神經胜肽 S 在壓力誘發小鼠古柯鹼癮頭之角色

The role of neuropeptide S in stress-induced cocaine relapse in mice

10.6342/NTU201703751

周昱仙

神經胜肽S ； 古柯鹼 ； 成癮 ； 復發 ； 壓力 ； Neuropeptide S ； cocaine ； addiction ； relapse ； stress

臺灣大學藥理學研究所學位論文

2017年

碩士

邱麗珠

英文

神經胜肽S (neuropeptide S)是由20個胺基酸所構成的胺基酸鏈，他所標的之受體為神經胜肽S受體 (neuropeptide S receptor, NPSR)。在小鼠大腦分布上，神經胜肽S的神經元主要分布在藍斑核周圍 (peri-locus coeruleus, peri-LC)和臂旁核(lateral parabrachial nucleus, PBN)當中。先前的研究已經指出，神經胜肽S和食慾素 (orexins)具備有類似的生理以及病理表現，其中便包括壓力引起的藥物回饋作用。已知結果如下：首先、壓力可以活化藍斑核周圍以及臂旁核當中的神經胜肽S神經元，並活化側下視丘 (lateral hypothalamus, LH)中的食慾素神經元；第二、透過側下視丘內注射神經胜肽S可活化食慾素神經元，並會引起求藥行為的產生；第三、在腹側背蓋區 (ventral tegmental area, VTA)當中，活化突觸後食慾素第一型受體 (orexin 1 receptor, OX1R)，會使磷脂酶C (PLC)和甘油二酯脂肪酶 (DAGL)使突觸後產生2-arachydonoylglycerol (2-AG) 。2-AG這類內生性大麻酯再逆行回突觸前激活內生性大麻酯受體 (cannabinoid 1 receptor ,CB1R)抑制GABA釋放，逐步可活化古柯鹼毒癮復發；第四、在VTA內注射食慾素A，進而活化上述OX1R-PLC-DAGL-2-AG-CB1R傳訊，使多巴胺型神經元活性上升觸發毒癮復發的行為。基於上述研究結果，我們假設神經胜肽S和壓力皆會透過內生性食慾素以及內生性大麻酯來帶起類似的機制，進而引起古柯鹼的毒癮復發。為了證實這個假說，在我們的實驗當中我們使用了場地制約性偏好測試(conditioned place preference，CPP)來進行古柯鹼渴望程度的測試。小鼠會先進行連續三天的古柯鹼制約訓練，接著再進行連續三天的強制戒斷訓練，之後我們給予小鼠三十分鐘的束縛性壓力，藉以引起古柯鹼毒癮的復發。在實驗結果中，我們使用一個選擇性神經胜肽S受體的拮抗劑─SHA 68，透過腹腔注射給予50 mg/kg，可以有效預防壓力引起的古柯鹼毒癮復發。此外，我們透過側腦室給予1 nmol的神經胜肽S可以在強制戒斷後的小鼠身上看到毒癮復發的現象，而這個現象同時也可以被選擇性食慾素第一型受體拮抗劑以及內生性大麻酯受體拮抗劑透過腹腔注射方式給予並抑制。透過酵素免疫分析法，我們發現透過側腦室給予1 nmol的神經胜肽S明顯的提升腹側背蓋區中食慾素Ａ的含量，同時我們也發現若在給予神經胜肽S之前先給予SHA 68可以預防此現象發生。最後，透過免疫螢光染色的結果顯示，側腦室外給予1 nmol的神經胜肽S可以顯著的增加在側下視丘，同時表現雙染c-Fos和食慾素A蛋白質的神經細胞表現密度。 總結，透過壓力，藍斑核周圍和臂旁核會釋放神經胜肽S來活化側下視丘中的食慾素神經元，並釋放食慾素。而食慾素則會在腹側背蓋區中活化食慾素第一型受體，引起由內生性大麻酯所帶來的去抑制機制，進而引起古柯鹼毒癮的求藥行為。如此，食慾素便是活化OX1R-PLC-DAGL-2-AG-CB1R傳訊，降低了周圍抑制性突觸所能釋放之GABA，使多巴胺型神經元活性上升而觸發毒癮復發的行為。

Neuropeptide S is a 20 amino-acid peptide identified as the endogenous ligand for the Neuropeptide S receptor (NPSR). In mice, NPS-containing neurons are localized in the peri-locus coeruleus (LC) and the parabrachial nucleus (PBN). Previous studies have demonstrated that NPS shares several similar pathophysiological roles as orexins, including stress-induced drug reward. First, stress can activate the NPS-containing neurons in the peri-LC and the PBN, and the orexin neurons in the lateral hypothalamic (LH). Second, intra-LH microinjection of NPS activates orexin neurons, and orexin neuron activation is associated with drug seeking. Third, activating postsynaptic orexin 1 receptors (OX1Rs), a family of Gq-protein coupled receptors, on VTA dopamine neurons leads to phospholipase (PLC) activation and generating diacylglycerol (DAG) that is converted into 2-arachidonoylglycerol (2-AG) by the DAG lipase (DAGL) inside the postsynaptic neuron. The generated 2-AG, an endocannabinoid, travels retrogradely across the synapse to inhibit GABA release by activating presynaptic CB1 receptors (CB1Rs), a disinhibition phenomenon. Fourth, intra-VTA microinjection of orexin A reinstated cocaine relapse via the OX1R-PLC-DAGL-2-AG-CB1R cascade-mediated dopaminergic disinhibition in the VTA. Based on previous study, we proposed that NPS and stress share a similar mechanism that involves endogenous orexin and endocannabinoid systems in inducing cocaine relapse. In order to validate the hypothesis, we use the conditioned place preference (CPP) to measure the cocaine craving of mice. Mice were trained to induced cocaine CPP by a 3-day bias-cocaine CPP training, and then to develop extinction by a 3 day-forced pairing with saline injections. Then a 30-min restraint stress was used to reinstate cocaine CPP in extinguished animals. We found that SHA 68 (50 mg/kg, i.p.), a selective NPSR antagonist, prevented the reinstatement of cocaine CPP. Besides, i.c.v. injection of NPS (1 nmol) induced the reinstatement of cocaine CPP in extinguished mice, and this effect was blocked by selective OX1R and CB1R antagonists, respectively. Through ELISA, we found that i.c.v. injection of NPS (1 nmol) significantly elevated the orexin A level in the VTA, and this effect was prevented by SHA 68. Finally, the immunofluorescence staining showed that , i.c.v. injection of NPS (1 nmol) significantly increased the density of the presence for double labelling to c-Fos and orexin A in the LH. During stress, NPS is released from the PBN or/and peri-LC in mice, to activate LH orexin neurons, releasing orexins. The released orexins may reinstate extinguished cocaine-seeking behavior via a disinhibition mechanism mediated by endocannabinoids generated after activation of the OX1R in the VTA. That is, orexins activate OX1R-PLC-DAGL-2-AG-CB1R signaling cascade, then inhibit GABA releasing. The inhibition of GABAergic synaptic neurotransmission onto dopaminergic neurons in the VTA results in activation of the mesolimbic dopaminergic circuitry, leading to relapse.

1. Adori, C., S. Barde, N. Bogdanovic, M. Uhlen, R. R. Reinscheid, G. G. Kovacs, and T. Hokfelt. 2015. 'Neuropeptide S- and Neuropeptide S receptor-expressing neuron populations in the human pons', Frontiers in Neuroanatomy, 9: 126.
   連結：
2. Aston-Jones, G., R. J. Smith, D. E. Moorman, and K. A. Richardson. 2009. 'Role of lateral hypothalamic orexin neurons in reward processing and addiction', Neuropharmacology, 56 Suppl 1: 112-21.
   連結：
3. Aston-Jones, G., R. J. Smith, G. C. Sartor, D. E. Moorman, L. Massi, P. Tahsili-Fahadan, and K. A. Richardson. 2010. 'Lateral hypothalamic orexin/hypocretin neurons: A role in reward-seeking and addiction', Brain Research, 1314: 74-90.
   連結：
4. Atwood, B. K., and K. Mackie. 2010. 'CB2: a cannabinoid receptor with an identity crisis', British Journal of Pharmacology, 160: 467-79.
   連結：
5. Baimel, C., S. E. Bartlett, L. C. Chiou, A. J. Lawrence, J. W. Muschamp, O. Patkar, L. W. Tung, and S. L. Borgland. 2015. 'Orexin/hypocretin role in reward: implications for opioid and other addictions', British Journal of Pharmacology, 172: 334-48.
   連結：
6. Berridge, C. W., R. A. Espana, and N. M. Vittoz. 2009. 'Hypocretin/orexin in arousal and stress', Brain Research.
   連結：
7. Borgland, S. L., S. A. Taha, F. Sarti, H. L. Fields, and A. Bonci. 2006. 'Orexin A in the VTA is critical for the induction of synaptic plasticity and behavioral sensitization to cocaine', Neuron, 49: 589-601.
   連結：
8. Cannella, N., D. Economidou, M. Kallupi, S. Stopponi, M. Heilig, M. Massi, and R. Ciccocioppo. 2009. 'Persistent increase of alcohol-seeking evoked by neuropeptide S: an effect mediated by the hypothalamic hypocretin system', Neuropsychopharmacology, 34: 2125-34.
   連結：
9. Cannella, N., M. Kallupi, H. W. Li, S. Stopponi, C. Cifani, R. Ciccocioppo, and M. Ubaldi. 2016. 'Neuropeptide S differently modulates alcohol-related behaviors in alcohol-preferring and non-preferring rats', Psychopharmacology (Berl), 233: 2915-24.
   連結：
10. Cannella, N., M. Kallupi, B. Ruggeri, R. Ciccocioppo, and M. Ubaldi. 2013. 'The role of the neuropeptide S system in addiction: focus on its interaction with the CRF and hypocretin/orexin neurotransmission', Progress in Neurobiology, 100: 48-59.
    連結：
11. Cheng, J. K., R. C. Chou, L. L. Hwang, and L. C. Chiou. 2003. 'Antiallodynic effects of intrathecal orexins in a rat model of postoperative pain', Journal of Pharmacology and Experimental Therapeutics, 307: 1065-71.
    連結：
12. Cheng, J. K., L. L. Hwang, R. C. Chou, and L. C. Chiou. 2005. 'Analgesic effects of orexins', Journal Neuropath Pain & Sympt Palliat, 1: 47-53 (invited review article).
    連結：
13. Citrome, L. 2014. 'Suvorexant for insomnia: a systematic review of the efficacy and safety profile for this newly approved hypnotic - what is the number needed to treat, number needed to harm and likelihood to be helped or harmed?', International Journal of Clinical Practice, 68: 1429-41.
    連結：
14. Clark, S. D., D. M. Duangdao, S. Schulz, L. Zhang, X. Liu, Y. L. Xu, and R. K. Reinscheid. 2011. 'Anatomical characterization of the neuropeptide S system in the mouse brain by in situ hybridization and immunohistochemistry', Journal of Comparative Neurology, 519: 1867-93.
    連結：
15. Cluderay, J. E., D. C. Harrison, and G. J. Hervieu. 2002. 'Protein distribution of the orexin-2 receptor in the rat central nervous system', Regulatory Peptides , 104: 131-44.
    連結：
16. Coleman, P. J., and J. J. Renger. 2010. 'Orexin receptor antagonists: a review of promising compounds patented since 2006', Expert Opinion on Therapeutic Patents, 20: 307-24.
    連結：
17. Cox, C. D., M. J. Breslin, D. B. Whitman, J. D. Schreier, G. B. McGaughey, M. J. Bogusky, A. J. Roecker, S. P. Mercer, R. A. Bednar, W. Lemaire, J. G. Bruno, D. R. Reiss, C. M. Harrell, K. L. Murphy, S. L. Garson, S. M. Doran, T. Prueksaritanont, W. B. Anderson, C. Tang, S. Roller, T. D. Cabalu, D. Cui, G. D. Hartman, S. D. Young, K. S. Koblan, C. J. Winrow, J. J. Renger, and P. J. Coleman. 2010. 'Discovery of the dual orexin receptor antagonist [(7R)-4-(5-chloro-1,3-benzoxazol-2-yl)-7-methyl-1,4-diazepan-1-yl][5-methy l-2-(2H-1,2,3-triazol-2-yl)phenyl]methanone (MK-4305) for the treatment of insomnia', Journal of Medicinal and Pharmaceutical Chemistry, 53: 5320-32.
    連結：
18. de Lecea, L. 2009. 'A decade of hypocretins: past, present and future of the neurobiology of arousal', Acta Physiologica (Oxf).
    連結：
19. De Vries, T. J., W. de Vries, M. C. Janssen, and A. N. Schoffelmeer. 2005. 'Suppression of conditioned nicotine and sucrose seeking by the cannabinoid-1 receptor antagonist SR141716A', Behavioural Brain Research, 161: 164-8.
    連結：
20. Drew, G. M., B. K. Lau, and C. W. Vaughan. 2009. 'Substance P drives endocannabinoid-mediated disinhibition in a midbrain descending analgesic pathway', Journal of Neuroscience, 29: 7220-9.
    連結：
21. Drew, G. M., V. A. Mitchell, and C. W. Vaughan. 2008. 'Glutamate spillover modulates GABAergic synaptic transmission in the rat midbrain periaqueductal grey via metabotropic glutamate receptors and endocannabinoid signaling', Journal of Neuroscience, 28: 808-15.
    連結：
22. Fadel, J., and A. Y. Deutch. 2002. 'Anatomical substrates of orexin-dopamine interactions: lateral hypothalamic projections to the ventral tegmental area', Neuroscience, 111: 379-87.
    連結：
23. Farooqui, A. A., D. K. Anderson, C. Flynn, E. Bradel, E. D. Means, and L. A. Horrocks. 1990. 'Stimulation of mono- and diacylglycerol lipase activities by bradykinin in neural cultures', Biochemical and Biophysical Research Communications, 166: 1001-9.
    連結：
24. Fattore, L., M. S. Spano, G. Cossu, S. Deiana, and W. Fratta. 2003. 'Cannabinoid mechanism in reinstatement of heroin-seeking after a long period of abstinence in rats', European Journal of Neuroscience, 17: 1723-6.
    連結：
25. Fronczek, R., C. R. Baumann, G. J. Lammers, C. L. Bassetti, and S. Overeem. 2009. 'Hypocretin/orexin disturbances in neurological disorders', Sleep Medicine Reviews, 13: 9-22.
    連結：
26. Furlong, T. M., D. M. Vianna, L. Liu, and P. Carrive. 2009. 'Hypocretin/orexin contributes to the expression of some but not all forms of stress and arousal', European Journal of Neuroscience, 30: 1603-14.
    連結：
27. Gregg, L. C., K. M. Jung, J. M. Spradley, R. Nyilas, R. L. Suplita, 2nd, A. Zimmer, M. Watanabe, K. Mackie, I. Katona, D. Piomelli, and A. G. Hohmann. 2012. 'Activation of type 5 metabotropic glutamate receptors and diacylglycerol lipase-alpha initiates 2-arachidonoylglycerol formation and endocannabinoid-mediated analgesia', Journal of Neuroscience, 32: 9457-68.
    連結：
28. Harris, G. C., and G. Aston-Jones. 2006. 'Arousal and reward: a dichotomy in orexin function', Trends in Neurosciences, 29: 571-7.
    連結：
29. Herring, W. J., E. Snyder, K. Budd, J. Hutzelmann, D. Snavely, K. Liu, C. Lines, T. Roth, and D. Michelson. 2012. 'Orexin receptor antagonism for treatment of insomnia: a randomized clinical trial of suvorexant', Neurology, 79: 2265-74.
    連結：
30. Hervieu, G. J., J. E. Cluderay, D. C. Harrison, J. C. Roberts, and R. A. Leslie. 2001. 'Gene expression and protein distribution of the orexin-1 receptor in the rat brain and spinal cord', Neuroscience, 103: 777-97.
    連結：
31. Hirose, M., S. Egashira, Y. Goto, T. Hashihayata, N. Ohtake, H. Iwaasa, M. Hata, T. Fukami, A. Kanatani, and K. Yamada. 2003. 'N-acyl 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline: the first orexin-2 receptor selective non-peptidic antagonist', Bioorganic & Medicinal Chemistry Letters, 13: 4497-9.
    連結：
32. Ho, Y. C., H. J. Lee, L. W. Tung, Y. Y. Liao, S. Y. Fu, S. F. Teng, H. T. Liao, K. Mackie, and L. C. Chiou. 2011. 'Activation of orexin 1 receptors in the periaqueductal gray of male rats leads to antinociception via retrograde endocannabinoid (2-arachidonoylglycerol)-induced disinhibition', Journal of Neuroscience, 31: 14600-10.
    連結：
33. Kallupi, M., G. de Guglielmo, N. Cannella, H. W. Li, G. Calo, R. Guerrini, M. Ubaldi, J. J. Renger, V. N. Uebele, and R. Ciccocioppo. 2013. 'Hypothalamic neuropeptide S receptor blockade decreases discriminative cue-induced reinstatement of cocaine seeking in the rat', Psychopharmacology (Berl), 226: 347-55.
    連結：
34. Kano, M., T. Ohno-Shosaku, Y. Hashimotodani, M. Uchigashima, and M. Watanabe. 2009. 'Endocannabinoid-mediated control of synaptic transmission', Physiological Reviews, 89: 309-80.
    連結：
35. Koob, G. F. 2009a. 'Brain stress systems in the amygdala and addiction', Brain Research, 1293: 61-75.
    連結：
36. ———. 2009b. 'Neurobiological substrates for the dark side of compulsivity in addiction', Neuropharmacology, 56 Suppl 1: 18-31.
    連結：
37. Korotkova, T. M., O. A. Sergeeva, K. S. Eriksson, H. L. Haas, and R. E. Brown. 2003. 'Excitation of ventral tegmental area dopaminergic and nondopaminergic neurons by orexins/hypocretins', Journal of Neuroscience, 23: 7-11.
    連結：
38. Lau, B. K., and C. W. Vaughan. 2008. 'Muscarinic modulation of synaptic transmission via endocannabinoid signalling in the rat midbrain periaqueductal gray', Molecular Pharmacology, 74: 1392-8.
    連結：
39. Lee, H. J., L. Y. Chang, Y. C. Ho, S. F. Teng, L. L. Hwang, K. Mackie, and L. C. Chiou. 2016. 'Stress induces analgesia via orexin 1 receptor-initiated endocannabinoid/CB1 signaling in the mouse periaqueductal gray', Neuropharmacology, 105: 577-86.
    連結：
40. Li, W., M. Chang, Y. L. Peng, Y. H. Gao, J. N. Zhang, R. W. Han, and R. Wang. 2009. 'Neuropeptide S produces antinociceptive effects at the supraspinal level in mice', Regulatory Peptides, 156: 90-5.
    連結：
41. Liao, H. T., H. J. Lee, Y. C. Ho, and L. C. Chiou. 2011. 'Capsaicin in the periaqueductal gray induces analgesia via metabotropic glutamate receptor-mediated endocannabinoid retrograde disinhibition', British Journal of Pharmacology, 163: 330-45.
    連結：
42. Asahi, S., S. Egashira, M. Matsuda, H. Iwaasa, A. Kanatani, M. Ohkubo, M. Ihara, and H. Morishima. 2003. 'Development of an orexin-2 receptor selective agonist, [Ala(11), D-Leu(15)]orexin-B', Bioorganic & Medicinal Chemistry Letters, 13: 111-3.
43. Brisbare-Roch, C., J. Dingemanse, R. Koberstein, P. Hoever, H. Aissaoui, S. Flores, C. Mueller, O. Nayler, J. van Gerven, S. L. de Haas, P. Hess, C. Qiu, S. Buchmann, M. Scherz, T. Weller, W. Fischli, M. Clozel, and F. Jenck. 2007. 'Promotion of sleep by targeting the orexin system in rats, dogs and humans', Nature Medicine, 13: 150-5.
44. Chemelli, R. M., J. T. Willie, C. M. Sinton, J. K. Elmquist, T. Scammell, C. Lee, J. A. Richardson, S. C. Williams, Y. Xiong, Y. Kisanuki, T. E. Fitch, M. Nakazato, R. E. Hammer, C. B. Saper, and M. Yanagisawa. 1999. 'Narcolepsy in orexin knockout mice: molecular genetics of sleep regulation', Cell, 98: 437-51.
45. Chiou, L. C., H. J. Lee, Y. C. Ho, S. P. Chen, Y. Y. Liao, C. H. Ma, P. C. Fan, J. L. Fuh, and S. J. Wang. 2010. 'Orexins/hypocretins: pain regulation and cellular actions', Current Pharmaceutical Design, 16: 3089-100.
46. de Lecea, L., T. S. Kilduff, C. Peyron, X. Gao, P. E. Foye, P. E. Danielson, C. Fukuhara, E. L. Battenberg, V. T. Gautvik, F. S. Bartlett, 2nd, W. N. Frankel, A. N. van den Pol, F. E. Bloom, K. M. Gautvik, and J. G. Sutcliffe. 1998. 'The hypocretins: hypothalamus-specific peptides with neuroexcitatory activity', Proceedings of the national academy of sciences of the united states of america, 95: 322-7.
47. De Vries, T. J., Y. Shaham, J. R. Homberg, H. Crombag, K. Schuurman, J. Dieben, L. J. Vanderschuren, and A. N. Schoffelmeer. 2001. 'A cannabinoid mechanism in relapse to cocaine seeking', Nature Medicine, 7: 1151-4.
48. Elphick, M. R., and M. Egertova. 2001. 'The neurobiology and evolution of cannabinoid signalling', Philosophical Transactions of the Royal Society of London Series Biological Sciences, 356: 381-408.
49. Kallupi, M., N. Cannella, D. Economidou, M. Ubaldi, B. Ruggeri, F. Weiss, M. Massi, J. Marugan, M. Heilig, P. Bonnavion, L. de Lecea, and R. Ciccocioppo. 2010. 'Neuropeptide S facilitates cue-induced relapse to cocaine seeking through activation of the hypothalamic hypocretin system', Proceedings of the national academy of sciences of the united states of america, 107: 19567-72.