Pronociceptive effect of 5-HT_1A receptor agonist on visceral pain involves spinal N-methyl-d-aspartate (NMDA) receptor

Abstract

The functional role of serotonergic 5-HT_1A receptors in the modulation of visceral pain is controversial. The objective of this study was to systematically examine the mechanism and site of action of a selective 5-HT_1A receptor agonist 8-hydroxy-2-(di-n-propylamino)-tetralin (DPAT) on visceral pain. In the behavioral model of visceral pain, systemic injection (5–250 μg/kg) of DPAT produced a significant increase in the viscero-motor response (VMR) to colorectal distension (CRD) and this effect was blocked by the selective 5-HT_1A receptor antagonist WAY-100135 (5 mg/kg, s.c.). Similarly, intrathecal (i.t.) injection (5 μmol) of DPAT into the lumbo-sacral (L6–S1) spinal cord produced a significant increase in VMR. The administration of N-methyl d-aspartate (NMDA) receptor antagonist AP5 (50 μg/kg) prior to DPAT injection completely blocked the pronociceptive effect of DPAT. Similarly, DPAT failed to increase VMR in rats chronically treated with NR1 subunit-targeted antisense oligonucleotide (ON), whereas the drug increased VMR in rats treated with mismatched-ON. Chronic i.t. injection of allylglycine (AG), a γ-amino decarboxylase (GAD) enzyme inhibitor, produced significant increase in VMRs, suggesting that the inhibition of GABA synthesis produces pronociception. In AG-treated rats, i.t. injection of DPAT failed to further increase in VMR, suggesting that the DPAT action is linked to GABA release. Similarly, WAY-100135 failed to attenuate VMR in AG-treated rats, suggesting that unlike DPAT, AG action is not via the activation of 5-HT_1A receptors. In electrophysiology experiments, DPAT (50 μg/kg) significantly increased the responses of spinal neurons to CRD, but did not influence the mechanotransduction property of CRD-sensitive pelvic nerve afferent fibers. The effect of DPAT on spinal neurons remained unaffected when tested in spinal-transected (C1–C2) rats. These results indicate that the 5-HT_1A receptor agonist DPAT produces pronociceptive effects, primarily via the activation of presynaptic 5-HT_1A receptors in GABAergic neuron to restrict GABA release and thereby disinhibits the excitatory glutamatergic neurons in the spinal cord.

Introduction

5-HT₁, a negative G-protein (Gi/Go)-coupled serotonin (5-HT) receptor, plays diverse and complex functions including cognition, emotion (fear and rage) and pain modulation (Millan and Colpaert, 1990, Millan et al., 1996, Kayser et al., 2007). Of five subclasses (5-HT_{1A, B, D, E, F}), 5-HT_1A is the most prevalent receptor in CNS. The specific binding of [³H]-8-hydroxy-2-(di-n-propylamino) tetralin ([³H]-DPAT), a 5-HT_1A receptor agonist, was detected in several regions of the rat brain. The major binding sites were found in the cortex, hippocampus, striatum, periaqueductal gray (PAG), rostroventral medulla (RVM) and spinal cord (Gozlan et al., 1983, Hall et al., 1985, Viisanen and Pertovaara, 2010). Although 5-HT_1A receptors are present both at pre- and post-synaptic sites, in some areas including the spinal cord the expression is predominantly at pre-synaptic sites. This pre-synaptic location of the receptor has complicated autoreceptor functions that regulate the release of specific neurotransmitters to modulate neuronal functions. For example, studies have documented that 5-HT_1A expressing neurons in the midbrain and spinal cord play a significant role in pain modulation by regulating the opioid release (Millan and Colpaert, 1990, Millan and Colpaert, 1991, Millan et al., 1996, Song et al., 2007). In the rat spinal cord, 5-HT_1A receptors are the major subclass accounting for 30–50% of the total population of 5-HT₁ (i.e., 5-HT_1A–F) and are primarily concentrated in the superficial laminae (I, III and IV) (Huang and Peroutka, 1987, Thor et al., 1990, Marlier et al., 1991, Laporte et al., 1996). The receptor exhibits a rapid increase in the expression under pathological conditions including peripheral inflammation and spinal cord injury (Zhang et al., 2002, Otoshi et al., 2009). Some studies indicate that the activation of 5-HT_1A receptors in the spinal cord and midbrain results in somatic antinociception (Gillet et al., 1985, Eide et al., 1988, El-Yassir et al., 1988, Giordano, 1989, El-Yassir and Fleetwood-Walker, 1990, Eide and Hole, 1991, Giordano and Rogers, 1992, Eide and Hole, 1993, Galeotti et al., 1997, Xu et al., 1994, Oyama et al., 1996, Gjerstad et al., 1996, Xiao et al., 2005, Wei and Pertovaara, 2006), whereas other studies report pronociceptive effect (Solomon and Gebhart, 1988, Zemlan et al., 1988, Crisp et al., 1991a, Crisp et al., 1991b, Alhaider and Wilcox, 1993, Ali et al., 1994, Zhang et al., 2001). Similarly, in the rodent model of visceral pain while one study indicates that the partial 5-HT_1A receptor agonist buspirone produces analgesia to noxious colon distension (Sivarao et al., 2004), a recent report indicates that 5-HT_1A antagonists (WAY-100635 and AZD7371) produce visceral analgesia in the same species (Lindström et al., 2009). Although these conflicting results are difficult to explain, it could be related to the subjective nature (visual estimate of abdominal contraction) of pain scoring; the strain of rats used and most importantly the physiological condition of the animal.

The objective of the present study was to examine the mechanism and the site of action of a selective 5-HT_1A receptor agonist DPAT in the rat model of visceral pain. We hypothesize that the pronociceptive effect of DPAT is due to activation of negative G-protein (Gi/Go)-coupled 5-HT_1A receptor located at the presynaptic terminals of GABAergic interneurons that results in hyperpolarization of cells and the reduction of γ-amino butyric acid (GABA) release. Since GABA primarily acts as an inhibitory neurotransmitter in the spinal cord and modulates the functions of excitatory glutamatergic neurons, the reduction of GABA release will disinhibit these neurons to produce hyperexcitation of spinal dorsal horn neurons resulting in visceral hyperalgesia.