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orphanin FQ and the nociceptin receptor ORL1 with substance P and calcitonin gene-related peptide expression in dorsal root ganglion of the rat
Neuroscience Letters 348 (2003) 190–194 www.elsevier.com/locate/neulet
Relationship of pronociceptin/orphanin FQ and the nociceptin receptor ORL1 with substance P and calcitonin gene-related peptide expression in dorsal root ganglion of the rat Joanna Mika, Yanzhang Li, Eberhard Weihe, Martin K.-H. Schafer* Department of Molecular Neuroscience, Institute of Anatomy and Cell Biology, Philipps University, Robert-Koch-Strasse 8, 35033 Marburg, Germany Received 30 April 2003; received in revised form 25 June 2003; accepted 25 June 2003
Abstract Recent evidence suggests a role of prepronociceptin/orphanin FQ (preproN/OFQ) derived neuropeptides in nociceptive signaling. Here, we examined the expression of preproN/OFQ and the nociceptin receptor ORL1 (opioid receptor like receptor 1) in the dorsal root ganglion (DRG) of the rat in relation to that of substance P (SP) and calcitonin gene-related peptide (CGRP). Double labeling in situ hybridization revealed a constitutive expression of preproN/OFQ in a distinct minor subpopulation of very small DRG neurons with no evidence for coexpression with either SP or CGRP. However, a major subpopulation of the preproN/OFQ-positive neurons showed direct juxtaposition to SP and CGRP containing neurons. ORL1 was abundantly expressed with a high degree of coexpression with SP (72%) and CGRP (82%) suggesting that N/OFQ may presynaptically modulate primary sensory nociceptive signaling. The DRG cell line F11 was found to express preproN/OFQ, but not ORL1, and, therefore, is well suited to study the mechanisms of N/OFQ gene regulation in vitro. q 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Dorsal root ganglion; F11; Sensory neuron; Neuropeptide; Pain; Nociceptin; Preprotachykinin; Calcitonin gene-related peptide
Nociceptin/orphanin FQ (N/OFQ) which is involved in nociceptive signaling [8,13] can have diverse effects depending on the place and route of administration and type of pain [16,17]. Depending on the species, proteolytic cleavage of prepronociceptin/orphanin FQ (preproN/OFQ) can generate additional neuropeptides such as nocistatin with effects opposite to N/OFQ [10]. There are conflicting reports with respect to the circuitry of neurons expressing preproN/OFQ and the nociceptin receptor, named opioid receptor like receptor 1 (ORL1). Spinal N/OFQ-positive neurons are predominantly located in the superficial dorsal horn, while ORL1 is highly expressed throughout spinal gray matter and in dorsal root ganglion (DRG) as well [8]. Rapid induction of N/OFQ has been reported in DRG after peripheral inflammation [1] with no evidence for a constitutive expression of N/OFQ. Inflammation-induced N/OFQ expression was seen in a subpopulation of nociceptive DRG neurons expressing the vanilloid receptor 1 (VR1) [6]. Recent evidence suggests minor constitutive *
Corresponding author. Tel.: þ 49-6421-2864036; fax: þ 49-64212868965. E-mail address: [email protected] (M.K.-H. Schafer).
expression of N/OFQ in DRG neurons [11]. It is not known whether N/OFQ or its receptor is expressed in primary afferents containing substance P (SP) and calcitonin generelated peptide (CGRP) which are well known to be involved in pain transmission. To determine whether N/OFQ could function as an autocrine or paracrine modulator of primary afferent neuropeptide signaling related to pain we examined the relationship of N/OFQ and ORL1 with SP and CGRP expressing primary afferents by double labeling in situ hybridization (ISH). In order to find a useful in vitro model to study the regulation of the N/OFQ system, we examined whether the sensory neuronal cell line F11 can express N/OFQ and ORL1. DRG (L5) and spinal cords were obtained from male Wistar rats (200 – 350 g) killed by exposure to CO2. Experiments were approved by the local Animal Research Committee in accordance with NIH guidelines. Tissues were processed for ISH and reverse transcription polymerase chain reaction (RT-PCR) analysis according to a standard protocol [14]. F11 cells (rat DRG/mouse neuroblastoma provided by Dr M.C. Fishman, Harvard Medical
0304-3940/03/$ - see front matter q 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/S0304-3940(03)00786-9
J. Mika et al. / Neuroscience Letters 348 (2003) 190–194
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Fig. 1. Distribution of prepronociceptin/orphanin FQ (preproN/OFQ) and ORL1 mRNA in rat spinal cord and DRG. X-Ray autoradiograms of frozen sections through spinal cord (A –D) and DRG (E–J) hybridized with 35S-labeled antisense riboprobes corresponding to preproN/OFQ mRNA (A,C,E,I) or ORL1 mRNA (B,D,G,J). Note specific signals for preproN/OFQ mRNA restricted to neuronal subpopulations located in superficial dorsal horn (A,C) and dorsolateral to the central canal (A) and in DRG (E,I). ORL1 mRNA was widely distributed in the dorsal (B,D) and ventral horn (B) and in the majority of primary afferent neurons (G,J). After hybridization with sense-strand probes no specific signals were observed (F,H). Exposure time of X-ray films: (A,E,F) 17 h; (B,G,H) 8 h. Exposure time for emulsion: (C,I) 14 days; (D,J) 7 days. Scale bar: (A,B) 500 mm; (C,D) 250 mm; (E– J) 200 mm.
School, Boston, MA) were grown in Ham’s F12 medium as described [12]. Cells were harvested at 70 – 80% confluence. Total RNA was isolated from DRGs and F11 cells using TRIzol Reagent (GibcoBRL) and DNase I treated. cDNA was synthesized from 2.5 mg total RNA using SUPERSCRIPT II (GibcoBRL) and oligo(dT)12-18 (1.25 mM, Amersham) in a total volume of 20 ml. PCR was performed using specific primers and AmpliTaq Gold DNA polymerase (Roche) under the following conditions: 5 min at 94 8C; ten cycles (94 8C for 15 s, 65 8C for 15 s [decrease 1 8C per cycle], 72 8C for 40 s) and 30 cycles (94 8C for 15 s, 55 8C for 15 s, 72 8C for 40 s) and 7 min at 72 8C. RT-PCR cloning into the pGEMT vectors (Promega) was performed to obtain specific probes for preproN/OFQ mRNA (nt. 199– 785; Accession number: S79730) and ORL1 mRNA (nt. 406 – 1186; Accession number: U01913). After sequence confirmation (Seqlab, Go¨ttingen) radioactive probes were generated by in vitro transcription using [35S]UTP as a label. Non-radioactive probes were generated for rat preprotachykinin-A (PPT-A) [2] and aCGRP (nt. 19 –354; Accession number: L00111) using digoxigenin (DIG)-UTP as a label. Single and double labeling ISH was performed as published previously [14]. Non-radioactive hybrids were detected with alkaline phosphatase-conjugated anti-DIG antibodies diluted to 1 unit/ml and 0.2 mM 5-bromo-4chloro-3-indolyl phosphate and nitroblue tetrazolium salt (Roche, Mannheim, Germany) yielding a blue precipitate after 16 h. Autoradiographic detection of 35S was carried out using photoemulsion NTB-2 (Eastman Kodak, Rochester,
NY) or K5 (Ilford). Exposure times were 7 –15 days. Cresyl violet was used as a counterstain. For microscopic analysis an Olympus AX-70 microscope (Hamburg, Germany) was used. The number of double-labeled cells was quantified for each DIG-labeled probe in L5 DRG from seven to eight rats. The numbers of DIG-positive and radiolabeled cells were counted in three sections per ganglion at the same magnification. The percentage of double-labeled cells was calculated and expressed as a percentage of radioactivelabeled vs. non-radioactive labeled cells. ISH analysis of N/OFQ and ORL1 revealed that both genes are expressed in spinal cord and DRG. In the spinal cord, N/OFQ expression was restricted to the superficial dorsal horn with highest mRNA levels in the substantia gelatinosa (Fig. 1A,C) and in neurons scattered in the deep dorsal horn and in the area dorsal to the central canal (Fig. 1A), while ORL1 mRNA exhibited an abundant expression pattern in all spinal layers (Fig. 1B,D) with ventral horn motoneurons exhibiting the highest levels (Fig. 1B). In contrast to a previous report [9] indicating the absence of ORL1 mRNA in lamina I of the cervico-thoracic spinal cord we detected ORL1 mRNA in many neurons of various size in lumbar lamina I (Fig. 1D). A recent study reported ORL1 mRNA expression in laminae I and II neurons of the medullary trigeminal nucleus [3]. In the DRG, low N/OFQ mRNA levels were detected in few very small neurons (Fig. 1E,I). In contrast, after 7 days of exposure ORL1 mRNA was seen in the majority of DRG neurons (Fig. 1G,J) with expression levels comparable to those of spinal motoneurons (Fig. 1B). Hybridization with sense probes did not
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Fig. 2. Co-existence pattern of pronociceptin/orphanin FQ (preproN/OFQ) and ORL1 mRNAs with SP and aCGRP mRNAs, respectively. High-magnification bright-field photomicrograph shows the simultaneous detection of preproN/OFQ mRNA (silver grains in A,B) or ORL1 mRNA (silver grains in C,D) and neuropeptide mRNAs (staining precipitate) coding for SP (A,C) or aCGRP (B,D) in neuronal subpopulations of rat DRG. ORL1 mRNA was present in many DRG neurons including SP- and aCGRP-positive neurons (arrow head in C,D). PreproN/OFQ transcripts are seen in few neurons of very small size (arrow in A,B), but not in SP-positive or aCGRP-positive primary afferents (arrow head in A,B). Note the close spatial relationship of preproN/OFQ containing perikarya with the small and medium size peptidergic neurons (stars in A,B). Exposure times were 7 days for ORL1 mRNA and 15 days for preproN/OFQ mRNA. Scale bar, 50 mm.
produce specific signals (Fig. 1F,H). To confirm the constitutive expression of N/OFQ and ORL1 mRNA in the DRG as shown by ISH, RT-PCR analysis of total RNA extracts from DRG was performed yielding strong bands of the predicted molecular size for proN/OFQ (587 bp) and for ORL1 mRNA (781 bp), respectively (see Fig. 3). Omission of reverse transcriptase in the RT-PCR reaction was used as a negative control. To examine the coexpression of N/OFQ or ORL1 mRNA with SP or CGRP we performed double labeling ISH. DRG sections hybridized with DIG-labeled cRNA probes for SP and aCGRP, respectively, showed their characteristic expression pattern. SP neurons (Fig. 2A,C) were of typical small to medium size, and aCGRP neurons (Fig. 2B,D) consisted of a wide variety of cell sizes as described previously [15]. Silver grains representing preproN/OFQ mRNA were never found over SP or aCGRP mRNApositive perikarya (Fig. 2A,B) demonstrating that preproN/ OFQ mRNA expressing neurons were strictly different from SP/aCGRP mRNA-positive neurons. However, we observed a large proportion of preproN/OFQ mRNApositive cell bodies located in immediate juxtaposition to SP mRNA containing neurons (42%) and to CGRP neurons
(50%) (Fig. 2A,B). ORL1 mRNA was found highly expressed in the vast majority of primary afferents (Fig. 1G,J) similar to the report by Neal et al. [9]. Consequently, there was a high degree of colocalization of ORL1 mRNA Table 1 Expression of pronociceptin/orphanin FQ (N/OFQ) and ORL1 mRNAs in cells of rat lumbar dorsal root ganglia (L5) expressing SP and aCGRP mRNAs Number of single-labeled cells per section
SP aCGRP
Number of doublelabeled cells 52 67
Neuropeptides 72 72%* 82 82%*
SP aCGRP
Number of doublelabeled cells – –
Neuropeptides 52 – 71 –
ORL1 120 144
43% þ 47% þ
N/OFQ 9 8
– –
*Percentage of neurons subpopulation expressing SP or aCGRP and ORL1. þPercentage of neurons subpopulation expressing ORL1 and SP or aCGRP.
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Fig. 3. RT-PCR analysis of prepronociceptin/orphanin FQ (preproN/OFQ) and ORL1 expression in rat DRG and the cell line F11. Electrophoretic separation of PCR reactions from rat DRG and F11 total RNA extracts shows the specific band for preproN/OFQ mRNA (587 bp) and ORL1 (781 bp). Note the constitutive expression of preproN/OFQ mRNA in both DRG and F11, but ORL1 only in DRG. GAPDH as a house keeping gene and a 100 bp ladder as a size marker were used.
with SP mRNA (Fig. 2C) as well as with aCGRP mRNA (Fig. 2D). Cell counts showed that 72% of SP mRNApositive and 82% of aCGRP mRNA-positive cells coexpressed ORL1 mRNA (Table 1). To determine whether the sensory cell line F11 expressed N/OFQ or ORL1 mRNAs we performed RT-PCR analysis. A strong band for preproN/OFQ mRNA (587 bp), but not for ORL1 mRNA was detected (Fig. 3) indicating that the F11 cell line is an in vitro model system to study the regulation of the preproN/OFQ gene, but not that of the ORL1 gene. The present study provides direct evidence that a minor subpopulation of small-sized rat primary afferent neurons express preproN/OFQ constitutively. Using double label ISH we could clearly demonstrate that preproN/OFQ expressing neurons are different from the peptidergic SP/CGRP-positive neurons and thus represent a distinct entity of very small DRG neurons. A transient induction of preproN/OFQ mRNA after peripheral inflammation was reported [1] which seemed to occur in one-third of VR1immunoreactive neurons [6]. Whether N/OFQ is also constitutively expressed in the non-peptidergic subpopulation of VR1-positive DRG neurons remains to be shown. As ORL1 is expressed in a high proportion of SP/CGRPpositive neurons and as a major subpopulation of N/OFQ neurons is located in juxtaposition to SP/CGRP-positive neurons we propose that N/OFQ released locally in the DRG may modulate in a paracrine manner SP/CGRP containing neurons expressing ORL1. Under the assumption that ORL1 is expressed at peripheral and central terminals in close vicinity to N/OFQ expressing terminals it can be envisaged that N/OFQ modulates both central and peripheral SP- or CGRP-mediated neurotransmission. In fact, evidence for a functional relationship of N/OFQ and SP neurons at the presynaptic site has been demonstrated previously. During neurogenic inflammation N/OFQ-mediated inhibition of SP and CGRP release from sensory nerve terminals was observed [4] and an indirect interaction of N/OFQ with
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SP by a N/OFQ-stimulated local release of SP was postulated [5]. In the dorsal horn, N/OFQ suppresses excitatory, but not glycinergic or GABAergic inhibitory synaptic transmission to substantia gelatinosa neurons [7]; this action is presynaptic in origin. Due to low abundance of both ORL1 and preproN/OFQ mRNAs it was not possible to employ non-radioactive probes to directly examine whether preproN/OFQ primary afferent neurons are endowed with ORL1 receptors allowing autocrine presynaptic modulation of transmitter – especially glutamate – release. However, this seems unlikely because ORL1 expression was observed mainly in neurons with cell sizes larger than those of the extremely small N/OFQ neurons. Interestingly, F11 cells which express high levels of preproN/OFQ lack ORL1 transcripts supporting the view that preproN/OFQ and ORL1 are indeed expressed in different subpopulations of DRG neurons. Nevertheless, F11 cells may be ideally suited to study gene regulation of preproN/OFQ in a sensory cell relevant for nociception. Taken together we suggest a specific role for N/OFQ in paracrine presynaptic modulation of peptidergic signaling at the level of the primary afferent neuron which may be particularly relevant for pain control.
Acknowledgements This research was supported by SFB 297 and BMBF FKZ:01GG9818. We are grateful to H. Hlawaty and B. Wiegand for excellent technical assistance and to T. Csepella for helping to prepare the manuscript.
References [1] T. Andoh, M. Itoh, Y. Kuraishi, Nociceptin gene expression in rat dorsal root ganglia induced by peripheral inflammation, NeuroReport 8 (1997) 2793–2796. [2] M.S. Carter, J.E. Krause, Structure, expression, and some regulatory mechanisms of the rat preprotachykinin gene encoding substance P, neurokinin A, neuropeptide K, and neuropeptide gamma, J. Neurosci. 10 (1990) 2203–2214. [3] C.A. Flores, P. Shughrue, S.L. Petersen, S.S. Mokha, Sex-related differences in the distribution of opioid receptor-like 1 receptor mRNA and colocalization with estrogen receptor mRNA in neurons of the spinal trigeminal nucleus caudalis in the rat, Neuroscience 118 (2003) 769–778. [4] Z. Helyes, J. Nemeth, E. Pinter, J. Szolcsanyi, Inhibition by nociceptin of neurogenic inflammation and the release of SP and CGRP from sensory nerve terminals, Br. J. Pharmacol. 121 (1997) 613–615. [5] M. Inoue, M. Kobayashi, S. Kozaki, A. Zimmer, H. Ueda, Nociceptin/ orphanin FQ-induced nociceptive responses through substance P release from peripheral nerve endings in mice, Proc. Natl. Acad. Sci. USA 95 (1998) 10949–10953. [6] M. Itoh, I. Takasaki, T. Andoh, H. Nojima, M. Tominaga, Y. Kuraishi, Induction by carrageenan inflammation of prepronociceptin mRNA in VR1-immunoreactive neurons in rat dorsal root ganglia, Neurosci. Res. 40 (2001) 227–233.
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[7] C. Luo, E. Kumamoto, H. Furue, J. Chen, M. Yoshimura, Nociceptin inhibits excitatory but not inhibitory transmission to substantia gelatinosa neurones of adult rat spinal cord, Neuroscience 109 (2002) 349–358. [8] J.C. Meunier, Nociceptin/orphanin FQ and the opioid receptor-like ORL1 receptor, Eur. J. Pharmacol. 340 (1997) 1–15. [9] C.R. Neal Jr., A. Mansour, R. Reinscheid, H.P. Nothacker, O. Civelli, H. Akil, S.J. Watson Jr., Opioid receptor-like (ORL1) receptor distribution in the rat central nervous system: comparison of ORL1 receptor mRNA expression with (125)I-[(14)Tyr]-orphanin FQ binding, J. Comp. Neurol. 412 (1999) 563–605. [10] E. Okuda-Ashitaka, S. Ito, Nocistatin: a novel neuropeptide encoded by the gene for the nociceptin/orphanin FQ precursor, Peptides 21 (2000) 1101–1109. [11] L.M. Pettersson, F. Sundler, N. Danielsen, Expression of orphanin FQ/nociceptin and its receptor in rat peripheral ganglia and spinal cord, Brain Res. 945 (2002) 266 –275. [12] D. Platika, M.H. Boulos, L. Baizer, M.C. Fishman, Neuronal traits of clonal cell lines derived by fusion of dorsal root ganglia neurons with
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neuroblastoma cells, Proc. Natl. Acad. Sci. USA 82 (1985) 3499–3503. R.K. Reinscheid, H. Nothacker, O. Civelli, The orphanin FQ/ nociceptin gene: structure, tissue distribution of expression and functional implications obtained from knockout mice, Peptides 21 (2000) 901– 906. M.K. Schafer, R. Day, In situ hybridization techniques to map processing enzymes, Methods Neurosci. 23 (1995) 16–23. Z. Wiesenfeld-Hallin, T. Hokfelt, J.M. Lundberg, W.G. Forssmann, M. Reinecke, F.A. Tschopp, J.A. Fischer, Immunoreactive calcitonin gene-related peptide and substance P coexist in sensory neurons to the spinal cord and interact in spinal behavioral responses of the rat, Neurosci. Lett. 52 (1984) 199 –204. X.J. Xu, J.X. Hao, Z. Wiesenfeld-Hallin, Nociceptin or antinociceptin: potent spinal antinociceptive effect of orphanin FQ/nociceptin in the rat, NeuroReport 7 (1996) 2092–2094. C.B. Zhu, X.D. Cao, S.F. Xu, G.C. Wu, Orphanin FQ potentiates formalin-induced pain behavior and antagonizes morphine analgesia in rats, Neurosci. Lett. 235 (1997) 37 –40.
Relationship of pronociceptin/orphanin FQ and the nociceptin receptor ORL1 with substance P and calcitonin gene-related peptide expression in dorsal root ganglion of the rat Joanna Mika, Yanzhang Li, Eberhard Weihe, Martin K.-H. Schafer* Department of Molecular Neuroscience, Institute of Anatomy and Cell Biology, Philipps University, Robert-Koch-Strasse 8, 35033 Marburg, Germany Received 30 April 2003; received in revised form 25 June 2003; accepted 25 June 2003
Abstract Recent evidence suggests a role of prepronociceptin/orphanin FQ (preproN/OFQ) derived neuropeptides in nociceptive signaling. Here, we examined the expression of preproN/OFQ and the nociceptin receptor ORL1 (opioid receptor like receptor 1) in the dorsal root ganglion (DRG) of the rat in relation to that of substance P (SP) and calcitonin gene-related peptide (CGRP). Double labeling in situ hybridization revealed a constitutive expression of preproN/OFQ in a distinct minor subpopulation of very small DRG neurons with no evidence for coexpression with either SP or CGRP. However, a major subpopulation of the preproN/OFQ-positive neurons showed direct juxtaposition to SP and CGRP containing neurons. ORL1 was abundantly expressed with a high degree of coexpression with SP (72%) and CGRP (82%) suggesting that N/OFQ may presynaptically modulate primary sensory nociceptive signaling. The DRG cell line F11 was found to express preproN/OFQ, but not ORL1, and, therefore, is well suited to study the mechanisms of N/OFQ gene regulation in vitro. q 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Dorsal root ganglion; F11; Sensory neuron; Neuropeptide; Pain; Nociceptin; Preprotachykinin; Calcitonin gene-related peptide
Nociceptin/orphanin FQ (N/OFQ) which is involved in nociceptive signaling [8,13] can have diverse effects depending on the place and route of administration and type of pain [16,17]. Depending on the species, proteolytic cleavage of prepronociceptin/orphanin FQ (preproN/OFQ) can generate additional neuropeptides such as nocistatin with effects opposite to N/OFQ [10]. There are conflicting reports with respect to the circuitry of neurons expressing preproN/OFQ and the nociceptin receptor, named opioid receptor like receptor 1 (ORL1). Spinal N/OFQ-positive neurons are predominantly located in the superficial dorsal horn, while ORL1 is highly expressed throughout spinal gray matter and in dorsal root ganglion (DRG) as well [8]. Rapid induction of N/OFQ has been reported in DRG after peripheral inflammation [1] with no evidence for a constitutive expression of N/OFQ. Inflammation-induced N/OFQ expression was seen in a subpopulation of nociceptive DRG neurons expressing the vanilloid receptor 1 (VR1) [6]. Recent evidence suggests minor constitutive *
Corresponding author. Tel.: þ 49-6421-2864036; fax: þ 49-64212868965. E-mail address: [email protected] (M.K.-H. Schafer).
expression of N/OFQ in DRG neurons [11]. It is not known whether N/OFQ or its receptor is expressed in primary afferents containing substance P (SP) and calcitonin generelated peptide (CGRP) which are well known to be involved in pain transmission. To determine whether N/OFQ could function as an autocrine or paracrine modulator of primary afferent neuropeptide signaling related to pain we examined the relationship of N/OFQ and ORL1 with SP and CGRP expressing primary afferents by double labeling in situ hybridization (ISH). In order to find a useful in vitro model to study the regulation of the N/OFQ system, we examined whether the sensory neuronal cell line F11 can express N/OFQ and ORL1. DRG (L5) and spinal cords were obtained from male Wistar rats (200 – 350 g) killed by exposure to CO2. Experiments were approved by the local Animal Research Committee in accordance with NIH guidelines. Tissues were processed for ISH and reverse transcription polymerase chain reaction (RT-PCR) analysis according to a standard protocol [14]. F11 cells (rat DRG/mouse neuroblastoma provided by Dr M.C. Fishman, Harvard Medical
0304-3940/03/$ - see front matter q 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/S0304-3940(03)00786-9
J. Mika et al. / Neuroscience Letters 348 (2003) 190–194
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Fig. 1. Distribution of prepronociceptin/orphanin FQ (preproN/OFQ) and ORL1 mRNA in rat spinal cord and DRG. X-Ray autoradiograms of frozen sections through spinal cord (A –D) and DRG (E–J) hybridized with 35S-labeled antisense riboprobes corresponding to preproN/OFQ mRNA (A,C,E,I) or ORL1 mRNA (B,D,G,J). Note specific signals for preproN/OFQ mRNA restricted to neuronal subpopulations located in superficial dorsal horn (A,C) and dorsolateral to the central canal (A) and in DRG (E,I). ORL1 mRNA was widely distributed in the dorsal (B,D) and ventral horn (B) and in the majority of primary afferent neurons (G,J). After hybridization with sense-strand probes no specific signals were observed (F,H). Exposure time of X-ray films: (A,E,F) 17 h; (B,G,H) 8 h. Exposure time for emulsion: (C,I) 14 days; (D,J) 7 days. Scale bar: (A,B) 500 mm; (C,D) 250 mm; (E– J) 200 mm.
School, Boston, MA) were grown in Ham’s F12 medium as described [12]. Cells were harvested at 70 – 80% confluence. Total RNA was isolated from DRGs and F11 cells using TRIzol Reagent (GibcoBRL) and DNase I treated. cDNA was synthesized from 2.5 mg total RNA using SUPERSCRIPT II (GibcoBRL) and oligo(dT)12-18 (1.25 mM, Amersham) in a total volume of 20 ml. PCR was performed using specific primers and AmpliTaq Gold DNA polymerase (Roche) under the following conditions: 5 min at 94 8C; ten cycles (94 8C for 15 s, 65 8C for 15 s [decrease 1 8C per cycle], 72 8C for 40 s) and 30 cycles (94 8C for 15 s, 55 8C for 15 s, 72 8C for 40 s) and 7 min at 72 8C. RT-PCR cloning into the pGEMT vectors (Promega) was performed to obtain specific probes for preproN/OFQ mRNA (nt. 199– 785; Accession number: S79730) and ORL1 mRNA (nt. 406 – 1186; Accession number: U01913). After sequence confirmation (Seqlab, Go¨ttingen) radioactive probes were generated by in vitro transcription using [35S]UTP as a label. Non-radioactive probes were generated for rat preprotachykinin-A (PPT-A) [2] and aCGRP (nt. 19 –354; Accession number: L00111) using digoxigenin (DIG)-UTP as a label. Single and double labeling ISH was performed as published previously [14]. Non-radioactive hybrids were detected with alkaline phosphatase-conjugated anti-DIG antibodies diluted to 1 unit/ml and 0.2 mM 5-bromo-4chloro-3-indolyl phosphate and nitroblue tetrazolium salt (Roche, Mannheim, Germany) yielding a blue precipitate after 16 h. Autoradiographic detection of 35S was carried out using photoemulsion NTB-2 (Eastman Kodak, Rochester,
NY) or K5 (Ilford). Exposure times were 7 –15 days. Cresyl violet was used as a counterstain. For microscopic analysis an Olympus AX-70 microscope (Hamburg, Germany) was used. The number of double-labeled cells was quantified for each DIG-labeled probe in L5 DRG from seven to eight rats. The numbers of DIG-positive and radiolabeled cells were counted in three sections per ganglion at the same magnification. The percentage of double-labeled cells was calculated and expressed as a percentage of radioactivelabeled vs. non-radioactive labeled cells. ISH analysis of N/OFQ and ORL1 revealed that both genes are expressed in spinal cord and DRG. In the spinal cord, N/OFQ expression was restricted to the superficial dorsal horn with highest mRNA levels in the substantia gelatinosa (Fig. 1A,C) and in neurons scattered in the deep dorsal horn and in the area dorsal to the central canal (Fig. 1A), while ORL1 mRNA exhibited an abundant expression pattern in all spinal layers (Fig. 1B,D) with ventral horn motoneurons exhibiting the highest levels (Fig. 1B). In contrast to a previous report [9] indicating the absence of ORL1 mRNA in lamina I of the cervico-thoracic spinal cord we detected ORL1 mRNA in many neurons of various size in lumbar lamina I (Fig. 1D). A recent study reported ORL1 mRNA expression in laminae I and II neurons of the medullary trigeminal nucleus [3]. In the DRG, low N/OFQ mRNA levels were detected in few very small neurons (Fig. 1E,I). In contrast, after 7 days of exposure ORL1 mRNA was seen in the majority of DRG neurons (Fig. 1G,J) with expression levels comparable to those of spinal motoneurons (Fig. 1B). Hybridization with sense probes did not
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Fig. 2. Co-existence pattern of pronociceptin/orphanin FQ (preproN/OFQ) and ORL1 mRNAs with SP and aCGRP mRNAs, respectively. High-magnification bright-field photomicrograph shows the simultaneous detection of preproN/OFQ mRNA (silver grains in A,B) or ORL1 mRNA (silver grains in C,D) and neuropeptide mRNAs (staining precipitate) coding for SP (A,C) or aCGRP (B,D) in neuronal subpopulations of rat DRG. ORL1 mRNA was present in many DRG neurons including SP- and aCGRP-positive neurons (arrow head in C,D). PreproN/OFQ transcripts are seen in few neurons of very small size (arrow in A,B), but not in SP-positive or aCGRP-positive primary afferents (arrow head in A,B). Note the close spatial relationship of preproN/OFQ containing perikarya with the small and medium size peptidergic neurons (stars in A,B). Exposure times were 7 days for ORL1 mRNA and 15 days for preproN/OFQ mRNA. Scale bar, 50 mm.
produce specific signals (Fig. 1F,H). To confirm the constitutive expression of N/OFQ and ORL1 mRNA in the DRG as shown by ISH, RT-PCR analysis of total RNA extracts from DRG was performed yielding strong bands of the predicted molecular size for proN/OFQ (587 bp) and for ORL1 mRNA (781 bp), respectively (see Fig. 3). Omission of reverse transcriptase in the RT-PCR reaction was used as a negative control. To examine the coexpression of N/OFQ or ORL1 mRNA with SP or CGRP we performed double labeling ISH. DRG sections hybridized with DIG-labeled cRNA probes for SP and aCGRP, respectively, showed their characteristic expression pattern. SP neurons (Fig. 2A,C) were of typical small to medium size, and aCGRP neurons (Fig. 2B,D) consisted of a wide variety of cell sizes as described previously [15]. Silver grains representing preproN/OFQ mRNA were never found over SP or aCGRP mRNApositive perikarya (Fig. 2A,B) demonstrating that preproN/ OFQ mRNA expressing neurons were strictly different from SP/aCGRP mRNA-positive neurons. However, we observed a large proportion of preproN/OFQ mRNApositive cell bodies located in immediate juxtaposition to SP mRNA containing neurons (42%) and to CGRP neurons
(50%) (Fig. 2A,B). ORL1 mRNA was found highly expressed in the vast majority of primary afferents (Fig. 1G,J) similar to the report by Neal et al. [9]. Consequently, there was a high degree of colocalization of ORL1 mRNA Table 1 Expression of pronociceptin/orphanin FQ (N/OFQ) and ORL1 mRNAs in cells of rat lumbar dorsal root ganglia (L5) expressing SP and aCGRP mRNAs Number of single-labeled cells per section
SP aCGRP
Number of doublelabeled cells 52 67
Neuropeptides 72 72%* 82 82%*
SP aCGRP
Number of doublelabeled cells – –
Neuropeptides 52 – 71 –
ORL1 120 144
43% þ 47% þ
N/OFQ 9 8
– –
*Percentage of neurons subpopulation expressing SP or aCGRP and ORL1. þPercentage of neurons subpopulation expressing ORL1 and SP or aCGRP.
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Fig. 3. RT-PCR analysis of prepronociceptin/orphanin FQ (preproN/OFQ) and ORL1 expression in rat DRG and the cell line F11. Electrophoretic separation of PCR reactions from rat DRG and F11 total RNA extracts shows the specific band for preproN/OFQ mRNA (587 bp) and ORL1 (781 bp). Note the constitutive expression of preproN/OFQ mRNA in both DRG and F11, but ORL1 only in DRG. GAPDH as a house keeping gene and a 100 bp ladder as a size marker were used.
with SP mRNA (Fig. 2C) as well as with aCGRP mRNA (Fig. 2D). Cell counts showed that 72% of SP mRNApositive and 82% of aCGRP mRNA-positive cells coexpressed ORL1 mRNA (Table 1). To determine whether the sensory cell line F11 expressed N/OFQ or ORL1 mRNAs we performed RT-PCR analysis. A strong band for preproN/OFQ mRNA (587 bp), but not for ORL1 mRNA was detected (Fig. 3) indicating that the F11 cell line is an in vitro model system to study the regulation of the preproN/OFQ gene, but not that of the ORL1 gene. The present study provides direct evidence that a minor subpopulation of small-sized rat primary afferent neurons express preproN/OFQ constitutively. Using double label ISH we could clearly demonstrate that preproN/OFQ expressing neurons are different from the peptidergic SP/CGRP-positive neurons and thus represent a distinct entity of very small DRG neurons. A transient induction of preproN/OFQ mRNA after peripheral inflammation was reported [1] which seemed to occur in one-third of VR1immunoreactive neurons [6]. Whether N/OFQ is also constitutively expressed in the non-peptidergic subpopulation of VR1-positive DRG neurons remains to be shown. As ORL1 is expressed in a high proportion of SP/CGRPpositive neurons and as a major subpopulation of N/OFQ neurons is located in juxtaposition to SP/CGRP-positive neurons we propose that N/OFQ released locally in the DRG may modulate in a paracrine manner SP/CGRP containing neurons expressing ORL1. Under the assumption that ORL1 is expressed at peripheral and central terminals in close vicinity to N/OFQ expressing terminals it can be envisaged that N/OFQ modulates both central and peripheral SP- or CGRP-mediated neurotransmission. In fact, evidence for a functional relationship of N/OFQ and SP neurons at the presynaptic site has been demonstrated previously. During neurogenic inflammation N/OFQ-mediated inhibition of SP and CGRP release from sensory nerve terminals was observed [4] and an indirect interaction of N/OFQ with
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SP by a N/OFQ-stimulated local release of SP was postulated [5]. In the dorsal horn, N/OFQ suppresses excitatory, but not glycinergic or GABAergic inhibitory synaptic transmission to substantia gelatinosa neurons [7]; this action is presynaptic in origin. Due to low abundance of both ORL1 and preproN/OFQ mRNAs it was not possible to employ non-radioactive probes to directly examine whether preproN/OFQ primary afferent neurons are endowed with ORL1 receptors allowing autocrine presynaptic modulation of transmitter – especially glutamate – release. However, this seems unlikely because ORL1 expression was observed mainly in neurons with cell sizes larger than those of the extremely small N/OFQ neurons. Interestingly, F11 cells which express high levels of preproN/OFQ lack ORL1 transcripts supporting the view that preproN/OFQ and ORL1 are indeed expressed in different subpopulations of DRG neurons. Nevertheless, F11 cells may be ideally suited to study gene regulation of preproN/OFQ in a sensory cell relevant for nociception. Taken together we suggest a specific role for N/OFQ in paracrine presynaptic modulation of peptidergic signaling at the level of the primary afferent neuron which may be particularly relevant for pain control.
Acknowledgements This research was supported by SFB 297 and BMBF FKZ:01GG9818. We are grateful to H. Hlawaty and B. Wiegand for excellent technical assistance and to T. Csepella for helping to prepare the manuscript.
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