School of Neuroscience for Thai Youth

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1.1 ¤ÇÒÁà»ç¹ÁÒáÅФÇÒÁÊӤѭ¢Í§»Ñ­ËÒ

ºÒÃÒ¤ÍÅà»ç¹ÊÒÃ㹡ÅØèÁ͹ؾѹ¸Øì¢Í§ chromone ·ÕèÊ¡Ñ´ä´é¨Ò¡ãºáÅд͡¢Í§ÊÁعä¾Ã¢ÕéàËÅç¡ (Hassanali-Walji et al., 1969)  ã¹µÓÃÒá¾·Âìá¼¹âºÃÒ³ä´éãªéÊèǹ¢Í§µé¹¢ÕéàËÅç¡à¾×èÍËÇѧ¼Å¡ÒÃÃÑ¡ÉÒ·ÕèᵡµèÒ§¡Ñ¹ àªè¹ ãªéÃÒ¡¶èÒ¾ÔÉä¢é ãªéµé¹ÃÑ¡ÉÒâä¼ÔÇ˹ѧáÅÐà»ç¹ÂÒÃкÒ Êèǹ¢Í§à»Å×Í¡ãªéá¡éÃÔ´Êմǧ·ÇÒÃáÅÐá¡é¡ÃÐÉÑ ãªéÂÍ´á¡éâäàºÒËÇÒ¹ áÅÐãªéãºá¡éÍÒ¡Òù͹äÁèËÅѺ à»ç¹µé¹ (Farnworth and Bunyapraphatsara, 1992)  ÁÕ¡ÒÃÈÖ¡ÉÒÄ·¸Ôì·Ò§àÀÊѪÇÔ·ÂҢͧºÒÃÒ¤ÍŵèÍÃкº»ÃÐÊÒ·Êèǹ¡ÅÒ§¾ºÇèÒºÒÃÒ¤ÍÅÊÒÁÒöŴ¾ÄµÔ¡ÃÃÁ¡ÒÃà¤Å×è͹äËÇ (locomotor activity) ¢Í§Ë¹Ù¶Õº¨Ñ¡ÃáÅÐ˹٢ÒÇ (ÍØäà ÍÃسÅѡɳì, 2490: ÍéÒ§¶Ö§ã¹ Thongsaard  et al., 1996; ¾Ô¡ØÅ ¨Ñ¹·Ãâ¸Ò, 2531; Sukma et al., 2002) áÅоºÇèÒàÁ×èÍãËéºÒÃÒ¤ÍÅã¹¢¹Ò´ 100-200 ÁÔÅÅÔ¡ÃÑÁµè͹éÓ˹ѡµÑÇ 1 ¡ÔâÅ¡ÃÑÁã¹Ë¹Ù¶Õº¨Ñ¡ÃÊÒÁÒöŴ¤ÇÒÁà¨çº»Ç´ä´é à¹×èͧ¨Ò¡ºÒÃÒ¤ÍÅÁÕ¼Åà¾ÔèÁÃдѺ nociceptive threshold µÒÁ¢¹Ò´·Õèà¾ÔèÁ¢Öé¹ (¾Ô¡ØÅ ¨Ñ¹·Ãâ¸Ò, 2531) àÁ×èÍ©Õ´ºÒÃÒ¤ÍÅã¹¢¹Ò´ 10 ÁÔÅÅÔ¡ÃÑÁµè͹éÓ˹ѡµÑÇ 1 ¡ÔâÅ¡ÃÑÁ·Ò§ªèͧ·éͧ˹٢ÒÇ áÅéÇ·´Êͺ´éÇ Elevated Plus maze ¾ºÇèÒÁռŷÓãËé˹٢ÒÇÅ´¤ÇÒÁà¤ÃÕ´â´ÂÍÍ¡Ä·¸Ôì¤ÅéÒ¡Ѻ diazepam µèÒ§¡Ñ¹·ÕèºÒÃÒ¤ÍÅäÁè·ÓãËéà¡Ô´ÍÒ¡Òçèǧ«ÖÁ¡ÅѺÂѧà¾ÔèÁ¾ÄµÔ¡ÃÃÁ¡ÒÃÊÓÃǨ (exploration ) áÅСÒõ×è¹µÑÇ (hyperlocomotor) «Ö觹Ѻà»ç¹¢éʹբͧºÒÃÒ¤ÍÅ·Õèà˹×Í¡ÇèÒÂÒ㹡ÅØèÁ diazepam ( Thongsaard  et al., 1996) ÍÂèÒ§äáçµÒÁÂѧÁÕÍÕ¡¡ÒÃÈÖ¡ÉÒ˹Öè§àÁ×èÍãËéºÒÃÒ¤ÍÅã¹¢¹Ò´ 10-20 ÁÔÅÅÔ¡ÃÑÁµè͹éÓ˹ѡµÑÇ 1 ¡ÔâÅ¡ÃÑÁá¡è˹ٶպ¨Ñ¡ÃäÁ辺ÇèÒÁÕÄ·¸ÔìÅ´¤ÇÒÁà¤ÃÕ´·Ñé§ã¹ Elevated Plus maze áÅÐ the shock-probe burying test (Fiorino et al., 1998; Sukma et al., 2002)  àÁ×èÍ·´Êͺ´éÇÂá͹äÎâ´ÃºÒÃÒ¤ÍÅäÎâ´Ã¤ÅÍäô쾺ÇèÒ·ÓãËé»ÃÔÁҳⴻÒÁչŴŧ·ÕèÊÁͧ˹ÙÊèǹ caudate áÅÐäÁ辺¡ÒÃà»ÅÕè¹á»Å§¢Í§»ÃÔÁÒ³ 3,4-dihydroxyphenylacetic acid (DOPAC) «Öè§à»ç¹ metabolite µÑÇ˹Ö觢ͧⴻÒÁÕ¹ áÊ´§ÇèҼŢͧá͹äÎâ´ÃºÒÃÒ¤ÍÅäÎâ´Ã¤ÅÍäôì·Õè·ÓãËéÃдѺⴻÒÁչŴŧäÁèÁÕ¤ÇÒÁÊÑÁ¾Ñ¹¸ì¡ÑºàÁá·ºÍÅÔ«ÖÁ¢Í§â´»ÒÁÕ¹áµèÍÒ¨Áռŵè͵ÑÇÃѺⴻÒÁÕ¹ 2 (¹À´Å ÍÔ¹·Ã·Ñµ, 2543)  ºÒÃÒ¤ÍÅÂѧÊÒÁÒöŴÃдѺⴻÒÁÕ¹¹Í¡à«ÅÅìã¹ÊÁͧ˹ÙÊèǹ striatumâ´Â¼èÒ¹µÑÇÃѺⴻÒÁÕ¹ 2 (Thongsaard et al., 1997; Sukma et al., 2002) ·Ñ駹ÕéÁÕÃÒ§ҹÇèÒ¡ÒáÃеØé¹µÑÇÃѺⴻÒÁÕ¹ 2 «Öè§à»ç¹ autoreceptor ÁռŷÓãËéªèͧâ¾á·Êà«ÕÂÁ (K⁺ channel) à»Ô´áÅзÓãËéªèͧá¤Åà«ÕÂÁ (Ca²⁺ channel) »Ô´ ¨Ö§·ÓãËéàÂ×èÍËØéÁà«ÅÅì»ÃÐÊÒ·â´»ÒÁÕ¹à¡Ô´ÊÀÒÇÐ hyperpolarization «Öè§ÁÕ¼Åä»à¾ÔèÁ¡Ò÷ӧҹ¢Í§â´»ÒÁÕ¹·Ãҹʻ͵àµÍÃì (dopamine transporter; DAT) (Sonders et al., 1997) «Ö觷Ó˹éÒ·Õ袹Êè§â´»ÒÁÕ¹¡ÅѺà¢éÒÊÙèà«ÅÅì»ÃÐÊÒ·â´»ÒÁÕ¹·ÓãËéâ´»ÒÁÕ¹·ÕèÍÂÙè¹Í¡à«ÅÅì»ÃÐÊÒ·â´»ÒÁչŴŧ 

            ¨Ò¡Ä·¸Ôì·Ò§àÀÊѪÇÔ·ÂҢͧºÒÃÒ¤ÍÅ·Õè·ÓãËé¾ÄµÔ¡ÃÃÁ¡ÒÃà¤Å×è͹äËǢͧ˹٢ÒÇŴŧ «Öè§ÁÕÃÒ§ҹÇèÒàÁ×èÍãËéÊÒà A68930 ·ÕèÁÕÄ·¸Ôìà»ç¹ agonist µè͵ÑÇÃѺⴻÒÁÕ¹ 1 ã¹¢¹Ò´ 0.9 - 15 äÁâ¤ÃâÁŵè͹éÓ˹ѡµÑÇ 1 ¡ÔâÅ¡ÃÑÁà¢éÒãµé¼ÔÇ˹ѧ  â´Âãªé¡Ò÷´Åͧ·ÕèÈÖ¡ÉҾĵԡÃÃÁ¡ÒÃà¤Å×è͹·Õè¢Í§Ë¹Ù¢ÒÇ·Õèà¡Ô´¢Öé¹àͧ (spontaneous locomotor activity) ã¹¾×é¹·Õèà»Ô´ (Open field testing) ¾ºÇèÒÊÒà A68930 ÊÒÁÒöáÊ´§Ä·¸ÔìʧºÃЧѺà¾ÔèÁ¢Öé¹ áÅÐÊÒà SCH23390 ·ÕèÁÕÄ·¸Ôìà»ç¹ antagonist  µè͵è͵ÑÇÃѺⴻÒÁÕ¹ 1 ÊÒÁÒöŴ¾ÄµÔ¡ÃÃÁ¹Õéä´é  (Salmi et  al., 2000)   ·Ñ駹Õé¡ÒáÃеØé¹µÑÇÃѺⴻÒÁÕ¹ 1  ·ÕèÊÁͧÊèǹ striatum ·ÓãËéà¾ÔèÁ¡ÒÃà¤Å×è͹·Õèâ´ÂÁÕ¡Åä¡¡ÒÃÍÍ¡Ä·¸Ôì¼èÒ¹·Ò§ nigrostriatal pathway (Bishop et  al., 2004) 

                ºÒÃÒ¤ÍÅ·ÓãËéÊѵÇì·´Åͧ¤ÅÒ¡ѧÇÅáÅЧèǧ«ÖÁÁÒ¡¢Öé¹ «Öè§ÁÕÃÒ§ҹÇèÒÂÒÁÕÄ·¸Ôìà»ç¹ agonist µè͵ÑÇÃѺⴻÒÁÕ¹ 2  ÊÒÁÒöÂѺÂÑ駡ÒÃà¡Ô´ ultrasonic vocalization ä´é áÊ´§ÇèÒÂÒ¡ÅØèÁ¹ÕéÁÕÄ·¸Ôì¤ÅÒ¡ѧÇÅ (Bartoszyk, 1998)  áÅШҡ¡Ò÷ÕèºÒÃÒ¤ÍÅÊÒÁÒö¡´¡ÒÃà¤Å×è͹·Õè«Ö觶١à˹ÕèÂǹÓâ´Â methamphetamine (Sukma et al., 2002) «Öè§ÊÒùÕéÍÍ¡Ä·¸Ôì·Õèâ´»ÒÁÕ¹·Ãҹʻ͵àµÍÃì ¼ÙéÇԨѨ֧¤Ò´ÇèÒºÒÃÒ¤ÍÅÍÍ¡Ä·¸ÔìµèÍÃкº»ÃÐÊÒ·â´»ÒÁÕ¹ã¹ÊÁͧÊèǹ¡ÅÒ§ áµè·Ñ駹ÕéÂѧäÁèÁÕ¡ÒÃÈÖ¡ÉÒ¡Åä¡¡ÒÃÍÍ¡Ä·¸Ôì¢Í§ºÒÃÒ¤Íŵè͵ÑÇÃѺⴻÒÁÕ¹áÅÐâ´»ÒÁÕ¹·Ãҹʻ͵àµÍÃì »ÃСͺ¡Ñºã¹»Ñ¨¨ØºÑ¹à·¤¹Ô¤ radioligand binding assay à»ç¹ÇÔ¸Õ·Õè¹ÔÂÁãªé㹡ÒÃÈÖ¡ÉÒÍѹµÃ¡ÔÃÔÂÒÃÐËÇèÒ§ÅÔ᡹´ì¡ÑºµÑÇÃѺ«Öè§à»ç¹à·¤¹Ô¤·ÕèÁÕ»ÃÐÊÔ·¸ÔÀÒ¾ ÁÕ¤ÇÒÁäÇÊÙ§ áÅÐÃÇ´àÃçÇ  ´Ñ§¹Ñé¹ Çѵ¶Ø»ÃÐʧ¤ì¢Í§â¤Ã§¡ÒûÃÔ­­Ò¹Ô¾¹¸ì¹Õé¨Ö§ÁØè§ÈÖ¡ÉÒà¡ÕèÂǡѺ¡Åä¡¡ÒÃÍÍ¡Ä·¸Ôì¢Í§ºÒäÍŵèÍÃкº»ÃÐÊÒ·â´»ÒÁÕ¹¨Ò¡¡ÒÃÇÑ´¤ÇÒÁªÍº¨Ñº ¢Í§ºÒÃÒ¤Íŵè͵ÑÇÃѺⴻÒÁÕ¹áÅÐâ´»ÒÁÕ¹·Ãҹʻ͵àµÍÃìâ´Âãªéà·¤¹Ô¤ radioligand binding assay

 

1.2 Çѵ¶Ø»ÃÐʧ¤ì¢Í§¡ÒÃÇÔ¨ÑÂ

               

                à¾×èÍÈÖ¡ÉÒ¡Åä¡¡ÒÃÍÍ¡Ä·¸Ôì¢Í§ºÒÃÒ¤ÍŵèÍÃкº»ÃÐÊ·â´»ÒÁÕ¹¨Ò¡¡ÒÃÇÑ´¤ÇÒÁªÍº¨Ñº¡ÑºµÑÇÃѺⴻÒÁÕ¹ 1 µÑÇÃѺⴻÒÁÕ¹ 2 áÅÐâ´»ÒÁÕ¹·Ãҹʻ͵àµÍÃì â´Âãªéà·¤¹Ô¤ radioligand binding assay

 

1.3 ¢Íºà¢µ¢Í§¡ÒÃÇÔ¨ÑÂáÅÐÇÔ¸Õ´Óà¹Ô¹¡ÒÃÇÔ¨ÑÂâ´ÂÂèÍ

 

¡ÒÃÇԨѤÃÑ駹Õéµéͧ¡ÒÃÇÑ´¤ÇÒÁªÍº¨Ñº¢Í§ºÒÃҤ͚ѺµÑÇÃѺã¹Ãкº»ÃÐÊÒ·â´»ÒÁÕ¹ã¹ÃдѺËÅÍ´·´Åͧ (in vitro) â´Âãªéà·¤¹Ô¤ radioligand binding assay â´ÂãªéÊÁͧ¢Í§Ë¹Ù¢ÒÇÊèǹ striatum àµÃÕÂÁàÁÁàºÃ¹·ÕèÁÕµÑÇÃѺⴻÒÁÕ¹ 1 áÅÐâ´»ÒÁÕ¹·Ãҹʻ͵àµÍÃì áÅÐãªéà«ÅÅìà¾ÒÐàÅÕé§ A9LhD2 ·Õè·ÓãËéÁÕµÑÇÃѺⴻÒÁÕ¹ 2 ¢Í§Á¹ØÉÂ캹àÂ×èÍËØéÁà«ÅÅìà¾×èÍàµÃÕÂÁàÁÁàºÃ¹¢Í§µÑÇÃѺⴻÒÁÕ¹ 2 ÇÔà¤ÃÒÐËì»ÃÔÁÒ³â»ÃµÕ¹·ÕèÍÂÙèã¹àÁÁàºÃ¹·ÕèàµÃÕÂÁä´éâ´ÂÇÔ¸Õ Bradford â´Âà·Õº¡ÑºÊÒÃÅÐÅÒ bovine serum albumin  ¨Ò¡¹Ñé¹ÇÑ´¤ÇÒÁªÍº¨Ñº¡ÑºµÑÇÃѺⴻÒÁÕ¹ 1 µÑÇÃѺⴻÒÁÕ¹ 2 áÅÐâ´»ÒÁÕ¹·Ãҹʻ͵àµÍÃì ·Ñ駹Õéä´éãªéÊÒõԴ©ÅÒ¡ÃѧÊÕ ÊÒÃÅÐÅÒºѿà¿ÍÃì áÅÐÊÀÒÇСÒ÷´Åͧ·Ñé§ÍسËÀÙÁÔáÅÐàÇÅÒ·ÕèàËÁÒÐÊÁ áÅéǹÓä»ÇÑ´»ÃÔÁÒ³ÃѧÊÕâ´Âãªéà¤Ã×èͧ liquid scintillation counter  áÅéǹӢéÍÁÙÅ·Õèä´éÁҤӹdzãËéÍÂÙèã¹ÃÙ»ÃéÍÂÅТͧ¡ÒÃÂѺÂÑ駡ÒèѺ¢Í§ radioligand ¡ÑºµÑÇÃѺ ËÒ¡ºÒÃÒ¤ÍÅãËé¤èÒÃéÍÂÅТͧ¡ÒÃÂѺÂÑé§ÁÕ¤èÒÊÙ§¡çáÊ´§ÇèÒºÒÃÒ¤ÍÅÁÕ¤ÇÒÁªÍº¨Ñº¡ÑºµÑÇÃѺÊÙ§

 

1.4 »ÃÐ⪹ì·Õè¤Ò´ÇèÒ¨Ðä´éÃѺ

 

1.       ä´é¢éÍÁÙŤÇÒÁªÍº¨Ñº¢Í§ºÒÃÒ¤Íŵè͵ÑÇÃѺⴻÒÁÕ¹ 1 µÑÇÃѺⴻÒÁÕ¹ 2 áÅÐâ´»ÒÁÕ¹

·Ãҹʻ͵àµÍÃìà¾×èÍãªé͸ԺÒ¡Åä¡¡ÒÃÍÍ¡Ä·¸Ôì¢Í§ºÒÃÒ¤ÍŵèÍÃкº»ÃÐÊÒ·â´»ÒÁÕ¹ã¹ÃдѺà«ÅÅì

2.       ä´é¢éÍÁÙÅ·Õèà»ç¹»ÃÐ⪹ìµèÍ¡ÒÃÈÖ¡ÉÒà¾ÔèÁàµÔÁà¾×èÍ¡ÒÃÇÔ¨ÑÂÊÁعä¾Ã¢ÕéàËÅç¡ÍÂèÒ§¤ÃºÇ§¨Ãà¾×èÍ¡ÒþѲ¹Òà»ç¹ÂÒÊÁعä¾Ãã¹Í¹Ò¤µ

 

 

º··Õè 2

àÍ¡ÊÒÃáÅЧҹÇԨѷÕèà¡ÕèÂÇ¢éͧ

 

2.1 ºÒÃÒ¤ÍÅ: ÊÒ÷ÕèÊ¡Ñ´ä´é¨Ò¡ÊÁعä¾Ã¢ÕéàËÅç¡

µé¹¢ÕéàËÅç¡à»ç¹ÊÁعä¾Ãä·Â·ÕèÁÕª×èÍÇÔ·ÂÒÈÒʵÃìÇèÒ Cassia siamea Lam. ¨Ñ´à»ç¹¾×ªã¹Ç§Èì Leguminosae (àµçÁ ÊÁԵԹѹ·¹ì, 2544) ¹Í¡¨Ò¡¹ÕéÂѧÁÕª×èÍàÃÕ¡㹷éͧ¶Ôè¹·ÕèᵡµèÒ§¡Ñ¹ àªè¹ ¢ÕéàËÅç¡á¡è¹ ¢ÕéàËÅ硺éÒ¹ ¢ÕéàËÅç¡ËÅǧ ¢ÕéàËÅç¡ãË­è ¼Ñ¡¨ÕéÅÕé áÅÐÂÐËÒ à»ç¹µé¹ Åѡɳзҧ¾Ä¡ÉÈÒʵÃì¢Í§µé¹¢ÕéàËÅç¡à»ç¹äÁéÂ×¹µé¹¢¹Ò´àÅ硶֧»Ò¹¡ÅÒ§ ¼ÅѴ㺠ÊÙ§»ÃÐÁÒ³ 8-15 àÁµÃ  ÅÓµé¹ÁÑ¡¤´§Íà»ç¹»ØèÁà»Å×Í¡ÊÕà·Ò¶Ö§ÊÕ¹éÓµÒÅ´Ó ÂÍ´Íè͹ÊÕá´§àÃ×èÍæ  ãº»ÃСͺà»ç¹áºº¢¹¹¡ àÃÕ§ÊÅѺ¡Ñ¹ ÁÕãºÂèÍ 5-12 ¤Ùè »ÅÒÂÊØ´ÁÕãºà´ÕÂÇ ãºÂèÍÂÃÙ»¢Íº¢¹Ò¹´éÒ¹º¹à¡ÅÕé§ ´Í¡ªèÍÊÕàËÅ×ͧÍÂÙèµÒÁ»ÅÒ¡Ôè§  ´Í¡¨ÐºÒ¹¨Ò¡â¤¹ªèÍä»Âѧ»ÅÒªèÍ ¡ÅÕºàÅÕé§ÁÕ 3-4 ¡ÅÕº ¡ÅÕº´Í¡ÁÕ 5 ¡ÅÕº à¡ÊõÑǼÙé 10 Íѹ ¼Åà»ç¹½Ñ¡áº¹ÂÒÇÁÕÊÕ¤ÅéÓ àÁÅç´ÃÙ»ä¢èÂÒÇẹÊÕ¹éÓµÒÅÍè͹àÃÕ§µÒÁ¢ÇÒ§ÁÕ 20-30 àÁÅç´ à¹×éÍäÁéÁÕÊÕ¹éÓµÒÅá¡èà¡×ͺ´Ó Êèǹ¢Í§´Í¡áÅÐ㺢ÕéàËÅç¡ãªéà»ç¹ÍÒËÒÃã¹ËÅÒ»ÃÐà·Èàªè¹ ä·Â ¾ÁèÒ ÍÔ¹à´Õ áÅÐÁÒàÅà«Õ à»ç¹µé¹ ã¹µÓÃÒ¡ÒÃá¾·Âìá¼¹ä·Âä´éÁÕ¡Òúѹ·Ö¡»ÃÐ⪹ì¢Í§¢ÕéàËÅç¡ã¹ËÅÒ´éÒ¹ àªè¹ ãªéá¡éÍÒ¡Ò÷éͧ¼Ù¡ ãªéá¡éÍÒ¡Òù͹äÁèËÅѺ ãªé·Ó¤ÇÒÁÊÐÍÒ´àÊé¹¼Á ·ÓãËé¼ÁªØèÁª×è¹à»ç¹à§Ò§ÒÁ äÁèÁÕÃѧᤠªèÇÂà¨ÃÔ­ÍÒËÒà ºÓÃا¹éÓ´Õ áÅкÓÃاâÅËÔµ à»ç¹µé¹ (ÊÁ¾Ã ÀÙµÔÂҹѹµì, 2542)

                ã¹»Õ ¾.È. 2485 «Öè§à»ç¹Âؤʧ¤ÃÒÁâÅ¡¤ÃÑ駷Õè 2 ¾ºÇèÒÂÒÃÑ¡ÉÒâä¢Ò´á¤Å¹à»ç¹ÍÂèÒ§ÁÒ¡ ÈÒʵÃÒ¨ÒÃÂì ¹¾.ÍÇ ࡵØÊÔ§Ëì ¨Ö§ä´é¾ÂÒÂÒÁ¤é¹ËÒÊÁعä¾Ãä·Â·Õèãªéà»ç¹ÂÒá¼¹âºÃÒ³ÍÂèÒ§á¾ÃèËÅÒÂÁÒÈÖ¡ÉÒÄ·¸Ôì·Ò§àÀÊѪÇÔ·ÂÒ áÅÐä´é¾ºÇèÒãºáÅд͡¢ÕéàËÅç¡·ÓãËéà¡Ô´ÍÒ¡Òçèǧ«ÖÁáÅÐÁÕ¾ÔɹéÍ¡ÇèÒÊÁعä¾Ãª¹Ô´Í×è¹æ·Õèä´éÈÖ¡ÉÒ µèÍÁÒ¨Ö§ÁÕ¡ÒÃÈÖ¡ÉÒÄ·¸Ôì·Ò§àÀÊѪÇÔ·ÂҢͧÊÒÃʡѴ㺢ÕéàËÅç¡ÍÕ¡¤ÃÑé§â´ÂãªéáÍÅ¡ÍÎÍÅìà»ç¹µÑÇ·ÓÅÐÅÒ ¾ºÇèÒÊÒÃÊ¡Ñ´¹ÕéÁÕÄ·¸Ôì¡´»ÃÐÊÒ·Êèǹ¡ÅÒ§ à¾ÔèÁ¤ÇÒÁµÖ§µÑǢͧ¡ÅéÒÁà¹×éÍàÃÕº áÅÐÁÕÄ·¸Ôì¢Ñº»ÑÊÊÒÇÐ (ÍØäà ÍÃسÅѡɳì, 2490; ÍéÒ§¶Ö§ã¹ ¾ÔªÑ µÑé§ÈÃÕÊÓàÃÔ§, 2540)

                ¨¹¡ÃзÑè§ã¹»Õ ¾.È. 2513 ¤³Ð¹Ñ¡ÇԨѨҡÁËÒÇÔ·ÂÒÅÑ Nottingham »ÃÐà·ÈÍѧ¡ÄÉä´éÃÒ§ҹÇèÒÊÒÁÒöʡѴÊÒê¹Ô´ãËÁè¨Ò¡ãº¢ÕéàËÅç¡ â´ÂÊÒùÕéà»ç¹Í¹Ø¾Ñ¹¸ì¢Í§ dioxaphenalene ·Õèà»ç¹ÊÒáÅØèÁ chromone ª¹Ô´Ë¹Öè§áÅÐä´éµÑ駪×èÍÇèÒºÒÃÒ¤ÍÅ «Öè§ÁÕª×èÍ·Ò§à¤ÁÕÇèÒ 3a,4-dihydro-3a,8-dihydroxy-2,5-dimethyl-1,4-dioxaphenalene áÅÐÁÕÊÙµÃâÁàÅ¡ØŤ×Í C₁₃H₁₂O₄ (Hassanali-Walji net  al., 1969)

 

               

 

               

 

ÃÙ»·Õè 1  â¤Ã§ÊÃéÒ§¢Í§ºÒÃÒ¤ÍÅ

 

Bycroft áÅФ³Ð (1970) ä´éàʹÍÇÔ¶ÕªÕÇÊѧà¤ÃÒÐËì¢Í§ºÒÃÒ¤ÍÅÇèÒà¡Ô´¨Ò¡¡ÒÃÃÇÁµÑǢͧËÁÙè acetate 7 âÁàÅ¡ØÅáÅéÇà¡Ô´»¯Ô¡ÔÃÔÂÒ¼èÒ¹·Ò§ polyketide pathway áÅéÇà¡Ô´à»ç¹ÊÒõÑÇ¡ÅÒ§·ÕèäÁèàʶÕÂèҡ¹Ñ鹨֧à¡Ô´»¯Ô¡ÔÃÔÂÒ cyclization  ä´éà»ç¹ºÒÃÒ¤ÍÅ µèÍÁÒÁÕ¼ÙéÈÖ¡ÉÒÊÒÃÊӤѭ¨Ò¡ãºÍè͹¢Í§µé¹¢ÕéàËÅ硺éÒ¹¾ºÇèÒÊÒÁÒöʡѴºÒÃÒ¤ÍÅ«Öè§à»ç¹¼ÅÖ¡ÃÙ»à¢çÁÊÕàËÅ×ͧä´é¤Ô´à»ç¹ÃéÍÂÅÐ 0.1 ¢Í§¹éÓ˹ѡ㺢ÕéàËÅç¡àÃÔèÁµé¹ (Chaichantipyuth, 1979)  áÅÐä´éÁÕ¼Ùé¾Ñ²¹ÒÇÔ¸ÕÊ¡Ñ´ÊÒúÒÃÒ¤ÍŨҡ㺢ÕéàËÅç¡áÅзÓãËéà¢éÁ¢é¹¢Öé¹´éÇÂÇÔ¸Õ pervaporation ·ÓãËéÊÒÁÒöʡѴºÒÃÒ¤ÍÅä´éà¾ÔèÁ¢Ö鹤Դà»ç¹ÃéÍÂÅÐ 0.8 ¢Í§¹éÓ˹ѡ㺢ÕéàËÅç¡àÃÔèÁµé¹ (¾ÔªÑ µÑé§ÈÃÕÊÓàÃÔ§, 2540)

                ºÒÃÒ¤ÍÅà»ç¹ÊÒ÷ÕèÁÕ¼ÅÖ¡ÊÕàËÅ×ͧá¡Áà¢ÕÂÇÊÒÁÒöÅÐÅÒÂä´éã¹¹éÓ ã¹ÊÀÒÇл¡µÔºÒÃÒ¤ÍÅÁÑ¡äÁè¤èÍÂàʶÕÂÃà¹×èͧ¨Ò¡à¡Ô´»¯Ô¡ÔÃÔÂÒ dehydration à¡Ô´à»ç¹ÊÒÃá͹äÎâ´ÃºÒÃÒ-¤ÍÅ (anhydrobarakol; C₁₃H₁₀O₃) «Öè§ÊÒùÕéà¡Ô´»¯Ô¡ÔÃÔÂҡѺ¡Ã´à¡Å×Í (HCl) ä´éà»ç¹à¡Å×Íá͹äÎâ´ÃºÒÃÒ¤ÍÅäÎâ´Ã¤ÅÍäôì (anhydrobarakol hydrochloride; C₁₃H₁₂ClO₃) ¹Í¡¨Ò¡¹ÕéºÒÃÒ¤ÍÅÂѧÊÒÁÒöà¡Ô´»¯Ô¡ÔÃÔÂÒÍÍ¡«Ôപѹ·ÕèËÁÙèäδÃÍ¡«ÔÅàÁ×èÍÍÂÙèã¹ÊÒÃÅÐÅÒ·ÕèÁÕ¹éÓà»ç¹µÑÇ·ÓÅÐÅÒ (Thongsaard et al., 2001) àÁ×èÍàµÃÕÂÁÊÒÃÅÐÅÒºÒÃÒ¤ÍÅãËéÍÂÙèã¹ÊÀÒÇТͧÊÒÃÅÐÅÒºѿà¿ÍÃì·Õè¤ÅéÒ¡Ѻã¹ÊÔè§ÁÕªÕÇÔµ (physiological buffer) ·ÕèÍسËÀÙÁÔ 37 ͧÈÒà«Åà«ÕÂÊ ¾ºÇèÒºÒÃÒ¤ÍÅÊÒÁÒö¤§µÑÇÍÂÙèä´éÍÂèÒ§¹éÍ 1 ªÑèÇâÁ§ (Thongsaard et al., 2001)

 

 

 

µÒÃÒ§·Õè 1   ¤Ø³ÊÁºÑµÔ¢Í§ºÒÃÒ¤ÍÅáÅÐ͹ؾѹ¸ì¢Í§Áѹ  (Thongsaard  et  al., 2001)

 

¢éÍà»ÃÕºà·Õº	ºÒÃÒ¤ÍÅ	á͹äÎâ´ÃºÒÃÒ¤ÍÅ	á͹äÎâ´ÃºÒÃÒ¤ÍÅäÎâ´Ã¤ÅÍäôì
ÊÙµÃà¤ÁÕ	C13H12O4	C13H10O3	C13H12ClO3
ÁÇÅâÁàÅ¡ØÅ	232	214	251.4
ÊÕ	Greenish yellow	Dark green	Lemon yellow
¨Ø´ËÅÍÁàËÅÇ	166-170 °C	163 °C	208-210 °C

 

2.2 Ä·¸Ôì¢Í§ºÒÃÒ¤ÍŵèÍÃкº»ÃÐÊÒ·Êèǹ¡ÅÒ§

 

                ºÒÃÒ¤ÍÅÁÕÄ·¸Ôì·Ò§àÀÊѪÇÔ·ÂÒàº×éͧµé¹·Õè¹èÒʹ㨾ÍÊÃØ»ä´é´Ñ§¹Õé àÁ×èÍãËéºÒÃÒ¤ÍÅá¡è˹ٶպ¨Ñ¡Ã¾ºÇèÒäÁèÊÒÁÒöµéҹķ¸Ôì·Õèà¡Ô´¨Ò¡ÂÒ¡ÃеØé¹Ãкº»ÃÐÊÒ·Êèǹ¡ÅÒ§«Öè§ä´éá¡è GABA antagonist (bicucullin áÅÐ picrotoxin) áÅÐ Glycine-mediated post-synaptic inhibition (strychnine) à¹×èͧ¨Ò¡äÁèÊÒÁÒöŴ¤èÒ convulsant dose-50 (CD₅₀)  áÅÐ lethal dose-50 (LD₅₀) ¢Í§ÂÒàËÅèÒ¹Õéä´é áÊ´§ÇèÒºÒÃÒ¤ÍÅäÁèä´éÁռżèÒ¹·Ò§Ãкº»ÃÐÊÒ· GABA ËÃ×Í Glycine (¾Ô¡ØÅ ¨Ñ¹·Ãâ¸Ò, 2531) µèÍÁÒ¾ºÇèÒºÒÃÒ¤ÍÅäÁèÊÒÁÒöµéҹķ¸Ôì·Õè·ÓãËéà¡Ô´¡Òêѡ¢Í§ bicucullin áÊ´§ÇèÒºÒÃÒ¤ÍÅäÁèä´éÁռżèÒ¹·Ò§ GABA_A Receptor (Thongsaard et al., 1996) ÁÕ¡ÒõÃǨ¨ÑººÒÃÒ¤ÍÅ·ÕèµÔ´ÊÒÃÃѧÊÕ¤×Í ¹²⁵I- barakol ã¹ÊÁͧ˹٢ÒÇâ´Âãªéà·¤¹Ô¤ receptor autoradiography ÊÒÁÒöàÃÕ§ÅӴѺ¤ÇÒÁ˹Òá¹è¹¢Í§ÊÕä´éµÒÁÅӴѺ´Ñ§¹Õé caudate-putamen, accumben nucleus, cerebral cortex, hippocampus, thalamic nuclei, granular cell, layer of cerebellum, inferior colliculus áÅÐ  substantia nigra  â´Âä´éàʹÍÇèÒµÓá˹觨Ѻ ¢Í§ºÒÃÒ¤ÍÅäÁèãªè·ÕèµÑÇÃѺⴻÒÁÕ¹ 1 µÑÇÃѺⴻÒÁÕ¹ 2 µÑÇÃѺ«ÕâÃâµ¹Ô¹áÅÐ µÑÇÃѺ GABA  (ÊÒ·ԾÂì à¾è§ÈÃÕ, 2539)

                àÁ×èÍãËéºÒÃÒ¤ÍÅá¡è˹ٶպ¨Ñ¡Ã¡è͹ãËéÊÒà 5-hydroxytryptophan ã¹¢¹Ò´ 200 ¹Ò⹡ÃÑÁµè͹éÓ˹ѡµÑÇ 1 ¡ÔâÅ¡ÃÑÁ¾ºÇèÒºÒÃÒ¤ÍÅÅ´Ä·¸Ôì¡ÒÃʺѴËÑǢͧ˹٢ÒÇ·ÕèªÑ¡¹Óâ´Â 5-hydroxytryptophan ¹Ñ蹤×ͺÒÃÒ¤ÍÅÊÒÁÒö¡´¡Ò÷ӧҹ¢Í§Ãкº»ÃÐÊÒ·«ÕâÃâµ¹Ô¹ (¾Ô¡ØÅ ¨Ñ¹·Ãâ¸Ò, 2531) ¹Í¡¨Ò¡¹ÕéºÒÃÒ¤ÍÅÂѧÊÒÁÒöµéҹķ¸Ôì 5-HT₂ receptor-mediated behaviour ã¹Ë¹Ù¶Õº¨Ñ¡ÃáÅÐÁÕÃÒ§ҹÇèÒ ritanserin «Öè§à»ç¹ 5-HT₂ receptor antagonist ÁÕÄ·¸Ôì¤ÅÒ¡ѧÇÅáÅмŨҡ¾ÄµÔ¡ÃÃÁ¹èÒ¨Ðà¡ÕèÂÇ¢éͧ¡ÑºÃкº»ÃÐÊÒ·«ÕâÃâµ¹Ô¹ (Thongsaard et al., 1996) áµèàÁ×èÍãªéà·¤¹Ô¤ High performance liquid chromatography ÃèÇÁ¡Ñº electrochemical detection (HPLC-ECD)  ÃèÇÁ¡Ñº Punching technique ÇÑ´¼Å¢Í§á͹äÎâ´ÃºÒÃÒ¤ÍÅäÎâ´Ã¤ÅÍäôìµèÍÃдѺ»ÃÔÁҳⴻÒÁÕ¹áÅЫÕâÃâµ¹Ô¹ã¹ÊÁͧ˹٢ÒÇ ¾ºÇèÒäÁèÁÕ¡ÒÃà»ÅÕè¹á»Å§¢Í§ÃдѺ«ÕâÃ⵹Թ㹺ÃÔàdzµèÒ§æ¢Í§ÊÁͧáÊ´§ÇèÒäÁèÁռŵèÍÃкº»ÃÐÊÒ·«ÕâÃâµ¹Ô¹ Êèǹ»ÃÔÁҳⴻÒÁÕ¹ÁÕ»ÃÔÁҳŴŧ·ÕèºÃÔàdz¤ÍÃìà´µÊèǹµé¹äÁ辺¡ÒÃà»ÅÕè¹á»Å§¢Í§»ÃÔÁÒ³ DOPAC «Öè§à»ç¹ metabolite µÑÇ˹Ö觢ͧ¡ÒÃÊÅÒÂâ´»ÒÁÕ¹ áÊ´§ÇèÒ¡ÒÃŴŧ¢Í§â´»ÒÁÕ¹äÁèà¡ÕèÂÇ¢éͧ¡ÑºàÁá·ºÍÅÔ«ÖÁ¢Í§â´»ÒÁÕ¹ (¹¾´Å ÍÔ¹·Ã·Ñµ, 2543)

                à¹×èͧ¨Ò¡ºÒÃÒ¤ÍÅÁÕá¹Çâ¹éÁÍÍ¡Ä·¸ÔìµèÍÃкº»ÃÐÊÒ·â´»ÒÁÕ¹¨Ö§ÁÕ¼ÙéÈÖ¡ÉÒÇԨѼŢͧºÒÃÒ¤ÍŵèÍÃкº»ÃÐÊÒ·â´»ÒÁÕ¹¤è͹¢éÒ§ÁÒ¡ ¾ºÇèÒºÒÃÒ¤ÍÅà¾ÔèÁ¾ÄµÔ¡ÃÃÁ¡ÒÃà¤Å×è͹·ÕèẺËÁع (rotation behavior) ã¹Ë¹Ù¢ÒÇ·Õè©Õ´ apomorphine ã¹¢¹Ò´ 1 ¹Ò⹡ÃÑÁµè͹éÓ˹ѡµÑÇ 1 ¡ÔâÅ¡ÃÑÁ áÊ´§ÇèÒºÒÃÒ¤ÍÅÊÒÁÒö¡ÃеØé¹Ãкº»ÃÐÊÒ·â´»ÒÁÕ¹ä´é (¾Ô¡ØÅ ¨Ñ¹·Ãâ¸Ò, 2531) áÅкÒÃÒ¤ÍÅã¹¢¹Ò´ 100 - 200 ÁÔÅÅÔ¡ÃÑÁµè͹éÓ˹ѡµÑÇ 1 ¡ÔâÅ¡ÃÑÁÊÒÁÒöŴ¤ÇÒÁà¨çº»Ç´ã¹Ë¹Ù¶Õº¨Ñ¡Ãâ´Âà¾ÔèÁ nociceptive threshold µÒÁ¢¹Ò´¢Í§ºÒÃÒ¤ÍÅ·Õèà¾ÔèÁ¢Öé¹ (¾Ô¡ØÅ ¨Ñ¹·Ãâ¸Ò, 2531) ÁÕÃÒ§ҹÇèÒÃкº»ÃÐÊÒ·â´»ÒÁÕ¹ÊÒÁÒö¤Çº¤ØÁ¡ÒÃÃѺÃÙé¤ÇÒÁà¨çº»Ç´ä´é â´ÂàÁ×èÍãËé quinpirole ·Õèà»ç¹ D₂-selective agonist  ÊÒÁÒöÂѺÂÑ駤ÇÒÁÃÙéÊÖ¡»Ç´·Õèà˹ÕèÂǹÓâ´ÂÊÒà formalin áÅÐÄ·¸Ôì¹ÕéÊÒÁÒöµéÒ¹ä´é´éÇ raclopride «Öè§à»ç¹ D₂  receptor antagonist (Taylor, 2003)

                àÁ×èÍ·´Êͺķ¸Ôì¤ÅÒ¡ѧÇÅâ´ÂãËéºÒÃÒ¤ÍÅã¹¢¹Ò´ 10 ÁÔÅÅÔ¡ÃÑÁµè͹éÓ˹ѡµÑÇ 1 ¡ÔâÅ¡ÃÑÁ·Ò§ªèͧ·éͧ˹٢ÒÇâ´Â·´Êͺ´éÇ Elevated Plus Maze ¾ºÇèÒÁÕ¼ÅÅ´¤ÇÒÁà¤ÃÕ´¢Í§Ë¹Ù¢ÒÇ â´ÂºÒÃÒ¤ÍÅÁÕÄ·¸Ôì¤ÅéÒ¡ѺÂÒ diazepam áµèäÁè·ÓãËéà¡Ô´ÍÒ¡Òçèǧ«ÖÁ¡ÅѺà¾ÔèÁ¾ÄµÔ¡ÃÃÁ¡ÒÃÊÓÃǨáÅÐáÊ´§¾ÄµÔ¡ÃÃÁµ×è¹µÑÇ «Ö觹Ѻà»ç¹¢éʹբͧºÒÃÒ¤ÍÅ·Õèà˹×Í¡ÇèÒÂÒ㹡ÅØèÁ diazepam (Thongsaard et  al., 1996)  ÍÂèÒ§äáçµÒÁÂѧÁÕÍÕ¡¡ÒÃÈÖ¡ÉÒ˹Ö觷Õè¢Ñ´áÂ駡ѹ¤×Í àÁ×èÍãËéºÒÃÒ¤ÍÅã¹¢¹Ò´ 10 - 20 ÁÔÅÅÔ¡ÃÑÁµè͹éÓ˹ѡµÑÇ 1 ¡ÔâÅ¡ÃÑÁã¹Ë¹Ù¶Õº¨Ñ¡ÃäÁ辺ÇèÒÁÕÄ·¸ÔìÅ´¤ÇÒÁà¤ÃÕ´·Ñé§ã¹¡Ò÷´Åͧ Elevated Plus Maze áÅÐ shock probe burying test (Fiorino et  al., 1998;  Sukma et  al., 2002)

 

 

2.3 Ä·¸Ôì·Ò§àÀÊѪÇÔ·ÂҢͧºÒÃÒ¤ÍŵèÍÃкºÍ×è¹æ

 

                ¹Í¡à˹×ͨҡķ¸ÔìµèÍÃкº»ÃÐÊÒ·Êèǹ¡ÅÒ§áÅéǺÒÃÒ¤ÍÅÂѧáÊ´§Ä·¸ÔìµèÍÃкºÍ×è¹æ àªè¹ àÁ×èÍãËéºÒÃÒ¤ÍÅã¹¢¹Ò´ 0.5 - 15 ÁÔÅÅÔ¡ÃÑÁµè͹éÓ˹ѡµÑÇ 1 ¡ÔâÅ¡ÃÑÁà¢éÒ·Ò§ËÅÍ´àÅ×Í´´ÓÁÕÄ·¸Ôì·ÓãËé¤ÇÒÁ´Ñ¹àÅ×ʹŴŧ·Ñ駤ÇÒÁ´Ñ¹«ÕÊâµÅÔ¡áÅФÇÒÁ´Ñ¹ä´áÍÊâµÅÔ¡ÍÂèÒ§ÁÕ¹ÑÂÊӤѭ·Ò§Ê¶ÔµÔ ºÒÃÒ¤ÍÅÁÕÄ·¸ÔìÅ´¤ÇÒÁ´Ñ¹ä´é·Ñé§ã¹Ë¹Ù¢ÒÇáÅÐã¹áÁÇ«Öè§ÁÕ¡Åä¡¡ÒÃÍÍ¡Ä·¸ÔìᵡµèÒ§¡Ñ¹ â´Âã¹Ë¹Ù¹èÒ¨ÐÍÍ¡Ä·¸Ôì¼èÒ¹ 2 ¡Å䡤×Í ¼èÒ¹·Ò§àÂ×èͺØà͹ⴷÕàÅÕÂÁ â´ÂºÒÃÒ¤ÍŨÐ仡ÃеØé¹à«ÅÅìà͹ⴷÕàÅÕÂÁãËéËÅÑè§ÊÒà endothelium-derived relaxing factor  «Ö觵èÍÁÒ¾ºÇèÒÊÒùÕé¤×Í nitric oxide (NO) «Ö觨Ðä»·ÓãËé¡ÅéÒÁà¹×éÍàÃÕº¢Í§ËÅÍ´àÅ×Í´¤ÅÒµÑÇ áÅÐ/ËÃ×ÍÍÒ¨ÍÍ¡Ä·¸Ôìâ´ÂµÃ§µèÍ¡ÅéÒÁà¹×éÍàÃÕº¢Í§ËÅÍ´àÅ×Í´ Êèǹã¹áÁǹèÒ¨ÐÍÍ¡Ä·¸Ôì¼èÒ¹·Ò§µÑÇÃѺ muscarinic à¹×èͧ¨Ò¡ atropine ÊÒÁÒöÂѺÂÑé§Ä·¸Ôì¢Í§ºÒÃÒ¤ÍÅ·ÕèÁÕµèÍ¡ÅéÒÁà¹×éÍàÃÕº¢Í§ËÅÍ´àÅ×Í´ä´é (¡Ô¨¨Ò ÊØÇÃó, 2535) àÁ×èÍãËéºÒÃÒ¤ÍÅ·Ò§ËÅÍ´àÅ×Í´´Ó¡è͹ãËé aconitine Ẻ bolus intravenous injection á¡è˹٢ÒÇ·Õèä´é·ÓãËéÊź ¾ºÇèÒÊÒÁÒöŴ¡ÒÃà¡Ô´ ventricular fibrillation áÅÐ ventricular tachycardia áÅÐ ÍѵÃÒ¡ÒõÒ¢ͧ˹٢ÒÇ·Õèà˹ÕèÂǹÓãËéà¡Ô´â´Â aconitine «Ö觤ҴÇèÒºÒÃÒ¤ÍÅÍÍ¡Ä·¸Ôìâ´ÂÅ´»ÃÔÁÒ³äÍÍ͹â«à´ÕÂÁ·ÕèÊÐÊÁÀÒÂã¹à«ÅÅì (Chen et al., 1999) áÅÐÁÕ¡ÒÃÈÖ¡ÉҼŢͧºÒÃÒ¤ÍŵèÍ¡Ò÷ӧҹ¢Í§à͹ä«Áì acetylcholinesterase (AChE) ·Ñ駪¹Ô´·ÕèÅÐÅÒÂÍÂÙèã¹à«ÅÅì (intracellular soluble AChE) áÅзÕèà¡ÒÐÍÂÙè¡ÑºàÂ×èÍËØéÁà«ÅÅì (membrane-bound AChE) ¾ºÇèÒºÒÃÒ¤ÍÅ·Õè¤ÇÒÁà¢éÁ¢é¹ã¹ªèǧ 0.05 - 1.0 ÁÔÅÅÔâÁÅÒÃìäÁèÁÕ¼ÅÂѺÂÑé§ÊÁÃö¹Ð¢Í§à͹ä«Áì·Ñé§ÊͧÃٻẺ (à©ÅÔÁÊÔ¹ à¾ÔèÁàµÔÁÊÔ¹, 2545)

                à¹×èͧ¨Ò¡¤¹ä·Â¹ÔÂÁºÃÔâÀ¤ÊÁعä¾Ãà¾ÔèÁ¢Öé¹ÁÒ¡ ͧ¤ì¡ÒÃàÀÊѪ¡ÃÃÁ¨Ö§ä´é¼ÅÔµÊÁعä¾Ã¢ÕéàËÅç¡ã¹Ãٻ᤻«ÙÅã¹à´×͹ÊÔ§ËÒ¤Á ¾.È. 2540  àÁ×è͹ÓÂÒÊÁعä¾Ã¹ÕéÁÒÈÖ¡ÉÒ¾ºÇèÒ·ÓãËéà¡Ô´¤ÇÒÁ§èǧËÅѺä´é§èÒ¢Öé¹ â´Â´Ù¨Ò¡¤Å×è¹ÊÁͧ·Õèà»ÅÕè¹á»Å§ä»ã¹ÍÒÊÒÊÁѤÃÊØ¢ÀÒ¾´Õâ´Â¾ºÇèÒ¡ÒõÃǨ¤Å×è¹ÊÁͧ·Õèà¡ÕèÂǡѺ¡ÒçèǧËÅѺ ( N₁P₁ amplitude)  3  µÓá˹è§Å´Å§ÍÂèÒ§ÁÕ¹ÑÂÊӤѭ·Ò§Ê¶ÔµÔ «Öè§áÊ´§ÇèÒÍÒÊÒÊÁѤçèǧÁÒ¡¢Öé¹ Êèǹã¹ÍÒÊÒÊÁѤ÷ÕèÁÕÍÒ¡Òù͹äÁèËÅѺÂÒ¢ÕéàËÅç¡·ÓãËé¤Ø³ÀÒ¾¡Òù͹¢Í§ÍÒÊÒÊÁѤôբÖé¹ àªè¹ ËÅѺä´éàÃçÇ¢Öé¹ µ×蹡ÅÒ§´Ö¡¹éÍÂŧáÅÐÃÙéÊÖ¡¾Ö§¾Í㨡Ѻ¡Òù͹ÁÒ¡ (Pooviboonsuk et  al., 2000) áµèÍÂèÒ§äáçµÒÁàÁ×è͵ԴµÒÁ¡ÒÃãªéÂÒÊÁعä¾ÃµèÍÁÒ¾ºÇèÒ·ÓãËéà¡Ô´ÀÒÇеѺÍÑ¡àʺà©Õº¾ÅѹÍÂèÒ§¹éÍÂã¹¼Ùé»èǨӹǹ 9 ÃÒÂã¹»Õ ¾.È. 2542  â´Â¤ÇÒÁÊÑÁ¾Ñ¹¸ì¢Í§ÀÒÇеѺÍÑ¡àʺ¨Ò¡ÂÒ¢ÕéàËÅ硨ѴÍÂÙèã¹ÃдѺ probable ¨¹¶Ö§¢Ñé¹ definite µÒÁࡳ±ìÁҵðҹ¡ÒÃÇÔ¹Ô¨©Ñ ËÃ×Í DILI scale (drug-induced liver injury scale) áÅÐÁÕ¼Ùé»èÇ 2 ÃÒÂä´é·´Åͧ¡Ô¹ÂÒ¢ÕéàËÅç¡«éÓãËÁèËÅѧÀÒÇеѺÍÑ¡àʺà©Õº¾Åѹ´Õ¢Öé¹áÅéǾºÇèÒà¡Ô´ÍÒ¡ÒâͧµÑºÍÑ¡àʺ«éÓÍÕ¡  áÊ´§ÇèҼŢͧÊÀÒÇеѺÍÑ¡àʺ¹Ñé¹à¡Ô´¢Ö鹨ҡÂÒÊÁعä¾Ã¢ÕéàËÅç¡  ¨Ö§·ÓãË餳СÃÃÁ¡ÒÃÍÒËÒÃáÅÐÂÒÊÑè§ÃЧѺ¡ÒüÅÔµÂÒàÁç´ãº¢ÕéàËÅç¡ (ÊÁºÑµÔ µÃÕ»ÃÐàÊÃÔ°ÊØ¢ áÅФ³Ð, 2543) ÍÂèÒ§äáçµÒÁÂѧäÁèÊÒÁÒöÊÃØ»ä´éÇèÒ¡ÒÃà¡Ô´ÀÒÇеѺÍÑ¡àʺà»ç¹¼Åâ´ÂµÃ§¢Í§ºÒÃÒ¤ÍÅ ËÃ×Íà¡Ô´¨Ò¡¼Å¢Í§ÊÒ÷Õèà»ç¹Í§¤ì»ÃСͺÍ×è¹æ «Öè§ÁÕ¢éͤÇõÃÐ˹ѡÇèÒµÃҺ㴷ÕèÂѧäÁè·ÃÒº¡Åä¡¡ÒÃÍÍ¡Ä·¸Ôì·Õèá·é¨ÃÔ§¢Í§ÂÒÊÁعä¾Ã¡çäÁè¤ÇÃÁÕ¡ÒÃãªéÂÒÊÁعä¾Ãã¹ÃдѺ¤ÅÔ¹Ô¡ (Hongsirinirachorn, 2003)

                ä´éÁÕ¡ÒÃÈÖ¡ÉÒ¤ÇÒÁà»ç¹¾ÔÉà©Õº¾Åѹ â´Â©Õ´ºÒÃÒ¤ÍÅ·Ò§ªèͧ·éͧ¢Í§Ë¹Ù¶Õº¨Ñ¡Ã ¾ºÇèÒºÒÃÒ¤ÍÅÁÕ¤ÇÒÁà»ç¹¾ÔɵèÓâ´ÂÁÕ¤èÒ convulsant dose-50 (CD₅₀) à·èҡѺ 296.17  ÁÔÅÅÔ¡ÃÑÁµèÍ¡ÔâÅ¡ÃÑÁ  áÅÐÁÕ¤èÒ lethal dose-50 (LD₅₀) à·èҡѺ 324.09 ÁÔÅÅÔ¡ÃÑÁµèÍ¡ÔâÅ¡ÃÑÁ (¾Ô¡ØÅ ¨Ñ¹·Ãâ¸Ò, 2531) Êèǹ¡Ò÷´Êͺ¤ÇÒÁà»ç¹¾ÔÉã¹ brine shrimp ¾ºÇèÒãËé¤èÒ LD₅₀ à·èҡѺ 930 ÁÔÅÅÔ¡ÃÑÁµèÍÁÔÅÅÔÅԵà (Sanan, 2000)     àÁ×èÍÈÖ¡ÉÒ¤ÇÒÁà»ç¹¾ÔɢͧºÒÃÒ¤ÍŵèÍà«ÅÅìÁÐàÃç§àÍçÁºÃÔâ;ÕÊÔºà¡éÒ´éÇÂÇÔ¸Õ XTT assay ¾ºÇèÒºÒÃÒ¤ÍÅáÊ´§¤ÇÒÁà»ç¹¾ÔɵèÍà«ÅÅìá»Ã¼Ñ¹µÃ§µÒÁ¤ÇÒÁà¢éÁ¢é¹áÅÐàÇÅÒ ã¹¢¹Ò´¤ÇÒÁà¢éÁ¢é¹äÁè¹éÍ¡ÇèÒ 0.8 ÁÔÅÅÔâÁÅÒÃì ¾ºÇèÒà«ÅÅìµÒ»ÃÐÁÒ³ 95% ÀÒÂã¹àÇÅÒ 72 ªÑèÇâÁ§ áÅз´Êͺ´éÇÂÇÔ¸Õ trypan blue dye exclusion assay ¾ºÇèÒºÒÃÒ¤ÍÅ·Õ袹Ҵ¤ÇÒÁà¢éÁ¢é¹ 0.8 áÅÐ 1.0  ÁÔÅÅÔâÁÅÒÃì áÊ´§¤ÇÒÁà»ç¹¾ÔɵèÍà«ÅÅìÁÐàÃç§àÍçÁºÃÔâ;ÕÊÔºà¡éÒâ´ÂÁըӹǹà«ÅÅì·ÕèÁÕªÕÇÔµ¹éÍ¡ÇèÒ¡ÅØèÁ¤Çº¤ØÁ»ÃÐÁÒ³ 63 - 76 % ËÅѧ¨Ò¡ä´éÃѺºÒÃÒ¤ÍŹҹ 36 - 72 ªÑè§âÁ§ áÅÐàÁ×èÍÈÖ¡ÉÒÅѡɳзҧÊѳ°Ò¹ÇÔ·ÂÒà«ÅÅìÁÐàÃç§àÍçÁºÃÔâ;ÕÊÔºà¡éÒ¾ºÇèÒÁÕ¢¹Ò´âµáÅкÇÁ¢Öé¹ àÁ×èÍÈÖ¡ÉÒÃٻẺ¡ÒõÒ¢ͧà«ÅÅì´éÇÂà·¤¹Ô¤ agarose gel electrophoresis ¾ºÇèÒºÒÃÒ¤ÍÅà˹ÕèÂǹÓãËéà«ÅÅìÁÐàÃç§àÍçÁºÃÔâ;ÕÊÔºà¡éÒµÒÂẺ apoptosis (à©ÅÔÁÊÔ¹ à¾ÔèÁàµÔÁÊÔ¹, 2545)

 

2.4 µÑÇÃѺⴻÒÁÕ¹

 

                â´»ÒÁÕ¹à»ç¹ÊÒÃÊ×èÍ»ÃÐÊÒ·¡ÅØèÁ catecholamines ·ÕèÊÃéÒ§ÁÒ¨Ò¡¡Ã´ÍÐÁÔâ¹ tyrosine ã¹ÊÁͧÁÕ»ÃÔÁҳⴻÒÁÕ¹»ÃÐÁÒ³ÃéÍÂÅÐ 80 ¢Í§ÊÒáÅØèÁ catecholamines ·Õè¶Ù¡ÊÃéÒ§¢Öé¹·Ñé§ËÁ´ àÁ×èÍâ´»ÒÁÕ¹¶Ù¡»Å´»Åèͨҡà«ÅÅì»ÃÐÊÒ·â´»ÒÁÕ¹áÅéǨÐÁռŵèÍÊÁͧÊèǹµèÒ§æã¹ËÅÒÂÇÔ¶Õ«Öè§ä´éá¡è (1) ÇÔ¶Õ nigrostriatal (2) ÇÔ¶Õ mesolimbic (3) ÇÔ¶Õ mesocortical áÅÐ (4) ÇÔ¶Õ tuberoinfundibular â´»ÒÁÕ¹ÍÍ¡Ä·¸Ôì¼èÒ¹µÑÇÃѺ·Õèà»ç¹â»ÃµÕ¹«Öè§ÍÂÙ躹àÂ×èÍËØéÁà«ÅÅì µÑÇÃѺⴻÒÁÕ¹·Ø¡ª¹Ô´¨Ñ´ÍÂÙè㹡ÅØèÁµÑÇÃѺ·Õè¨ÑºÍÂÙè¡Ñºâ»ÃµÕ¹¨Õ (G-protein-coupled receptor) µÑÇÃѺⴻÒÁÕ¹¾ºä´é·Ñ駷ÕèàÂ×èÍËØéÁà«ÅÅì»ÃÐÊÒ·¡è͹¨Ø´»ÃÐÊÒ¹»ÃÐÊÒ· (presynaptic neuron) áÅзÕèàÂ×èÍËØéÁà«ÅÅì»ÃÐÊÒ·ËÅѧ¨Ø´»ÃÐÊÒ¹»ÃÐÊÒ· (postsynaptic neuron) ¡ÒáÃеØé¹µÑÇÃѺ·ÕèàÃÕ¡ÇèÒ autoreceptor ¨ÐÁÕ¼ÅÂѺÂÑ駡ÒÃÊѧà¤ÃÒÐËìâ´»ÒÁÕ¹ â´Âà¡Ô´¼èÒ¹¡ÒÃÂѺÂÑ駡Ò÷ӧҹ¢Í§à͹ä«Áì tyrosine hydroxylase ¹Í¡¨Ò¡¹ÕéÂѧÂѺÂÑ駡ÒÃËÅÑè§â´»ÒÁÕ¹¨Ò¡¶Ø§à¡çº·ÕèÍÂÙèã¹à«ÅÅì»ÃÐÊÒ·¡è͹¨Ø´»ÃÐÊÒ¹»ÃÐÊÒ·  áÅÐÅ´ firing rate ¢Í§à«ÅÅì»ÃÐÊÒ·â´»ÒÁÕ¹ÍÂèÒ§ÁÕ¹ÑÂÊӤѭ·Ò§Ê¶ÔµÔ (Tarazi, 2001)

ã¹Âؤááæä´éáºè§µÑÇÃѺⴻÒÁÕ¹ÍÍ¡à»ç¹ 2 ¡ÅØèÁËÅÑ¡â´Âãªé¤ÇÒÁÊÒÁÒö㹡ÒáÃеØé¹ËÃ×ÍÂѺÂÑ駡Ò÷ӧҹ¢Í§à͹ä«Áì adenylyl cyclase à»ç¹à¡³±ì㹡ÒèѴ¡ÅØèÁ«Öè§ÊÒÁÒöáºè§ä´éà»ç¹¡ÅØèÁµÑÇÃѺⴻÒÁÕ¹ 1 (dopamine 1 - like receptors) áÅСÅØèÁµÑÇÃѺⴻÒÁÕ¹ 2 (dopamine 2 - like receptors) µèÍÁÒÁÕ¡ÒÃÈÖ¡ÉÒà¾ÔèÁàµÔÁ¨Ö§¾ºÇèÒ¡ÅØèÁµÑÇÃѺⴻÒÁÕ¹ 1ä´éá¡èµÑÇÃѺⴻÒÁÕ¹ 1 áÅеÑÇÃѺⴻÒÁÕ¹ 5 «Öè§ÊÒÁÒö¡ÃеØ鹡Ò÷ӧҹ¢Í§à͹ä«Áì adenylyl cyclase ·ÓãËé¼ÅÔµ cyclic-adenosine monophosphate (cAMP) à¾ÔèÁÁÒ¡¢Öé¹  µÑÇÃѺⴻÒÁÕ¹ 1  ¾ºä´é·ÕèÊÁͧÊèǹ  dorsal striatum (caudate-putamen), nucleus accubens, olfactory tubercle, cerebral cortex  áÅÐ amygdala  µÑÇÃѺⴻÒÁÕ¹ 5 ¾ºä´é·ÕèÊÁͧÊèǹ  hippocampus, lateral mammillary nucleus áÅÐ parafascicular nucleus ã¹ÊÁͧÊèǹ thalamus

ÊÓËÃѺ¡ÅØèÁµÑÇÃѺⴻÒÁÕ¹ 2 ä´éá¡è µÑÇÃѺⴻÒÁÕ¹ 2 µÑÇÃѺⴻÒÁÕ¹ 3 áÅеÑÇÃѺⴻÒÁÕ¹ 4 «Öè§àÁ×èͶ١¡ÃеØ鹨ÐãËé¼ÅÂѺÂÑ駡Ò÷ӧҹ¢Í§à͹ä«Áì adenylyl cyclase µÑÇÃѺⴻÒÁÕ¹ 2 ¾ºä´é·ÕèÊÁͧÊèǹ  dorsal striatum, olfactory tubercle,  nucleus accubens, substantia nigra pars compacta áÅÐ ventral tegmental area ¹Í¡¨Ò¡¹ÕéµÑÇÃѺⴻÒÁÕ¹ 2 Âѧ¾ºä´éã¹ÍÇÑÂÇÐÍ×è¹æ ä´éá¡è  ´Ç§µÒªÑé¹ retina  äµÃкºäËÅàÇÕ¹àÅ×Í´ áÅÐ pituitary gland  µÑÇÃѺⴻÒÁÕ¹ 3 ¾ºä´é·ÕèÊÁͧÊèǹ  islands of Calleja, hypothalamus, thalamus áÅÐ cerebellum   áÅеÑÇÃѺⴻÒÁÕ¹ 4  ¾ºä´é·ÕèÊÁͧÊèǹ  frontal cortex, amygdala, olfactory bulb, hippocampus, hypothalamus áÅÐ mesenchephalon  (Vallone et al., 2000)

 

2.5 â´»ÒÁÕ¹·Ãҹʻ͵àµÍÃì

 

                â´»ÒÁÕ¹·Ãҹʻ͵àµÍÃìà»ç¹â»ÃµÕ¹ÊӤѭ·ÕèÍÂÙèºÃÔàdzàÂ×èÍËØéÁà«ÅÅì¢Í§à«ÅÅì»ÃÐÊÒ·â´»ÒÁÕ¹ ·Ó˹éÒ·Õè´Ù´¡ÅѺⴻÒÁÕ¹·ÕèËÅÑè§ÍÍ¡ÁÒã¹ä«á¹»Êìà¢éÒÊÙèà«ÅÅì»ÃÐÊÒ·¡è͹ä«á¹»Êì â´»ÒÁÕ¹

·Ãҹʻ͵àµÍÃìà»ç¹â»ÃµÕ¹ÅÓàÅÕ§ÊÒÃÊ×èÍ»ÃÐÊÒ·â´»ÒÁÕ¹·ÕèµéͧÍÒÈÑÂäÍÍ͹â«à´ÕÂÁáÅÐäÍÍ͹¤ÅÍäôì㹡Ò÷ӧҹ â¤Ã§ÊÃéÒ§âÁàÅ¡ØŢͧⴻÒÁÕ¹·Ãҹʻ͵àµÍÃìÁÕ´éÒ¹»ÅÒ N-terminus áÅлÅÒ C-terminus ÍÂÙèÀÒÂã¹à«ÅÅì (Mortenson and Amara, 2003)  áÅоºÇèÒâ´»ÒÁÕ¹·Ãҹʻ͵àµÍÃì·ÕèÍÂÙ躹àÂ×èÍËØéÁà«ÅÅì¨Ð¨Ñº¡Ñ¹à»ç¹ dimer (Javitch, 2003)

ÁÕ¡ÒÃÈÖ¡ÉÒ¡ÒáÃШÒµÑǢͧⴻÒÁÕ¹·Ãҹʻ͵àµÍÃìã¹ÍÇÑÂÇеèÒ§æã¹Ë¹Ù¢ÒÇâ´ÂãªéÇÔ¸Õ Immunohistochemical analysis ¾ºâ´»ÒÁÕ¹·Ãҹʻ͵àµÍÃìã¹Ãкº»ÃÐÊÒ·Êèǹ¡ÅÒ§ anterior pituitary gland, Auerbach’s nervous system, Meisner’s nervous system, adrenal medulla áÅÐ »Í´ (Mitsuma et al., 1998) â´Â·ÑèÇä»áÅéǺÃÔàdzÊÁͧ·ÕèãªéÈÖ¡ÉÒâ´»ÒÁÕ¹·Ãҹʻ͵àµÍÃì¤×ͺÃÔàdz dorsal striatum áÅÐ nucleus accumbens «Öè§ÁÕ»ÃÔÁҳⴻÒÁÕ¹·Ãҹʻ͵àµÍÃì¤è͹¢éҧ˹Òá¹è¹ (Gulley and Zahniser, 2003; Jiao et al., 2003)  àÁ×èÍÈÖ¡ÉÒ˹ٶպ¨Ñ¡Ã·Õè¶Ù¡µÑ´ÂչⴻÒÁÕ¹·Ãҹʻ͵àµÍÃì (DAT knockout mice) ¾ºÇèÒ¡ÒÃÊѧà¤ÃÒÐËìâ´»ÒÁÕ¹·ÕèÍÒÈÑÂà͹ä«Áì tyrosine hydroxylase à¡Ô´ä´é¹éÍÂŧ ¡ÒÃà¡çºâ´»ÒÁչ㹶اà¡çºÅ´Å§ ÁÕ¡ÒûŴ»ÅèÍÂâ´»ÒÁչŴŧ áÅÐÁÕ¡Òõ×è¹µÑÇà¾ÔèÁ¢Öé¹ (Gulley and Zahniser, 2003)

 

µÒÃÒ§·Õè 2  ¤Ø³ÊÁºÑµÔ¢Í§â´»ÒÁÕ¹·Ãҹʻ͵àµÍÃìã¹ÊѵÇì species µèÒ§æ (Chen and Reith, 2000)

 

Species	ª×èÍàÃÕ¡	¨Ó¹Ç¹¡Ã´ÍÐÁÔâ¹	homology to hDAT (%)
human	hDAT	620	100
rat	rDAT	619	92
mouse	mDAT	617	88

 

                ¡ÒÃÈÖ¡ÉÒã¹ rat dopamine transporter (rDAT) áÅÐ human dopamine transporter (hDAT) Êèǹã˭辺ÇèÒ¡Òâ¹Êè§â´»ÒÁÕ¹ÍÒÈÑ stoichiometry ·ÕèÁÕÍѵÃÒÊèǹ¢Í§â´»ÒÁÕ¹ 1 âÁàÅ¡ØÅ äÍÍ͹â«à´ÕÂÁ 2 äÍÍ͹áÅÐäÍÍ͹¤ÅÍäôì 1 âÁàÅ¡ØÅ  «Öè§ÁÕ¡Åä¡·ÑèÇ令×ÍäÍÍ͹â«à´ÕÂÁ 2 äÍÍ͹¨Ñº¡Ñºâ´»ÒÁÕ¹·Ãҹʻ͵àµÍÃì¡è͹ µÒÁ´éÇÂâ´»ÒÁÕ¹ 1 âÁàÅ¡ØÅ ¨Ò¡¹Ñé¹äÍ͹¤ÅÍäôì 1 äÍÍ͹¨Ö§ÁҨѺ¡Ñºâ´»ÒÁÕ¹·Ãҹʻ͵àµÍÃì áµè·Ñ駹ÕéÁÕ¼ÙéàʹÍÇèÒÍÒ¨à¡Ô´·Ò§ËÅÒ¡Åä¡·Ñ駨ҡ¡Ò÷Õèâ´»ÒÁÕ¹¨Ñº¡è͹äÍÍ͹â«à´ÕÂÁ ËÃ×ÍäÍÍ͹â«à´ÕÂÁ¨Ñº¡è͹µÒÁ´éÇÂâ´»ÒÁÕ¹ áÅéÇäÍÍ͹â«à´ÕÂÁÍÕ¡âÁàÅ¡ØÅà¢éҨѺⴻÒÁÕ¹·Ãҹʻ͵àµÍÃì ¨Ò¡¹Ñé¹äÍ͹¤ÅÍäôì¨Ö§¨Ð¨Ñºâ´»ÒÁÕ¹·Ãҹʻ͵àµÍÃìà»ç¹ÅӴѺÊØ´·éÒ (Schenk et  al., 2003)

 

2.6 à·¤¹Ô¤  radioligand binding assay

 

                à·¤¹Ô¤ radioligand binding assay à»ç¹·Õè¹ÔÂÁãªéÈÖ¡ÉÒº·ºÒ·¡ÒèѺ¢Í§ ligand ¡ÑºµÑÇÃѺ¢Í§ÊÒÃÊ×èÍ»ÃÐÊÒ·áÅÐÎÍÃìâÁ¹ËÅÒª¹Ô´ ¹ÑºÇèÒà»ç¹à·¤¹Ô¤·ÕèÁÕ¤ÇÒÁÊӤѭ㹡Òäé¹ËÒÊÒÃãËÁè·ÕèÁÕÄ·¸ÔìµèÍÃкº»ÃÐÊÒ·Êèǹ¡ÅÒ§«Ö觤è͹¢éÒ§ÃÇ´àÃçÇáÅÐÁÕ¤ÇÒÁäÇÊÙ§ (Zhu et al., 1996) â´ÂãªéÊÒ÷ÕèµÔ´©ÅÒ¡´éÇÂäÍâ«â·»¡ÑÁÁѹµÃѧÊÕ àªè¹ ³H  áÅÐ ¹²⁵I  «Öè§ÊÒùÕéÊÒÁÒö·Õè¨Ð¨Ñº¡ÑºµÑÇÃѺä´éÍÂèÒ§¨Óà¾ÒÐ ¡ÒõԴ©ÅÒ¡´éÇ  äÍâ«â·»¡ÑÁÁѹµÃѧÊÕ·ÓãËéÊÒÁÒöµÃǨ¨Ñº»ÃÔÁÒ³ÊÒÃä´éã¹ÃдѺ femtomol áµè·Ñ駹Õé¨ÐµéͧÁÕ¡ÒÃÊѧà¤ÃÒÐËì radioligand ·ÕèÁÕËÁÙèà¤ÁÕ·ÕèÊÒÁÒöà»Åè§áʧ fluorescence ãËéÊÒÁÒöµÃǨ¨Ñºä´é

                liquid scintillation counting à»ç¹ÇÔ¸Õ¡ÒõÃǨ¨Ñº¡ÒûŴ»ÅèÍÂ͹ØÀҤີéÒ·ÕèÁÕ»ÃÐÊÔ·¸ÔÀÒ¾ÊÙ§ áÅÐÍÒ¨ãªéÇÑ´¡ÒûŴ»ÅèÍÂÃѧÊÕá¡ÁÁÒä´é´éÇ àÁ×èÍÊÒõÑÇÍÂèÒ§·ÕèÁÕ radioligand ¡ÃШÒµÑÇã¹µÑÇ·ÓÅÐÅÒ·ÕèàÃÕ¡ÇèÒ liquid scintillation solvent  «Öè§ÁÕÊÒ÷ÕèÊÒÁÒöà»Åè§áʧ fluorescence à»ç¹Í§¤ì»ÃСͺÍÂÙè  äÍâ«â·»¡ÑÁÁѹµÃѧÊÕ·ÕèÍÂÙèã¹âÁàÅ¡ØŢͧ radioligand ¨Ð»Å´»ÅèÍÂ͹ØÀҤີéÒ¾ÃéÍÁ¡ÑºÊÙ­àÊÕ¾Åѧ§Ò¹ãËé¡ÑºµÑÇ·ÓÅÐÅÒ ·ÓãËéâÁàÅ¡ØŵÑÇ·ÓÅÐÅÒÂÍÂÙèã¹Ê¶Ò¹Ð¡ÃеØé¹áÅÐà¡Ô´¡ÒûŴ»Åè;Åѧ§Ò¹ãËé¡ÑºâÁàÅ¡ØŵÑÇ·ÓÅÐÅÒÂÍÕ¡ª¹Ô´Ë¹Ö觫Öè§ÊÒÁÒö»Å´»ÅèÍÂáʧ fluorescence ·Õè¨Ð¶Ù¡µÃǨ¨Ñºâ´Â photomultiplier tube áÅéÇà»ÅÕè¹ÊÑ­­Ò³ÃÙ»áʧ·Õèà¡Ô´¢Öé¹ãËéÍÂÙèã¹ÃÙ» electrical pulse ·Õèà»ç¹ÊÑ´Êèǹâ´ÂµÃ§¡Ñº»ÃÔÁÒ³áʧ fluorescence ·Õèà¡Ô´¢Öé¹

㹡ÒÃÈÖ¡ÉÒ´éÇÂà·¤¹Ô¤ radioligand binding assay »ÃСͺ´éÇ¢Ñ鹵͹ËÅÑ¡ 2 Êèǹ ¤×Í

1.       ¡ÒÃàµÃÕÂÁàÁÁàºÃ¹·ÕèÁÕµÑÇÃѺ·Õèʹã¨ÈÖ¡ÉÒ

2.       ¡ÒúèÁ radioligand ¡ÑºàÁÁàºÃ¹ã¹ÊÀÒÇзÕèÁÕáÅÐäÁèÁÕÊÒ÷Õè·Ó¡ÒÃÈÖ¡ÉÒ (test compounds)

¡ÒÃàµÃÕÂÁàÁÁàºÃ¹¨ÐµéͧàÅ×Í¡ºÃÔàdzà¹×éÍàÂ×èͧ͢ÊѵÇì·ÕèÁÕ»ÃÔÁÒ³µÑÇÃѺ·Õè·Ó¡ÒÃÈÖ¡ÉÒÍÂÙè»ÃÔÁÒ³ÁÒ¡ áµèÍÂèÒ§äáçµÒÁàÁÁàºÃ¹·ÕèàµÃÕÂÁä´é¡çÁÕͧ¤ì»ÃСͺÍ×è¹æ¹Í¡à˹×ͨҡµÑÇÃѺ·Õè·Ó¡ÒÃÈÖ¡ÉÒ ¡ÒèѺ¡Ñ¹ÃÐËÇèÒ§ radioligand ¡ÑºµÑÇÃѺàÃÕ¡ÇèÒ “specific binding” áµè¶éÒ radioligand ¨Ñº¡ÑºµÓá˹è§Í×è¹·ÕèäÁèãªéµÑÇÃѺ¨ÐàÃÕ¡ÇèÒ “nonspecific binding” â´Â¡ÒèѺẺ nonspecific binding à¡Ô´ä´éËÅÒÂ¡Ã³Õ àªè¹ ¨Ñº¡ÑºµÑÇÃѺª¹Ô´Í×è¹æ  à͹ä«Áì áÅÐâ»ÃµÕ¹·ÕèàÂ×èÍËØéÁà«ÅÅì à»ç¹µé¹ ËÃ×ÍÍÒ¨à¡Ô´¨Ò¡ radioligand ÅÐÅÒÂÍÂÙèã¹Í§¤ì»ÃСͺ¾Ç¡ä¢ÁѹËÃ×Ͷ١´Ù´à¢éÒä»Âѧ intact cell ËÃ×Í membrane vesicle áÅÐÍÒ¨¨Ðà¡Ô´ä´éã¹¢Ñ鹵͹¡ÒáÃͧá¡ bound ligand ÍÍ¡¨Ò¡ free ligand (Foreman and Johnson, 1996)

                ¡ÒûÃÐàÁÔ³¤èÒ nonspecific binding ·Óä´éâ´ÂÇÑ´¡ÒèѺ¢Í§ radioligand ã¹ÊÀÒÇзÕèÁÕÊÒ÷Õè¨ÑºÍÂèÒ§¨Óà¾ÒСѺµÑÇÃѺ¹Ñé¹æâ´Âãªé¤ÇÒÁà¢éÁ¢é¹¢Í§ÊÒôѧ¡ÅèÒÇã¹¢¹Ò´¤ÇÒÁà¢éÁ¢é¹ 100-1000 ¢Í§¤èÒ dissociation equilibrium constant (Kd) «Ö觷ÓãËéÊÒôѧ¡ÅèÒÇÊÒÁÒö»Ô´¡Ñ鹡ÒèѺ¢Í§ radioligand µè͵ÑÇÃѺä´é·Ñé§ËÁ´ áÅÐàÁ×èͼèÒ¹¢Ñ鹵͹¡ÒáÃͧáÅéÇÊÒÁÒöÇÑ´»ÃÔÁÒ³ÃѧÊÕ·Õè»Å´»Åèͨҡ radioligand ä´éáÊ´§ÇèÒà¡Ô´¡ÒèѺÍÂèÒ§äÁè¨Óà¾ÒСѺͧ¤ì»ÃСͺÍ×è¹æ ·ÕèäÁèãªèµÑÇÃѺ·Õè·Ó¡ÒÃÈÖ¡ÉÒ ¤èÒ¡ÒèѺ¢Í§ radioligand ·ÕèäÁè¨Óà¾ÒÐàÁ×èÍà·Õº¡Ñº¡ÒèѺ·Ñé§ËÁ´ (total binding) ¤ÇÃÁÕ¤èÒäÁèà¡Ô¹ÃéÍÂÅÐ 5 (Katz et  al., 2000)

 

                Specific binding  =  [Total binding ] – [Nonspecific binding]

                     %  inhibition  =  100 – [%  specific binding]

 

                㹡óÕÊÒ÷Õèµéͧ¡Ò÷´ÊͺÁÕ¤ÇÒÁªÍº¨Ñº¡ÑºµÑÇÃѺ ËÅѧ¨Ò¡¹Ó仺èÁµÒÁÊÀÒÇзÕèàËÁÒÐÊÁáÅéÇ ÊÒ÷Õèµéͧ¡Ò÷´Êͺ·ÕèÊÒÁÒöá¢è§¢Ñ¹ radioligand ¨Ñº¡ÑºµÑÇÃѺ·ÕèÈÖ¡ÉÒä´éÁÒ¡ àÁ×èͼèÒ¹¢Ñ鹵͹¡ÒáÃͧâ´Âãªéà¤Ã×èͧ¡ÃͧÃкºÊØ­­Ò¡ÒÈ µÑÇÃѺ¨Ð¶Ù¡¨Ñº´éÇÂÊÒ÷Õèµéͧ¡Ò÷´ÊͺáÅÐÊèǹ˹Ö觶١¨Ñº´éÇ radioligand ¶éÒÊÒ÷Õèµéͧ¡Ò÷´ÊͺÁÕ¤ÇÒÁªÍº¨Ñº¡ÑºµÑÇÃѺÊÙ§¡ç¨ÐÁÕ¤ÇÒÁÊÒÁÒö㹡ÒÃÂѺÂÑ駡ÒèѺÃÐËÇèÒ§ radioligand ¡ÑºµÑÇÃѺä´éÁÒ¡ àÁ×èͤӹdzã¹ÃÙ» % inhibition ¡ç¨Ð·ÓãËéÁÕ¤èÒÊÙ§

 

 

 

 

 

 

 

 

 

º··Õè 3

ÇÔ¸Õ´Óà¹Ô¹¡ÒÃÇÔ¨ÑÂ

 

3.1 ÊÒÃà¤ÁÕáÅÐÍØ»¡Ã³ì

 

3.1.1 ÊÒÃà¤ÁÕ

 

                Barakol ä´éÃѺ¤ÇÒÁ͹Øà¤ÃÒÐËì¨Ò¡¹ÔÊÔµºÑ³±ÔµÈÖ¡ÉÒ ÀÒ¤ÇÔªÒªÕÇà¤ÁÕ ¤³ÐàÀÊѪÈÒʵÃì    ¨ØÌÒŧ¡Ã³ìÁËÒÇÔ·ÂÒÅÑÂ

                Bovine serum albumin (Cat No. 20K0946)

                Butaclamol (Cat. No. 022k4606, Sigma, Germany)

                C₁₂H₂₂O₁₁ (Cat No. 23501087, Merck, Germany)

                Dimethyl sulfoxide (DMSO) (Cat No. 89H3636, Sigma, Germany)

                GBR12909 (Cat. No. 083k4611, Sigma, Germany)

                [³H]Raclopride (Cat. No. NET 975, Perkin Elmer, USA)

                [³H]SCH23390 (Cat. No. TRK 876, Amersham, USA)

                [³H]WIN35428 (Cat. No. 3499313, Perkin Elmer, USA)

                MgCl₂ 6H₂O (Cat No. 313K776332, Merck, Germany)

                NaCl (Cat No. K31445404, Merck, Germany)

                NaH₂PO₄ 2H₂O (Cat No. 8042, Riedel-de Haen, AG)

                Na₂HPO₄ (Fluka, AG)

                Nomifensine (Cat. No. 102k4129, Sigma, Germany)

                Polyethyleneimine (Cat No. 072K0159, Sigma, Germany)

                Raclopride (Cat. No. 091k1322, Sigma, Germany)

                Tris-HCl (Cat No. 054K5413, Sigma, Germany)

                SCH23390 (Cat. No. 072k4604, Sigma, Germany)

                Ultima Gold Cocktail (Cat. No. 6013329, Perkin Elmer, USA)

 

                3.1.2 à¤Ã×èͧÁ×ÍáÅÐÍØ»¡Ã³ì

 

                à¤Ã×èͧº´à¹×éÍàÂ×èÍ ÂÕèËéÍ Cole-palmer ÃØè¹ Glass-Col 099C, USA

                à¤Ã×èͧ»Ñè¹  ÂÕèËéÍ Hettich zentrifugen ÃØè¹ EBA 12 áÅÐ ÃØè¹12R , Germany

                à¤Ã×èͧ»Ñè¹ high speed centrifuge ÂÕèËéÍ HITACHI ÃØè¹ SCR 20B, Japan

                à¤Ã×èͧ Spectrophotometer ÂÕèËéÍ Spectronic®  ÃØè¹ Genesys 5, UK

                à¤Ã×èͧ¡ÃͧÃкºÊØ­­Ò¡ÒÈ 1225 Sampling Manifold ÂÕèËéÍ Millipore, USA

                à¤Ã×èͧ liquid scintillation counter ÂÕèËéÍ Wallac 1409/1411 ÃØè¹ SYSTEM 1400™, Finland

                ¡ÃдÒÉ¡Ãͧ Whatman GF/B àÊé¹¼èÒ¹ÈÙ¹Âì¡ÅÒ§ 24 ÁÔÅÅÔàÁµÃ Whatman  international                  Ltd, England

 

3.2 ¡ÒÃàµÃÕÂÁÊѵÇì·´Åͧ áÅÐÇÔ¸Õá¡ÊÁͧ¨Ò¡Ë¹Ù¢ÒÇ

 

ÊѵÇì·´Åͧ·Õèãªéä´éÃѺ¤ÇÒÁ͹Øà¤ÃÒÐËì¨Ò¡¹ÔÊÔµËÅÑ¡ÊٵûÃÔ­­Ò´Øɮպѳ±Ôµ ÀÒ¤ÇÔªÒàÀÊѪÇÔ·ÂÒ ¤³ÐàÀÊѪÈÒʵÃì  ¨ØÌÒŧ¡Ã³ìÁËÒÇÔ·ÂÒÅÑ  â´Âãªé˹٢ÒÇÊÒ¾ѹ¸Øì Wistar à¾È¼Ùé·Õèä´é¨Ò¡ÊӹѡÊѵÇì·´ÅͧáËè§ªÒµÔ ÁËÒÇÔ·ÂÒÅÑÂÁËÔ´Å ÈÒÅÒÂÒ ¨Ñ§ËÇÑ´¹¤Ã»°Á â´Â¹Ó˹٢ÒÇÁҾѡ·ÕèËéͧÊѵÇì·´Åͧ  ¤³ÐàÀÊѪÈÒʵÃì  ¨ØÌÒŧ¡Ã³ìÁËÒÇÔ·ÂÒÅÑ àÅÕé§äÇé㹡çàÅÕé§˹٨ӹǹ 1 µÑǵè͡ç¾ÃéÍÁ¡ÑºÁÕÇÑÊ´ØÃͧ¹Í¹ ãËéÍÒËÒÃáÅйéÓµÒÁ»¡µÔ áÅÐä´é»¯ÔºÑµÔµèÍÊѵÇì·´ÅͧµÒÁ¨ÃÃÂÒºÃó¡ÒÃãªéÊѵÇì·´Åͧ¢Í§ÊÀÒÇÔ¨ÑÂáË觪ҵԵÅÍ´ÃÐÂÐàÇÅÒ¡ÒÃÈÖ¡ÉÒÇԨѠ

¹Ó˹٢ÒÇ·Õèà¾Ô觵ÒÂÁÒá¡àÍÒÊÁͧÊèǹ striatum â´ÂÃÐËÇèÒ§á¡ÊÁͧµéͧáªèÊÁͧ  ã¹ phosphate buffer saline ·ÕèàÂ繨ѴµÅÍ´àÇÅÒ áÅéǹӫҡÊѵÇì·´Åͧä»à¡çºäÇéã¹µÙéáªèÊѵÇì·´Åͧ·ÕèÁÕÍسËÀÙÁÔ -20 °C  ¢Í§ÀÒ¤ÇÔªÒàÀÊѪÇÔ·ÂÒ ¤³ÐàÀÊѪÈÒʵÃì  ¨ØÌÒŧ¡Ã³ìÁËÒÇÔ·ÂÒÅÑ à¾×èÍÃÍ´Óà¹Ô¹¡ÒáӨѴ«Ò¡ÊѵÇì·´Åͧâ´ÂÇÔ¸Õ¡ÒÃà¼ÒµèÍä»

 

 

 

3.3 ¡ÒÃàµÃÕÂÁàÁÁàºÃ¹·ÕèÁÕµÑÇÃѺⴻÒÁÕ¹áÅÐâ´»ÒÁÕ¹·Ãҹʻ͵àµÍÃì

                3.3.1 ¡ÒÃàµÃÕÂÁàÁÁàºÃ¹¨Ò¡ÊÁͧ˹٢ÒǺÃÔàdz striatum

                               

                3.3.1.1 ¡ÒÃàµÃÕÂÁàÁÁàºÃ¹·ÕèÁÕµÑÇÃѺⴻÒÁÕ¹ 1

 

á¡ÊÁͧ˹٢ÒǺÃÔàdz striatum ¹éÓ˹ѡÃÇÁ 3.45 ¡ÃÑÁ áÅéÇàµÔÁŧ㹺ѿà¿ÍÃìàÂç¹ Tris-HCl 50  ÁÔÅÅÔâÁÅÒÃì pH 7.4 »ÃÔÁҵà 40 ÁÔÅÅÔÅԵà  ¹ÓÁÒº´´éÇÂà¤Ã×èͧº´à¹×éÍàÂ×èÍ·Õè¤ÇÒÁàÃçÇ 70 ÃͺµèÍ¹Ò·Õ áÅлÑè¹´éÇÂà¤Ã×èͧ»Ñè¹  ¤ÇÒÁàÃçÇ 800 x g  ¹Ò¹10 ¹Ò·Õ ·Õè 4 ͧÈÒà«Åà«ÕÂÊ ¨Ò¡¹Ñé¹á¡àÍÒÊèǹ·Õèà»ç¹¹éÓãʪÑ鹺¹ÁÒ»Ñè¹´éÇÂà¤Ã×èͧ»Ñè¹ high speed centrifuge ·Õè¤ÇÒÁàÃçÇ 18,000 ÃͺµèÍ¹Ò·Õ ¹Ò¹ 15 ¹Ò·Õ ·Õè 4 ͧÈÒà«Åà«ÕÂÊ áÅÐ¹Ó pellet (3.39 ¡ÃÑÁ) ·Õèä´éÁÒÅÐÅÒÂ㹺ѿà¿ÍÃìàÂç¹ Tris-HCl 50 ÁÔÅÅÔâÁÅÒÃì pH 7.4 »ÃÔÁҵà 35 ÁÔÅÅÔÅԵà áÅéǺ´·Õè¤ÇÒÁàÃçÇ 70 ÃͺµèÍ¹Ò·Õ  ¨Ò¡¹Ñé¹¹Óä»»Ñè¹·Õè¤ÇÒÁàÃçÇ 18,000 ÃͺµèÍ¹Ò·Õ ¹Ò¹ 15 ¹Ò·Õ ·Õè 4 ͧÈÒà«Åà«ÕÂÊ á¡àÍÒÊèǹ·Õèà»ç¹¹éÓãÊ·Ôé§ä» áÅéÇ¹Ó pellet (3.27 ¡ÃÑÁ) ·Õèä´éÁÒÅÐÅÒÂ㹺ѿà¿ÍÃìàÂç¹ Tris-HCl 50 ÁÔÅÅÔâÁÅÒÃì pH 7.4 »ÃÔÁҵà 35 ÁÔÅÅÔÅԵà º´·Õè¤ÇÒÁàÃçÇ 70 ÃͺµèÍ¹Ò·Õ áÅлÑè¹·Õè¤ÇÒÁàÃçÇ 18,000 ÃͺµèÍ¹Ò·Õ ¹Ò¹ 15 ¹Ò·Õ ·Õè 4 ͧÈÒà«Åà«ÕÂÊ àÍÒÊèǹ·Õèà»ç¹¹éÓãÊ·Ôé§ä» ¹Ó pellet (3.35 ¡ÃÑÁ) ·Õèä´éÅÐÅÒÂ㹺ѿà¿ÍÃìàÂç¹·Õè»ÃСͺ´éÇ Tris-HCl 50 ÁÔÅÅÔâÁÅÒÃì  NaCl 120 ÁÔÅÅÔâÁÅÒÃì áÅÐ MgCl₂ 2 ÁÔÅÅÔâÁÅÒÃì pH 7.4 »ÃÔÁҵà 11 ÁÔÅÅÔÅԵà º´´éÇÂà¤Ã×èͧº´à¹×éÍàÂ×èÍ·Õè¤ÇÒÁàÃçÇ 70 ÃͺµèÍ¹Ò·Õ ¨Ò¡¹Ñ鹤ӹdz¤èÒ»ÃÔÁÒ³â»ÃµÕ¹·ÕèÍÂÙèã¹àÁÁàºÃ¹´éÇÂÇÔ¸Õ Bradford áÅéÇ aliquot ãËéÁÕ¤ÇÒÁà¢éÁ¢é¹ 10 ÁÔÅÅÔ¡ÃÑÁµèÍÁÔÅÅÔÅÔµÃáÅÐà¡çºäÇé·ÕèÍسËÀÙÁÔ -80 ͧÈÒà«Åà«ÕÂÊ

 

3.3.1.2 ¡ÒÃàµÃÕÂÁàÁÁàºÃ¹·ÕèÁÕâ´»ÒÁÕ¹·Ãҹʻ͵àµÍÃì

 

á¡ÊÁͧ˹٢ÒǺÃÔàdz striatum ¹éÓ˹ѡÃÇÁ 3.68 ¡ÃÑÁ áÅéÇàµÔÁŧ㹺ѿà¿ÍÃìàÂç¹·Õè»ÃСͺ´éÇ Tris-HCl 50  ÁÔÅÅÔâÁÅÒÃìáÅЫÙâ¤ÃÊ 0.32 âÁÅÒÃì pH 7.4 »ÃÔÁҵà 50 ÁÔÅÅÔÅԵà  ¹ÓÁÒº´´éÇÂà¤Ã×èͧº´à¹×éÍàÂ×èÍ·Õè¤ÇÒÁàÃçÇ 70 ÃͺµèÍ¹Ò·Õ áÅлÑè¹´éÇÂà¤Ã×èͧ high speed centrifuge ·Õè¤ÇÒÁàÃçÇ 3,300 ÃͺµèÍ¹Ò·Õ ¹Ò¹ 10 ¹Ò·Õ ·Õè 4 ͧÈÒà«Åà«ÕÂÊ ¨Ò¡¹Ñé¹àÍÒÊèǹ·Õèà»ç¹¹éÓãÊÁÒ»Ñè¹´éÇÂà¤Ã×èͧ high speed centrifuge ·Õè¤ÇÒÁàÃçÇ 13800 ÃͺµèÍ¹Ò·Õ ¹Ò¹ 20 ¹Ò·Õ ·Õè 4 ͧÈÒà«Åà«ÕÂÊ ¹Ó pellet (3.39 ¡ÃÑÁ) ·Õèä´éÅÐÅÒÂ㹺ѿà¿ÍÃìàÂç¹·Õè»ÃСͺ´éÇ Tris-HCl 50  ÁÔÅÅÔâÁÅÒÃìáÅЫÙâ¤ÃÊ 0.32 âÁÅÒÃì pH 7.4 »ÃÔÁҵà 50 ÁÔÅÅÔÅԵà º´´éÇÂà¤Ã×èͧº´à¹×éÍàÂ×èÍ·Õè¤ÇÒÁàÃçÇ 70 ÃͺµèÍ¹Ò·Õ ¨Ò¡¹Ñ鹤ӹdz»ÃÔÁÒ³â»ÃµÕ¹·ÕèÍÂÙèã¹àÁÁàºÃ¹´éÇÂÇÔ¸Õ Bradford áÅéÇà¡çºäÇé·ÕèÍسËÀÙÁÔ -80 ͧÈÒà«Åà«ÕÂÊ

 

            3.3.2 ¡ÒÃàµÃÕÂÁàÁÁàºÃ¹¨Ò¡à«ÅÅìà¾ÒÐàÅÕé§  A9LhD2L

 

            3.3.2.1 à«ÅÅìà¾ÒÐàÅÕé§ A9LhD2

 

                §Ò¹ÇԨѹÕéä´éãªéà«ÅÅìà¾ÒÐàÅÕé§ A9L cell line hD2 subclone #18 (ATCC^® Catalog No. CRL-10225) «Öè§à»ç¹à«ÅÅì fibroblast  ¢Í§Ë¹Ù¶Õº¨Ñ¡Ã·ÕèÁÕª×èÍÇÔ·ÂÒÈÒʵÃìÇèÒ Mus musculus  strain: C3H/An à¾È¼Ùé·ÕèÁÕÍÒÂØ 100 Çѹ «Öè§ä´é¶Ù¡ transfect ´éÇÂÂÕ¹¢Í§µÑÇÃѺⴻÒÁÕ¹ 2 ¢Í§Á¹ØÉÂì (human dopamine 2 receptor gene) (ATCC, 2005)  áÅÐÁÕÃÒ§ҹ¶Ö§¡ÒÃãªéà«ÅÅìà¾ÒÐàÅÕ駹Õéà»ç¹áºº¨ÓÅͧà¾×èÍÈÖ¡ÉҼŢͧÊÒÃà¤ÁÕ㹡Òêѡ¹ÓãËéà¡Ô´¾ÔɵèÍàÁá·ºÍÅÔ«ÖÁã¹à«ÅÅì¢Í§ÊѵÇìàÅÕé§ÅÙ¡´éǹÁ (Yang, 2005) ÊÓËÃѺ¢Ñ鹵͹¡ÒÃàÅÕé§à«ÅÅìä´éãªéÍÒËÒÃàÅÕé§à«ÅÅì Dulbecco’s modified Eagle’s medium ·ÕèÁÕͧ¤ì»ÃСͺ¤×Í L-glutamine 4 mM, glucose 4.5 g/L, sodium bicarbonate 1.5 g/L, sodium pyruvate 1.0 mM áÅÐ  fetal bovine serum ÃéÍÂÅÐ 10 â´ÂàÅÕé§à«ÅÅì¹Õéã¹µÙéºèÁÀÒÂãµéºÃÃÂÒ¡ÒÈ·ÕèÁÕ¡êÒ« CO₂ à¢éÁ¢é¹ÃéÍÂÅÐ  5 áÅÐÁÕÍسËÀÙÁÔ 37 ͧÈÒà«Åà«ÕÂÊ (ATCC, 2005) 

 

3.2.2.2 ¡ÒÃàµÃÕÂÁàÁÁàºÃ¹·ÕèÁÕµÑÇÃѺⴻÒÁÕ¹ 2  (dopamine 2 receptor)  

 

à«ÅÅìà¾ÒÐàÅÕé§ A9LhD2L ¨Ó¹Ç¹ 6.1 x 10⁸ à«ÅÅì ¹ÓÁÒàµÔÁ㹺ѿà¿ÍÃìàÂç¹ Tris-HCl 50 ÁÔÅÅÔâÁÅÒÃì pH 7.4 »ÃÔÁҵà 50 ÁÔÅÅÔÅԵà  ¹ÓÁÒº´´éÇÂà¤Ã×èͧº´à¹×éÍàÂ×èÍ·Õè¤ÇÒÁàÃçÇ 70 ÃͺµèÍ¹Ò·Õ áÅлÑè¹´éÇÂà¤Ã×èͧ high speed centrifuge ·Õè¤ÇÒÁàÃçÇ 18,000 ÃͺµèÍ¹Ò·Õ áÅÐ¹Ó pellet (3.51 ¡ÃÑÁ) ·Õèä´éÅÐÅÒÂ㹺ѿà¿ÍÃìàÂç¹ Tris-HCl 50 ÁÔÅÅÔâÁÅÒÃì pH 7.4 »ÃÔÁҵà 35 ÁÔÅÅÔÅԵà º´·Õè¤ÇÒÁàÃçÇ 70 ÃͺµèÍ¹Ò·Õ  áÅéÇ»Ñè¹·Õè¤ÇÒÁàÃçÇ 18,000 ÃͺµèÍ¹Ò·Õ ¹Ò¹ 15 ¹Ò·Õ ·Õè 4 ͧÈÒà«Åà«ÕÂÊ àÍÒÊèǹ·Õèà»ç¹¹éÓãÊ·Ôé§ä» ¹Ó pellet (2.48 ¡ÃÑÁ) ·Õèä´éÅÐÅÒÂ㹺ѿà¿ÍÃìàÂç¹ Tris-HCl 50 ÁÔÅÅÔâÁÅÒÃì pH 7.4 »ÃÔÁҵà 8 ÁÔÅÅÔÅԵà º´´éÇÂà¤Ã×èͧº´à¹×éÍàÂ×èÍ·Õè¤ÇÒÁàÃçÇ 70 ÃͺµèÍ¹Ò·Õ ¨Ò¡¹Ñ鹤ӹdz»ÃÔÁÒ³â»ÃµÕ¹·ÕèÍÂÙèã¹àÁÁàºÃ¹´éÇÂÇÔ¸Õ Bradford áÅéÇ aliquot ãËéÁÕ¤ÇÒÁà¢éÁ¢é¹ 5 ÁÔÅÅÔ¡ÃÑÁµèÍÁÔÅÅÔÅÔµÃáÅÐà¡çºäÇé·ÕèÍسËÀÙÁÔ -80 ͧÈÒà«Åà«ÕÂÊ

 

3.4 ¡ÒÃÇÔà¤ÃÒÐËì»ÃÔÁÒ³â»ÃµÕ¹ã¹àÁÁàºÃ¹â´ÂÇÔ¸Õ Bradford

               

                ÇÔà¤ÃÒÐËì»ÃÔÁÒ³â»ÃµÕ¹·ÕèÍÂÙèã¹àÁÁàºÃ¹â´Âà·Õº¡ÑºÊÒÃÅÐÅÒ stock solution ¢Í§ bovine serum albumin  ¤ÇÒÁà¢éÁ¢é¹ 1 ÁÔÅÅÔ¡ÃÑÁµèÍÁÔÅÅÔÅԵà ¨Ò¡¡ÒÃàµÔÁ Bradford reagent à¾×èÍãËé·Ó»¯Ô¡ÔÃÔÂҡѺ¡Ã´ÍÐÁÔâ¹·ÕèÍÂÙèã¹â»ÃµÕ¹·Õèµéͧ¡ÒÃÇÔà¤ÃÒÐËì â´Â·Ó 2-fold dilution ãËéä´é¤ÇÒÁà¢éÁ¢é¹ 0.25, 0.125, 0.0625, 0.03125 áÅÐ 0.015625 ÁÔÅÅÔ¡ÃÑÁµèÍÁÔÅÅÔÅԵà â´Â·Ó¤ÇÒÁà¢éÁ¢é¹ÅÐ 2 ËÅÍ´ ËÅÍ´ÅÐ 250 äÁâ¤ÃÅԵà  à¨×ͨҧàÁÁàºÃ¹·ÕèàµÃÕÂÁä´éâ´Â»Ô໵ÁÒ 10 äÁâ¤ÃÅÔµÃáÅéÇàµÔÁ¹éÓ¡ÅÑè¹»ÃÔÁҵà 990 äÁâ¤ÃÅԵà ¨Ò¡¹Ñé¹»Ô໵ÁÒàµÔÁ 2 ËÅÍ´ ËÅÍ´ÅÐ 250 äÁâ¤ÃÅԵà  ·Ó blank â´Âãªé¹éÓ¡ÅÑè¹»ÃÔÁҵà 250 äÁâ¤ÃÅԵèӹǹ 2 ËÅÍ´ àµÔÁ Bradford reagent 2.5 ÁÔÅÅÔÅÔµÃŧ㹷ءËÅÍ´·´Åͧ à¡çºäÇéã¹·ÕèÁ×´ 10 ¹Ò·Õ áÅéǹÓä»ÇÑ´¤èÒ¡Òôٴ¡Å×¹áʧ·Õè¤ÇÒÁÂÒǤÅ×è¹ 595 nm â´Âãªéà¤Ã×èͧ Spectrophotometer ¨Ò¡¹Ñé¹ÊÃéÒ§¡ÃÒ¿ÁҵðҹÃÐËÇèÒ§¤ÇÒÁà¢éÁ¢é¹¢Í§ BSA standard solution áÅФèÒ¡Òôٴ¡Å×¹áʧ·Õè¤ÇÒÁÂÒǤÅ×è¹ 595 nm ¤Ó¹Ç³ËÒ¤èÒ¤ÇÒÁà¢éÁ¢é¹¢Í§â»ÃµÕ¹¨Ò¡ÊÁ¡Ò÷Õèä´é¨Ò¡¡ÃÒ¿Áҵðҹ

 

3.5 ¡ÒÃÇÑ´¤ÇÒÁªÍº¨Ñº¢Í§ºÒÃÒ¤Íŵè͵ÑÇÃѺⴻÒÁÕ¹áÅÐâ´»ÒÁÕ¹·Ãҹʻ͵        àµÍÃìâ´Âãªéà·¤¹Ô¤ Radioligand binding assay

            3.5.1 ¡ÒÃÇÑ´¤ÇÒÁªÍº¨Ñº¢Í§ºÒÃÒ¤Íŵè͵ÑÇÃѺⴻÒÁÕ¹ 1

 

                àµÃÕÂÁÊÒÃÅÐÅÒºÒÃÒ¤ÍÅãËéÁÕ¤ÇÒÁà¢éÁ¢é¹àÃÔèÁµé¹à·èҡѺ 2, 20, 200 áÅÐ 2000 äÁâ¤ÃâÁÅÒÃì  àÁ×èÍàµÔÁÊÒÃÅÐÅÒºÒÃÒ¤ÍÅ 50 äÁâ¤ÃÅԵà ŧã¹ËÅÍ´·´ÅͧãËéÁÕ»ÃÔÁÒµÃÃÇÁà»ç¹ 1,000 äÁâ¤ÃÅԵèÐä´é¤ÇÒÁà¢éÁ¢é¹ÊØ´·éÒ¢ͧÊÒÃÅÐÅÒºÒÃÒ¤ÍÅà»ç¹ 0.1, 1, 10 áÅÐ 100  äÁâ¤ÃâÁÅÒÃì µÒÁÅӴѺ  ÊÒÃÅÐÅÒ¢ͧÅÔ᡹´ì·ÕèµÔ´©ÅÒ¡ÃѧÊÕ¤×ÍÊÒÃÅÐÅÒ [³H]SCH23390 ·ÕèÁÕ¤ÇÒÁà¢éÁ¢é¹ÊØ´·éÒÂà»ç¹ 5 x 10^-8 âÁÅÒÃì (radioactive concentration à·èҡѺ 1 mCi/ml, specific activity à·èҡѺ  71 Ci/mmol) ãªéÊÒÃÅÐÅÒ butaclamol ·ÕèÁÕ¤ÇÒÁà¢éÁ¢é¹ÊØ´·éÒÂà»ç¹ 5 x 10^-6 âÁÅÒÃì à¾×èÍËÒ¤èÒ non-specific binding  áÅÐãªéÊÒÃÅÐÅÒ SCH23390 ·ÕèÁÕ¤ÇÒÁà¢éÁ¢é¹ÊØ´·éÒÂà»ç¹ 5 x 10^-10 âÁÅÒÃì  (IC₅₀à·èҡѺ

 0.5 x 10^-9 âÁÅÒÃì) à»ç¹ positive control  ÊÒÃÅÐÅÒºѿà¿ÍÃì·Õèãªé»ÃСͺ´éÇ Tris-HCl 50 ÁÔÅÅÔâÁÅÒÃì  NaCl 120 ÁÔÅÅÔâÁÅÒÃì áÅÐ MgCl₂ 2 ÁÔÅÅÔâÁÅÒÃì ·ÕèÁÕ¤èÒ pH 7.4 à¾×èÍ»ÃѺ»ÃÔÁҵ蹤ú 1,000 äÁâ¤ÃÅԵ÷ءËÅÍ´·´Åͧ ãªé¡ÃдÒÉ¡Ãͧ Whatman GF/B ·Õèä´éáªèã¹ÊÒÃÅÐÅÒ 0.3% polyethyleneimine ¨¹ÍÔèÁµÑÇà»ç¹àÇÅÒÍÂèÒ§¹éͤÃÖ觪ÑèÇâÁ§ ÊÓËÃѺ¡ÒáÃͧä´éãªéà¤Ã×èͧ¡ÃͧÃкºÊØ­­Ò¡ÒÈ 1225 Sampling Manifold  áÅÐãªéà¤Ã×èͧ liquid scintillation counter à¾×è͹Ѻ»ÃÔÁÒ³ÃѧÊÕ·Õè»Å´»ÅèÍÂÍÍ¡ÁÒ¨Ò¡ÊÒÃÃѧÊÕ·ÕèµÔ´ÍÂÙè¡ÑºÅÔ᡹´ì·Õè¨Ñº¡ÑºµÑÇÃѺⴻÒÁÕ¹ 1 «Ö觤éÒ§ÍÂÙ躹¡ÃдÒÉ¡Ãͧ

                ã¹¢Ñé¹ááä´éàµÔÁ 10% DMSO 50 äÁâ¤ÃÅԵüÊÁ¡Ñº¹éÓ¡ÅÑè¹»ÃÔÁҵà 50 äÁâ¤ÃÅÔµÃà¾×èÍãªéà»ç¹ solvent control  àµÔÁ butaclamol 10^-4âÁÅÒÃì »ÃÔÁҵà 50 äÁâ¤ÃÅԵüÊÁ¡Ñº¹éÓ¡ÅÑè¹»ÃÔÁҵà 50 äÁâ¤ÃÅԵà à¾×èͤӹdzËÒ non-specific binding  àµÔÁ SCH23390 1 x 10^-8 âÁÅÒÃì »ÃÔÁҵà 50 äÁâ¤ÃÅԵüÊÁ¡Ñº¹éÓ¡ÅÑè¹»ÃÔÁҵà 50 äÁâ¤ÃÅÔµÃÊÓËÃѺà»ç¹ positive control áÅÐàµÔÁÊÒÃÅÐÅÒºÒÃÒ¤ÍŤÇÒÁà¢éÁ¢é¹ 2, 20, 200 áÅÐ 2000 äÁâ¤ÃâÁÅÒÃì »ÃÔÁҵà 50 äÁâ¤ÃÅԵüÊÁ¡Ñº 10 % DMSO »ÃÔÁҵà 50 äÁâ¤ÃÅÔµÃã¹áµèÅФÇÒÁà¢éÁ¢é¹ áÅéÇ»ÃѺ»ÃÔÁҵôéǺѿà¿ÍÃì·ÕèÁÕÍسËÀÙÁÔà·èҡѺÍسËÀÙÁÔËéͧ ¨Ò¡¹Ñé¹»Ô໵ [³H]SCH23390 ¤ÇÒÁà¢éÁ¢é¹ 5  x 10^-9 âÁÅÒÃì »ÃÔÁҵà 100 äÁâ¤ÃÅԵà ŧ㹷ءËÅÍ´·´Åͧ áÅлÔ໵àÁÁàºÃ¹¨Ò¡ÊÁͧ˹٢ÒǺÃÔàdz striatum »ÃÔÁҵà 75 äÁâ¤ÃÅԵà (»ÃÔÁÒ³â»ÃµÕ¹ 750 äÁâ¤Ã¡ÃÑÁ) ŧ㹷ءËÅÍ´·´Åͧ ¼ÊÁãËéà¢éҡѹáÅéǺèÁ·ÕèÍسËÀÙÁÔ 25 ͧÈÒà«Åà«ÕÂÊ ¹Ò¹ 30 ¹Ò·Õ ¡ÃͧÊÒÃÅÐÅÒÂã¹áµèÅÐËÅÍ´·´Åͧ¼èÒ¹¡ÃдÒÉ¡Ãͧ Whatman GF/B â´Âãªéà¤Ã×èͧ¡ÃͧÃкºÊØ­­Ò¡ÒÈ 1225 Sampling Manifold áÅéÇÅéÒ§´éÇÂÊÒÃÅÐÅÒºѿà¿ÍÃì Tris-HCl 50 ÁÔÅÅÔâÁÅÒÃì pH 7.4 ·ÕèàÂ繨Ѵ ¨Ó¹Ç¹ 3 ¤ÃÑé§ ¤ÃÑé§ÅÐ 3 ÁÔÅÅÔÅԵà ¨Ò¡¹Ñé¹à¡çº¡ÃдÒÉ¡Ãͧ Whatman GF/B ã¹ vial ·ÕèºÃèØÊÒÃÅÐÅÒ scintillant 5 ÁÔÅÅÔÅԵà ¹Ó vial áµèÅТǴÁÒà¢ÂèÒ¹Ò¹ 3 ¹Ò·Õ  áÅéÇ¹Ó vial ·Ñé§ËÁ´ä»µÃǨÇÑ´»ÃÔÁÒ³ÃѧÊÕ´éÇÂà¤Ã×èͧ liquid scintillation counter

 

                3.5.2 ¡ÒÃÇÑ´¤ÇÒÁªÍº¨Ñº¢Í§ºÒÃÒ¤Íŵè͵ÑÇÃѺⴻÒÁÕ¹ 2

 

                                ÊÒÃÅÐÅÒ¢ͧÅÔ᡹´ì·ÕèµÔ´©ÅÒ¡ÃѧÊÕ¤×ÍÊÒÃÅÐÅÒ [³H]Raclopride ·ÕèÁÕ¤ÇÒÁà¢éÁ¢é¹ÊØ´·éÒÂà»ç¹  2 x 10^-9 âÁÅÒÃì (radioactive concentration à·èҡѺ 1 mCi/ml, specific activity à·èҡѺ 87 Ci/mmol)  ãªéÊÒÃÅÐÅÒ butaclamol ·ÕèÁÕ¤ÇÒÁà¢éÁ¢é¹ÊØ´·éÒÂà»ç¹  1 x 10^-4 âÁÅÒÃì à¾×èÍËÒ¤èÒ non-specific binding áÅÐãªéÊÒÃÅÐÅÒ raclopride ·ÕèÁÕ¤ÇÒÁà¢éÁ¢é¹ÊØ´·éÒÂà»ç¹  1 x 10^-8 âÁÅÒÃì (IC₅₀à·èҡѺ7.12 x 10^-9 âÁÅÒÃì) à»ç¹ positive control  ÊÒÃÅÐÅÒºѿà¿ÍÃì·Õèãªé»ÃСͺ´éÇ Tris-HCl 50 ÁÔÅÅÔâÁÅÒÃì NaCl 120 ÁÔÅÅÔâÁÅÒÃì áÅÐ MgCl₂ 2 ÁÔÅÅÔâÁÅÒÃì ·ÕèÁÕ¤èÒ pH 7.4  à¾×èÍ»ÃѺ»ÃÔÁҵ蹤ú 1,000 äÁâ¤ÃÅԵ÷ءËÅÍ´·´Åͧ

                ã¹¢Ñé¹ááä´éàµÔÁ 10% DMSO 50 äÁâ¤ÃÅԵüÊÁ¡Ñº¹éÓ¡ÅÑè¹»ÃÔÁҵà 50 äÁâ¤ÃÅÔµÃà¾×èÍãªéà»ç¹ solvent control àµÔÁ butaclamol  2 x 10^-3 âÁÅÒÃì »ÃÔÁҵà 50 äÁâ¤ÃÅԵüÊÁ¡Ñº¹éÓ¡ÅÑè¹»ÃÔÁҵà 50 äÁâ¤ÃÅԵà à¾×èͤӹdzËÒ non-specific binding  àµÔÁ raclopride  2 x 10^-7 âÁÅÒÃì »ÃÔÁҵà 50 äÁâ¤ÃÅԵüÊÁ¡Ñº¹éÓ¡ÅÑè¹»ÃÔÁҵà 50 äÁâ¤ÃÅÔµÃÊÓËÃѺà»ç¹ positive control áÅÐàµÔÁÊÒÃÅÐÅÒºÒÃÒ¤ÍŤÇÒÁà¢éÁ¢é¹ 2, 20, 200 áÅÐ 2000 äÁâ¤ÃâÁÅÒÃì »ÃÔÁҵà 50 äÁâ¤ÃÅԵüÊÁ¡Ñº 10% DMSO »ÃÔÁҵà 50 äÁâ¤ÃÅÔµÃã¹áµèÅФÇÒÁà¢éÁ¢é¹ áÅéÇ»ÃѺ»ÃÔÁҵôéǺѿà¿ÍÃì·ÕèÁÕÍسËÀÙÁÔà·èҡѺÍسËÀÙÁÔËéͧ ¨Ò¡¹Ñé¹»Ô໵ [³H]Raclopride ¤ÇÒÁà¢éÁ¢é¹ 20 x 10^-9  âÁÅÒÃì »ÃÔÁҵà 100 äÁâ¤ÃÅԵà ŧ㹷ءËÅÍ´·´Åͧ áÅлÔ໵àÁÁàºÃ¹ãËéÁÕ»ÃÔÁÒ³â»ÃµÕ¹ 200 äÁâ¤Ã¡ÃÑÁ ŧ㹷ءËÅÍ´·´Åͧ ¼ÊÁãËéà¢éҡѹ ºèÁ·ÕèÍسËÀÙÁÔ 25 ͧÈÒà«Åà«ÕÂÊ ¹Ò¹ 30 ¹Ò·Õ ¡ÃͧÊÒÃÅÐÅÒÂã¹áµèÅÐËÅÍ´·´Åͧ¼èÒ¹¡ÃдÒÉ¡Ãͧ Whatman GF/B ·Õèä´éáªèã¹ÊÒÃÅÐÅÒ 0.3% polyethyleneimine ¨¹ÍÔèÁµÑÇà»ç¹àÇÅÒÍÂèÒ§¹éͤÃÖ觪ÑèÇâÁ§ ãªéà¤Ã×èͧ¡ÃͧÃкºÊØ­­Ò¡ÒÈ 1225 Sampling Manifold áÅéÇÅéÒ§´éÇÂÊÒÃÅÐÅÒºѿà¿ÍÃì Tris-HCl 50 ÁÔÅÅÔâÁÅÒÃì pH 7.4 ·ÕèàÂ繨Ѵ ¨Ó¹Ç¹ 3 ¤ÃÑé§ ¤ÃÑé§ÅÐ 3 ÁÔÅÅÔÅԵà ¨Ò¡¹Ñé¹à¡çº¡ÃдÒÉ¡Ãͧ Whatman GF/B ã¹ vial ·ÕèºÃèØÊÒÃÅÐÅÒ scintillant 5 ÁÔÅÅÔÅԵà ¹Ó vial áµèÅТǴÁÒà¢ÂèÒ¹Ò¹ 3 ¹Ò·Õ  áÅéÇ¹Ó vial ·Ñé§ËÁ´ä»µÃǨÇÑ´»ÃÔÁÒ³ÃѧÊÕ´éÇÂà¤Ã×èͧ liquid scintillation counter

 

                3.5.3 ¡ÒÃÇÑ´¤ÇÒÁªÍº¨Ñº¢Í§ºÒÃÒ¤ÍŵèÍâ´»ÒÁÕ¹·Ãҹʻ͵àµÍÃì

               

                 ÊÒÃÅÐÅÒ¢ͧÅÔ᡹´ì·ÕèµÔ´©ÅÒ¡ÃѧÊÕ¤×ÍÊÒÃÅÐÅÒ [³H]WIN35428 ¤ÇÒÁà¢éÁ¢é¹ 36 x 10 ^-9âÁÅÒÃì (radioactive concentration à·èҡѺ 1 mCi/ml, specific activity à·èҡѺ 86 Ci/mmol)  ãªéÊÒÃÅÐÅÒ nomifensine ¤ÇÒÁà¢éÁ¢é¹ÊØ´·éÒÂà»ç¹ 1 x 10^-4 âÁÅÒÃì à¾×èÍËÒ¤èÒ non-specific binding áÅÐãªéÊÒÃÅÐÅÒ GBR12909 ¤ÇÒÁà¢éÁ¢é¹ÊØ´·éÒÂà»ç¹ 1 x 10^-7 âÁÅÒÃì áÅÐ 1 x 10^-8 âÁÅÒÃì (IC₅₀à·èҡѺ 2.8 x 10^-9 âÁÅÒÃì) à»ç¹ positive control  ÊÒÃÅÐÅÒºѿà¿ÍÃì·Õèãªé¤×Í ÊÒÃÅÐÅÒ sodium phosphate buffer  ¤ÇÒÁà¢éÁ¢é¹  32 ÁÔÅÅÔâÁÅÒÃì  ·ÕèÁÕ¤èÒ pH 7.4  à¾×èÍ»ÃѺ»ÃÔÁҵ蹤ú 1,000 äÁâ¤ÃÅԵ÷ءËÅÍ´·´Åͧ

                ã¹¢Ñé¹ááä´éàµÔÁ 10% DMSO 50 äÁâ¤ÃÅԵüÊÁ¡Ñº¹éÓ¡ÅÑè¹»ÃÔÁҵà 50 äÁâ¤ÃÅÔµÃà¾×èÍà¾×èÍãªéà»ç¹ solvent control àµÔÁ nomifensine  2 x 10^-3 âÁÅÒÃì »ÃÔÁҵà 50 äÁâ¤ÃÅԵüÊÁ¡Ñº¹éÓ¡ÅÑè¹»ÃÔÁҵà 50 äÁâ¤ÃÅԵà à¾×èͤӹdzËÒ non-specific binding  áÅÐàµÔÁ GBR12909 ¤ÇÒÁà¢éÁ¢é¹ 2x10^-6 âÁÅÒÃìáÅÐ 2x10^-7 âÁÅÒÃì   »ÃÔÁҵà 50 äÁâ¤ÃÅԵüÊÁ¡Ñº¹éÓ¡ÅÑè¹»ÃÔÁҵà 50 äÁâ¤ÃÅÔµÃÊÓËÃѺà»ç¹ positive control  ¨Ò¡¹Ñé¹àµÔÁÊÒÃÅÐÅÒºÒÃÒ¤ÍŤÇÒÁà¢éÁ¢é¹ 2, 20, 200 áÅÐ 2000 äÁâ¤ÃâÁÅÒÃì »ÃÔÁҵà 50 äÁâ¤ÃÅԵüÊÁ¡Ñº 10 % DMSO »ÃÔÁҵà 50 äÁâ¤ÃÅÔµÃã¹áµèÅФÇÒÁà¢éÁ¢é¹ áÅéÇ»ÃѺ»ÃÔÁҵôéǺѿà¿ÍÃì·ÕèÁÕÍسËÀÙÁÔà·èҡѺËéͧÍسËÀÙÁÔ ¨Ò¡¹Ñé¹»Ô໵ [³H]WIN35428 ¤ÇÒÁà¢éÁ¢é¹ 36 ¹Òâ¹âÁÅÒÃì »ÃÔÁҵà 100 äÁâ¤ÃÅԵà ŧ㹷ءËÅÍ´·´Åͧ áÅлÔ໵àÁÁàºÃ¹ãËéÁÕ»ÃÔÁÒ³â»ÃµÕ¹ 750 äÁâ¤Ã¡ÃÑÁ ŧ㹷ءËÅÍ´·´Åͧ ¼ÊÁãËéà¢éҡѹ ºèÁ·ÕèÍسËÀÙÁÔ 25 ͧÈÒà«Åà«ÕÂÊ ¹Ò¹ 15 ¹Ò·Õ ¡ÃͧÊÒÃÅÐÅÒÂã¹áµèÅÐËÅÍ´·´Åͧ¼èÒ¹¡ÃдÒÉ¡Ãͧ Whatman GF/B ·Õèä´éáªèã¹ÊÒÃÅÐÅÒ 0.3% polyethyleneimine ¨¹ÍÔèÁµÑÇà»ç¹àÇÅÒÍÂèÒ§¹éͤÃÖ觪ÑèÇâÁ§ ãªéà¤Ã×èͧ¡ÃͧÃкºÊØ­­Ò¡ÒÈ 1225 Sampling Manifold áÅéÇÅéÒ§´éÇÂÊÒÃÅÐÅÒºѿà¿ÍÃì·ÕèàÂ繨Ѵ ¨Ó¹Ç¹ 3 ¤ÃÑé§ ¤ÃÑé§ÅÐ 3 ÁÔÅÅÔÅԵà ¨Ò¡¹Ñé¹à¡çº¡ÃдÒÉ¡Ãͧ Whatman GF/B ã¹ vial ·ÕèºÃèØÊÒÃÅÐÅÒ scintillant 5 ÁÔÅÅÔÅԵà ¹Ó vial áµèÅТǴÁÒà¢ÂèÒ¹Ò¹ 3 ¹Ò·Õ  áÅéÇ¹Ó vial ·Ñé§ËÁ´ä»µÃǨÇÑ´»ÃÔÁÒ³ÃѧÊÕ´éÇÂà¤Ã×èͧ liquid scintillation counter

 

 

µÒÃÒ§·Õè 3  ÊÃØ»ÊÀÒÇСÒÃÇÑ´¤ÇÒÁªÍº¨Ñºµè͵ÑÇÃѺⴻÒÁÕ¹áÅÐâ´»ÒÁÕ¹·Ãҹʻ͵àµÍÃìâ´Âãªé        à·¤¹Ô¤ Radioligand binding assay

 

	Radioligands	Nonspecific binding	Positive control	Binding buffer	Incubation
µÑÇÃѺⴻÒÁÕ¹ 1	[3H]SCH23390	Butaclamol	SCH23390	50 mM Tris-HCl, 120 mM NaCl, 2 mM MgCl2  pH 7.4	25 °C, 30 min
µÑÇÃѺⴻÒÁÕ¹ 2	[3H]Raclopride	Butaclamol	Raclopride	50 mM Tris-HCl, 120 mM NaCl, 2 mM MgCl2  pH 7.4	25 °C, 30 min
â´»ÒÁÕ¹·ÃÒ¹ ʻ͵àµÍÃì	[3H]WIN35428	Nomifensine	GBR12909	35 mM Phosphate buffer pH 7.4	25 °C, 15 min

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

º··Õè 4

¼Å¡ÒÃÇÔ¨ÑÂ

 

4.1 ¡ÒÃàµÃÕÂÁàÁÁàºÃ¹

4.1.1 àÁÁàºÃ¹·ÕèÁÕµÑÇÃѺⴻÒÁÕ¹ 1

 

àÁÁàºÃ¹¢Í§ÊÁͧ˹٢ÒÇÊèǹ striatum ·ÕèàµÃÕÂÁä´é«Öè§ÁÕ¹éÓ˹ѡ 3.45 ¡ÃÑÁ àÁ×è͹ÓÁÒÇÔà¤ÃÒÐËìËÒ»ÃÔÁÒ³â»ÃµÕ¹â´ÂÇÔ¸Õ Bradford ¾ºÇèÒÇÑ´¤èÒà©ÅÕè¡Òôٴ¡Å×¹áʧ·Õè¤ÇÒÁÂÒǤÅ×è¹ 595 nm ä´éà·èҡѺ 0.210  áÅÐàÁ×è͹ÓÁҤӹdzËÒ¤èÒ¤ÇÒÁà¢éÁ¢é¹¢Í§â»ÃµÕ¹¨Ò¡ÊÁ¡Ò÷Õèä´é¨Ò¡¡ÃÒ¿Áҵðҹä´éà·èҡѺ 10.95 ÁÔÅÅÔ¡ÃÑÁµèÍÁÔÅÅÔÅԵà ´Ñ§ÃÙ»·Õè 2 ¨Ò¡¹Ñé¹¹ÓàÁÁàºÃ¹¹ÕéÁÒà¨×ͨҧ´éÇÂÊÒÃÅÐÅÒºѿà¿ÍÃìà¾×èÍãËéä´é¤ÇÒÁà¢éÁ¢é¹ÊØ´·éÒÂà»ç¹ 10 ÁÔÅÅÔ¡ÃÑÁµèÍÁÔÅÅÔÅԵà áÅéǹÓÊÒÃá¢Ç¹ÅÍ¢ͧµÑÇÃѺº¹àÂ×èÍËØéÁà«ÅÅì仼èÒ¹ä¹âµÃਹàËÅÇ»ÃÐÁÒ³ 1 ¹Ò·Õ ¨Ò¡¹Ñ鹨֧¹Óä»à¡çº·ÕèÍسËÀÙÁÔ - 80 ͧÈÒà«Åà«ÕÂÊ

 

ÃÙ»·Õè 2   ¡ÃÒ¿ÁҵðҹáÊ´§¤ÇÒÁÊÑÁ¾Ñ¹¸ìÃÐËÇèÒ§¤ÇÒÁà¢éÁ¢é¹¢Í§ BSA standard solution                                           áÅФèÒ¡Òôٴ¡Å×¹áʧ·Õè¤ÇÒÁÂÒǤÅ×è¹ 595 nm

 

 

4.1.2 àÁÁàºÃ¹·ÕèÁÕµÑÇÃѺⴻÒÁÕ¹ 2

 

àÁÁàºÃ¹¨Ò¡à«ÅÅìà¾ÒÐàÅÕé§ A9LhD2L ¨Ó¹Ç¹ 6.074 x 10⁸ cell  àÁ×èÍÇÔà¤ÃÒÐËìËÒ»ÃÔÁÒ³â»ÃµÕ¹â´ÂÇÔ¸Õ Bradford ¾ºÇèÒÇÑ´¤èÒà©ÅÕè¡Òôٴ¡Å×¹áʧ·Õè¤ÇÒÁÂÒǤÅ×è¹ 595 nm ä´éà·èҡѺ 0.412 áÅÐàÁ×è͹ÓÁҤӹdzËÒ¤èÒ¤ÇÒÁà¢éÁ¢é¹¢Í§â»ÃµÕ¹â´Âà·Õº¨Ò¡ÊÁ¡Ò÷Õèä´é¨Ò¡¡ÃÒ¿Áҵðҹä´éà·èҡѺ 19.87 ÁÔÅÅÔ¡ÃÑÁµèÍÁÔÅÅÔÅԵà ¨Ò¡¹Ñé¹¹ÓàÁÁàºÃ¹¹ÕéÁÒà¨×ͨҧ´éÇÂÊÒÃÅÐÅÒºѿà¿ÍÃìà¾×èÍãËéä´é¤ÇÒÁà¢éÁ¢é¹ÊØ´·éÒÂà»ç¹ 5 ÁÔÅÅÔ¡ÃÑÁµèÍÁÔÅÅÔÅԵà áÅéǹÓÊÒÃá¢Ç¹ÅÍ¢ͧµÑÇÃѺº¹àÂ×èÍËØéÁà«ÅÅì仼èÒ¹ä¹âµÃਹàËÅÇ»ÃÐÁÒ³ 1 ¹Ò·Õ ¨Ò¡¹Ñ鹨֧¹Óä»à¡çº·ÕèÍسËÀÙÁÔ - 80 ͧÈÒà«Åà«ÕÂÊ

 

4.1.3 àÁÁàºÃ¹·ÕèÁÕ â´»ÒÁÕ¹·Ãҹʻ͵àµÍÃì

 

àÁÁàºÃ¹¢Í§ÊÁͧ˹٢ÒÇÊèǹ striatum ·ÕèÁÕ¹éÓ˹ѡ 3.68 ¡ÃÑÁ àÁ×èÍÇÔà¤ÃÒÐËìËÒ»ÃÔÁÒ³â»ÃµÕ¹â´ÂÇÔ¸Õ Bradford ¾ºÇèÒÇÑ´¤èÒà©ÅÕè¡Òôٴ¡Å×¹áʧ ·Õè¤ÇÒÁÂÒǤÅ×è¹ 595 nm ä´éà·èҡѺ 0.150 áÅÐàÁ×è͹ÓÁҤӹdzËÒ¤èÒ¤ÇÒÁà¢éÁ¢é¹¢Í§â»ÃµÕ¹¨Ò¡ÊÁ¡Ò÷Õèä´é¨Ò¡¡ÃÒ¿Áҵðҹä´éà·èҡѺ 8.76 ÁÔÅÅÔ¡ÃÑÁµèÍÁÔÅÅÔÅԵà áÅéǹÓÊÒÃá¢Ç¹ÅÍ¢ͧµÑÇÃѺº¹àÂ×èÍËØéÁà«ÅÅì仼èÒ¹ä¹âµÃਹàËÅÇ»ÃÐÁÒ³ 1 ¹Ò·Õ ¨Ò¡¹Ñ鹨֧¹Óä»à¡çº·ÕèÍسËÀÙÁÔ - 80 ͧÈÒà«Åà«ÕÂÊ

 

4.2 ¡ÒÃÇÑ´¤ÇÒÁªÍº¨Ñº¢Í§ºÒÃÒ¤Íŵè͵ÑÇÃѺⴻÒÁÕ¹â´Âãªéà·¤¹Ô¤ Radioligand  binding assay

 

4.2.1 ¡ÒÃÇÑ´¤ÇÒÁªÍº¨Ñº¢Í§ºÒÃÒ¤Íŵè͵ÑÇÃѺⴻÒÁÕ¹ 1

 

àÁ×èÍ¹Ó vial ·Ñé§ËÁ´ä»µÃǨÇÑ´»ÃÔÁÒ³ÃѧÊÕ´éÇÂà¤Ã×èͧ liquid scintillation counter ÇÑ´»ÃÔÁÒ³ÃѧÊÕã¹Ë¹èÇ count per minute (cpm)  áÅФӹdz¤èÒ % inhibition  ¾ºÇèÒÊÒÃÅÐÅÒ  SCH23390 «Öè§ãªéà»ç¹ positive control ·Õè¤ÇÒÁà¢éÁ¢é¹ÊØ´·éÒÂà»ç¹ 0.5 ¹Òâ¹âÁÅÒÃì áÅÐÊÒÃÅÐÅÒºÒÃÒ¤ÍÅ·Õè¤ÇÒÁà¢éÁ¢é¹ÊØ´·éÒÂà»ç¹ 0.1, 1, 10 áÅÐ 100 äÁâ¤ÃâÁÅÒÃì ãËé¤èÒà©ÅÕè % inhibition à·èҡѺ 37.8, -4.6, 5.7, -6.8, áÅÐ -0.7 µÒÁÅӴѺ  ´Ñ§áÊ´§ã¹µÒÃÒ§·Õè 4

 

µÒÃÒ§·Õè 4  ¤èÒà©ÅÕè % inhibition ¢Í§ºÒÃÒ¤ÍÅ㹡ÒÃÂѺÂÑ駡ÒèѺÃÐËÇèÒ§ [³H]SCH23390 ¡Ñº  µÑÇÃѺⴻÒÁÕ¹ 1 (n=9)

 

 

                ¨Ò¡µÒÃÒ§áÊ´§ÇèÒºÒÃÒ¤ÍÅ·Õè¤ÇÒÁà¢éÁ¢é¹ 0.1, 1, 10 áÅÐ 100 äÁâ¤ÃâÁÅÒÃì äÁèÊÒÁÒöÂѺÂÑ駡ÒèѺÃÐËÇèÒ§ [³H]SCH23390 ¡ÑºµÑÇÃѺⴻÒÁÕ¹ 1

 

ÃÙ»·Õè 3   á¼¹ÀÙÁÔáÊ´§¤èÒ  % inhibition ¢Í§ºÒÃÒ¤ÍÅ㹡ÒÃÂѺÂÑ駡ÒèѺÃÐËÇèÒ§[³H]SCH23390 ¡Ñº  µÑÇÃѺⴻÒÁÕ¹ 1 (n=9)

4.2.2 ¡ÒÃÇÑ´¤ÇÒÁªÍº¨Ñº¢Í§ºÒÃÒ¤Íŵè͵ÑÇÃѺⴻÒÁÕ¹ 2

 

                àÁ×èÍ¹Ó vial ·Ñé§ËÁ´ä»µÃǨÇÑ´»ÃÔÁÒ³ÃѧÊÕ´éÇÂà¤Ã×èͧ liquid scintillation counter ÇÑ´»ÃÔÁÒ³ÃѧÊÕã¹Ë¹èÇ  cpm  áÅФӹdz¤èÒ % inhibition  ¾ºÇèÒÊÒÃÅÐÅÒ raclopride ·Õèãªéà»ç¹ positive control ·Õè¤ÇÒÁà¢éÁ¢é¹ÊØ´·éÒÂà»ç¹ 10 ¹Òâ¹âÁÅÒÃì áÅÐÊÒÃÅÐÅÒºÒÃÒ¤ÍÅ·Õè¤ÇÒÁà¢éÁ¢é¹ÊØ´·éÒÂà»ç¹ 0.1, 1, 10 áÅÐ 100 äÁâ¤ÃâÁÅÒÃì ãËé¤èÒà©ÅÕè % inhibition à·èҡѺ  71.5, -14.1, -9.4, -11.4, áÅÐ -0.4 µÒÁÅӴѺ  ´Ñ§áÊ´§ã¹µÒÃÒ§·Õè 5

               

µÒÃÒ§·Õè  5  ¤èÒà©ÅÕè % inhibition ¢Í§ºÒÃÒ¤ÍÅ㹡ÒÃÂѺÂÑ駡ÒèѺÃÐËÇèÒ§ [³H]Raclopride ¡Ñº µÑÇÃѺⴻÒÁÕ¹ 2 (n = 5)

 

 

                ¨Ò¡µÒÃÒ§áÊ´§ÇèÒºÒÃÒ¤ÍÅ·Õè¤ÇÒÁà¢éÁ¢é¹ 0.1, 1, 10 áÅÐ 100 äÁâ¤ÃâÁÅÒÃì äÁèÊÒÁÒöÂѺÂÑ駡ÒèѺÃÐÇèÒ§ [³H]Raclopride ¡ÑºµÑÇÃѺⴻÒÁÕ¹ 2

 

 

 

 

 

 

ÃÙ»·Õè 4  á¼¹ÀÙÁÔáÊ´§¤èÒ  % inhibition ¢Í§ºÒÃÒ¤ÍÅ㹡ÒÃÂѺÂÑ駡ÒèѺÃÐËÇèÒ§[³H]Raclopride ¡Ñº µÑÇÃѺⴻÒÁÕ¹ 2

 

 

4.3 ¡ÒÃÇÑ´¤ÇÒÁªÍº¨Ñº¢Í§ºÒÃÒ¤ÍŵèÍâ´»ÒÁÕ¹·Ãҹʻ͵àµÍÃìâ´Âãªéà·¤¹Ô¤ Radioligand  binding assay

 

àÁ×èÍ¹Ó vial ·Ñé§ËÁ´ä»µÃǨÇÑ´»ÃÔÁÒ³ÃѧÊÕ´éÇÂà¤Ã×èͧ liquid scintillation counter ÇÑ´»ÃÔÁÒ³ÃѧÊÕã¹Ë¹èÇ cpm áÅФӹdz¤èÒ % inhibition  ¾ºÇèÒÊÒÃÅÐÅÒ GBR12909 ·Õè¤ÇÒÁà¢éÁ¢é¹ÊØ´·éÒÂà»ç¹ 10 ¹Òâ¹ âÁÅÒÃì (10 à·èҢͧ¤èÒ IC₅₀)ãËé¤èÒà©ÅÕè % inhibition à·èҡѺ -12.3 áÅÐÊÒÃÅÐÅÒ GBR12909 ·Õè¤ÇÒÁà¢éÁ¢é¹ÊØ´·éÒÂà»ç¹ 100 ¹Òâ¹ âÁÅÒÃì ãËé¤èÒà©ÅÕè % inhibition à·èҡѺ 48.6 ´Ñ§áÊ´§ã¹µÒÃÒ§·Õè 6

 

 

 

 

 

 

 

 

µÒÃÒ§·Õè 6  ¤èÒà©ÅÕè % Inhibition ¢Í§ºÒÃÒ¤ÍÅ㹡ÒÃÂѺÂÑ駡ÒèѺÃÐËÇèÒ§ [³H]WIN35,428 ¡Ñºâ´»ÒÁÕ¹·ÃÒʻ͵àµÍÃì (n = 2)

 

 

¨Ò¡µÒÃÒ§áÊ´§ÇèÒÊÒÃÅÐÅÒ GBR12909 ·Õè¤ÇÒÁà¢éÁ¢é¹ 10 ¹Òâ¹âÁÅÒÃì«Öè§à»ç¹¤èÒ¤ÇÒÁà¢éÁ¢é¹·Õè 10 à·èҢͧ¤èÒ IC₅₀ äÁèÊÒÁÒöÂѺÂÑ駡ÒèѺ¢Í§ [³H]WIN35428 (dopamine transporter blocker) µèÍâ´»ÒÁÕ¹·Ãҹʻ͵àµÍÃì â´Â·Õè GBR12909 ·Õè¤ÇÒÁà¢éÁ¢é¹ 10 ¹Òâ¹âÁÅÒÃì¤ÇèÐãËé¤èÒ % inhibition ·ÕèÁÒ¡¡ÇèÒ 50 áÊ´§ÇèÒÊÀÒÇСÒ÷´Åͧà¾×èÍÇÑ´¤ÇÒÁªÍº¨Ñº¢Í§ºÒÃҤ͚ѺⴻÒÁÕ¹ÂѧäÁèàËÁÒÐÊÁ ´Ñ§¹Ñ鹨֧äÁèÊÒÁÒöÊÃØ»¼Å¤ÇÒÁªÍº¨Ñº¢Í§ºÒÃÒ¤ÍŵèÍâ´»ÒÁÕ¹·ÃҹʻÍÃìµàµÍÃìä´é áÅÐà¹×èͧ¨Ò¡äÍÍ͹â«à´ÕÂÁ¡ÑºäÍÍ͹¤ÅÍäôìÁÕ¤ÇÒÁÊӤѭµèÍ¡ÒèѺÃÐËÇèÒ§ÅÔ᡹´ì¡Ñºâ´»ÒÁÕ¹ ·Ãҹʻ͵àµÍÃì ´Ñ§¹Ñé¹ÊÒÃÅÐÅÒºѿà¿ÍÃì·Õèãªé·´Åͧ¨Ö§à»ç¹»Ñ¨¨ÑÂÊӤѭ·Õèµéͧ¤Ó¹Ö§¶Ö§à¾×è;Ѳ¹ÒÊÀÒÇСÒ÷´Åͧ·ÕèàËÁÒÐÊÁµèÍä»

 

º··Õè 5

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ºÒÃÒ¤ÍÅà»ç¹ÊÒÃ͹ؾѹ¸ì¢Í§ chromone ·ÕèÊ¡Ñ´ä´é¨Ò¡ÊÁعä¾Ã¢ÕéàËÅç¡  ºÒÃÒ¤ÍÅÊÒÁÒöáÊ´§Ä·¸Ôì·Ò§àÀÊѪÇÔ·ÂÒ·Õèà¡ÕèÂÇ¢éͧ¡ÑºÃкº»ÃÐÊÒ·â´»ÒÁÕ¹ §Ò¹ÇԨѹÕé¨Ö§ä´éÇÑ´¤ÇÒÁªÍº¨Ñº¢Í§ºÒÃÒ¤Íŵè͵ÑÇÃѺⴻÒÁÕ¹ 1  áÅÐâ´»ÒÁÕ¹·Ãҹʻ͵àµÍÃì·ÕèÍÂÙèã¹àÁÁàºÃ¹«Öè§àµÃÕÂÁ¨Ò¡ÊÁͧ¢Í§Ë¹Ù¢ÒǺÃÔàdz striatum  áÅеÑÇÃѺⴻÒÁÕ¹ 2 ·ÕèÍÂÙèã¹àÁÁàºÃ¹«Öè§àµÃÕÂÁ¨Ò¡à«ÅÅìà¾ÒÐàÅÕé§ A9LhD2 â´Âãªéà·¤¹Ô¤ radioligand  binding assay 

¨Ò¡¼Å¡ÒÃÇԨѹÕéáÊ´§ãËéàËç¹ÇèÒºÒÃÒ¤ÍÅ·Õè¤ÇÒÁà¢éÁ¢é¹ 0.1, 1, 10 áÅÐ 100 äÁâ¤ÃâÁÅÒÃì äÁèÊÒÁÒöÂѺÂÑ駡ÒèѺ¢Í§ [³H]SCH23390 «Öè§à»ç¹ antagonist ¡ÑºµÑÇÃѺⴻÒÁÕ¹ 1 ·Ñ駹Õéä´éÇÑ´¤ÇÒÁªÍº¨Ñº¢Í§ SCH23390 ¤ÇÒÁà¢éÁ¢é¹ÊØ´·éÒÂà·èҡѺ¤èÒ IC₅₀ (0.5 ¹Òâ¹âÁÅÒÃì) à¾×èÍà»ç¹ positive control ¾ºÇèÒãËé¤èÒ % inhibition à©ÅÕèÂà·èҡѺ 37.8 «Öè§ã¡Åéà¤Õ§¡Ñº¤èÒ¡ÒÃÂѺÂÑ駵ÒÁ·Äɮդ×Í»ÃÐÁÒ³ÃéÍÂÅÐ 50 áÅкÒÃÒ¤ÍÅ·Õè¤ÇÒÁà¢éÁ¢é¹´Ñ§¡ÅèÒÇ¢éÒ§µé¹¡çäÁèÊÒÁÒöÂѺÂÑ駡ÒèѺ¢Í§ [³H]Raclopride «Öè§ÊÒùÕéà»ç¹ antagonist µè͵ÑÇÃѺⴻÒÁÕ¹ 2 ·Ñ駹Õé¼ÙéÇÔ¨ÑÂä´éÇÑ´¤ÇÒÁªÍº¨Ñº¢Í§ raclopride (IC50 à·èҡѺ 7.12  ¹Òâ¹âÁÅÒÃì) ·ÕèÁÕ¤ÇÒÁà¢éÁ¢é¹ÊØ´·éÒÂà·èҡѺ¤èÒ  10 ¹Òâ¹âÁÅÒÃì à¾×èÍà»ç¹ positive control ¾ºÇèÒãËé¤èÒ % inhibition à©ÅÕèÂà·èҡѺ 71.5 «Öè§áÊ´§ÇèҼšÒÃÇԨѤÃÑ駹ÕéÁÕ¤ÇÒÁ¹èÒàª×èͶ×Í

¨Ò¡¼Å¡Ò÷´Åͧ¢Í§ÊÒ÷Õèãªéà»ç¹ positive control µè͵ÑÇÃѺáÊ´§ãËéàËç¹ÇèÒºÒÃÒ¤ÍÅäÁèÁÕ¤ÇÒÁªÍº¨Ñº¡ÑºµÑÇÃѺⴻÒÁÕ¹ 1 áÅеÑÇÃѺⴻÒÁÕ¹ 2 áÅÐà¤ÂÁÕ¡ÒÃÈÖ¡ÉÒ·Õèãªé [¹²⁵I]barakol ¾ºÇèÒµÓá˹觨Ѻ¢Í§ºÒÃÒ¤ÍÅäÁèãªè·ÕèµÑÇÃѺⴻÒÁÕ¹ 1  µÑÇÃѺⴻÒÁÕ¹ 2  µÑÇÃѺ«ÕâÃâµ¹Ô¹ áÅÐ µÑÇÃѺ GABA  (ÊÒ·ԾÂì à¾è§ÈÃÕ, 2539)  áÅШҡ¼Å¡ÒÃÇԨѷÕ辺ÇèÒºÒÃÒ¤ÍÅÊÒÁÒöŴÃдѺⴻÒÁÕ¹¹Í¡à«ÅÅì»ÃÐÊÒ·ã¹ÊÁͧ˹ÙÊèǹ striatum (Thongsaard et al., 1997) ¨Ö§à»ç¹ä»ä´éÇèÒºÒÃÒ¤ÍÅÍÒ¨ÍÍ¡Ä·¸ÔìÅ´¡ÒÃËÅÑè§â´»ÒÁÕ¹¼èÒ¹¡Åä¡·ÕèäÁèà¡ÕèÂÇ¢éͧ¡ÑºµÑÇÃѺⴻÒÁÕ¹ 1 áÅеÑÇÃѺⴻÒÁÕ¹ 2 â´ÂÍÒ¨ÁռŵèÍ K⁺ channel áÅÐ Ca²⁺ Channel (Thongsaard et al., 1997) «Ö觡ÒáÃеØé¹ K⁺ channel ·ÓãËéäÍÍ͹â¾á·Êà«ÕÂÁà¢éÒà«ÅÅìÁÒ¡¢Öé¹à»ç¹¼ÅãËéàÂ×èÍËØéÁà«ÅÅì»ÃÐÊÒ·à¡Ô´ÊÀÒ¾ hyperpolarization Ê觼ÅãËé»ÃÔÁÒ³äÍÍ͹á¤Å«ÕÂÁ·Õèà¢éÒà«ÅÅì»ÃÐÊÒ·¼èÒ¹·Ò§ Ca²⁺ Channel Ŵŧ ¨Ö§·ÓãËé¡ÒûŴ»ÅèÍÂâ´»ÒÁչŴ¹éÍÂŧ (Iannazzo et  al., 1997) «Ö觡ÒÃËÅÑè§â´»ÒÁÕ¹¢Í§à«ÅÅì»ÃÐÊÒ·â´»ÒÁÕ¹¶Ù¡¤Çº¤ØÁ´éÇ 3 ¡Åä¡ËÅÑ¡¤×Í ¡Åä¡áá¡ÒâѺÍÍ¡´éÇÂÇÔ¸Õ exocytosis «Ö觵éͧÍÒÈÑÂäÍÍ͹á¤Åà«ÕÂÁ ¡Å䡵èÍÁÒ¤×Í¡Òâ¹Êè§Âé͹¡ÅѺ (reversal transport) ¼èҹⴻÒÁÕ¹·Ãҹʻ͵àµÍÃì áÅСÅä¡ÊØ´·éÒÂâ´»ÒÁÕ¹Âѧ¶Ù¡¤Çº¤ØÁ¡ÒûŴ»ÅèÍÂä´é·ÕèºÃÔàdz»ÅÒ»ÃÐÊÒ·Êèǹ dendrite â´ÂÍÒÈѵÑÇÃѺ (Hobson  et  al.,  2003)  ¹Í¡à˹×ͨҡµÑÇÃѺⴻÒÁÕ¹ 2 ·Õèà»ç¹ autoreceptor «Öè§ÍÂÙè·Õè presynaptic neuron áÅéÇÂѧÁÕµÑÇÃѺÍÕ¡ 2 ª¹Ô´·Õè¨Ñ´ÍÂÙè㹡ÅØèÁà´ÕÂǡѹ ¤×Í µÑÇÃѺⴻÒÁÕ¹ 3  áÅеÑÇÃѺⴻÒÁÕ¹ 4  «Ö觡ÒáÃеØé¹µÑÇÃѺ´Ñ§¡ÅèÒÇÁÕ¼ÅÂѺÂÑ駡ÒÃÊѧà¤ÃÒÐËìâ´»ÒÁÕ¹â´Âà¡Ô´¼èÒ¹¡ÒÃÂѺÂÑ駡Ò÷ӧҹ¢Í§à͹ä«Áì tyrosine hydroxylase ¹Í¡¨Ò¡¹ÕéÂѧÊÒÁÒöÂѺÂÑ駡ÒÃËÅÑè§â´»ÒÁÕ¹¨Ò¡¶Ø§à¡çº·ÕèÍÂÙèã¹à«ÅÅì»ÃÐÊÒ·¡è͹¨Ø´»ÃÐÊÒ¹»ÃÐÊÒ· áÅÐÅ´ firing rate ¢Í§à«ÅÅì»ÃÐÊÒ·â´»ÒÁÕ¹ (Tarazi, 2001)

ÁÕ§Ò¹ÇÔ¨ÑÂÅèÒÊØ´·Õè·´Êͺâ´ÂãËé methamphetamine «Öè§à»ç¹ dopamine transporter substrate á¡è˹٢ÒÇã¹¢¹Ò´ 1 ÁÔÅÅÔ¡ÃÑÁµè͹éÓ˹ѡµÑÇ 1 ¡ÔâÅ¡ÃÑÁ¾ºÇèÒ·ÓãËé˹٢ÒÇáÊ´§¾ÄµÔ¡ÃÃÁà¤Å×è͹·ÕèÁÒ¡¢Öé¹ (hyperlocomotor activity) áÅÐàÁ×èÍãËéºÒÃÒ¤ÍÅã¹¢¹Ò´ 25 - 100 ÁÔÅÅÔ¡ÃÑÁµè͹éÓ˹ѡµÑÇ 1 ¡ÔâÅ¡ÃÑÁ¡è͹·Õè¨ÐãËé methamphetamine ¾ºÇèÒºÒÃÒ¤ÍÅÊÒÁÒö¡´¾ÄµÔ¡ÃÃÁ¡ÒÃà¤Å×è͹·Õè¹Õéä´é (Sukma  et al., 2002) §Ò¹ÇԨѤÃÑ駹Õé¨Ö§¤Ò´ÇèÒºÒÃÒ¤ÍŨÐÁÕ¼ÅÅ´Ä·¸Ôì¢Í§ methamphetamine ¼èÒ¹¡ÒÃà¡Ô´ÍѹµÃ¡ÔÃÔÂҡѺⴻÒÁÕ¹·Ãҹʻ͵àµÍÃì ËÃ×ÍÍÒ¨à¡Ô´¼èÒ¹¡Åä¡Í×蹫Öè§à¤ÂÁÕÃÒ§ҹÇèÒÂÒ¡ÅØèÁ amphetamines ¡ÃеØé¹ãËéà«ÅÅì»ÃÐÊÒ·â´»ÒÁÕ¹ÊÃéҧ͹ØÁÙÅÍÔÊÃСÅØèÁ reactive oxygen species (ROS)áÅÐ reactive nitrogen species (RNS) à¾ÔèÁÁÒ¡¢Öé¹ (Pubill et al., 2004) ¢³Ðà´ÕÂǡѹ¡çÁÕ¡ÒÃÈÖ¡ÉÒ·Õè¹ÓºÒÃÒ¤ÍÅÁÒ·´Êͺ´éÇÂÇÔ¸Õ DPPH radical scavenging activity ¾ºÇèÒºÒÃÒ¤ÍÅÁÕÄ·¸ÔìµéÒ¹ÍÍ¡«Ôപѹ (antioxidative activity) ÁÒ¡¡ÇèÒ butylated hydroxyl toluene (BHT) ·Õèãªéà»ç¹ÊÒÃÁҵðҹ (Sanan, 2000) ¨Ö§à»ç¹ä»ä´éÇèÒºÒÃÒ¤ÍÅÍÒ¨ÍÍ¡Ä·¸ÔìÅ´¡ÒÃà¤Å×è͹äËÇ·Õèà˹ÕèÂǹÓâ´Â methamphetamine â´ÂÅ´»ÃÔÁҳ͹ØÁÙÅÍÔÊÃзÕèà¡Ô´¢Öé¹ ´Ñ§¹Ñ鹺ÒÃÒ¤ÍÅÍÒ¨ÁÕá¹Çâ¹éÁ·ÕèÊÒÁÒö¨Ñº¡Ñºâ´»ÒÁÕ¹·Ãҹʻ͵àµÍÃì«Öè§ÁÕ˹éÒ·Õèà¡çºâ´»ÒÁÕ¹¡ÅѺà¢éÒã¹à«ÅÅì»ÃÐÊÒ·Ê觼ÅãËéÃдѺⴻÒÁÕ¹¹Í¡à«ÅÅì»ÃÐÊҷŴŧ¨Ö§·ÓãËé˹٢ÒÇ·Õèä´éÃѺºÒÃÒ¤ÍÅáÊ´§¾ÄµÔ¡ÃÃÁ¡ÒÃà¤Å×è͹äËÇŴŧ

                §Ò¹ÇԨѤÃÑ駹Õéä´é·Ó¡ÒÃÇÑ´¤ÇÒÁªÍº¨Ñº¢Í§ºÒÃÒ¤ÍŵèÍâ´»ÒÁÕ¹·Ãҹʻ͵àµÍÃìâ´ÂãªéÊÒÃradioligand ¤×Í [³H]WIN35428 â´Âãªé GBR 12909 ·ÕèÁդسÊÁºÑµÔà»ç¹ dopamine transporter blocker ·Õè¤ÇÒÁà¢éÁ¢é¹ 10 ¹Òâ¹âÁÅÒÃì «Öè§à»ç¹¤èÒ¤ÇÒÁà¢éÁ¢é¹»ÃÐÁÒ³ 10 à·èҢͧ¤èÒ IC₅₀ (2.8  ¹Òâ¹âÁÅÒÃì)  à¾×èÍà»ç¹ positive control ·Ñ駹Õéä´é·´ÊͺËÒÊÀÒÇСÒ÷´Åͧ·ÕèàËÁÒÐÊÁ·Ñ駡ÒÃà»ÅÕè¹á»Å§ÊÒÃÅÐÅÒºѿà¿ÍÃì ÍسËÀÙÁÔáÅÐÃÐÂÐàÇÅÒ·ÕèãªéºèÁ (äÁèä´éáÊ´§¢éÍÁÙÅ) ¾ºÇèÒÊÀÒÇСÒ÷´Åͧ·Õè´Õ·ÕèÊØ´¾ºÇèÒ GBR 12909 ãËé¤èÒ % inhibition µèÍ [³H]WIN35428 à·èҡѺ -15.4 «Ö觤èÒ % inhibition ·Õè¤ÇèÐà»ç¹µéͧÁÒ¡¡ÇèÒÃéÍÂÅÐ 50 ¨Ò¡¼Å¡Ò÷´Åͧ´Ñ§¡ÅèÒÇáÊ´§ÇèÒÊÀÒÇСÒ÷´Åͧ¢Í§¡ÒÃÇÑ´¤ÇÒÁªÍº¨Ñº¢Í§ºÒÃҤ͚ѺⴻÒÁÕ¹·Ãҹʻ͵àµÍÃìâ´Âãªéà·¤¹Ô¤ radioligand binding assay ÂѧäÁèàËÁÒÐÊÁ ¤³Ð¼ÙéÇԨѨ֧äÁèÊÒÁÒöÊÃØ»¼Å¤ÇÒÁªÍº¨Ñº¢Í§ºÒÃÒ¤ÍŵèÍâ´»ÒÁÕ¹·ÃҹʻÍÃìµàµÍÃìä´é  áÅÐà¹×èͧ¨Ò¡¡Ò÷ӧҹ¢Í§â´»ÒÁÕ¹·Ãҹʻ͵àµÍÃìµéͧÍÒÈÑ·Ñé§äÍÍ͹â«à´ÕÂÁáÅÐäÍÍ͹¤ÅÍäôìÃèÇÁ´éÇÂ㹡Òôٴ¡ÅѺⴻÒÁÕ¹à¢éÒÊÙèà«ÅÅì»ÃÐÊÒ·¡è͹¨Ø´»ÃÐÊÒ¹»ÃÐÊÒ· ´Ñ§¹Ñé¹»ÃÔÁÒ³äÍÍ͹´Ñ§¡ÅèÒÇã¹ÊÒÃÅÐÅÒºѿà¿ÍÃì·Õèãªé㹡Ò÷´Åͧ¨Ö§à»ç¹»Ñ¨¨ÑÂ˹Ö觷ÕèÊӤѭ㹡ÒÃÈÖ¡ÉÒ¤ÇÒÁ ªÍº¨Ñº¢Í§ºÒÃҤ͚ѺⴻÒÁÕ¹·Ãҹʻ͵àµÍÃì

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·Ãҹʻ͵àµÍÃì·Õèä´é¨Ò¡ÊÁͧ¢Í§Ë¹Ù¢ÒǺÃÔàdz striatum  áÅеÑÇÃѺⴻÒÁÕ¹ 2 ·Õèä´é¨Ò¡à«ÅÅìà¾ÒÐàÅÕé§ A9LhD2 â´Âãªéà·¤¹Ô¤ radioligand  binding assay  ¨Ò¡¼Å¡ÒÃÇԨѤÃÑ駹ÕéáÊ´§ãËéàËç¹ÇèÒºÒÃÒ¤ÍÅ·Õè¤ÇÒÁà¢éÁ¢é¹ 0.1, 1, 10 áÅÐ 100 äÁâ¤ÃâÁÅÒÃì äÁèÁÕ¤ÇÒÁªÍº¨Ñº¡ÑºµÑÇÃѺⴻÒÁÕ¹ 1  áÅеÑÇÃѺⴻÒÁÕ¹ 2  à¹×èͧ¨Ò¡ÊÀÒÇСÒ÷´Åͧ㹡ÒÃÇÑ´¤ÇÒÁªÍº¨Ñº¢Í§ºÒÃҤ͚ѺⴻÒÁÕ¹·ÃÒ¹

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