www.elsevier.com / locate / bres
Research report
3Autoradiographic localization of [ H]nociceptin binding sites in the rat
brain
a b a ,
*
´
Sebastien Florin , Jean-Claude Meunier , Jean Costentin
a
´ ´ ´ ´
Unite de Neuropsychopharmacologie experimentale(CNRS UMR 6036), Institut Federatif de Recherche Multidisciplinaire des Peptides (IFRMP 23), ´ ´
Faculte de Medecine et de Pharmacie de Rouen, 22 Boulevard Gambetta, 76183 Rouen, Cedex, France
b
´ ´
Unite de Neuropharmacologie Moleculaire, Institut de Pharmacologie et Biologie Structurale (CNRS UPR), 205 route de Narbonne, 31077 Toulouse, Cedex, France
Accepted 20 June 2000
Abstract
The binding sites of nociceptin (also named orphanin FQ), the endogenous ligand of ORL1 (opiate receptor like 1), were localized in rat brain, using an autoradiographic procedure. High levels of binding were observed in the cingulate, retrosplenial, perirhinal, insular and occipital cortex, anterior and posteromedial cortical amygdaloid nuclei, basolateral amygdaloid nucleus, amygdaloid complex, posterior hippocampus, dorsal endopiriform, central medial thalamic, paraventricular, rhomboid thalamic, suprachiasmatic, ventromedial hypo-thalamic nuclei, mammillary complex, superficial gray layer of the superior colliculus, locus coeruleus, dorsal raphe nucleus. More moderate labelling was observed in the prefrontal, fronto–parietal, temporal, piriform cortex, dentate gyrus, anterior olfactory nucleus, olfactory tubercle, shell of nucleus accumbens, claustrum, lateral septum, laterodorsal thalamic, medial habenular, subthalamic, reuniens thalamic nuclei, subiculum, periaqueductal grey matter and pons. A lower binding site density was observed in the anterior and medial hippocampus, olfactory bulb, caudate putamen, the core of the nucleus accumbens, medial septum, ventrolateral, ventroposterolateral and mediodorsal thalamic nuclei, lateral and medial geniculate nuclei, hypothalamic area, substantia nigra, ventral tegmentum area and interpedoncular nucleus. A moderate and similar labelling was found in the dorsal and ventral horn of the spinal cord. No labelling was apparent in the corpus callosum. Thus, it appears that the ORL1 receptor is particularly abundant in the cerebral cortex, limbic system of the rat brain and some areas involved in pain perception. 2000 Elsevier Science B.V. All rights reserved.
Theme: Neurotransmitters, Modulators, transporters, and receptors
Topic: Regional localization of receptors transmitters
3
Keywords: ORL1 receptor; [ H]nociceptin; Autoradiography; Rat brain
1. Introduction [2,3,7,11,40,41] and mouse [17], particularly the limbic
and cortical regions and some areas involved in pain The ORL1 (opiate receptor like 1) receptor has been perception. It has been suggested that the ORL1 receptor cloned from human [17], rat [2,3,7,11,40,41] and mouse plays an important role in several central functions such as brain [17,20]. It is a member of the G protein-coupled cognitive, mnemonic, attentional processes, locomotor receptor family and its sequence displays high similarity activity and nociception. This latter function was evi-with those of them-,d- andk-opioid receptors. However, denced in mice after repeated administration of an anti-this receptor does not bind any of the established opiate sense oligonucleotide to ORL1 mRNA, through an in-ligands with high affinity [2,3,7,11,17,20,40,41]. In situ crease in the nociceptive threshold in the hot-plate test hybridization studies have shown that ORL1 transcripts are [15]. The endogenous agonist of the ORL1 receptor has widely distributed in the central nervous system of the rat been isolated and sequenced from rat brain [15] and porcine hypothalamus [24]. It is a 17 amino acid residue peptide which closely resembles the opioid peptide dynor-*Corresponding author. Tel.: 133-2-3514-8593; fax: 1
33-2-3514-phin A [15,24]. It is processed in vivo from a larger 8603.
E-mail address: neuro.psyphar@univ-rouen.fr (J. Costentin). precursor, prepronociceptin (PPNOC), whose cDNA
tests [24]. Hence, this peptide was named nociceptin 3. Results
reflecting to its apparent pronociceptive properties [15].
However, Reinscheid et al. [24] named it orphanin FQ. Nociceptin binding sites are widely distributed in the Nociceptin stimulates horizontal and vertical locomotor central nervous system of the rat (Fig. 1). Moreover, they activities, exploratory behaviour in mice [5], food intake in display three levels of labelling corresponding to a high rats [23,34]; it elicits anxiolytic effects [10] and impairs (136–80 fmol / mg tissue eq.), moderate (80–40 fmol / mg
spatial learning [27]. tissue eq.) and low (,40 fmol / mg tissue Eq.) binding site
In the present study, we analysed the localization of densities (Table 1). Many telencephalic regions showed ORL1 receptor in the rat brain using radiolabelled heavy labelling (Table 1). In the cortex, the pattern of
3
[ H]nociceptin. labelling was essentially the same in all neocortical and
allocortical regions. Indeed, a dense labelling was observed in cingulate, retrosplenial, perirhinal, insular and occipital
2. Materials and methods cortex. The labelling of the prefrontal, fronto–parietal,
temporal and piriform cortex is less dense.
Male Sprague Dawley rats, 200 g, were purchased from A high density of sites was found in several amygdaloid `
Charles River (St Aubin les Elbeuf, France) and used in nuclei such as the anterior and posteromedial cortical this study. They were housed four per Makrolon box (L: nuclei and the basolateral nucleus. In the hippocampus 38 cm, W: 20 cm, H: 10 cm), with free access to standard formation, a moderate labelling was seen in the dentate semi-synthetic laboratory diet and tap water, under con- gyrus, and a lower density of site was observed in the trolled conditions (temperature: 22618C; 0700–1900 h anterior and medial hippocampus while a higher labelling
light / dark cycle). was seen in the posterior hippocampus.
Six rats were sacrificed by decapitation and their brains Labelling of the olfactory system was more diffuse. The were quickly removed on ice, and frozen in isopentane at anterior olfactory nucleus and the olfactory tubercle were 2308C. Frontal sections (20 mm) were cut, using a fairly labelled and the olfactory bulb is lightly labelled. cryotome (Leica), and thaw-mounted onto chrome alum– Dense labelling was detected in the endopiriform nu-gelatin-coated slides. Tissue sections were dehydrated at cleus. The lateral septum showed moderate binding, as did 48C for 2 h and stored at 2308C until processing. the shell of nucleus accumbens and claustrum. The medial Brain sections were preincubated at 378C for 15 min in a septum and the core of nucleus accumbens are moderately buffer (Tris–HCl 50 mM, BSA 0.1% pH 7.4). They were labelled. Finally, the caudate putamen was only labelled to then incubated for 3 h at room temperature in the same a limited extent.
buffer with a protease inhibitor (bacitracin 0.5 mg / ml) and With the exception of the central medial nucleus, which 3
2 nm [ H]nociceptin (spec. Act. 23 Cl / mmol, CNRS, UPR was highly labelled, most thalamic nuclei such as the 9062, Toulouse, France) to assess total binding. Non- laterodorsal, ventrolateral, ventroposterolateral and specific binding was evaluated on adjacent sections incu- mediodorsal thalamic nuclei, the lateral and medial genicu-bated under identical conditions in the presence of 1 mm late nuclei showed moderate to low binding. In most nuclei unlabelled nociceptin (H. Mazarguil, CNRS, UPR 9062, of the perithalamic and subthalamic areas, moderate to Toulouse, France). The slides were rinsed four times (3 high labelling was observed, especially in the subthalamic, min) at 48C in the Tris–HCl 50 mm, BSA 0.1% (pH 7.4) medial habenular, reuniens, rhomboid nuclei and paraven-buffer and dried under a cold air stream. tricular nucleus. In the hypothalamus, the suprachiasmatic, The sections were apposed onto Amersham Hyperfilm ventromedial nuclei and the mammillary complex were 3
[ H], for 9 weeks, with tritiated standard strips (Amer- highly labelled. The remainder of the hypothalamus
pos-sham, Les Ulis, France). sessed few nociceptin binding sites.
Quantitative analysis of the autoradiograms was per- A high degree of labelling was observed in the superfi-formed using a computerized image analysis system cial grey layer of the superior colliculus. Moderate binding (Biocom Bio 500). Autoradiographic image analysis was was seen in the subiculum and the periaqueductal grey carried out in 55 brain regions described in the rat brain matter. The substantia nigra, the ventral tegmentum area atlas of Paxinos and Watson [22]. The averaged optical and the interpedoncular nucleus were only labelled to a density values from a minimum of three consecutive limited degree.
Fig. 1. Distribution of [3H]nociceptin binding sites in the brain (A, B, D, E, F) and in the spinal cord of the rat (G). The non-specific binding is illustrated on the section C. AC, anterior commissure; Acb, nucleus accumbens; AcbSh, shell of the nucleus accumbens; AM, amygdaloid complex; BLA, basolateral amygdaloid nucleus; CC, corpus callosum; Cg, cingulate cortex; CM, central medial thalamic nucleus; CP, caudate putamen; DH, dorsal horn of the spinal cord; DR, dorsal raphe nucleus; En, endopiriform nucleus; FP, fronto–parietal cortex; Hi, hippocampus; IC, insular cortex; LC, locus coeruleus; LS, lateral septum; MHb, medial habenular nucleus; Pir, piriform cortex; PRh, perirhinal cortex; PV, paraventricular thalamic nucleus; Tu, olfactory tubercle; VH, ventral horn of the spinal cord; VMH, ventrolateral hypothalamic nucleus; VPL, ventroposterolateral thalamic nucleus.
4. Discussion cortical regions. Our data are also in agreement with those
may modulate a variety of central functions. Such func- itory modulator, regulating synaptic transmission and tions may include the regulation of learning and memory, synaptic plasticity in the hippocampus. This suggests that neuroendocrine control, modulation of input signals par- activation of ORL1 receptors may play an important role ticipating in integration of visual, auditory, nociceptive, in synaptic plasticity involved in learning and memory olfactory sensory functions as well as in the integration of [8,44]. Furthermore amygdala, which is also known to be central outputs important for the control of movement and involved in learning and memory, is highly labelled. Meis
motor processing. and Pape [14] showed that the rat lateral and central
The intracerebroventricular injection of nociceptin in- amygdala responded to nociceptin with an increase in duces hyperalgesia [15,24], stimulates or decreases inwardly rectifying potassium conductance, resulting in an locomotor activity [5,24], increases food intake [23,34] impairment in cell excitability.
and impairs spatial learning [27]. Thus of particular Finally, the ventromedial nucleus of hypothalamus, relevance is the neuroanatomic localization of nociceptin known to participate in the control of food intake, showed binding sites in areas of the central nervous system very high labelling. Central injection [23] or microinjec-concerned with pain perception, the control of movement, tion into the ventromedial hypothalamic nucleus [34] of food intake, learning and memory. With regard to the nociceptin significantly increased food intake in satiated processing of nociceptive information, nociceptin binding rats. Lee et al. [13] showed that nociceptin hyperpolarises sites are present in the dorsal horn of spinal cord. This data neurones in the rat ventromedial hypothalamus.
is in agreement with several studies which showed im- The neuroanatomical distribution of nociceptin binding munostaining of nociceptin in the dorsal horn of spinal sites is similar to that of them- andk-opioid receptors but cord [4,12,25,29,30]. Stanfa et al. [33] reported that, reveals strikingly differences with the d receptors pattern intrathecally injected, nociceptin inhibited C-fibre evoked throughout the rat brain [35]. The d receptors are of wind-up and post-discharge of dorsal horn neurones, insignificant density in amygdala, hippocampus, thalamus, indicating that nociceptin acts as an inhibitory peptide at central grey and locus coeruleus where nociceptin binding the spinal level. Shu et al. [31] showed that nociceptin sites, m- and k-receptors are found. Moreover, striatum inhibited glutamate-induced currents in isolated rat spinal contains a high density of m-, d- and k-opioid receptors dorsal horn neurones; nociceptin might contribute to while there is a low nociceptin binding site density. nociceptive transmission in the spinal level. These data are Thus, nociceptin binding sites are widely distributed in agreement with behavioural studies which showed that throughout the rat brain with a pattern which closely intrathecal administration of nociceptin induced analgesia resembles that of them- andk-opioid receptors. According in several nociception tests [36,37,42,43]. to this localization, the nociceptin / ORL1 system could In supraspinal nuclei, nociceptin binding sites are pres- participate, in addition to various functions already iden-ent in relay structures modulating nociceptive transmission tified, in many others.
including the periaqueductal grey matter and thalamic nuclei. Vaughan et al. [39] showed nociceptin action on the membrane properties of rat periaqueductal grey neurones
Acknowledgements
in vitro. Moreover, microinjection of nociceptin in the periaqueductal grey attenuated the tail flick inhibition
This work was supported by BIOMED (contract no. produced by morphine [19]. This is consistent with the
BMH4-CT97-2317). studies carried out by Mogil et al. [16] who concluded that
nociceptin reverses supraspinal morphine antinociception. Several areas of the central nervous system concerned
with motor control showed the presence of nociceptin References binding sites. These sites are distributed in motor cortex
and subcortical motor nuclei in the mesencephalon. They [1] B. Anton, J. Fein, T. To, X. Li, L. Silberstein, C.J. Evans, include structures innervated by dopaminergic, noradrener- Immunohistochemical localization of ORL1 in the central nervous gic and serotoninergic systems such as the substantia nigra, system of the rat, J. Comp. Neurol. 368 (1996) 229–251.
[2] J.R. Bunzow, C. Saez, M. Mortrud, C. Bouvier, J.T. Williams, M. the locus coeruleus and dorsal raphe nucleus, respectively.
Low, D.K. Grandy, Molecular cloning and tissue distribution of a Studies revealed that nociceptin inhibited noradrenaline
of a novel member of the opioid receptor gene family, FEBS Lett. orphan opioid receptor ORL1, stimulates feeding in rats,
Neurore-347 (1994) 279–283. port 20 (1996) 369–371.
[4] N.J. Dun, S.L. Dun, L.L. Hwang, Nociceptin-like immunoreactivity [24] R.K. Reinscheid, H.P. Nothacker, A. Bourson, A. Ardati, R.A. in autonomic nuclei of the rat spinal cord, Neurosci. Lett. 234 Henningsen, J.R. Bunzow, D.K. Grandy, H. Langen Jr., F.J. (1997) 95–98. Monsma, O. Civelli, Orphanin FQ: a neuropeptide that activates an opioid-like G-protein-coupled receptor, Science 270 (1995) 792– [5] S. Florin, C. Suaudeau, J.-C. Meunier, J. Costentin, Nociceptin
794. stimulates locomotion and exploratory behaviour in mice, Eur. J.
Pharmacol. 317 (1996) 9–13. [25] M. Riedl, S. Shuster, L. Vulchanova, J. Wang, H.H. Loli, R. Elde, Orphanin FQ / nociceptin–immunoreactive nerve fibers parallel those [6] S. Florin, I. Leroux-Nicollet, J.-C. Meunier, J. Costentin,
Au-3
containing endogenous opioids in rat spinal cord, Neuroreport 7 toradiographic localization of [ H]nociceptin binding sites from
(1996) 1369–1372. telencephalic to mesencephalic regions of the mouse brain,
Neuro-sci. Lett. 230 (1997) 1–4. [26] H. Saito, K. Maruyama, T. Saido, S. Kawashima, N23K, a gene transiently up-regulated during neural differentiation, encodes a [7] K. Fukuda, S. Kato, K. Mon, M. Nishi, H. Takeshima, N. Iwabe, T.
precursor protein for a newly identified neuropeptide nociceptin, Miyata, T. Houtani, T. Sugimoto, cDNA cloning and regional
Biochem. Biophys. Res. Comm. 217 (1995) 539–545. distribution of a novel member of the opioid receptor family, FEBS
Lett. 343 (1994) 42–46. [27] J. Sandin, J. Georgieva, P.A. Schott, S.O. Ogren, L. Terenius, Nociceptin / orphanin FQ microinjected into hippocampus impairs [8] Y. Goda, M. Mutneja, Memory mechanisms: the nociceptin
con-spatial learning in rats, Eur. J. Neurosci. 9 (1997) 194–197. nection, Curr. Biol. 8 (1998) R889–R891.
[28] E. Schlicker, S. Werthweim, M. Kathmann, U. Bauer, Nociceptin [9] T. Houtani, M. Nishi, H. Takeshima, T. Nukuda, S. Tetsuo,
inhibits noradrenaline release in the mouse brain cortex via pre-Structure and regional distribution of nociceptin / orphanin FQ
synaptic ORL1 receptors, Naunyn. Schmiedebergs Arch. Pharmacol. precursor, Biochem. Biophys. Res. Comm. 219 (1996) 714–719.
358 (1998) 418–422. [10] F. Jenck, J.L. Moreau, J.R. Martin, C.J. Kilpatrick, R.K. Reinscheid
[29] R. Schuligoi, R. Amann, P. Angelberger, B.A. Peskar, Determi-Jr., F.J. Monsma, H.P. Nothacker, O. Civelli, Orphanin FQ acts as
nation of nociceptin-like immunoreactivity in the rat dorsal spinal an anxiolytic to attenuate behavioral responses to stress, Proc. Natl.
cord, Neurosci. Lett. 224 (1997) 136–138. Acad. Sci. USA 94 (1997) 14854–14858.
¨ ¨
[30] S. Schulz, M. Schreff, D. Nuß, C. Gramsch, V. Hollt, Nociceptin / [11] J.E. Lachowicz, Y. Shen Jr., F.J. Monsma, D.R. Sibley, Molecular
orphanin FQ and opioid peptides show overlapping distribution but cloning of a novel G protein-coupled receptor related to the opiate
not co-localization in pain-modulatory brain regions, Neuroreport 7 receptor family, J. Neurochem. 64 (1994) 34–40.
(1996) 3021–3025. [12] C.C. Lai, S.Y. Wu, S.L. Dun, N.J. Dun, Nociceptin-like
immuno-reactivity in the rat dorsal horn and inhibition of substantia [31] Y.S. Shu, Z.Q. Zhao, M.Y. Li, G.M. Zhou, Orphanin FQ / nociceptin gelatinosa neurons, Neuroscience 81 (1997) 887–891. modulates glutamate- and kainic acid-induced currents in acutely [13] K. Lee, J.R. Nicholson, A.T. McKnight, Nociceptin hyperpolarises isolated rat spinal dorsal horn neurons, Neuropeptides 32 (1998)
neurones in the rat ventromedial hypothalamus, Neurosci. Lett. 239 567–571.
(1997) 37–40. [32] L.J. Sim, S.R. Childers, Anatomical distribution of m, d, and k
-35
[14] S. Meis, H.C. Pape, Postsynaptic mechanisms underlying respon- opioid and nociceptin / orphanin FQ-stimulated [ S]guanylyl-59 -o-siveness of amygdaloid neurons to nociceptin / orphanin FQ, J. (gamma-thio)-triphosphate binding in guinea pig brain, J. Comp. Neurosci. 18 (1998) 8133–8144. Neurol. 386 (1997) 562–572.
[15] J.-C. Meunier, C. Mollereau, L. Toll, C. Suaudeau, C. Moisand, P. [33] L.C. Stanfa, V. Chapman, N. Kerr, A.H. Dickenson, Inhibitory action Alvinerie, J.L. Butour, J.C. Guillemot, P. Ferrara, B. Monsarrat, H. of nociceptin on spinal dorsal horn neurones of the rat, in vivo, Eur. Mazarguil, G. Vassart, M. Parmentier, J. Costentin, Isolation and J. Pharmacol. 118 (1996) 1875–1877.
structure of the endogenous agonist of opioid receptor-like ORL1 [34] T.R. Stratford, M.R. Holahan, A.E. Kelly, Injections of nociceptin receptor, Nature 377 (1995) 532–535. into nucleus accumbens shell or ventromedial hypothalamic nucleus [16] J.S. Mogil, J.E. Grisel, G. Zhangs, J.K. Belknap, D.K. Grandy, increase food intake, Neuroreport 20 (1997) 423–426.
Functional antagonism of m-, d- and k-opioid antinociception by [35] A. Tempel, R.S. Zukin, Neuroanatomical patterns of them,d, and orphanin FQ, Neurosci. Lett. 214 (1996) 131–134. k-opioid receptors of rat brain as determined by quantitative in vitro [17] C. Mollereau, M. Parmentier, P. Mailleux, J.L. Butour, C. Moisand, autoradiography, Proc. Natl. Acad. Sci. USA 84 (1987) 4308–4312. P. Chalon, D. Caput, G. Vassart, J.C. Meunier, ORL1, a novel [36] J.H. Tian, W. Xu, Y. Fang, J.S. Mogil, J.E. Grisel, D.K. Grandy, J.S. member of the opioid receptor family: cloning, functional expres- Han, Bidirectional modulatory effect of orphanin FQ on morphine-sion and localization, FEBS Lett. 341 (1994) 3333–3338. induced analgesia: antagonism in brain and potentiation in spinal [18] C. Mollereau, M.T. Simons, P. Soularue, F. Linus, G. Vassart, J.-C. cord of the rat, Brit. J. Pharmacol. 120 (1997) 676–680.
Meunier, M. Parmentier, Structure, tissue distribution and chromo- [37] J.H. Tian, W. Xu, W. Zhang, Y. Fang, J.E. Grisel, J.S. Mogil, D.K. somal localization of the prepronociceptin gene, Proc. Natl. Acad. Grandy, J.S. Han, Involvement of endogenous orphanin FQ in Sci. USA 93 (1996) 8666–8670. electroacupuncture-induced analgesia, NeuroReport 20 (1997) 497– [19] M.M. Morgan, J.E. Grisel, C.S. Robbins, D.K. Grandy, Antinocicep- 500.
tion mediated by the periaqueductal gray is attenuated by orphanin [38] C.W. Vaughan, M.J. Christie, Increase by the ORL1 receptor (opioid
1
FQ, Neuroreport 8 (1997) 3431–3434. receptor-like 1) ligand, nociceptin, of inwardly rectifying K [20] M. Nishi, H. Takeshima, M. Mon, K. Nakagawara, T. Takeuchi, conductance in dorsal raphe nucleus neurons, Brit. J. Pharmacol.
Structure and chromosomal mapping of genes for the mouse k- 117 (1996) 1609–1611.
opioid receptor and an opioid receptor homologue (MOR-C), [39] C.W. Vaughan, S.L. Ingram, M.J. Christie, Actions of the ORL1 Biochem. Biophys. Res. Comm. 205 (1994) 1353–1357. ligand nociceptin on membrane properties of rat periaqueductal gray [21] H.P. Nothacker, R.K. Reinscheid, A. Mansour, R.A. Henningsen, A. neurons in vitro, J. Neurosci. 17 (1997) 996–1003.
Ardati Jr., F.J. Monsma, S.J. Watson, O. Civelli, Primary structure [40] J.B. Wang, P.S. Johnson, Y. Imai, A.M. Persico, B.A. Ozenberger, and tissue distribution of the orphanin FQ precursor, Proc. Natl. Sci. C.M. Eppler, G.R. Uhl, cDNA cloning of an orphan opiate receptor USA 93 (1996) 8677–8682. gene family member and its splice variant, FEBS Lett. 348 (1994) [22] G. Paxinos, C. Watson, The Rat Brain in Stereotaxie Coordinates, 75–79.
![Fig. 1. Distribution of [3H]nociceptin binding sites in the brain (A, B, D, E, F) and in the spinal cord of the rat (G)](https://thumb-ap.123doks.com/thumbv2/123dok/3140030.1382875/3.612.101.496.63.558/fig-distribution-nociceptin-binding-sites-brain-spinal-cord.webp)
