Elsevier

Brain Research

Volume 1070, Issue 1, 27 January 2006, Pages 90-100
Brain Research

Research Report
Catecholaminergic activation of G-protein coupling in rat spinal cord: Further evidence for the existence of dopamine and noradrenaline receptors in spinal grey and white matter

https://doi.org/10.1016/j.brainres.2005.10.101Get rights and content

Abstract

[35S]GTPγS autoradiography of slide-mounted tissue sections was used to examine G-protein coupling in the rat spinal cord, as stimulated by dopamine, the D1 receptor agonist SKF 38393, noradrenaline, and noradrenaline in the presence of the alpha adrenoceptor antagonist, phentolamine. Measurements were obtained from the different laminae of spinal grey and from the dorsal, lateral, and ventral columns of white matter, at cervical, thoracic, and lumbar levels. At every level, there was a relatively strong basal incorporation of GTPγS in laminae II–III > lamina IV–X of spinal grey, even in presence of DPCPX to block endogenous activation by adenosine A1 receptors. Dopamine, and to a lesser degree SKF 38393, but not the D2 receptor agonist quinpirole, stimulated G-protein coupling in laminae IV–X. Both dopamine and SKF 38393 also induced a weak but significant activation throughout the white matter. In both grey and white matter, the activation by dopamine was markedly reduced in presence of a selective D1 receptor antagonist. Noradrenaline strongly stimulated coupling throughout the spinal grey at all levels, an effect that was uniformly reduced in the presence of phentolamine. With or without phentolamine, there was also significant stimulation by noradrenaline in the white matter. Under the same experimental conditions, alpha 1, alpha 2, and beta adrenergic receptor agonists failed to activate GTPγS incorporation in either grey or white matter. However, in the presence of selective alpha 1 or alpha 2 receptor antagonist, significant reductions of noradrenaline-stimulated GTPγS incorporation were observed in both grey and white matter. The beta antagonist propanolol reduced GTPγS incorporation in grey matter only. Thus, the results confirmed the existence of D1 dopamine receptors and of alpha 1, alpha 2, and beta adrenergic receptors in the grey matter of rat spinal cord. In white matter, they strongly suggested the presence of dopamine D1, and of alpha 1 and alpha 2 adrenergic receptors on glia and/or microvessels, that might be activated by diffuse transmission in vivo.

Introduction

The rat spinal cord receives catecholamine inputs from both dopamine (DA) and noradrenaline (NA) neurons. Studies combining injection of various fluorescent retrograde tracers with tyrosine hydroxylase (Hökfelt et al., 1979) or dopamine-β-hydroxylase immunocytochemistry (Westlund et al., 1981) have indicated that the cell bodies of origin of the DA innervation are located in the caudal diencephalon (A11 group), whereas the NA projection arises from cell bodies in the A5–A7 groups of the pons, i.e., the locus coeruleus, subcoeruleus and Kolliker–Fuse nucleus (Westlund et al., 1983). Following on early fluorescence histochemical studies (Olson et al., 1973, Nygren and Olson, 1977, Björklund and Skagerberg, 1979a, Björklund and Skagerberg, 1979b), light and electron microscopic immunocytochemical studies using antibodies against DA, dopamine-β-hydroxylase, or NA have revealed distinct distributional patterns and ultrastructural features for the DA and NA innervations of rat spinal cord (DA: Yoshida and Tanaka, 1988, Shirouzu et al., 1990, Ridet et al., 1992, Holstege et al., 1996; NA: Hagihira et al., 1990, Rajaofetra et al., 1992, Ridet et al., 1993). DA immunoreactive axon terminals are sparsely distributed in laminae III–X of the spinal grey, i.e., in dorsal horn, intermediolateral cell column, ventral horn, and around the central canal. There are only few immunoreactive terminals in laminae I and II, irrespective of the segmental level examined. NA innervation is more diffuse and abundant, with terminals in all laminae at every level, most numerous in laminae I and II of dorsal horn, the intermediolateral cell column, the motoneuron areas of the ventral horn, and around the central canal.

In keeping with the distribution of the DA terminals, convergent ligand binding, immunocytochemical and in situ hybridization data indicate the presence of D1 and D2 receptors in the deep layers of dorsal horn, the intermediolateral cell column, and in the vicinity of motoneurons (Dubois et al., 1986, Bouthenet et al., 1987, Yokohama et al., 1994, van Dijken et al., 1996), and of D3 receptors in the dorsal horn (Levant and McCarson, 2001). According to these studies, the D1 receptor sites are most numerous in the ventral horn, whereas the highest density of D2 sites is in laminae II–III of the dorsal horn. Alpha 1 and alpha 2 adrenergic receptors have been detected in all regions of rat spinal grey (Roudet et al., 1993, Roudet et al., 1994, Pieribone et al., 1994), with a predominance of alpha 1 subtypes in the ventral horn and alpha 2 subtypes in the superficial layers of dorsal horn. Beta 1 and beta 2 adrenergic receptors appear to be uniformly distributed throughout the spinal grey (Patterson and Hanley, 1987, Schrader and Grobecker, 1989). It has also been reported that DA and NA receptors are expressed by spinal cord astrocytes, at least in explant cultures of rat spinal cord (Hösli and Hösli, 1982, Hösli and Hösli, 1987).

All DA and NA receptors belong to the G-protein-coupled receptor superfamily. Among the three DA subtypes present in spinal cord, D1 binds to Gs proteins that stimulate adenylyl cyclase, and also to Gq proteins that activate phospholipase C to regulate intracellular calcium levels (Wang et al., 1995, Undie et al., 2000). D2 and D3 receptors bind to various inhibitory G-proteins (Gi1, Gi2, Gi3, Goa, Gob) that inhibit adenylyl cyclase, activate K+ channels, and stimulate mitogenesis (Watts et al., 1998, Oak et al., 2001). Different families of adrenergic receptors similarly couple to different G-proteins that modulate intracellular events. All alpha 1 adrenergic receptors bind to Gq proteins that activate phospholipase C or other signaling cascades (Koshimizu et al., 2003). The various alpha 2 receptor subtypes bind to G-proteins that inhibit adenylyl cyclase, activate K+ channels, and inhibit voltage-gated Ca2+ channels (Saunders and Limbird, 1999). All beta adrenergic receptors couple to G-proteins that stimulate adenylyl cyclase (Johnson, 1998).

In view of this detailed knowledge and the presumed implication of both the DA and NA inputs in the control of nociceptive, autonomic and motor function, it was of interest to relate innervation patterns, distribution of receptors and G-protein signaling by these two systems in the rat spinal cord. For this purpose, we used guanosine 5′-O-(γ-[35S]thio)triphosphate) autoradiography to visualize and measure the efficacy of G-protein coupling stimulated by DA, NA, and several of their agonists, in the different layers of grey matter and in the white matter, at cervical, thoracic and lumbar levels of adult rat spinal cord.

Section snippets

Concentration–response relationship for DA, the D1 receptor agonist, SKF 38393, NA alone, and NA in presence of the alpha adrenoceptor antagonist, phentolamine

As measured in the grey matter of lumbar spinal cord, DA stimulated [35S]GTPγS incorporation in a concentration-dependent manner, with a maximal effect at 1 mM (Fig. 1A). A similar, albeit lower, concentration-dependent stimulation was observed with SKF 38393, also reaching its maximum at 1 mM. The stimulation by NA was also concentration-dependent, with a maximal effect near 1 mM (Fig. 1B). In presence of 1 mM phentolamine, the NA activation was significantly reduced but not abolished, and

Methodological considerations

[35S]GTPγS incorporation was used here for the first time to determine the anatomical location and efficacy of G-protein coupling activated by DA and NA receptors in adult rat spinal cord. The main experiments were carried out at three segmental levels, to take into account possible differences in density of catecholamine innervation and/or distribution of catecholaminergic receptors.

In these experiments, stimulation of G-protein coupling by DA (400 μM), SKF 38393 (200 μM), and quinpirole (200

Chemicals

Dopamine hydrochloride (3-hydroxy-tyramine hydrochloride), R(+)-SKF-38393 hydrochloride, LY 171,555 (−)-quinpirole hydrochloride, SCH 23390, raclopride, L(−) noradrenaline HCl, phentolamine mesylate, methoxamine HCl, (R)-(−)-phenylephrine hydrochloride, clonidine hydrochloride, guanabenz acetate, (−)-isoprenaline hydrochloride, terbutaline hemisulfate, benoxathian hydrochloride, RX 821002, propranolol hydrochloride, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), guanosine 5′-diphosphate (GDP), and

Acknowledgments

This study was supported by grants MOP then NRF 3544 (L.D.), and MOP-38094 (T.A.R.), from the Canadian Institutes of Health Research, as well as a team grant of FCAR/FRSQ (ER-70713). The expert technical assistance of Jeanne Lavoie (histology) and Claude Gauthier (photography) is also acknowledged.

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