- Email: [email protected]
orphanin FQ receptor-knockout mice
Brain Research 783 Ž1998. 236–240
Research report
Enhancement of spatial attention in nociceptinrorphanin FQ receptor-knockout mice Takayoshi Mamiya a , Yukihiro Noda a , Miyuki Nishi b , Hiroshi Takeshima b , Toshitaka Nabeshima a
a, )
Department of Neuropsychopharmacology and Hospital Pharmacy, Nagoya UniÕersity School of Medicine, 65 Tsuruma-cho, Showa-ku, Nagoya 466, Japan b Department of Pharmacology, Faculty of Medicine, UniÕersity of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan Accepted 18 November 1997
Abstract We isolated genes for the opioid receptor homologue MOR-C, namely nociceptin receptor Ždesignated alternatively as orphanin FQ receptor. and generated nociceptin receptor-knockout mice. Previously, we have reported that the nociceptin system appears to participate in the regulation of the auditory system. However, the behavior of the nociceptin receptor-knockout mice has yet to be fully characterized. In the present study, we investigated changes in several behavioral performances in mice which lack nociceptin receptor. Nociceptive thresholds of nociceptin receptor-knockout mice were unchanged in the hot-plate and electric foot-shock tests as well as tail-flick and acetic acid-induced writhing tests compared to those of wild-type mice. The nociceptin receptor-knockout mice did not show any behavioral changes in the elevated plus-maze task. Surprisingly, in the water-finding test, the nociceptin receptor-knockout mice showed an enhanced retention of spatial attention Žlatent learning. compared to wild-type mice. In a biochemical study, dopamine content in the frontal cortex was lower in nociceptin receptor-knockout mice than wild-type mice. These results suggest that nociceptin receptor plays an important role in spatial attention by regulating the dopaminergic system in the brain. q 1998 Elsevier Science B.V. Keywords: Nociceptin receptor; Knockout mice; Water-finding test; Spatial attention Žlatent learning.; Dopamine content
1. Introduction Soon after the cloning of the d opioid receptor w6,11x, the receptor for unknown ligand belonging to the opioid receptor family has been reported from several groups and designated independently as ORL1, ROR-C, LC132 and C3 w2,7,12,14x. This receptor exhibited 68% homology with m receptor, 67% with d receptor, 66% with k receptor, and 32% with somatostatin receptor, and is abundantly expressed in rat and mouse brain w5x. Recently, the endogenous ligand for this receptor has been isolated and named nociceptin or orphanin FQ w16,24x. Nociceptin is derived from a larger precursor which shows sequence similarity to the opioid peptide precursors, particularly pre-prodynorphin w9,15,21x. Several studies have reported the physiological functions of nociceptin. For example, in contrast to the opioid peptides with analgesic effects, Abbreviations: NE: norepinephrine; DA: dopamine; DOPAC: 3,4-dihydroxyphenylacetic acid; HVA: homovanillic acid ) Corresponding author. Fax: q 81-52-744-2979; E-mail: [email protected] 0006-8993r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 1 4 0 6 - 6
nociceptin induces hyperalgesia w16,24x and allodynia w22x, when administered intracerebroventricularly Ži.c.v.. and intrathecally, respectively. Nociceptin or orphanin FQ inhibits adenylyl cyclase activity in cells transfected with ORL1 w16x or LC132 w24x. Furthermore, nociceptin was shown to increase Kq conductance in rat dorsal raphe neurons in vitro w27x, and to inhibit voltage-dependent Ca2q channel currents in the human neuroblastoma cell line, SH-SY5Y w4x. In situ hybridization analysis w2,3,7,14x and immunohistochemistry w1x revealed distribution of ORL1 in many areas of the central nervous system. However, specific antagonists for nociceptin receptor are not available, and the physiological roles of nociceptin have yet to be elucidated at the whole-animal level. One approach would be to produce knockout mice lacking the nociceptin receptor by means of gene targeting and analyze the physiological phenotype of the mutants. Recently, we have reported that the nociceptin system appears to participate in the regulation of the auditory system w19x. To characterize the role of nociceptin receptor in whole animals, we investigated several behavioral performances in nociceptin receptor-knockout mice.
T. Mamiya et al.r Brain Research 783 (1998) 236–240
237
2. Materials and methods 2.1. Animals We used male nociceptin receptor-knockout and wildtype mice Ž9–12 weeks old. which have been reported w19x. The animals were housed in a controlled environment Ž23 " 18C, 50 " 5% humidity. and allowed food and water ad libitum. The room lights were off between 2000 and 0800. All experimental protocols were conducted with due regard for the Japanese Experimental Animal Research Association standards as defined in the Guidelines for Animal Experiments Ž1987., and were approved in advance by the Animal Research Committee at Nagoya University. 2.2. EleÕated plus-maze test The elevated plus-maze was a modification of the apparatus and consisted of two open Ž25 = 8 = 0.5 cm. and two closed arms Ž25 = 8 = 20 cm. emanating from a common central platform Ž8 = 8 cm. to form a plus shape w13x. The entire apparatus was elevated to a height of 50 cm above floor level. Testing commenced by placing a mouse on the central platform of the maze facing an open arm, and a standard 5-min test duration was employed. Conventional parameters consisted of the frequency of open arms and closed arms, total arm entries, and the amount of time spent by mice in the open arms, on the central platform and in the closed arms of the maze. These data were also used to calculate percent open arm entries wi.e., Žopen arm entriesrtotal arm entries. = 100x and percent time spent in open and closed arms wi.e., Žopen timeropen and closed time. = 100x. 2.3. NociceptiÕe test Nociception was determined using the hot-plate and electric foot-shock tests w17x.
Fig. 1. ŽA. Percent open arm entries and percent time spent in open arms on the elevated plus-maze test in wild-type Žqrq. and knockout Žyry. mice. ŽB. Response to nociceptive Žthermal and electric. stimuli in wild-type Žqrq. and knockout Žyry. mice. The numbers in the column represent the number of mice used.
Fig. 2. Performance in the water-finding test in wild-type Žqrq. and knockout Žyry. mice. ) P - 0.05, ) ) P - 0.01 vs. corresponding naive group, †P - 0.05 vs. wild-type control mice ŽStudent’s t-test.. The numbers in the column represent the number of mice used.
In the hot-plate test Ž55 " 18C., hot-plate latency represents time elapsed until the mice licked their hind paws andror jumping. In the electric foot-shock test, we used a transparent acrylic rectangular cage Ž23 = 28 = 12 cm. equipped with a metal wire floor. The shock intensity was raised stepwise manually from 0.1 to 2.0 mA in 0.1 mA increments Ž20-s interval. until a flinch was observed ŽShockgenerator NS-SG01, Neuroscience, Tokyo, Japan.. 2.4. Water-finding task The apparatus and procedure were constructed as described previously w10,18x. The test consisted of two trials: a training trial Žthe 1st day. and retention trial Žthe 2nd day.. Briefly, the apparatus consisted of an open field with an alcove in the middle of one of the long walls of the enclosure. The floor of the open field was divided into 15 identical squares for measuring locomotor activity. A drinking tube was inserted into the center of the alcove ceiling with its tip 5 cm Žin the training trial. or 7 cm Žin the retention trial. above the floor. In the training trial, mice not deprived of water were placed individually into one corner of the open field. Each mouse was allowed 3 min to explore the environment; the 3 min were counted from the time the mouse started to explore. During this time, ambulation was measured by counting the number of times the animals crossed from one square to another in the open field Žlocomotion count.. The frequency of touching, sniffing, or licking of the water tube in the alcove Žnumber of approaches. was also recorded. Animals that did not start exploring after 3 min had elapsed or that did not find the drinking tube during the 3-min exploratory period were omitted from the retention trial. Non-trained mice Žnaive mice. were prepared for comparison with the trained mice Žcontrol mice. in terms of their ability to find the water source in the same environment. The mice were immediately returned to their home cages after the training trial and were again individually placed on the test apparatus 24 h later for a single retention trial. The mice were deprived of water for 24 h before the retention trial. In the retention trial, mice were
T. Mamiya et al.r Brain Research 783 (1998) 236–240
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Table 1 NE, DOPAC, DA and HVA contents and DOPACrDA and HVArDA ratios in the frontal cortex, hippocampus and striatum in wild-type Žqrq . and knockout Žyry . mice Region
Phenotype
N
Frontal cortex
qrq yry qrq yry qrq yry
5 8 5 8 5 8
Hippocampus Striatum
Contents Žngrg wet tissue.
Ratio
NE
DOPAC
DA
HVA
DOPACrDA
HVArDA
419 " 18 387 " 16 499 " 34 480 " 37 120 " 18 117 " 7
88 " 6 86 " 5 40 " 8 49 " 1 3371 " 322 3347 " 172
94 " 15 64 " 4 ) 32 " 8 41 " 6 18 476 " 1472 17 295 " 627
123 " 6 120 " 13 154 " 6 138 " 9 1455 " 108 1456 " 120
0.92 " 0.23 1.36 " 0.09 1.20 " 0.16 1.21 " 0.25 0.19 " 0.02 0.19 " 0.01
1.30 " 0.37 1.87 " 0.24 4.82 " 0.55 3.41 " 0.38 0.08 " 0.005 0.08 " 0.004
)
P - 0.05 vs. wild-type mice ŽStudent’s t-test.. N represents the number of mice used.
individually placed in the same corner of the apparatus as that used during the training trial. The time until the mouse moved out of the corner was measured as the starting latency. In addition, the time taken to enter the alcove Žentering latency. and the time between entering the alcove and drinking the water Žfinding latency. were scored. Thus, the drinking latency consisted of the sum of the entering and finding latencies. In other words, memory acquisition was able to be assessed by recording four latencies Žstarting, entering, finding and drinking., ambulation counts and the number of approaches. 2.5. Measurement of monoamine contents Animals were killed by decapitation. The brain was rapidly removed and the frontal cortex, hippocampus and striatum were dissected out on an ice-cold plate. Each tissue sample was quickly frozen in dry ice and stored in a deep freezer at y808C until assayed. The contents of norepinephrine ŽNE., dopamine ŽDA., 3,4-dihydroxyphenylacetic acid ŽDOPAC. and homovanillic acid ŽHVA. in the three regions were determined using high-performance liquid chromatography with electrochemical detection w20x. 2.6. Statistical analysis All results were expressed as the mean " S.E.M. for each group. Statistical analysis of the difference between groups was assessed with Student’s t-test. P - 0.05 was taken as the significant level of difference.
3. Results 3.1. EleÕated plus-maze test Fig. 1A shows the performance on the elevated plusmaze test in wild-type and knockout mice. Total arm entries and closed arm entries were not significantly different between the two groups Ždata not shown.. No significant differences were detected in percent open arm entries
and percent time spent in open arms between wild-type and knockout mice. 3.2. NociceptiÕe test Nociceptive thresholds in two nociceptive tests are shown in Fig. 1B. No measurable difference in the antinociceptive thresholds to thermal and electric stimuli was observed between wild-type and knockout mice. 3.3. Water-finding test In the training trial of the water-finding test, there was no significant difference in any latency, the ambulation counts and the number of approaches between wild-type and knockout mice Ždata not shown.. Fig. 2 shows the results of the performance in the retention trial in wild-type and knockout mice. No measurable difference was observed between both naive mice. The finding and drinking latencies in wild-type and knockout mice were shorter than those in the corresponding naive mice. The entering latency was shorter in wild-type than in the corresponding naive mice, but not in knockout mice. Furthermore, the finding latency of knockout mice was significantly shorter than that of wild-type mice. 3.4. Difference in monoamines and DOPACr DA and HVA r DA ratio The contents of NE, DOPAC, DA and HVA and DOPACrDA and HVArDA ratios in three different regions in wild-type and knockout mice are shown in Table 1. DA content in the frontal cortex of knockout mice was significantly low compared to that of wild-type mice. The contents and ratios in other regions did not significantly differ between knockout and wild-type mice.
4. Discussion We have previously reported generation and characterization of mice lacking the nociceptin receptor w19x. No
T. Mamiya et al.r Brain Research 783 (1998) 236–240
obvious morphological abnormalities could be detected in the knockout mice. Further, the knockout mice did not differ from wild-type littermates in health, growth and reproduction. As shown previously, a statistical analysis revealed no significant difference between wild-type and knockout mice in locomotor activity w19x. The state of anxiety in knockout mice was evaluated by elevated plus-maze. Animals with high anxiety show low percent open arm entries and low percent time spent in open arms in this task w13x. For example, benzodiazepines increased these parameters w13x. Ordinarily, unstressed animals will spend approximately 15–25% of the trial in the open arms of the maze w8,23,26x. However, because of the lighting for our environment, wild-type mice spent less than 10% of the trial on the open arms of the maze in our experiment. We did not detect any difference in performances on the elevated plus-maze between wild-type and knockout mice. Taken together, these findings suggest that nociceptin receptor is involved in neither anxiety nor anti-anxiety. Since the nociceptin receptor has been reported to be involved in nociceptive response w16,24x, we compared the response to some nociceptive stimuli in wild-type and knockout mice. However, no measurable difference in the nociceptive thresholds to thermal and electric stimuli was observed between wild-type and knockout mice. These results were consistent with our previous reports w19,22x. It is suggested that these knockout mice do not have any obvious behavioral and anatomical abnormalities. A regional distribution of mRNA for the nociceptin precursor has been shown in the brain and spinal cord w9,15,21x. Expression levels of nociceptin receptor mRNA are high in the periaqueductal gray, dorsal raphe nucleus, locus coeruleus and the dorsal horn of the spinal cord w2x. An in situ hybridization study indicated that mRNA for ORL1 is expressed in cortical and limbic regions of the brain, suggesting that nociceptin receptor plays an important role in cognitive, mnemonic and attentional processes w12x. Recently, immunohistochemical localization of ORL1 in rat brain was reported, with intense and specific immunolabelling observed in the hippocampus, brainstem and cortex w1x which were relevant to learning and memory. Furthermore, nociceptinrorphanin FQ has been reported to induce the impairment of spatial learning w25x and inhibits synaptic transmission and long-term potentiation w28x in the hippocampus. Thus, there is a possibility that nociceptin andror nociceptin receptor are involved in learning and memory. Our present study examined the ability of learning and memory, particularly latent learning, in the knockout mice. Water-finding test has been used as an index of latent learning w10,18x. In the task, we did not use any motivation to train animals, since non water-deprived animals were trained in the apparatus with the drinking tube. However, when animals were deprived of water before the retention test, animals which had previously experienced the apparatus exhibited shorter en-
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tering, finding and drinking latencies than the non-experienced naive animals in the retention trial. In other words, control mice entered the alcove and found the drinking tube more quickly than did naive mice. In the present study, neither the latencies or ambulation counts, nor the number of approaches in the training trial, differed significantly between wild-type and knockout mice. Interestingly, in the retention trial, the finding latency of knockout mice was significantly shortened compared to wild-type mice, indicating that the knockout mice found the drinking tube more quickly than wild-type mice. These results suggest that the latent learning ability of knockout mice was better than that of wild-type mice. The mechanism of the enhancement in latent learning is unknown. Possibly, stress such as thirstiness may release endogenous excess nociceptin and impair the ability of latent learning in wild-type mice. Such stress-induced nociceptin may inhibit the latent-learning function through nociceptin receptor, although we cannot currently measure endogenously released nociceptin during this task. Ichihara et al. w10x have reported that dopamine receptor agonists impair latent learning in the water-finding test in mice, suggesting that activation of the dopaminergic system impairs the ability of latent-learning ability. In the present biochemical study, dopamine contents in the frontal cortex of knockout mice were significantly low compared to those of wild-type mice. HVArDA ratio, one of the parameters of dopamine turnover in the hippocampus, tended to be low in knockout compared to wild-type mice. These results suggest that the function of the dopaminergic system in knockout mice was reduced compared to that of wild-type mice. Alternatively, the enhancement of latent learning may result from the suppression of dopaminergic function in knockout mice. In addition, our findings suggest nociceptin system may regulate stimulatively the dopaminergic system. However, further studies are required to clarify the role of nociceptin andror nociceptin receptor in learning and memory.
5. Conclusion Our behavioral analysis reveals that the nociceptin receptor-knockout mice display a significant enhancement of latent learning, although no obvious morphological or behavioral abnormalities could be detected. Our results suggest that nociceptin receptor plays an important role in latent learning by modulating the dopaminergic system in the brain.
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w2x J.B. Bunzow, C. Saez, M. Mortrud, C. Bouvier, J.T. Williams, M. Low, D.K. Grandy, Molecular cloning and tissue distribution of a putative member of the rat opioid receptor gene family that is not a m , d or k opioid receptor type, FEBS Lett. 347 Ž1994. 284–288. w3x Y. Chen, Y. Fan, J. Liu, A. Mestek, M. Tian, C.A. Kozak, L. Yu, Molecular cloning, tissue distribution and chromosomal localization of a novel member of the opioid receptor gene family, FEBS Lett. 347 Ž1994. 279–283. w4x M. Connor, A. Yeo, G. Henderson, Nociceptin inhibits Ca2q channel currents and elevates intracellular Ca2q in the SH-SY5Y human neuroblastoma cell line, Br. J. Pharmacol. 118 Ž1996. 205–207. w5x C.T. Dooley, R.A. Houghten, Orphanin FQ: receptor binding and analog structure activity relationships in rat brain, Life Sci. 59 Ž1996. PL23–PL29. w6x C.J. Evans, D.E. Keith, H. Morrison, K. Magendzo, R.H. Edwards, Cloning of a delta opioid receptor by functional expression, Science 258 Ž1992. 1952–1955. w7x K. Fukuda, S. Kato, K. Mori, M. Nishi, H. Takeshima, N. Iwabe, T. Miyata, T. Houtani, T. Sugimoto, cDNA cloning and regional distribution of a novel member of the opioid receptor family, FEBS Lett. 343 Ž1994. 42–46. w8x L.A. Hilakivi, R.G. Lister, Correlations between behavior of mice in Porsolt’s swim test and in tests of anxiety, locomotion, and exploration, Behav. Neural Biol. 53 Ž1990. 153–159. w9x T. Houtani, M. Nishi, H. Takeshima, T. Nukada, T. Sugimoto, Structure and regional distribution of nociceptionrorphanin FQ precursor, Biochem. Biophys. Res. Commun. 219 Ž1996. 714–719. w10x K. Ichihara, T. Nabeshima, T. Kameyama, Dopaminergic agonists impair latent learning in mice: possible modulation by noradrenergic function, J. Pharmacol. Exp. Ther. 264 Ž1993. 122–128. w11x B.L. Kieffer, K. Befort, C. Gaveriaux-Ruff, C.G. Hirth, The d-opioid receptor: isolation of a cDNA by expression cloning and pharmacological characterization, Proc. Natl. Acad. Sci. U.S.A. 89 Ž1992. 12048–12052. w12x J.E. Lachowicz, Y. Shen, F.J. Monsma Jr., D.R. Sibley, Molecular cloning of a novel G protein-coupled receptor related to the opiate receptor family, J. Neurochem. 64 Ž1995. 34–40. w13x R.G. Lister, The use of a plus-maze to measure anxiety in the mouse, Psychopharmacology 92 Ž1987. 180–185. w14x C. Moullereau, M. Parmentier, P. Mailleux, J.L. Butour, C. Moisand, P. Chalon, D. Caput, G. Vssart, J.C. Meunier, ORL1, a novel member of the opioid receptor family: cloning, functional expression and localization, FEBS Lett. 341 Ž1994. 33–38. w15x C. Moullereau, M.J. Simons, P. Soularue, F. Liners, G. Vssart, J.C. Meunier, M. Parmentier, Structure, tissue distribution, and chromosomal localization of the prepronociceptin gene, Proc. Natl. Acad. Sci. U.S.A. 93 Ž1996. 8666–8670. w16x J.C. Meunier, C. Mollereau, L. Toll, C. Suaudeau, C. Moisand, P. Alvinerle, J.-L. Butour, J.-C. Guillemot, P. Ferrara, B. Monsarrat, H.
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Mazargull, G. Vassart, M. Parmentier, J. Costentin, Isolation and structure of the endogenous agonist of opioid receptor-like ORL1 receptor, Nature 377 Ž1995. 532–535. T. Nabeshima, S. Yamada, A. Sugimoto, K. Matsuno, T. Kameyama, Comparison of tizanidine and morphine with regard to tolerance-developing ability to antinociceptive action, Pharmacol. Biochem. Behav. 25 Ž1986. 835–841. T. Nabeshima, K. Ichihara, Measurement of dissociation of amnesic and behavioral effects of drugs in mice, in: P.M. Conn ŽEd.., Paradigms for the Study of Behavior, Methods in Neurosciences, Vol. 14, Academic Press, San Diego, CA, USA, 1993, pp. 217–229. M. Nishi, T. Houtani, Y. Noda, T. Mamiya, K. Sato, T. Doi, J. Kuno, H. Takeshima, T. Nukada, T. Nabeshima, T. Yamashita, T. Noda, T. Sugimoto, Unstrained nociceptive response and disregulation of hearing ability in mice lacking the nociceptinrorphanin FQ receptor, EMBO J. 16 Ž1997. 1858–1864. A. Nitta, Y. Furukawa, K. Hayashi, M. Hiramatsu, T. Kameyama, T. Nabeshima, Denervation of dopaminergic neurons with 6-hydroxydopamine increases nerve growth factor content in rat brain, Neurosci. Lett. 144 Ž1992. 152–156. H.P. Nothacker, R.K. Reinscheid, A. Mansour, R.A. Henningsen, A. Ardati, F.J. Monsma Jr., S.J. Watoson, O. Civelli, Primary structure and tissue distribution of the orphanin FQ precursor, Proc. Natl. Acad. Sci. U.S.A. 93 Ž1996. 8677–8682. E. Okuda-Ashitaka, S. Tachibana, T. Houtani, T. Minami, Y. Masu, M. Nishi, H. Takeshima, T. Sugimoto, S. Ito, Identification and characterization of an endogenous ligand for opioid receptor homologue ROR-C: its involvement in allodynic response to innocuous stimulus, Mol. Brain Res. 43 Ž1996. 96–104. S. Pellow, P. Chopin, S.E. File, M. Briley, Validation of open: closed arm entries in an elevated plus-maze as a measure of anxiety in the rat, J. Neurosci. Methods 14 Ž1985. 149–167. R.K. Reinscheid, H.P. Nothacker, A. Bourson, A. Ardati, R.A. Henningsen, J.R. Bunzow, D.K. Grandy, H. Langen, F.J. Monsma Jr., O. Civelli, Orphanin FQ: a neuropeptide that activates an opioid-like G protein-coupled receptor, Science 270 Ž1995. 792–794. J. Sandin, J. Georgieva, P.A. Schott, S.O. Ogren, L. Terenius, Nociceptinrorphanin FQ microinjected into hippocampus impairs spatial learning in rats, Eur. J. Neurosci. 9 Ž1997. 194–197. L. Singh, M.J. Field, J. Hughes, R. Menzies, R.J. Oles, C.A. Vass, G.N. Woodruff, The behavioural properties of CI-988, a selective cholecystokinin B antagonist, Br. J. Pharmacol. 104 Ž1991. 239–245. C.W. Vaughan, M.J. Christie, Increase by the ORL1 receptor Žopioid receptor-like1. ligand, nociceptin, of inwardly rectifying Kq conductance in dorsal raphe nucleus neurones, Br. J. Pharmacol. 117 Ž1996. 1609–1611. T.P. Yu, J. Fein, T. Phan, C.J. Evans, C.W. Xie, Orphanin FQ inhibits synaptic transmission and long-term potentiation in rat hippocampus, Hippocampus 7 Ž1997. 88–94.
Research report
Enhancement of spatial attention in nociceptinrorphanin FQ receptor-knockout mice Takayoshi Mamiya a , Yukihiro Noda a , Miyuki Nishi b , Hiroshi Takeshima b , Toshitaka Nabeshima a
a, )
Department of Neuropsychopharmacology and Hospital Pharmacy, Nagoya UniÕersity School of Medicine, 65 Tsuruma-cho, Showa-ku, Nagoya 466, Japan b Department of Pharmacology, Faculty of Medicine, UniÕersity of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan Accepted 18 November 1997
Abstract We isolated genes for the opioid receptor homologue MOR-C, namely nociceptin receptor Ždesignated alternatively as orphanin FQ receptor. and generated nociceptin receptor-knockout mice. Previously, we have reported that the nociceptin system appears to participate in the regulation of the auditory system. However, the behavior of the nociceptin receptor-knockout mice has yet to be fully characterized. In the present study, we investigated changes in several behavioral performances in mice which lack nociceptin receptor. Nociceptive thresholds of nociceptin receptor-knockout mice were unchanged in the hot-plate and electric foot-shock tests as well as tail-flick and acetic acid-induced writhing tests compared to those of wild-type mice. The nociceptin receptor-knockout mice did not show any behavioral changes in the elevated plus-maze task. Surprisingly, in the water-finding test, the nociceptin receptor-knockout mice showed an enhanced retention of spatial attention Žlatent learning. compared to wild-type mice. In a biochemical study, dopamine content in the frontal cortex was lower in nociceptin receptor-knockout mice than wild-type mice. These results suggest that nociceptin receptor plays an important role in spatial attention by regulating the dopaminergic system in the brain. q 1998 Elsevier Science B.V. Keywords: Nociceptin receptor; Knockout mice; Water-finding test; Spatial attention Žlatent learning.; Dopamine content
1. Introduction Soon after the cloning of the d opioid receptor w6,11x, the receptor for unknown ligand belonging to the opioid receptor family has been reported from several groups and designated independently as ORL1, ROR-C, LC132 and C3 w2,7,12,14x. This receptor exhibited 68% homology with m receptor, 67% with d receptor, 66% with k receptor, and 32% with somatostatin receptor, and is abundantly expressed in rat and mouse brain w5x. Recently, the endogenous ligand for this receptor has been isolated and named nociceptin or orphanin FQ w16,24x. Nociceptin is derived from a larger precursor which shows sequence similarity to the opioid peptide precursors, particularly pre-prodynorphin w9,15,21x. Several studies have reported the physiological functions of nociceptin. For example, in contrast to the opioid peptides with analgesic effects, Abbreviations: NE: norepinephrine; DA: dopamine; DOPAC: 3,4-dihydroxyphenylacetic acid; HVA: homovanillic acid ) Corresponding author. Fax: q 81-52-744-2979; E-mail: [email protected] 0006-8993r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 1 4 0 6 - 6
nociceptin induces hyperalgesia w16,24x and allodynia w22x, when administered intracerebroventricularly Ži.c.v.. and intrathecally, respectively. Nociceptin or orphanin FQ inhibits adenylyl cyclase activity in cells transfected with ORL1 w16x or LC132 w24x. Furthermore, nociceptin was shown to increase Kq conductance in rat dorsal raphe neurons in vitro w27x, and to inhibit voltage-dependent Ca2q channel currents in the human neuroblastoma cell line, SH-SY5Y w4x. In situ hybridization analysis w2,3,7,14x and immunohistochemistry w1x revealed distribution of ORL1 in many areas of the central nervous system. However, specific antagonists for nociceptin receptor are not available, and the physiological roles of nociceptin have yet to be elucidated at the whole-animal level. One approach would be to produce knockout mice lacking the nociceptin receptor by means of gene targeting and analyze the physiological phenotype of the mutants. Recently, we have reported that the nociceptin system appears to participate in the regulation of the auditory system w19x. To characterize the role of nociceptin receptor in whole animals, we investigated several behavioral performances in nociceptin receptor-knockout mice.
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2. Materials and methods 2.1. Animals We used male nociceptin receptor-knockout and wildtype mice Ž9–12 weeks old. which have been reported w19x. The animals were housed in a controlled environment Ž23 " 18C, 50 " 5% humidity. and allowed food and water ad libitum. The room lights were off between 2000 and 0800. All experimental protocols were conducted with due regard for the Japanese Experimental Animal Research Association standards as defined in the Guidelines for Animal Experiments Ž1987., and were approved in advance by the Animal Research Committee at Nagoya University. 2.2. EleÕated plus-maze test The elevated plus-maze was a modification of the apparatus and consisted of two open Ž25 = 8 = 0.5 cm. and two closed arms Ž25 = 8 = 20 cm. emanating from a common central platform Ž8 = 8 cm. to form a plus shape w13x. The entire apparatus was elevated to a height of 50 cm above floor level. Testing commenced by placing a mouse on the central platform of the maze facing an open arm, and a standard 5-min test duration was employed. Conventional parameters consisted of the frequency of open arms and closed arms, total arm entries, and the amount of time spent by mice in the open arms, on the central platform and in the closed arms of the maze. These data were also used to calculate percent open arm entries wi.e., Žopen arm entriesrtotal arm entries. = 100x and percent time spent in open and closed arms wi.e., Žopen timeropen and closed time. = 100x. 2.3. NociceptiÕe test Nociception was determined using the hot-plate and electric foot-shock tests w17x.
Fig. 1. ŽA. Percent open arm entries and percent time spent in open arms on the elevated plus-maze test in wild-type Žqrq. and knockout Žyry. mice. ŽB. Response to nociceptive Žthermal and electric. stimuli in wild-type Žqrq. and knockout Žyry. mice. The numbers in the column represent the number of mice used.
Fig. 2. Performance in the water-finding test in wild-type Žqrq. and knockout Žyry. mice. ) P - 0.05, ) ) P - 0.01 vs. corresponding naive group, †P - 0.05 vs. wild-type control mice ŽStudent’s t-test.. The numbers in the column represent the number of mice used.
In the hot-plate test Ž55 " 18C., hot-plate latency represents time elapsed until the mice licked their hind paws andror jumping. In the electric foot-shock test, we used a transparent acrylic rectangular cage Ž23 = 28 = 12 cm. equipped with a metal wire floor. The shock intensity was raised stepwise manually from 0.1 to 2.0 mA in 0.1 mA increments Ž20-s interval. until a flinch was observed ŽShockgenerator NS-SG01, Neuroscience, Tokyo, Japan.. 2.4. Water-finding task The apparatus and procedure were constructed as described previously w10,18x. The test consisted of two trials: a training trial Žthe 1st day. and retention trial Žthe 2nd day.. Briefly, the apparatus consisted of an open field with an alcove in the middle of one of the long walls of the enclosure. The floor of the open field was divided into 15 identical squares for measuring locomotor activity. A drinking tube was inserted into the center of the alcove ceiling with its tip 5 cm Žin the training trial. or 7 cm Žin the retention trial. above the floor. In the training trial, mice not deprived of water were placed individually into one corner of the open field. Each mouse was allowed 3 min to explore the environment; the 3 min were counted from the time the mouse started to explore. During this time, ambulation was measured by counting the number of times the animals crossed from one square to another in the open field Žlocomotion count.. The frequency of touching, sniffing, or licking of the water tube in the alcove Žnumber of approaches. was also recorded. Animals that did not start exploring after 3 min had elapsed or that did not find the drinking tube during the 3-min exploratory period were omitted from the retention trial. Non-trained mice Žnaive mice. were prepared for comparison with the trained mice Žcontrol mice. in terms of their ability to find the water source in the same environment. The mice were immediately returned to their home cages after the training trial and were again individually placed on the test apparatus 24 h later for a single retention trial. The mice were deprived of water for 24 h before the retention trial. In the retention trial, mice were
T. Mamiya et al.r Brain Research 783 (1998) 236–240
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Table 1 NE, DOPAC, DA and HVA contents and DOPACrDA and HVArDA ratios in the frontal cortex, hippocampus and striatum in wild-type Žqrq . and knockout Žyry . mice Region
Phenotype
N
Frontal cortex
qrq yry qrq yry qrq yry
5 8 5 8 5 8
Hippocampus Striatum
Contents Žngrg wet tissue.
Ratio
NE
DOPAC
DA
HVA
DOPACrDA
HVArDA
419 " 18 387 " 16 499 " 34 480 " 37 120 " 18 117 " 7
88 " 6 86 " 5 40 " 8 49 " 1 3371 " 322 3347 " 172
94 " 15 64 " 4 ) 32 " 8 41 " 6 18 476 " 1472 17 295 " 627
123 " 6 120 " 13 154 " 6 138 " 9 1455 " 108 1456 " 120
0.92 " 0.23 1.36 " 0.09 1.20 " 0.16 1.21 " 0.25 0.19 " 0.02 0.19 " 0.01
1.30 " 0.37 1.87 " 0.24 4.82 " 0.55 3.41 " 0.38 0.08 " 0.005 0.08 " 0.004
)
P - 0.05 vs. wild-type mice ŽStudent’s t-test.. N represents the number of mice used.
individually placed in the same corner of the apparatus as that used during the training trial. The time until the mouse moved out of the corner was measured as the starting latency. In addition, the time taken to enter the alcove Žentering latency. and the time between entering the alcove and drinking the water Žfinding latency. were scored. Thus, the drinking latency consisted of the sum of the entering and finding latencies. In other words, memory acquisition was able to be assessed by recording four latencies Žstarting, entering, finding and drinking., ambulation counts and the number of approaches. 2.5. Measurement of monoamine contents Animals were killed by decapitation. The brain was rapidly removed and the frontal cortex, hippocampus and striatum were dissected out on an ice-cold plate. Each tissue sample was quickly frozen in dry ice and stored in a deep freezer at y808C until assayed. The contents of norepinephrine ŽNE., dopamine ŽDA., 3,4-dihydroxyphenylacetic acid ŽDOPAC. and homovanillic acid ŽHVA. in the three regions were determined using high-performance liquid chromatography with electrochemical detection w20x. 2.6. Statistical analysis All results were expressed as the mean " S.E.M. for each group. Statistical analysis of the difference between groups was assessed with Student’s t-test. P - 0.05 was taken as the significant level of difference.
3. Results 3.1. EleÕated plus-maze test Fig. 1A shows the performance on the elevated plusmaze test in wild-type and knockout mice. Total arm entries and closed arm entries were not significantly different between the two groups Ždata not shown.. No significant differences were detected in percent open arm entries
and percent time spent in open arms between wild-type and knockout mice. 3.2. NociceptiÕe test Nociceptive thresholds in two nociceptive tests are shown in Fig. 1B. No measurable difference in the antinociceptive thresholds to thermal and electric stimuli was observed between wild-type and knockout mice. 3.3. Water-finding test In the training trial of the water-finding test, there was no significant difference in any latency, the ambulation counts and the number of approaches between wild-type and knockout mice Ždata not shown.. Fig. 2 shows the results of the performance in the retention trial in wild-type and knockout mice. No measurable difference was observed between both naive mice. The finding and drinking latencies in wild-type and knockout mice were shorter than those in the corresponding naive mice. The entering latency was shorter in wild-type than in the corresponding naive mice, but not in knockout mice. Furthermore, the finding latency of knockout mice was significantly shorter than that of wild-type mice. 3.4. Difference in monoamines and DOPACr DA and HVA r DA ratio The contents of NE, DOPAC, DA and HVA and DOPACrDA and HVArDA ratios in three different regions in wild-type and knockout mice are shown in Table 1. DA content in the frontal cortex of knockout mice was significantly low compared to that of wild-type mice. The contents and ratios in other regions did not significantly differ between knockout and wild-type mice.
4. Discussion We have previously reported generation and characterization of mice lacking the nociceptin receptor w19x. No
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obvious morphological abnormalities could be detected in the knockout mice. Further, the knockout mice did not differ from wild-type littermates in health, growth and reproduction. As shown previously, a statistical analysis revealed no significant difference between wild-type and knockout mice in locomotor activity w19x. The state of anxiety in knockout mice was evaluated by elevated plus-maze. Animals with high anxiety show low percent open arm entries and low percent time spent in open arms in this task w13x. For example, benzodiazepines increased these parameters w13x. Ordinarily, unstressed animals will spend approximately 15–25% of the trial in the open arms of the maze w8,23,26x. However, because of the lighting for our environment, wild-type mice spent less than 10% of the trial on the open arms of the maze in our experiment. We did not detect any difference in performances on the elevated plus-maze between wild-type and knockout mice. Taken together, these findings suggest that nociceptin receptor is involved in neither anxiety nor anti-anxiety. Since the nociceptin receptor has been reported to be involved in nociceptive response w16,24x, we compared the response to some nociceptive stimuli in wild-type and knockout mice. However, no measurable difference in the nociceptive thresholds to thermal and electric stimuli was observed between wild-type and knockout mice. These results were consistent with our previous reports w19,22x. It is suggested that these knockout mice do not have any obvious behavioral and anatomical abnormalities. A regional distribution of mRNA for the nociceptin precursor has been shown in the brain and spinal cord w9,15,21x. Expression levels of nociceptin receptor mRNA are high in the periaqueductal gray, dorsal raphe nucleus, locus coeruleus and the dorsal horn of the spinal cord w2x. An in situ hybridization study indicated that mRNA for ORL1 is expressed in cortical and limbic regions of the brain, suggesting that nociceptin receptor plays an important role in cognitive, mnemonic and attentional processes w12x. Recently, immunohistochemical localization of ORL1 in rat brain was reported, with intense and specific immunolabelling observed in the hippocampus, brainstem and cortex w1x which were relevant to learning and memory. Furthermore, nociceptinrorphanin FQ has been reported to induce the impairment of spatial learning w25x and inhibits synaptic transmission and long-term potentiation w28x in the hippocampus. Thus, there is a possibility that nociceptin andror nociceptin receptor are involved in learning and memory. Our present study examined the ability of learning and memory, particularly latent learning, in the knockout mice. Water-finding test has been used as an index of latent learning w10,18x. In the task, we did not use any motivation to train animals, since non water-deprived animals were trained in the apparatus with the drinking tube. However, when animals were deprived of water before the retention test, animals which had previously experienced the apparatus exhibited shorter en-
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tering, finding and drinking latencies than the non-experienced naive animals in the retention trial. In other words, control mice entered the alcove and found the drinking tube more quickly than did naive mice. In the present study, neither the latencies or ambulation counts, nor the number of approaches in the training trial, differed significantly between wild-type and knockout mice. Interestingly, in the retention trial, the finding latency of knockout mice was significantly shortened compared to wild-type mice, indicating that the knockout mice found the drinking tube more quickly than wild-type mice. These results suggest that the latent learning ability of knockout mice was better than that of wild-type mice. The mechanism of the enhancement in latent learning is unknown. Possibly, stress such as thirstiness may release endogenous excess nociceptin and impair the ability of latent learning in wild-type mice. Such stress-induced nociceptin may inhibit the latent-learning function through nociceptin receptor, although we cannot currently measure endogenously released nociceptin during this task. Ichihara et al. w10x have reported that dopamine receptor agonists impair latent learning in the water-finding test in mice, suggesting that activation of the dopaminergic system impairs the ability of latent-learning ability. In the present biochemical study, dopamine contents in the frontal cortex of knockout mice were significantly low compared to those of wild-type mice. HVArDA ratio, one of the parameters of dopamine turnover in the hippocampus, tended to be low in knockout compared to wild-type mice. These results suggest that the function of the dopaminergic system in knockout mice was reduced compared to that of wild-type mice. Alternatively, the enhancement of latent learning may result from the suppression of dopaminergic function in knockout mice. In addition, our findings suggest nociceptin system may regulate stimulatively the dopaminergic system. However, further studies are required to clarify the role of nociceptin andror nociceptin receptor in learning and memory.
5. Conclusion Our behavioral analysis reveals that the nociceptin receptor-knockout mice display a significant enhancement of latent learning, although no obvious morphological or behavioral abnormalities could be detected. Our results suggest that nociceptin receptor plays an important role in latent learning by modulating the dopaminergic system in the brain.
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