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Nociceptin-like immunoreactivity in the rat dorsal horn and inhibition of substantia gelatinosa neurons
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Neuroscience Vol. 81, No. 4, pp. 887–891, 1997 Copyright ? 1997 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306–4522/97 $17.00+0.00 S0306-4522(97)00251-0
Letter to Neuroscience NOCICEPTIN-LIKE IMMUNOREACTIVITY IN THE RAT DORSAL HORN AND INHIBITION OF SUBSTANTIA GELATINOSA NEURONS C. C. LAI, S. Y. WU, S. L. DUN and N. J. DUN* Department of Anatomy and Neurobiology, Medical College of Ohio, 3000 Arlington Avenue, Toledo, OH 43614, U.S.A. Key words: orphanin FQ, opioid peptide, primary afferent transmission, dorsal horn neurons.
Nociceptin, also referred to as orphanin FQ, is believed to be the endogenous ligand for the ORL1.8,9,12 Nociceptin, when injected intracerebroventricularly to mice, produced hyperalgesia in behavioral tests.8,12 Recent studies have demonstrated the presence of ORL1 transcript in the spinal cord, and ORL1-like immunoreactivity has been localized to nerve fibers and somata throughout the spinal cord.1,16 Here, we report the localization of nociceptin-like immunoreactivity to fiber-like elements of the superficial layers of the rat dorsal horn by immunohistochemical techniques. Whole-cell recordings from substantia gelatinosa neurons in transverse lumbar spinal cord slices of 22–26-day-old rats showed that exogenous nociceptin at low concentrations (100–300 nM) depressed excitatory postsynaptic potentials evoked by stimulation of dorsal rootlets without causing an appreciable change of resting membrane potentials and glutamate-evoked depolarizations. At a concentration of 1 µM, nociceptin hyperpolarized substantia gelatinosa neurons and suppressed spike discharges. The hyperpolarizing and synaptic depressant action of nociceptin was not reversed by the known opioid receptor antagonist naloxone (1 µM). Our result provides evidence that nociceptin-like peptide is concentrated in nerve fibers of the rat dorsal horn and that it may serve as an inhibitory transmitter within the substantia gelatinosa. ? 1997 IBRO. Published by Elsevier Science Ltd. Immunohistochemical procedures used in this study were essentially the same as described previously.2,3 *To whom correspondence should be addressed at: Department of Pharmacology, East Tennessee State University, P.O. Box 70577, Johnson City, TN 37614, U.S.A. (present address). Abbreviations: EGTA, ethylene glycolbis(aminoethylether) tetra-acetate; EPSP, excitatory postsynaptic potential; HEPES, N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid; NOCI-LI, nociceptin-like immunoreactivity; PBS, phosphate-buffered saline; SG, substantia gelatinosa.
Sprague–Dawley rats, 25–30 days old, of either sex (Zivic Miller, Zelienople, PA) were anesthetized with ketamine (70 mg/kg) and intracardially perfused with chilled phosphate-buffered saline (PBS) followed by freshly prepared 4% paraformaldehyde in PBS. Transverse spinal cord sections of 40 µm were prepared using a Vibratome and processed for nociceptin-like immunoreactivity (NOCI-LI) by the standard avidin–biotin complex method as described.2,3 Nociceptin antisera were a rabbit polyclonal directed against the rat nociceptin peptide (Phoenix Pharmaceuticals Inc., Mountain View, CA). The nociceptin-antisera, which were used at a dilution of 1:1000–2000 with 0.4% Triton X-100 and 1% bovine serum albumin in PBS, exhibited no cross-reactivity with [Met]enkephalin, dynorphin A and â-endorphin (Phoenix Pharmaceuticals Inc.). Two sets of control experiments were performed. First, nociceptin antisera were omitted in the staining procedures from randomly selected sections. Second, spinal sections were processed with nociceptinantisera pre-absorbed overnight with the peptide nociceptin (1 µM; Peninsula Laboratories, Belmont, CA). Procedures for obtaining 500-µm transverse lumbar spinal cord slices from 20–25-day-old rats have been described.17,18 The slice was held in place between two nylon grids and superfused with a Krebs solution of the following composition (in mM): 127 NaCl, 2.0 KCl, 1.2 KH2PO4, 2.4 CaCl2, 1.3 MgCl2, 26 NaHCO3 and 10 glucose; the solution was saturated with 95% O2 and 5% CO2. Patch electrodes filled with a solution containing (mM) 130 K+ gluconate, 1 MgCl2, 1 CaCl2, 4 ATP, 10 EGTA and 10 HEPES had a resistance of 3–5 MÙ. All experiments were carried out at room temperature (21&1)C). Viewed under a dissecting microscope with transmitted illumination, the substantia gelatinosa (SG) was readily discernable as a
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Fig. 1. Photomicrographs through a section of rat lumbar spinal cord labeled with nociceptin-antisera and a spinal section processed with nociceptin-antisera pre-absorbed with nociceptin (1 µM) overnight. (A) A low magnification showing that nociceptin-like immunoreactivity is largely confined to the superficial layers of the dorsal horn and around the central canal. (B) A higher magnification showing that nociceptin-like immunoreactivity is concentrated in networks of fiber-like elements around the central canal (cc) area. (C) A higher magnification showing that nociceptin-like immunoreactivity is concentrated in superficial layers of dorsal horn; fewer fibers are noted in the deeper laminae. (D) A control section showing no nociceptin-immunoreactivity. Scale bar=500 µm (A,D)=250 µm (B, C).
translucent band across the top of the dorsal horn. Whole-cell patch recordings were made from unidentified SG neurons. A bipolar concentric stimulating electrode (Fredrick Haer Co.) was placed close to the dorsal root entry zone to orthodromically activate SG neurons. Nociceptin was added to the Krebs solution and applied to the spinal slices by superfusion. In some experiments, nociceptin (300 µM) was ejected by pressure (40 p.s.i., 0.5–1 s pulse duration) from a micropipette positioned above the recording neuron with the use of a Picospritizer (General Valve Co.). Glutamate (10 mM) was applied to the recording neuron by pressure ejection (40 p.s.i., 10–30 ms pulse duration). Results are expressed as mean&S.D. and analysed with paired Student’s t-test. Examination of spinal cord sections from four rats labeled with nociceptin antisera revealed dense networks of NOCI-LI fiber-like elements occupying the first II-III laminae of the length of the spinal cord (Fig. 1A). Fewer NOCI-LI nerve fibers were seen in the deeper laminae (Fig. 1C); moderately dense
networks of NOCI-LI fibers were noted around the central canal (Fig. 1B). In the ventral horn, NOCI-LI fibers were sparse. A few slightly labeled NOCI-LI somata, generally of small diameter, were noted in the dorsal horn. Sections from control experiments were free of positive staining (Fig. 1D). Whole-cell recordings in current-clamp mode were obtained from SG neurons. These neurons had a mean resting membrane potential of "63&5.3 mV and input resistance of 950&38 MÙ (n=47). A single electrical stimulus applied to the dorsal root entry zone elicited in SG neurons an excitatory postsynaptic potential (EPSP) and/or inhibitory postsynaptic potential, as has been reported.19 To minimize the interference from inhibitory synaptic responses, the GABAA receptor antagonist bicuculline (10 µM) and glycine receptor blocker strychnine (1 µM) were routinely included in the Krebs solution. SG neurons that responded to electrical stimulations with a short latency (0.8–2 ms) EPSP were selected for further study. Nociceptin applied to spinal cord slices by superfusion produced two distinct effects that were
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Fig. 2. Depression of EPSPs but not glutamate-induced depolarizations by nociceptin in a rat substantia gelatinosa neuron. EPSPs (small upward deflections) were elicited by electrical stimulation of dorsal rootlets, and glutamate-depolarizations (larger upward deflections) were evoked by pressure application of glutamate from a micropipette positioned above the recording neuron. Top trace is a continuous chart recording; lower traces are averaged EPSPs (eight consecutive signals) taken at times marked on the continuous chart recording. Superfusion of nociceptin (300 nM), as indicated by a solid bar, depressed the EPSPs by ~30% but not the glutamate-induced depolarizations; the synaptic depressant effect was reversible upon wash.
concentration-dependent. At lower concentrations (100 or 300 nM), nociceptin depressed the EPSPs in all 17 SG neurons; the mean reduction was 13.2&4.1% (n=10, P<0.05) and 29.2&2.3% (n=7, P<0. 05). The averaged response of eight consecutive EPSPs taken at the end of 5 min superfusion of nociceptin was compared statistically with the averaged EPSP taken immediately prior to the start of superfusion. Synaptic depression was not accompanied by a detectable change of resting membrane potential and input resistance in these neurons. Nociceptin (30 nM) caused no detectable change of EPSPs in four SG neurons tested. While depressing the EPSPs, nociceptin (300 nM) had no significant effect on the depolarizations induced by exogenously applied glutamate in the same SG; a representative experiment is shown in Fig. 2. The mean amplitude of EPSPs was reduced to 63& 3.9% of control value (n=6; P<0.05) by nociceptin (300 nM), whereas the mean glutamatedepolarizations were 97&8% (n=6; P>0.05) of control responses. Prior incubation of the spinal slices with the opiate receptor antagonist naloxone (1 µM) did not prevent the synaptic depressant action of nociceptin in six cells tested. EPSPs were abolished by the non-NMDA receptor antagonist 6-cyano7-nitroquinoxaline-2,3-dione (1 µM), as has been reported.19 Nociceptin (1 µM) by superfusion or by pressure ejection caused a hyperpolarization that varied from several to over 15 mV in different SG neurons (n=22). A population of SG neurons displayed spontaneous ongoing activities. Nociceptin hyperpolarized and effectively eliminated ongoing discharges in these neurons (Fig. 3A). Nociceptin hyperpolarizations were associated with a decrease of membrane resistance (Fig. 3B). The response was made larger and smaller upon depolarization or hyperpolarization
and had a mean reversal potential of "95&4.3 mV (n=3), which is close to the calculated K+ equilibrium potential ("94 mV at 21)C). Nociceptin-induced hyperpolarization persisted in a tetrodotoxin (0.3 µM)containing Krebs solution (n=4) and was naloxoneinsensitive in three cells tested. Similar to the pattern of distribution reported in two recent studies,11,13 our immunohistochemical studies show that NOCI-LI was concentrated in fiber-like elements of the superficial layers of the rat dorsal horn. Although the distribution profile of NOCI-LI in the rat dorsal horn appears to be similar to that of other opioid peptides, enkephalin and dynorphin, 4,7 the large majority of NOCI-LI nerve fibers do not overlap with enkephalin- or dynorphincontaining fibers in the superficial layers of the dorsal horn.11,13 These findings, together with the earlier observations of the presence of ORL1 transcript and ORL1-like immunoreactivity in the spinal cord,1,16 indicate that nociceptin may constitute a distinct opioid system in the rat spinal cord. Insofar as the action is concerned, exogenous nociceptin may inhibit the activity of SG neurons by two mechanisms that are concentration-related. At low concentrations (¦300 nM), nociceptin attenuated excitatory synaptic transmission. This is consistent with recent observations that nociceptin depresses dorsal root-evoked ventral root potentials in hemisected rat spinal cord in vitro and discharges of dorsal horn neurons in vivo.5,14 The site and mechanism of synaptic depressant action of nociceptin remains to be clarified. The observations that synaptic depression is not accompanied by a detectable change of the resting membrane potential, input resistance and glutamate-depolarization suggest that the peptide may act presynaptically to decrease excitatory transmitter release, thereby a diminution of excitatory synaptic potentials. The suggestion that
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Fig. 3. Hyperpolarizations of substantia gelatinosa neurons by nociceptin. (A) Superfusion of nociceptin (1 µM) hyperpolarized this neuron and suppressed ongoing neuronal activities. (B) Pressure application of nociceptin (initial upward deflection denotes pressure ejection artifact) elicited a hyperpolarization associated with a decrease of membrane resistance, as indicated by a reduction (approximately 40%) of hyperpolarizing electrotonic potentials (small downward deflections) at the resting membrane potential of "62 mV. The response became smaller at more negative potentials and nullified at the membrane potential of about "92 mV; further hyperpolarization resulted in a depolarization. A plot of the amplitude of responses and membrane potential is shown to the right; the line intersects at about "94 mV, which is close to the reversal potential.
nociceptin may inhibit glutamate release from primary afferent fibers is in line with the recent demonstration of inhibition of K+ evoked glutamate release from rat cerebrocortical slices by the peptide.10 A presynaptic inhibitory action has been proposed to explain the synaptic depressant action of metenkephalin in dorsal horn neurons.6 In the latter study, met-enkephalin appears to reduce Ca2+ entry into presynaptic nerve terminals, thereby causing a reduction of transmitter release. It is not known whether nociceptin decreases transmitter release by reducing Ca2+ entry or by increasing K+ conductance of the terminal membrane. At high concentrations (¦1 µM) or by pressure ejections, nociceptin hyperpolarized SG neurons and suppressed spike discharges. Hyperpolarizations persisted in a TTX-containing solution, indicating that the peptide acted directly on SG neurons. The hyperpolarizations were accompanied by a decrease in membrane resistance and reversed near the calculated K+ equilibrium potential, suggesting that nociceptin may hyperpolarize SG neurons by increasing a K+ conductance. The specific type of K+ conductance affected by nociceptin is not known. The peptide
has been reported to hyperpolarize dorsal raphe neurons by increasing an inwardly rectifying K+ current.15 The synaptic depressant and hyperpolarizing action of nociceptin was insensitive to the opioid receptor antagonist naloxone. Our finding is in agreement with several recent studies that ORL receptors differ from other opioid receptors in their resistance to naloxone.5,10,14,15 Other than naloxoneinsensitive, the action of nociceptin appears to be similar to that of met-enkephalin, which has been found to hyperpolarize rat SG neurons and inhibit synaptic transmission.6,20 Our results and those of more recent studies5,14 suggest that in the dorsal horn nociceptin may act as an inhibitory transmitter in a manner similar to that of enkephalin, implying that the peptide may produce analgesia at the spinal level. In contrast, earlier studies indicated that nociceptin when administered intracerebroventricularly may produce hyperalgesia in mice.8,12 Differences in species and experimental conditions notwithstanding, nociceptin may produce hyperalgesia by acting on ORL1 of medullary neurons.
Nociceptin and dorsal horn neurons
In conclusion, the present study shows that nociceptin-like immunoreactivity is present in fiberlike elements of superficial layers of the rat dorsal horn and that the peptide applied exogenously to the spinal slices inhibits the activity of SG neurons by
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either depressing excitatory postsynaptic potentials or hyperpolarizing the neurons. Acknowledgements—This study was supported by NIH Grants NS18710 and HL51314.
REFERENCES
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Anton B., Fein J., To T., Li X., Silberstein L. and Evans C. J. (1996) Immunohistochemical localization of ORL-1 in the central nervous system of the rat. J. comp. Neurol. 368, 229–251. Dun N. J., Dun S. L., Wu S. Y., Fo¨rstermann U., Schmidt H. H. H. W. and Tseng L. F. (1993) Nitric oxide synthase immunoreactivity in the rat, mouse, cat and squirrel monkey spinal cord. Neuroscience 54, 845–857. Dun N. J., Miyazaki T., Tang H. and Dun E. C. (1996) Pituitary adenylate cyclase activating polypeptide immunoreactivity in the rat spinal cord and medulla: Implication of sensory and autonomic functions. Neuroscience 73, 677–686. Elde R., Ho¨kfelt T., Johanasson O. and Terenius L. (1976) Immunohistochemical studies using antibodies to leucine–enkephalin: initial observation on the nervous system of the rat. Neuroscience 1, 349–351. Faber E. S. L., Chamber J. P., Evans R. H. and Henderson G. (1996) Depression of glutamatergic transmission by nociceptin in the neonatal rat hemisected spinal cord preparation in vitro. Br. J. Pharmac. 119, 189–190. Hori Y., Endo K. and Takahashi T. (1992) Presynaptic inhibitory action of enkephalin on excitatory transmission in superficial dorsal horn neurons. J. Physiol., Lond. 450, 673–685. Khachaturian H., Watson S. L., Lewis M. E., Coy D., Goldstein A. and Akil H. (1982) Dynorphin immunocytochemistry in the rat central nervous system. Peptides 3, 941–954. Meunier J.-C., Mollereau C., Toll L., Suaudeau C., Moisand C., Alvinerie P., Butour J.-L., Guillemot J.-C., Ferrera P., Monserrat B., Mazarguil M, Vassart G., Parmentier M. and Constintin J. (1995) Isolation and structure of the endogenous agonists of opioid receptor-like ORL1 receptor. Nature 377, 532–535. Mollereau C., Pamentier M., Maileux R., Butour J.-L., Moisand C., Chalon P., Caput D., Vassart G. and Meunier J.-C. (1994) ORL1, a novel member of the opioid receptor family. Fedn Eur. biochem. Socs Lett. 341, 33–38. Nicol B., Lambert D. G., Rowbotham D. J., Smart D. and McKnight A. T. (1996) Nociceptin induced inhibition of K+ evoked glutamate release from rat cerebrocortical slices. Br. J. Pharmac. 119, 1081–1083. Reidl M., Shuster S., Vulchanova S., Wang J., Loh H. H. and Elde R. (1996) OrphaninFQ/nociceptin-immunoreactive nerve fibers parallel those containing endogenous opioids in rat spinal cord. NeuroReport 7, 1369–1372. Reinscheid R. K., Nothacker H.-P., Bourson A., Ardati A., Henningson R. A., Bunzow J. R., Grandy D. K., Langen H., Monsma F. J. Jr and Civelli O. (1995) Orphanin FQ: a neuropeptide that activated an opioid-like G protein-coupled receptor. Science 270, 792–794. Schulz S., Schreff M., Nu¨B D., Gramsch C. and Ho¨llt V. (1996) Nociceptin/orphanin FQ and opioid peptides show overlapping distribution but not co-localization in pain-modulatory brain regions. NeuroReport 7, 3021–3025. Stanfa L. C., Chapman V., Kerr N. and Dickenson A. H. (1996) Inhibitory action of nociceptin on spinal dorsal horn neurones of the rat in vivo. Br. J. Pharmac. 118, 1875–1877. Vaughan C. W. and Christie M. J. (1996) Increase by the ORL1 receptor (opioid receptor-like1) ligand, nociceptin, of inwardly rectifying K conductance in dorsal raphe nucleus neurones. Br. J. Pharmac. 117, 1609–1611. Wick M. J., Minnerath S. R., Lin X., Elde R., Law P. Y. and Loh H.-H. (1994) Isolation of a novel cDNA encoding a putative membrane receptor with high homology to the cloned µ, ä and ê opioid receptors. Molec. Brain Res. 117, 1609–1611. Wu S. Y. and Dun N. J. (1993) Excitatory amino acids depress synaptic currents in neonate rat sympathetic preganglionic neurons. J. Neurophysiol. 69, 203–2038. Wu S. Y. and Dun N. J. (1996) Potentiation of IPSCs by nitric oxide in immature rat sympathetic preganglionic neurones in vitro. J. Physiol., Lond. 495, 479–490. Yoshimura Y. and Nishi S. (1993) Blind patch-clamp recordings from substantia gelatinosa neurons in adult rat spinal cord slices: pharmacological properties of synaptic currents. Neuroscience 53, 519–526. Yoshimura Y. and North R. A. (1983) Substantia gelatinosa neurones hyperpolarized in vitro by enkephalin. Nature 305, 529–530. (Accepted 16 May 1997)
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Neuroscience Vol. 81, No. 4, pp. 887–891, 1997 Copyright ? 1997 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306–4522/97 $17.00+0.00 S0306-4522(97)00251-0
Letter to Neuroscience NOCICEPTIN-LIKE IMMUNOREACTIVITY IN THE RAT DORSAL HORN AND INHIBITION OF SUBSTANTIA GELATINOSA NEURONS C. C. LAI, S. Y. WU, S. L. DUN and N. J. DUN* Department of Anatomy and Neurobiology, Medical College of Ohio, 3000 Arlington Avenue, Toledo, OH 43614, U.S.A. Key words: orphanin FQ, opioid peptide, primary afferent transmission, dorsal horn neurons.
Nociceptin, also referred to as orphanin FQ, is believed to be the endogenous ligand for the ORL1.8,9,12 Nociceptin, when injected intracerebroventricularly to mice, produced hyperalgesia in behavioral tests.8,12 Recent studies have demonstrated the presence of ORL1 transcript in the spinal cord, and ORL1-like immunoreactivity has been localized to nerve fibers and somata throughout the spinal cord.1,16 Here, we report the localization of nociceptin-like immunoreactivity to fiber-like elements of the superficial layers of the rat dorsal horn by immunohistochemical techniques. Whole-cell recordings from substantia gelatinosa neurons in transverse lumbar spinal cord slices of 22–26-day-old rats showed that exogenous nociceptin at low concentrations (100–300 nM) depressed excitatory postsynaptic potentials evoked by stimulation of dorsal rootlets without causing an appreciable change of resting membrane potentials and glutamate-evoked depolarizations. At a concentration of 1 µM, nociceptin hyperpolarized substantia gelatinosa neurons and suppressed spike discharges. The hyperpolarizing and synaptic depressant action of nociceptin was not reversed by the known opioid receptor antagonist naloxone (1 µM). Our result provides evidence that nociceptin-like peptide is concentrated in nerve fibers of the rat dorsal horn and that it may serve as an inhibitory transmitter within the substantia gelatinosa. ? 1997 IBRO. Published by Elsevier Science Ltd. Immunohistochemical procedures used in this study were essentially the same as described previously.2,3 *To whom correspondence should be addressed at: Department of Pharmacology, East Tennessee State University, P.O. Box 70577, Johnson City, TN 37614, U.S.A. (present address). Abbreviations: EGTA, ethylene glycolbis(aminoethylether) tetra-acetate; EPSP, excitatory postsynaptic potential; HEPES, N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid; NOCI-LI, nociceptin-like immunoreactivity; PBS, phosphate-buffered saline; SG, substantia gelatinosa.
Sprague–Dawley rats, 25–30 days old, of either sex (Zivic Miller, Zelienople, PA) were anesthetized with ketamine (70 mg/kg) and intracardially perfused with chilled phosphate-buffered saline (PBS) followed by freshly prepared 4% paraformaldehyde in PBS. Transverse spinal cord sections of 40 µm were prepared using a Vibratome and processed for nociceptin-like immunoreactivity (NOCI-LI) by the standard avidin–biotin complex method as described.2,3 Nociceptin antisera were a rabbit polyclonal directed against the rat nociceptin peptide (Phoenix Pharmaceuticals Inc., Mountain View, CA). The nociceptin-antisera, which were used at a dilution of 1:1000–2000 with 0.4% Triton X-100 and 1% bovine serum albumin in PBS, exhibited no cross-reactivity with [Met]enkephalin, dynorphin A and â-endorphin (Phoenix Pharmaceuticals Inc.). Two sets of control experiments were performed. First, nociceptin antisera were omitted in the staining procedures from randomly selected sections. Second, spinal sections were processed with nociceptinantisera pre-absorbed overnight with the peptide nociceptin (1 µM; Peninsula Laboratories, Belmont, CA). Procedures for obtaining 500-µm transverse lumbar spinal cord slices from 20–25-day-old rats have been described.17,18 The slice was held in place between two nylon grids and superfused with a Krebs solution of the following composition (in mM): 127 NaCl, 2.0 KCl, 1.2 KH2PO4, 2.4 CaCl2, 1.3 MgCl2, 26 NaHCO3 and 10 glucose; the solution was saturated with 95% O2 and 5% CO2. Patch electrodes filled with a solution containing (mM) 130 K+ gluconate, 1 MgCl2, 1 CaCl2, 4 ATP, 10 EGTA and 10 HEPES had a resistance of 3–5 MÙ. All experiments were carried out at room temperature (21&1)C). Viewed under a dissecting microscope with transmitted illumination, the substantia gelatinosa (SG) was readily discernable as a
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Fig. 1. Photomicrographs through a section of rat lumbar spinal cord labeled with nociceptin-antisera and a spinal section processed with nociceptin-antisera pre-absorbed with nociceptin (1 µM) overnight. (A) A low magnification showing that nociceptin-like immunoreactivity is largely confined to the superficial layers of the dorsal horn and around the central canal. (B) A higher magnification showing that nociceptin-like immunoreactivity is concentrated in networks of fiber-like elements around the central canal (cc) area. (C) A higher magnification showing that nociceptin-like immunoreactivity is concentrated in superficial layers of dorsal horn; fewer fibers are noted in the deeper laminae. (D) A control section showing no nociceptin-immunoreactivity. Scale bar=500 µm (A,D)=250 µm (B, C).
translucent band across the top of the dorsal horn. Whole-cell patch recordings were made from unidentified SG neurons. A bipolar concentric stimulating electrode (Fredrick Haer Co.) was placed close to the dorsal root entry zone to orthodromically activate SG neurons. Nociceptin was added to the Krebs solution and applied to the spinal slices by superfusion. In some experiments, nociceptin (300 µM) was ejected by pressure (40 p.s.i., 0.5–1 s pulse duration) from a micropipette positioned above the recording neuron with the use of a Picospritizer (General Valve Co.). Glutamate (10 mM) was applied to the recording neuron by pressure ejection (40 p.s.i., 10–30 ms pulse duration). Results are expressed as mean&S.D. and analysed with paired Student’s t-test. Examination of spinal cord sections from four rats labeled with nociceptin antisera revealed dense networks of NOCI-LI fiber-like elements occupying the first II-III laminae of the length of the spinal cord (Fig. 1A). Fewer NOCI-LI nerve fibers were seen in the deeper laminae (Fig. 1C); moderately dense
networks of NOCI-LI fibers were noted around the central canal (Fig. 1B). In the ventral horn, NOCI-LI fibers were sparse. A few slightly labeled NOCI-LI somata, generally of small diameter, were noted in the dorsal horn. Sections from control experiments were free of positive staining (Fig. 1D). Whole-cell recordings in current-clamp mode were obtained from SG neurons. These neurons had a mean resting membrane potential of "63&5.3 mV and input resistance of 950&38 MÙ (n=47). A single electrical stimulus applied to the dorsal root entry zone elicited in SG neurons an excitatory postsynaptic potential (EPSP) and/or inhibitory postsynaptic potential, as has been reported.19 To minimize the interference from inhibitory synaptic responses, the GABAA receptor antagonist bicuculline (10 µM) and glycine receptor blocker strychnine (1 µM) were routinely included in the Krebs solution. SG neurons that responded to electrical stimulations with a short latency (0.8–2 ms) EPSP were selected for further study. Nociceptin applied to spinal cord slices by superfusion produced two distinct effects that were
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Fig. 2. Depression of EPSPs but not glutamate-induced depolarizations by nociceptin in a rat substantia gelatinosa neuron. EPSPs (small upward deflections) were elicited by electrical stimulation of dorsal rootlets, and glutamate-depolarizations (larger upward deflections) were evoked by pressure application of glutamate from a micropipette positioned above the recording neuron. Top trace is a continuous chart recording; lower traces are averaged EPSPs (eight consecutive signals) taken at times marked on the continuous chart recording. Superfusion of nociceptin (300 nM), as indicated by a solid bar, depressed the EPSPs by ~30% but not the glutamate-induced depolarizations; the synaptic depressant effect was reversible upon wash.
concentration-dependent. At lower concentrations (100 or 300 nM), nociceptin depressed the EPSPs in all 17 SG neurons; the mean reduction was 13.2&4.1% (n=10, P<0.05) and 29.2&2.3% (n=7, P<0. 05). The averaged response of eight consecutive EPSPs taken at the end of 5 min superfusion of nociceptin was compared statistically with the averaged EPSP taken immediately prior to the start of superfusion. Synaptic depression was not accompanied by a detectable change of resting membrane potential and input resistance in these neurons. Nociceptin (30 nM) caused no detectable change of EPSPs in four SG neurons tested. While depressing the EPSPs, nociceptin (300 nM) had no significant effect on the depolarizations induced by exogenously applied glutamate in the same SG; a representative experiment is shown in Fig. 2. The mean amplitude of EPSPs was reduced to 63& 3.9% of control value (n=6; P<0.05) by nociceptin (300 nM), whereas the mean glutamatedepolarizations were 97&8% (n=6; P>0.05) of control responses. Prior incubation of the spinal slices with the opiate receptor antagonist naloxone (1 µM) did not prevent the synaptic depressant action of nociceptin in six cells tested. EPSPs were abolished by the non-NMDA receptor antagonist 6-cyano7-nitroquinoxaline-2,3-dione (1 µM), as has been reported.19 Nociceptin (1 µM) by superfusion or by pressure ejection caused a hyperpolarization that varied from several to over 15 mV in different SG neurons (n=22). A population of SG neurons displayed spontaneous ongoing activities. Nociceptin hyperpolarized and effectively eliminated ongoing discharges in these neurons (Fig. 3A). Nociceptin hyperpolarizations were associated with a decrease of membrane resistance (Fig. 3B). The response was made larger and smaller upon depolarization or hyperpolarization
and had a mean reversal potential of "95&4.3 mV (n=3), which is close to the calculated K+ equilibrium potential ("94 mV at 21)C). Nociceptin-induced hyperpolarization persisted in a tetrodotoxin (0.3 µM)containing Krebs solution (n=4) and was naloxoneinsensitive in three cells tested. Similar to the pattern of distribution reported in two recent studies,11,13 our immunohistochemical studies show that NOCI-LI was concentrated in fiber-like elements of the superficial layers of the rat dorsal horn. Although the distribution profile of NOCI-LI in the rat dorsal horn appears to be similar to that of other opioid peptides, enkephalin and dynorphin, 4,7 the large majority of NOCI-LI nerve fibers do not overlap with enkephalin- or dynorphincontaining fibers in the superficial layers of the dorsal horn.11,13 These findings, together with the earlier observations of the presence of ORL1 transcript and ORL1-like immunoreactivity in the spinal cord,1,16 indicate that nociceptin may constitute a distinct opioid system in the rat spinal cord. Insofar as the action is concerned, exogenous nociceptin may inhibit the activity of SG neurons by two mechanisms that are concentration-related. At low concentrations (¦300 nM), nociceptin attenuated excitatory synaptic transmission. This is consistent with recent observations that nociceptin depresses dorsal root-evoked ventral root potentials in hemisected rat spinal cord in vitro and discharges of dorsal horn neurons in vivo.5,14 The site and mechanism of synaptic depressant action of nociceptin remains to be clarified. The observations that synaptic depression is not accompanied by a detectable change of the resting membrane potential, input resistance and glutamate-depolarization suggest that the peptide may act presynaptically to decrease excitatory transmitter release, thereby a diminution of excitatory synaptic potentials. The suggestion that
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Fig. 3. Hyperpolarizations of substantia gelatinosa neurons by nociceptin. (A) Superfusion of nociceptin (1 µM) hyperpolarized this neuron and suppressed ongoing neuronal activities. (B) Pressure application of nociceptin (initial upward deflection denotes pressure ejection artifact) elicited a hyperpolarization associated with a decrease of membrane resistance, as indicated by a reduction (approximately 40%) of hyperpolarizing electrotonic potentials (small downward deflections) at the resting membrane potential of "62 mV. The response became smaller at more negative potentials and nullified at the membrane potential of about "92 mV; further hyperpolarization resulted in a depolarization. A plot of the amplitude of responses and membrane potential is shown to the right; the line intersects at about "94 mV, which is close to the reversal potential.
nociceptin may inhibit glutamate release from primary afferent fibers is in line with the recent demonstration of inhibition of K+ evoked glutamate release from rat cerebrocortical slices by the peptide.10 A presynaptic inhibitory action has been proposed to explain the synaptic depressant action of metenkephalin in dorsal horn neurons.6 In the latter study, met-enkephalin appears to reduce Ca2+ entry into presynaptic nerve terminals, thereby causing a reduction of transmitter release. It is not known whether nociceptin decreases transmitter release by reducing Ca2+ entry or by increasing K+ conductance of the terminal membrane. At high concentrations (¦1 µM) or by pressure ejections, nociceptin hyperpolarized SG neurons and suppressed spike discharges. Hyperpolarizations persisted in a TTX-containing solution, indicating that the peptide acted directly on SG neurons. The hyperpolarizations were accompanied by a decrease in membrane resistance and reversed near the calculated K+ equilibrium potential, suggesting that nociceptin may hyperpolarize SG neurons by increasing a K+ conductance. The specific type of K+ conductance affected by nociceptin is not known. The peptide
has been reported to hyperpolarize dorsal raphe neurons by increasing an inwardly rectifying K+ current.15 The synaptic depressant and hyperpolarizing action of nociceptin was insensitive to the opioid receptor antagonist naloxone. Our finding is in agreement with several recent studies that ORL receptors differ from other opioid receptors in their resistance to naloxone.5,10,14,15 Other than naloxoneinsensitive, the action of nociceptin appears to be similar to that of met-enkephalin, which has been found to hyperpolarize rat SG neurons and inhibit synaptic transmission.6,20 Our results and those of more recent studies5,14 suggest that in the dorsal horn nociceptin may act as an inhibitory transmitter in a manner similar to that of enkephalin, implying that the peptide may produce analgesia at the spinal level. In contrast, earlier studies indicated that nociceptin when administered intracerebroventricularly may produce hyperalgesia in mice.8,12 Differences in species and experimental conditions notwithstanding, nociceptin may produce hyperalgesia by acting on ORL1 of medullary neurons.
Nociceptin and dorsal horn neurons
In conclusion, the present study shows that nociceptin-like immunoreactivity is present in fiberlike elements of superficial layers of the rat dorsal horn and that the peptide applied exogenously to the spinal slices inhibits the activity of SG neurons by
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either depressing excitatory postsynaptic potentials or hyperpolarizing the neurons. Acknowledgements—This study was supported by NIH Grants NS18710 and HL51314.
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