CeA project to ventrolateral PAG (Gray and Magnuson, 1992). CeA-PAG projection neurons have been shown to contain corticotropin-releasing factor (CRF), galanin, neurotensin or substance P (Gray and Magnuson, 1987, Gray and Magnuson, 1992, Gray, 1993). When microinjected into PAG, neurotensin, galanin or substance P produces a potent analgesic effect (Al-Rodhan et al., 1991, Wang et al., 2000, Rosen et al., 2004). CRF is believed to induce supraspinal analgesia by activating brainstem descending antinociceptive circuitry (Vit et al., 2006). Our previous study demonstrated that neurotensin exerts a direct excitatory effect on PAG-RVM projection neurons (Li et al., 2001a). Electrophysiological recording studies also reported that CRF or substance P depolarizes and excites PAG neurons (Bowers et al., 2003, Drew et al., 2005). Thus, peptidergic CeA-PAG projection neurons play an important role in supraspinal pain modulation by regulating the activity of PAG neurons and PAG-RVM descending antinociceptive pathway.

When supraspinally applied, N/OFQ and NST induce hyperalgesic and antinociceptive effects, respectively (Meunier et al., 1995; Reinscheid et al., 1995, Nakagawa et al., 1999, Okuda-Ashitaka and Ito, 2000). Immunocytochemical staining and in situ hybridization studies showed that ppN/OFQ mRNA and mRNA or protein of NOP receptor are highly expressed in CeA (Anton et al., 1996, Mollereau et al., 1996, Nothacker et al., 1996, Boom et al., 1999, Houtani et al., 2000). CeA-PAG-RVM descending pain-modulating pathway is the supraspinal action site of opioid- or cannabinoid-induced analgesia (Manning, 1998, Manning et al., 2003). We hypothesized that N/OFQ and NST regulate spinal nociceptive transmission by directly modulating the activity of CeA neurons projecting to PAG and that opposite effects of N/OFQ and NST on supraspinal pain modulation result from their opposing effects on the excitability of CeA-PAG projection neuron. To test this hypothesis, we investigated electrophysiological effects of N/OFQ and NST on retrogradely labeled CeA-PAG projection neurons in brain slice and identified N/OFQ- or NST-responsive peptidergic CeA-PAG projection neurons.