臺灣博碩士論文加值系統

中文摘要
中腦導水管周圍灰質(periaqueductal gray, PAG)及頭腹內側延腦(rostral ventromedial medulla, RVM) 為腦幹下行鎮痛路徑之重要構造，而中縫大核(nucleus raphe magnus)為頭腹內側延腦重要神經核。活體動物研究已証實若將微量之神經降壓素注入導水管周圍灰質或中縫大核後，可產生強烈之止痛作用。神經降壓素可能是經由興奮PAG-RVM此一下行鎮痛路徑及中縫大核而產生止痛作用。所以我們結合全細胞膜箝定電生理記錄(whole cell patch-clamp recordings)、單細胞反轉錄-聚合酶鏈鎖反應(single cell RT-PCR)及細胞內鈣離子測定等實驗方法，以導水管周圍灰質投射至頭腹內側延腦 (PAG-RVM)及中縫大核之神經元為研究對象，以探討神經降壓素於這些神經元產生止痛作用之離子及分子機轉。
先以逆行性之螢光染色劑DiIC18微注入白鼠之頭腹內側延腦內，即離導水管周圍灰質神經元後，再選取呈螢光反應之PAG-RVM投射神經元進行實驗。電流箝定實驗顯示，神經降壓素可對PAG-RVM神經元產生去極化作用並引發動作電位。電位箝定記錄則顯示神經降壓素乃藉打開不受電位影響之非選擇性陽離子通道而將PAG-RVM神經元興奮。第一亞型神經降壓素受體(NTR-1) 之專一性拮抗劑SR 48692及可同時拮抗NTR-1與第二亞型神經降壓素受體(NTR-2)之非專一性拮抗劑SR 142948A不能阻止神經降壓素對於PAG-RVM神經元之興奮作用。將GDP-b-S或anti-Gaq/11抗血清透析至PAG-RVM投射神經元後，可將神經降壓素引起陽離子電流之作用阻斷。以使用fura-2之螢光測定法發現神經降壓素可將儲存於PAG-RVM神經元細胞器內之鈣離子快速的釋出。神經降壓素誘發之陽離子電流亦可被IP3之拮抗劑肝素(heparin)或鈣離子之快速螯合劑 BAPTA阻斷。這些結果均顯示神經降壓素藉活化一種新亞型受體增加鈣離子依賴型非選擇性陽離子電導而對PAG-RVM投射神經元產生去極化及興奮作用。
即離後之中大核神經元可發現有二種細胞亞型，分別為主要(primary)之血清素性(serotonergic)神經元，及次要(secondary)之非血清素性神經元。於電流箝定實驗中，神經降壓素可對血清素性神經元產生去極化作用而激發其動作電位。電位箝定實驗顯示神經降壓素乃經由促進對電位不敏感的非選擇性陽離子電導興奮血清素性神經元。SR48692及SR142948A均不能阻止神經降壓素對於血清素性神經元之興奮作用。將GDP-b-S或anti-Gaq/11灌流入神經元內後，可抑制神經降壓素引起之陽離子電流。利用fura-2之螢光測定研究顯示神經降壓素誘發鈣離子從細胞器中被釋出。將肝素或BAPTA透析入血清素性神經元內後，神經降壓素引起之陽離子電流會被阻斷。這些結果顯示神經降壓素可能經由一種新亞型的神經降壓素受體來促進中縫大核血清素性神經元之陽離子電導。至於神經降壓素與Gaq/11蛋白之偶聯機轉則是因IP3之產生而將鈣離子從細胞器中被釋放，致使非選擇性之陽離子電導被活化。
於生物體內，類鴉片藥品及胜肽可經由活化中縫大核的g-亞型類鴉片受體而引起鎮痛作用，此鎮痛作用被認為是經由對g-胺基丁酸性(GABAergic)中繼神經元產生過極化作用而消除對中縫大核投射至脊髓之神經元的抑制所達成。為直接証明類鴉片胜肽可對中縫大核的g-胺基丁酸性神經元產生此抑制作用，結合全細胞膜箝定電生理記錄及單細胞反轉錄-聚合酶鏈鎖反應等實驗方法，發現專一性mu-亞型類鴉片受體致效劑DAMGO([D-Ala2,N-methyl-Phe4,Gly-ol5]enkephalin)會對表現出glutamate decarboxylase (GAD67)mRNA之中縫大核g-胺基丁酸性神經元產生過極化作用。被鴉片藥品或胜的持續或重複作用後，mu-亞型類鴉片受體會產生去敏作用(desensitization)，去敏作用之產生機轉可能為G蛋白-偶聯受體磷酸化(G protein-coupled receptor kinase, GRK)對此類受體之磷酸化 (phosphorylation)所致。為進一步了解GRKs於中縫大核g-胺基丁酸性神經元被類鴉片胜肽作用後所產生之去敏作用過程中所扮演之角色，我們先利用單細胞反轉錄-聚合酶鏈鎖反應分析對DAMGO有反應的中縫大核g-胺基丁酸性神經元其GRK2及GRK3表現情形，發現只有GRK2表現，再於全細胞電生理記錄時，先將一個合成胜肽加進玻璃微電極內溶液中，此胜肽與GRK2上與 Gbg 結合之區域結構(domain)相同，結果發現其可抑制Gbg對GRK2之活化作用而使去敏作用減低。這些結果顯示DAMGO經由打開向內整流性鉀離子通道而選擇性的對中縫大核之g-胺基丁酸性神經元產生過極化作用，而GRK2則可參與調控中縫大核的g-胺基丁酸性神經元上mu-亞型類鴉片受體的短期去敏作用。

Abstract
The midbrain periaqueductal gray (PAG) has projection to the rostral ventromedial medulla (RVM) and this circuit is an important descending antinociceptive pathway in the brain stem. The nucleus raphe magnus (NRM) is an essential component of the rostral ventromedial medulla. Microinjection of neurotensin (NT) into the PAG or NRM has been shown to produce a potent analgesic effect. To test the hypothesis that NT induces the analgesic effect by activating this descending antinociceptive pathway and the NRM, and to investigate the ionic and molecular mechanisms by which neurotensin achieve this effect , we combined whole cell patch-clamp recordings, single cell RT-PCR and intracellular calcium measurements to study the effect of neurotensin on PAG neurons that project to RVM (PAG-RVM) and NRM neurons.
PAG-RVM neurons were identified by microinjecting DiIC18, a retrograde tracing dye, into the rat RVM. Subsequently, fluorescently labeled PAG-RVM neurons were acutely dissociated and selected for whole cell patch-clamp recordings. During current-clamp recordings, neurotensin depolarized retrogradely labeled PAG-RVM neurons and evoked action potentials. Voltage-clamp recordings indicated that neurotensin excited PAG-RVM neurons by opening the voltage-insensitive and non-selective cation channels. Both SR48692, a selective antagonist of subtype 1 neurotensin receptor (NTR-1), and SR 142948A, a non-selective antagonist of NTR-1 and subtype 2 neurotensin receptor (NTR-2) failed to prevent neurotensin from exciting PAG-RVM neurons. Neurotensin failed to evoke cationic currents after internally perfusing PAG-RVM projection neurons with GDP-b-S or anti-Gaq/11 antiserum. Cellular Ca2+ fluorescence measurement using fura-2 indicated that neurotensin rapidly induced Ca2+ release from intracellular stores of PAG-RVM neurons. Neurotensin-evoked cationic currents were blocked by heparin, an IP3 receptor antagonist, and 1,2-bis(2-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid (BAPTA), a fast chelator of Ca2+.
Two subtypes of neurons, primary serotonergic and secondary non-serotonergic cells, were identified from acutely dissociated neurons of the nucleus raphe magnus(NRM). During current-clamp recordings, NT depolarized NRM serotonergic neurons and evoked action potentials. Voltage-clamp recordings showed that NT excited serotonergic neurons by enhancing a voltage-insensitive and non-selective cationic conductance. Both SR 48692 and SR 142948A, failed to prevent neurotensin from exciting NRM serotonergic neurons. Neurotensin-evoked cationic currents were inhibited by the intracellular administration of GDP--S or dialyzing serotonergic neurons with anti-Gaq/11 antibody. Cellular imaging study using fura-2 showed that NT induced calcium release from the intracellular store. Neurrotensin-evoked current was blocked after the internal perfusion of heparin, an IP3 receptor antagonist, or BAPTA, a fast Ca2+ chelator.
It is concluded that neurotensin enhancement of the cationic conductance of PAG-RVM and NRM serotonergic neurons is mediated by a novel subtype of neurotensin receptors. The coupling mechanism via Gaq/11 proteins is likely to involve the generation of IP3 and subsequent IP3-evoked Ca2+ release from intracellular stores results in activating the non-selective cationic conductance.
The NRM sends projections to spinal dorsal horn and inhibits nociceptive transmission. Analgesic effect produced by -opioid receptor agonists including morphine partially results from activating the NRM-spinal cord pathway. It is generally believed that mu-opioid receptor agonists disinhibit spinally projecting neurons of the NRM and produce analgesia by hyperpolarizing GABAergic interneurons.
In the present study, whole-cell patch-clamp recordings combined with single-cell RT-PCR analysis were used to test the hypothesis that DAMGO ([D-Ala2,N-methyl-Phe4,Gly-ol5]enkephalin), a specific mu-opioid receptor agonist, selectively hyperpolarizes NRM neurons expressing mRNA of glutamate decarboxylase (GAD67). Homologous desensitization of mu-opioid receptors in NRM neurons could result in the development of morphine-induced tolerance. G protein-coupled receptor kinase (GRK) is believed to mediate opioid receptor desensitization in vivo. Therefore, we also investigated the involvement of GRK in mediating homologous desensitization of DAMGO-induced electrophysiological effects on NRM neurons by using two experimental strategies. First, single-cell RT-PCR assay was used to study the expressison of GRK2 and GRK3 mRNAs in individual DAMGO-responsive NRM neurons. Whole-cell recording was also performed with an internal solution containing the synthetic peptide , which corresponds to G bg-binding domain of GRK and inhibits Gbg activation of GRK. Our results suggest that DAMGO selectively hyperpolarizes GABAergic neurons by opening inwardly rectifying K+ channels and that GRK2 mediates homologous short-term homologous desensitization of mu-opioid receptors in NRM GABAergic neurons.

目錄
授權書…………………………………………………………………...iii
誌謝……………………………………………………………………...iv
簡寫表………………………………………………………………….viii
中文摘要………………………………………………………………...ix
英文摘要………………………………………………………………..xiii
第一章 緒論……………………………………………………………1
1-1 研究背景及文獻回顧………………………………………….2
1-2 研究目的……………………………………………………...10
第二章 實驗材料與方法………………………………………...…...13
2-1 大白鼠導水管周圍灰質投射至頭腹內側延腦神經元
之逆行染色及即離………………………………………… .14
2-2 大白鼠中縫大核神經元之即離……………………………...15
2-3 全細胞電位與電流箝定記錄………………………………...16
2-4 溶液成分……………………………………………………...17
2-5 即離中縫大核血清素性神經元之免疫螢光染色…………...18
2-6 Anti-Gaq/11抗體之細胞內灌流……………………………….19
2-7 神經元之單細胞反轉錄-聚合酶鏈鎖反應………………….19
2-8 細胞內鈣離子之測定………………………………………...22
2-9 GRK2胜肽之合成……………………………………………23
2-10 統計方法……………………………………………………...24
2-11 藥品…………………………………………………………...24
第三章 結果(I)
神經降壓素對投射至頭腹內側延腦的導水管周圍灰質
( PAG-RVM ) 神經元之調控作用………………………………25
3-1 PAG-RVM投射神經元之辨識……………………………….29
3-2 神經降壓素經由促進非選擇性陽離子電導活化PAG-RVM
投射神經元……………………………………………………30
3-3 Gaq/11蛋白將神經降壓素受體偶聯至PAG-RVM投射神經元
的陽離子通道………………………………………………….33
3-4 神經降壓素經由IP3引起鈣離子釋放而促進陽離子電導…..36
3-5 討論……………………………………………………………38
第四章 結果(II)
神經降壓素對中縫大核血清素性神經元之調控作用…….….44
4-1 即離中縫大核神經元後兩種亞型細胞之辨識……………...46
4-2 神經降壓素藉增加非選擇性陽離子電導而對中縫大核
血清素性神經元產生興奮作用……………………………...47
4-3 Gq/11蛋白將神經降壓素受體偶聯至中縫大核
血清素性神經元之陽離子通道…………………………….. 50
4-4 神經降壓素藉IP3誘發鈣離子釋放而增加陽離子電導…….52
4-5 討論…………………………………………………………...53
第五章 結果(III)
第二亞型G蛋白-偶聯受體磷酸化(G protein-coupled
receptor kinase 2, GRK2)對中縫大核g-胺基丁酸性(GABAergic)
神經元上mu-亞型類鴉片受體去敏作用之調控…………………...59
5-1 DAMGO經由促進向內整流性鉀離子電導而對中縫大核
-胺基丁酸性神經元產生過極化作用……………………….64
5-2 GRK2參與中縫大核g-胺基丁酸性神經元mu-亞型類鴉片
受體之短期同質性去敏作用………………………………….66
5-3 討論……………………………………………………………71
第六章 總結及展望…………………………………………………....78
圖表……………………………………………………………………..82
參考文獻……………………………………………………………… 110

參考文獻
Al-Rodhan, N.R.F., Richelson, E., Gilbert, J.A., McCormick, D.J., Kanba, K.S., Pfenning, M.A., Nelson, A., Larson, E.W. and Yaksh, T.L., 1991. Structure-antinociceptive activity of neurotensin and some novel analogues in the periaqueductal gray region of the brainstem. Brain Research 557, 227-235.
Alessi, D. R., Cuenda, A., Cohen, P., Dudley, D. T. and Saltie, A. R., 1995. PD 098059 is a specific inhibitor of the activation of mitogen-activated protein kinase kinase in vitro and in vivo. Journal of Biological Chemistry 270, 27489-27494.
Appleyard, S.M., Celver, J., Pineda, V., Kovor, A., Wayman, G.A. and Chavkin, C., 1999. Agonist-dependent desensitization of the mu-opioid receptor by G protein receptor kinase and -arrestin. Journal of Biological Chemistry 274, 23802-23807.
Appleyard, S. M., Patterson, T. A., Jin, W. Z. and Chavkin, C., 1997. Agonist-induced phosphorylation of the mu-opioid receptor. Journal of Neurochemistry 69, 2405-2412.
Arriza, J. L., Dawson, T. M., Simerly, R. B., Martin, L. J., Caron, M. G., Snyder ,S. H. and Lefkowitz, R. J., 1992. The G-protein-coupled receptor kinases ARK1 and ARK2 are widely distributed at synapses in rat brain. Journal of Neuroscience 12, 4045-4055.
Azami, J., Llewelen, M. D. and Roberts, M. H. T., 1982. The contribution of nucleus reticularis paragigantocellularis and nucleus raphe magnus to the analgesia produced by systematically administered morphine, investigated with the microinjection technique. Pain 12, 229-246.
Bandler, R. and Shipley, M.T., 1994. Columnar organization in the midbrain periaqueductal gray: modules for emotional expression? Trends in Neurosciences 17: 379- 388.
Barry, P.H. and Lynch, J.W., 1991. Liquid junction potentials and small cell effects in patch-clamp analysis. Journal of Membrane Biology 121, 101-117.
Basbum, A. I. and Fields, H. L., 1984. Endogenous pain control systems, brainstem spinal pathways and endorphin circuitry. Annual Reviews of Neuroscience. 7, 309-338.
Behbehani, M. M., 1992. Physiological mechanisms of the analgesic effect of neurotensin. Annals of New York Academy of Science 668, 253-265.
Behbehani, M.M., Park, M.R. and Clement, M.E., 1988. Interactions between the lateral hypothalamus and the periaqueductal gray. Journal of Neuroscience 8, 2780-2787.
Behbehani, M.M. and Pert, A., 1984. A mechanism for the analgesic effect of neurotensin as revealed by behavioral and electrophysiological techniques. Brain Research 324, 35-42.
Behbehani, M.M., Shipley, M.T. and McLean, J.H., 1987. Effect of neurotensin on neurons in the periaqueductal gray. Journal of Neuroscience 7, 2035-2041.
Beitz, A.J., 1982. The sites of origin of brain stem neurotensin and serotonin projections to the rodent nucleus raphe magnus. Journal of Neuroscience 2, 829-842.
Beitz, A.J., Mullett, M.A. and Weiner, L.L., 1983. The periaqueductal gray projections to the rat spinal trigeminal, raphe magnus, gigantocellular pars alpha and paragigantocellular nuclei arise from separate neurons. Brain Research 288, 307-314.
Bitner, R. S., Nikkel, A. L., Curzon, P., Crneric, S. P., Bannon, A. W. and Decker, M. W., 1998 . Role of the nucleus raphe magnus in antinociception produced by ABT-594: immediate early gene responses possibly linked to neuronal nicotinic acetylcholine receptors on serotonergic neurons. Journal of Neuroscience 18, 5426-5432.
Boekhoff, I., Inglese, J., Schleicher, S., Koch, W. J., Lefkowitz, R. J. and Breer, H., 1994. Olfactory desensitization requires membrane targeting of receptor kinase mediated by -subunits of heterotrimeric G proteins. Journal of Biological Chemistry 269, 37-40.
Botto, J. M., Charbry, J., Sarret, P., Vincent, J. P. and Mazella, J., 1998. Stable expression of the mouse lavocabastine-sensitive neurotensin receptor in HEK 293 cell line: binding properties, photoaffinity labeling, and internalization mechanism. Biochemecial and Biophysical Research Communications 243, 585-590.
Bowker, R. M. and Dilts, R. P., 1998. Distribution of -opioid receptors in the nucleus raphe magnus and nucleus gigantocellularis: a quantitative autoradiographic study. Neuroscience Letters 88, 247-252.
Brunton, J. and Charpak, S., 1998. mu-opioid peptides inhibit thalamic neurons. Journal of Neuroscience 18, 1671-1678.
Bunemann, M. and Hosey, M. M., 1999. G-protein coupled receptor kinases as modulators of G-protein signalling. Journal of Physiology 517, 5-23.
Burd, A. L., El-Kouhen, R., Loh, H. H. and Law, P. Y., 1998. Identification of serine 356 and serine 363 as the amino acid involved in etorphine-induced down-regulation of the mu-opioid receptor. Journal of Biological Chemistry 273, 34488-34495.
Caraway, R.E. and Ferris, C.F., 1983. The isolation of a new hypotensive peptide, neurotensin, from bovine hypothalamus. Journal of Biological Chemistry, 248, 6854-6861
Caraway, R.E. and Leeman, S.E., 1996. Characterization of radioimmunoassayable neurotensin in the rat: Its differential distribution in the central nervous system, small intestine, and stomach. Journal of Biological Chemistry 251, 7045-7051.
Chakrabarti, S., Law, P. Y. and Loh, H. H., 1998. Distinct differences between morphine- and [D-Ala2,N-MePhe4,Gly-ol5]enkephalin- mu-opioid receptor complexes demonstrated by cyclic AMP-dependent protein kinase phosphorylation. Journal of Neurochemistry 71, 231 - 239.
Chieng, B. and Christie, M.J., 1994. Hyperpolarization by opioids acting on mu-receptors of a subpopulation of rat periaqueductal gray neurons in vitro. British Journal of Pharmacology 113, 121-128.
Congar, P., Leinekugel, X., Ben-Ari, Y. and Crepel, V., 1997. A long-lasting calcium-activated nonselective cationic current is generated by synaptic stimulation or exogenous activation of group 1 metabotropic glutamate receptors in CA1 pyramidal neurons. Journal of Neuroscience 17, 5366-5379.
Cruzblanca, H., Koh, D.S. and Hille, B., 1998. Bradykinin inhibits M current via phospholipase C and Ca2+ release from IP3-sensitive Ca2+ stores in rat sympathetic neurons. Proceedings of the National Academy of Sciences of the USA 95, 7151-7156.
Cvejic, S., Trapaidze, N., Cyr, C. and Devi, L. A., 1996. Thr353, located within the COOH-terminal tail of the  opiate receptor, is involved in receptor down-regulation. Journal of Biological Chemistry 271, 4073-4076.
Diverse-Pierluissi, M., Inglese, J., Stoffe, R. H., Lefkowitz, R. J. and Dunlap, K., 1996. G protein-coupled receptor kinase mediates desensitization of norepinephrine-induced Ca++ channel inhibition. Neuron 16, 579-585.
Dubuc, I., Costentin, J., Terranova, J.P., Barnouin, M.C., Soubrie, P., Lefur, G., Rostene, W. and Kitabgi, P., 1994. The nonpeptide neurotensin antagonist, SR 48692, used as a tool to reveal putative neurotensin receptor subtypes. British Journal of Pharmacology 112, 352-354.
Dubuc, I., Sarret, P., Labb-Jullie, C., Botto, J.M., Honore, E., Bourdel, E., Martinez, J., Costentin, J., Vincent, J.P., Kitabgi, P. and Mazella, J., 1999. Identification of the receptor subtype involved in the analgesic effect of neurotensin. Journal of Neuroscience 19, 503-510.
Emson, P.C., Goedert, M., Willams, B., Nikovic, M. and Hunt, S.P., 1982. The regional distribution and chromatographic characterization of neurotensin-like immunoreactivity in the rat central nervous system. Advances in Biochemical Psychopharmacology 33, 477-85.
Exton, J.H., 1997. Cell signalling through guanine-nucleotide-binding regulatory proteins and phospholipase. European Journal of Biochemistry 243, 10-20.
Fang, F. G., Moreau, J. L. and Fields, H. L., 1987. Dose-dependent antinociceptive action of neurotensin microinjected into the rostroventromedial medulla of the rat. Brain Research 420, 171-174.
Farkas, R.H., Nakajima, S. and Nakajima, Y., 1994. Neurotensin excites basal forebrain cholinergic neurons: Ionic and signal-transduction mechanisms. Proceedings of the National Academy of Sciences of the USA 91, 2853-2857.
Ffrench-Mullen, J.M.H., Plata-Salaman, C.R., Buckley, N.J. and Danks, P., 1994. Muscarinic modulation by a G-protein -subunit of delayed rectifier K+ current in rat ventromedial hypothalamic neurons. Journal of Physiology 474, 21-26.
Fields, H. L., Heinricher, M. M. and Mason, P., 1991. Neurotransmitters in nociceptive modulatory circuits. Annual Reviews of Neuroscience 14, 219-245.
Fiorillo, C. D. and Williams, J. T., 1996. Opioid desensitization: Interaction with G-protein-coupled receptors in the locus coeruleus. Journal of Neuroscience 16, 1479-1485.
Gray, T.S. and Magnuson, D.J., 1992. Peptide immunoreactive neurons in the amygdala and the bed nucleus of the stria terminalis project to the midbrain central grays in the rat. Peptides 13, 451-460.
Grudt, T. J. and Williams, J. T., 1994. mu-opioid agonists inhibit spinal trigeminal substantia gelatinosa neurons in guinea pig and rat. Journal of Neuroscience 14, 1646-1654.
Grynkiewicz, G., Poenie, M. and Tsien, R., 1985. A new generation of calcium indicators with greatly improved fluorescence properties. Journal of Biological Chemistry 260, 3440-3450.
Gully, D., Canton, M., Boigegrain, R., Jeanjean, F., Molimard, J.C., Poncelet, M., Gueudet, C., Heaulme, M., Leyris, R., Brouard, A., Pelaprat, D., Labb-Jullie, C., Mazella, J., Soubrie, P., Maffrand, J.P., Rostene, W., Kitabgi, P. and Le Fur, G., 1993. Biochemical and pharmacological profile of a potent and selective nonpeptide antagonist of neurotensin receptor. Proceedings of the National Academy of Sciences of the USA 90, 65-69.
Gully, D., Labeeuw, B., Boigegrain, R., Oury-Donat, F., Bachy, A., Poncelet, M., Steinberg, R., Suaud- Chagny, M.F., Santucci, V., Vita, N., Pecceu, F., Labb-Jullie, C., Kitabgi, P., Soubrie, P., Le Fur, G. and Maffrand, J.P., 1997. Biochemical and pharmacological activities of SR 14298A, a new potent neurotensin antagonist. Journal of Pharmacological and Experimental Therapeutics 280, 802-810.
Haga, T., Haga, K. and Kameyama, K., 1994. G protein-coupled receptor kinases. Journal of Neurochemistry 63, 400-412.
Haley, J.E., Abogadie, F.C., Delmas, P., Dayrell, M., Vallis, Y., Milligan, G., Caufield, M.P., Brown, D.A. and Buckley, N.J., 1998. The  subunit of Gq contributes to muscarinic inhibition of potassium current in sympathetic neurons. Journal of Neuroscience 18, 4521-4531.
Hamil, O.P., Marty, A., Neher, E., Sakmann, B. and Sigworth, F., 1981. Improved patch-clamp technique for high resolution current recording from cells and cell-free membrane patches. Pflugers Archieves 391, 85-100.
Hammond, D. L. and Yaksh, T. L., 1984. Antagonism of stimulation produced antinociception by intrathecal administration of methysergide or phentolamine. Brain Research 298, 329-337.
Hawes, B. E., Luttrell, L. M., van Biesen, T. and Lefkowitz, R. L.,1996. Phosphatidy l — inositol 3-kinase is an early intermediate in the G bg-mediated mitogen-activated protein kinase signaling pathway. Journal of Biological Chemistry 271, 12133-12136.
Helper, J.R. and Gilman, A.G., 1992. G proteins. Trends in Biochemical Sciences 17, 383-387.
Herlitze, S., Garcia, D.E., Mackie, K., Hille, B., Scheuer, T. and Catterall, W.A., 1996. Modulation of Ca2+ channels by G-protein  subunits. Nature 380, 258-262.
Hille, B., 1994. Modulation of ion-channel function by G-protein-coupled receptors. Trends in Neurosciences 17, 531-535.
Hudson, P.M. and Lumb, B.M., 1996. Neurons in the midbrain periaqueductal gray send collateral projections to nucleus raphe magnus and the rostral ventrolateral medulla in the rat. Brain Research 733, 138-141.
Ikeda, S.R., 1996. Voltage-dependent modulation of N-type calcium channels by G-proteinsubunits. Nature 380, 255-258.
Jennes, L.S., Stump, W. and Kalivas, P.W., 1982. Neurotensin: topographic distribution in the rat brain by immunohistochemistry. Journal of Comparative Neurology 210, 211-224.
Jiang, Z.G., Pessia, M. and North, R.A., 1994. Neurotensin excitation of rat ventral tegmental neurons. Journal of Physiology (London) 474, 119-129.
Jolas, T. and Aghajanian, G.K., 1996. Neurotensin excitation of serotonergic neurons in the dorsal raphe nucleus of the rat in vitro. European Journal of Neuroscience 8, 153-161.
Julien, J. F., Samama P. and Mallet, J., 1990. Rat brain glutamic acid decarboxylase sequence deduced from a cloned cDNA. Journal of Neurochemistry 54, 703-706.
Kangrga, I.M. and Loewy, A.D., 1995. Whole-cell recordings from visualized C1 adrenergic bulbospinal neurons: ionic mechanisms underlying vasomotor tone. Brain Research 670, 215-232.
Kessler, J. P., Moyse., E., Kitabgi, P., Vincent, J. P. and Beaudet, A.,1987. Distribution of neurotensin binding sites in the caudal brainstem of the rat: a light microscopic radioautographic study. Neuroscience 23, 189-198.
Kirkpatrick, K. and Bourque, C.W., 1995. Effects of neurotensin on rat supraoptic neurons in vitro. Journal of Physiology 482, 373-381.
Kobayashi, R.M., Brown, M.R. and Vale, W., 1982. Regional distribution of neurotensin and somatostatin in rat brain. Brain Research 126, 584-588.
Koch, W. J., Hawes, B. E., Inglese, J., Luttrell, L. M. and Lefkowitz, R. J., 1994. Cellular expression of the carboxyl terminus of a G protein-coupled receptor kinase attenuate G-mediated signaling. Journal of Biological Chemistry 269, 6193-6197.
Koch, W. J., Inglese, J., Stone, W. C. and Lefkowitz, R. J., 1993. The binding site for thebg subunits of heterotrimeric G proteins on the a-adrenergic receptor kinase. Journal of Biological Chemistry 268, 8256-8260.
Kovoor, A., Celver, J.P., Wu, A. and Chavkin, C., 1998. Agonist-induced homologous desensitization of mu-opioid receptors mediated by G protein-coupled receptor kinase is dependent on agonist efficacy. Molecular Pharmacology 54, 704-711.
Kramer, H. A. and Simon, E. J., 2000. Role of protein kinase C (PKC) in agonist-induced mu-opioid receptor down-regulation : I. PKC translocation to the membrane of SH-SY5Y neuroblastoma cells is induced by -opioid agonists. Journal of Neurochemistry 72, 585-593.
Krupnick, J. G. and Benovic J. L., 1998. The role of receptor kinases and arrestins in G protein coupled receptor regulation. Annual Reviews of Pharmacology and Toxicology 38, 289-319.
Lakos, S. and Basbaum, A.I., 1988. An ultrastructural study of the projections from the midbrain periaqueductal gray to spinally projecting, serotonin-immunoreactive neurons of the medullary nucleus raphe magnus in the rat. Brain Research 443, 383-388.
LaMorte, V.J., Harootunian, A.T., Spiegel, A.M., Tsien, R.Y. and Feramisco, J.R., 1993. Mediation of growth factor induced DNA synthesis and calcium mobilization by Gq and Gi2. Journal of Cell Biology 121, 91-99.
Law, P. Y. and Loh, H. H., 1999. Regulation of opioid receptor activities. Journal of Pharmacology and Experimental Therapeutics 289, 607-624.
Lee, K., Dixon, A.K., Gonzalez, I., Stevens, E.B., Mcnulty, S., Oles, R., Richardson, P.J., Pinnock, R.D. and Singh, L., 1999. Bombesin-like peptides depolarize rat hippocampal interneurons through interaction with subtype 2 bombesin receptors. Journal of Physiology 518, 791-802.
Manberg, P.J., Youngblood, W.W., Nemeroff, C.B., Rossor, M., Iversen, L.L., Prange, A.J. and Kizer, J.S., 1982. Regional distribution of neurotensin in human brain. Journal of Neurochemistry, 38, 1777-1780.
Mason, P., Strassman, A. and Maciewicz, R., 1985. Pontomedullary raphe neurons: monosynaptic excitation from midbrain sites that suppress the jaw opening reflex. Brain Research 329, 384-389.
Mazella, J., Botto, J.M., Guillemare, E., Coppola, T., Sarret, P. and Vincent, J.P., 1996. Structure, functional expression, and cerebral localization of the levocabastine-sensitive neurotensin /neuromedin N receptor from mouse brain. Journal of Neuroscience 16, 5613-5620.
Menetrey, D. and Basbaum, A. I., 1987. The distribution of substance P-, enkephalin- and dynorphin-immunoreactive neurons in the medulla of the rat and their contribution of bulbospinal pathway. Neuroscience 23, 173-187.
Meng, J. D., Manning, B. H., Martin, W. J. and Fields, H. L.,1998. An analgesia circuit activated by cannabinoids. Nature 395, 381-383.
Milligan, G., 1993. Regional distribution and quantitative measurement of the phosphoinositidase C-linked guanine nucleotide binding proteins G11 and Gq in rat brain. Journal of Neurochemistry 61, 845-851.
Morgan, M.M., Heinricher, M.M. and Fields, H.L., 1992. Circuitry linking opioid -sensitive nociceptive modulatory systems in periaqueductal gray and spinal cord with rostral ventromedial medulla. Neuroscience 47, 863-871.
Mullaney, I., Mitchell, F. M., McCallum, J., Buckley, N. J. and Milligan, G.,1993. The human muscarinic M1 acetylcholine receptor, when expressed in CHO cells, activates and downregulates both Gq and G11 equally and non-selectively. FEBS Letters 324, 241-245.
Murthy, K.S. and Makhlouf, G.M., 1994. Vasoactive intestinal peptide/pituitary adenylate cyclase-activating peptide-dependent activation of membrane-bound NO synthase in smooth muscle mediated by pertussis toxin-sensitive Gi1-2. Journal of Biological Chemistry 269, 15977-15980.
Naranjo, J. R., Arnedo, A., Molinero, M. T. and Del Rio, J., 1989. Involvement of spinal monoaminergic pathways in nociception produced by substance P and neurotensin in rodents. Neuropharmacology 28, 291-298.
Nestler, E.J., 1996. Under siege: The brain on opiates. Neuron 16, 897-900.
Offermanns, S., Heiler, E., Spicher, K. and Schultz, G., 1994. Gq and G11 are concurrently activated by bombesin and vasopressin in Swiss 3T3 cells. FEBS Letters 349, 201-204.
Osborne, B.P. and Williams, J.T., 1995. Characterization of acute homologous desensitization of the  mu opioid receptor-induced currents in locus coeruleus neurons. British Journal of Pharmacology 115, 925-932.
Osborne, B.P., Vaughan, C.W., Wilson, H.I. and Christie, M.J., 1996. Opioid inhibition of rat periaqueductal gray neurons with identified projections to rostral ventromedial medulla in vivo. Journal of Physiology (London) 490, 383-389.
Ozaita, A., Escriba, P.V., Ventayol, P., Murga, C., Mayor, F., Jr. and Garcia-Sevilla, J.A., 1998. Regulation of G protein-coupled receptor kinase 2 in brains of opiate-treated rats and human opiate addicts. Journal of Neurochemistry 70, 1249-1257.
.
Pan, Z. Z., Williams, J. T. and Osborne, P. B., 1990. Opioid actions on single nucleus raphe magnus neurons from rat and guinea-pig in vitro. Journal of Physiology 427, 516-532.
Pan, Z. Z., Wessendorf, M. W. and Williams, J. T., 1993. Modulation by serotonin of the neurons in rat nucleus raphe magnus in vitro. Neuroscience 54, 421-429
Pan, Z. Z., Tershner, S. A. and Fields, H. L., 1997. Cellular mechanism for anti-analgesic action of agonists of the -opioid receptor. Nature 389, 382-385
Patridge, L.D., Muller, T.H. and Swadulla, D., 1994. Calcium-activated non-selective channels in the nervous system. Brain Research Reviews 19, 319-325.
Pei, G., Kieffer, B. L., Lefkowitz, R. J. and Freedman, N. J., 1995. Agonist-dependent phosphorylation of the mouse -opioid receptor: Involvement of G protein-coupled receptor kinases but not protein kinase C. Molecular Pharmacology 48, 173-177
Pitcher, J. A., Freedman, N. J. and Lefkowitz, R. J., 1998. G protein-coupled receptor kinases. Annual Reviews of Biochemistry 67, 653-692.
Polakiewicz, R. D., Schieferl, S. M., Dorner, L. F., Kansra, V. and Comb, M. J., 1998. A mitogen-activated protein kinase pathway is required for mu-opioid receptor desensitization. Journal of Biological Chemistry 273, 12402-12406.
Puttfarcken, P., Werling, L. L. and Cox, B. M., 1998. Effects of chronic morphine exposure on opioid inhibition of adenylyl cyclase in 7315C cell membranes: a useful model for the study of tolerance at mu opioid receptor. Molecular Pharmacology 33, 520-527..
Raynor, K., Kong, H., Hines J., Kong, G., Benovic, J., Yasuda, K., Bell, G. I. and Reisine, T., 1994. Molecular mechanisms of agonist-induced desensitization of the cloned mouse kappa opioid receptor. Journal of Pharmacology and Experimental Therapeutics 270, 1381-1386.
Reichling, D.B. and Basbaum, A.I., 1990. Contribution of brainstem GABAergic circuitry to descending antinociceptive controls: I. GABA-immunoreactive projection neurons in the periaquaductal gray and nucleus raphe magnus. Journal of Comparative Neurology 302, 370-377.
Reisine, T. and Bell, G. I., 1993. Molecular biology of the opiate receptors. Trends in Neurosciences 16, 384-389.
Renno, W.M., Mullet, M.A. and Beitz, A.J., 1992. Systemic morphine reduces GABA release in the lateral but not the medial portion of the midbrain periaqueductal gray of the rat. Brain Research 594, 221-232.
Rizvi, T.A., Enis, M., Behbehani, M.M. and Shipley, M.T., 1991. Connections between the central nucleus of the amygdala and the midbrain periaqueductal gray: topography and reciprocity. Journal of Comparative Neurology 303, 121-131.
Rossi, G.C., Pasternak, G.W. and Bodner, R.J., 1994.  and  opioid synergy between the periaqueductal gray and the rostro-ventral medulla. Brain Research 665, 85-93.
Sanchez, D. and Ribas, J., 1991. Properties and ionic basis of the action potentials in the periaqueductal gray neurons of the guinea-pig. Journal of Physiology (London) 440, 167-187.
Sandkuhler, J. and Gebhart, G.F., 1984. Relative contributions of the nucleus raphe magnus and adjacent medullary reticular formation to the inhibition by stimulation in the periaqueductal gray of a spinal nociceptive reflex in the pentobarbital-anesthetized rat. Brain Research 305, 77-87.
Schmidt, H., Schulz, S., Klutzny, M., Koch, T., Handel, M. and Holt, V., 2000. Involvement of mitogen-activated protein kinase in agonist-induced phosphorylation of the mu-opioid receptor in HEK 293 cells. Journal of Neurochemistry 74, 414-422.
Shenker, A., Goldsmith, P., Unson, C.G. and Spiegel, A.M., 1991. The G protein coupled to the thromboxane A2 receptors in human platelet is a member of the novel Gq family. Journal of Biological Chemistry 266, 9309-9313.
Shipley, M.T., Mclean, J.H. and Behbehani, M.M., 1987. Heterogeneous distribution of neurotensin-like immunoreactive neurons and fibers in the midbrain periaqueductal gray of the rat. Journal of Neuroscience 7, 2025-2034.
Simon, M.I., Strathmann, M.P. and Gautam, N., 1991. Diversity of G proteins in signal transduction. Science 252, 802-808.
Skagerberg, S. and Bjorklund, A., 1985. Topographic principles of the spinal projections of serotonergic and non-serotonergic brainstem neurons in the rat. Neuroscience 15, 445-480.
Sternweis, P.C. and Smircka, A.V., 1992. Regulation of phospholipase C by G proteins. Trends in Biochemical Sciences 17, 502-506.
Stiller, C.O., Bergquist, J., Beck, O., Ekman, R. and Brodin, E., 1996. Local administration of morphine decreases the extracellular level of GABA in the periaqueductal gray matter of freely moving rats. Neuroscience Letters 209, 165-168.
Stiller, C.O., Gustafsson, H., Fried, K. and Brodin, E., 1997. Opioid-induced release of neurotensin in the periaqueductal gray matter of freely moving rats. Brain Research 774, 149-158.
Strathmann, M.P. and Simon, M.I., 1990. G protein diversity: a distinct class of asubunits is present in vertebrates and invertebrates. Proceedings of the National Academy of Sciences of the USA 87, 9113-9117.
Tanaka, K., Masu, M. and Nakanishi, S., 1990. Structure and functional expression of the cloned rat neurotensin receptor. Neuron 4, 847-854.
Thomas, A.P. and Delaville, F., 1991. The use of fluorescent indicators for measurements of cytosolic-free calcium concentration in cell populations and single cells. In: Cellular Calcium: A Practical Approach (edited by McCormack, J.G. and Cobbold, P.H.), New York: Oxford University Press, 1- 54.
Trudeau, L.E., 2000. Neurotensin regulates intracellular calcium in ventral tegmental area astrocytes: evidence for the involvement of multiple receptors. Neuroscience 97, 293-302.
Uhl, G.R., 1990. Neurotensin receptors. In: Handbook of Chemical Neuroanatomy (edited by Bjorklund, A., Hokfelt, T. and Kuhar, M.J.), Amsterdam: Elsevier Science Publishers. Vol.9, pp.443-453.
Urban, M. O. and Gebhart, G. F., 1997. Characterization of biphasic modulation of spinal nociceptive transmission by neurotensin in the rat rostral ventromedial medulla. Journanal of Neurophysiology 78, 1550-1562.
Vaughan, C.W., Ingram, S.L., Connor, M.A. and Christie, M.J., 1997. How opioids inhibit GABA-mediated neurotransmission. Nature 390, 611-614.
Vincent, J. P., Mazella, J. and Kitabgi, P., 1999. Neurotensin and neurotensin receptors. Trends in Pharmacologcal Sciences 20, 302-309.
Vita, N., Oury-Donat, F., Chalon, P., Guillemot, M., Kaghad, M., Bachy, A., Thurneyssen, O., Garcia, S., Poinot-Chazel, C., Casellas, P., Keane, P., Le Fur, G., Maffrand, J. P., Soubrie, P., Caput, D. and Ferrara, P., 1998. Neurotensin is an antagonist of the human neurotensin NT2 receptor expressed in Chinese hamster ovary cells. European Journal of Pharmacology 360, 265-272.
Wang, H.L., 1997. A site-directed mutagenesis study on the conserved alanine residue in the distal third intracellular loops of CCKB and neurotensin receptors. British Journal of Pharmacology 121, 310-316.
Wang, H. L., 2000. A cluster of Ser/Thr residues at the C-terminus of mu-opioid receptor is required for G protein-coupled receptor kinase 2-mediated desensitization. Neuropharmacology 39, 353-363.
Wang, H.L., Cheng, W.T., Hsu, C.Y., Huang, P.C., Chow, Y.W. and Li, A.H., 2002. Identification of two C-terminal amino acids, Ser355 and Thr357, required for short-term homologous desensitization of mu-opioid receptors. Biochemical Pharmacology 7339, 1-10.
Wang, H. L. and Wu, T., 1996. Gaq/11 mediates neurotensin excitation of substantia nigra dopaminergic neurons. Molecular Brain Research 36, 29-36.
Whistler, J. L. and von Zastrow, M., 1998. orphine-activated opioid receptors elude desensitization by b-arrestin. Proceedings of the National Academy of Sciences of the USA 95, 9914-9919.
Williams, F.G. and Beitz, A.J., 1989. A quantitative ultrastructural analysis of neurotensin-like immunoreactive terminals in the midbrain periaqueductal gray: analysis of their possible relationship to periaqueductal gray-raphe magnus projection neurons. Neuroscience 29, 121-134.
Williams, F.G. and Beitz, A.J., 1993. Chronic pain increases brainstem proneuro - tensin/neuromedin-N mRNA expression: a hybridization-histochemical and immunohistochemical study using three different rat models for chronic nociception. Brain Research 611, 87-102.
Williams, F.G., Mullet, M.A. and Beitz, A.J., 1995. Basal release of Met-enkephalin and neurotensin in the ventrolateral periaqueductal gray matter of the rat: a microdialysis study of antinociceptive circuits. Brain Research 690, 207-216.
Wu, T., Li, A. and Wang, H.L., 1995. Neurotensin increases the cationic conductance of rat substantia nigra dopaminergic neurons through the inositol (1,4,5) trisphosphate - calcium pathway. Brain Research 683, 242-250.
Wu, T. and Wang, H.L., 1996. Gaq/11 mediates cholecystokinin activation of the cationic conductance in rat substantia nigra dopaminergic neurons. Journal of Neurochemistry 66, 1060- 1066.
Wu, T. and Wang, H.L., 1997. The excitatory effect of cholecystokinin on rat neostriatal neurons: ionic and molecular mechanisms. European Journal of Pharmacology 66, 1060-1066.
Yaksh, T. L. and Wilson, T. R., 1979. Spinal serotonin terminal system mediates antinociception. Journal of Pharmacology and Experimental Therapeutics 220, 266-277.
Yamada, M., Yamada, M., Lombet, A., Forgez, P. and Rostene, W., 1998. Distinct functional characteristics of levocabastine sensitive rat neurotensin NT2 receptor expressed in Chinese hamster ovary cells. Life Science 62, 375-380.
Yu, Y., Zhang, L., Yin, X., Sun, H., Uhl, G. R. and Wang, J. B., 1997.  Opioid receptor phosphorylation, desensitization, and ligand efficacy. Journal of Biological Chemistry 272, 28869- 28874.
Zhang, J., Ferguson, S. S. G., Barak, L. S., Bodduluri, S. R., Laporte, S. A., Law, P. Y. and Caron, M. G., 1998. Role for G protein-coupled receptor kinsase in agonist-specific regulation of -opioid receptor responsiveness. Proceedings of the National Academy of Sciences of the USA 95, 7157-7162.
Zhang, L., Yu, Y., Seamus, M., Weight, F. F., Uhl, G. R. and Wang, J. B., 1996. Differential opiate receptor phosphorylation and desensitization induced by agonists and phorbol esters. Journal of Biological Chemistry 271, 11449-11454.