藥物成癮或依賴的個體即使在長期戒治後，若暴露於與藥物使用相關的環境，仍可能產生強烈的用藥動機（Craving），甚至造成戒癮失敗（Relapse）。因此，藥物強化行為及產生欣快感的作用，與其使用藥物環境線索連結所形成的長期記憶是濫用藥物的一個重要因素。我們運用兩種心理運動刺激劑（甲基安非他命和古柯鹼），在小鼠C57BL/6Nj品系中引發明顯且長期持續的場地偏好行為，作為藥物和使用環境造成長期記憶的實驗模型。本研究旨在探討在心理運動刺激劑形成了制約場地偏好記憶後，提取這種記憶所涉的胞內訊息傳遞分子。利用經過三次環境線索與甲基安非他命或古柯鹼配對形成並表現場地偏好的小鼠，其在不同的腦區域（包括前額葉、伏隔核、紋狀體、杏仁核、海馬回）中，和相同藥物劑量處理但不與環境線索配對的小鼠（不表現場地偏好的小鼠），以西方墨漬法，比較三種和長期記憶相關的訊息傳遞分子：蛋白質激酶A(PKA)，環腺嘌呤單磷酸反應單元結合蛋白質(CREB)，以及鈣・調鈣蛋白激酶第Ⅱ型 (CaMKⅡ)磷酸化與總量的差異。
實驗結果顯示（1）形成古柯鹼場地偏好的小鼠的CaMKⅡ磷酸化蛋白質量在杏仁核顯著的高於不表現場地偏好的小鼠。利用腦內注射抑制CaMKⅡ活化的KN-62到杏仁核，發現會抑制場地偏好的形成；（2）形成甲基安非他命場地偏好的小鼠較不表現場地偏好的小鼠在伏隔核的CREB總量有減少的趨勢，但在杏仁核呈現較高的PKA。神經核中蛋白質總量的改變但蛋白質的磷酸化程度卻不改變，暗示了兩個同時存在的可能性：（1）因蛋白質總量的調控需要時間，故總量的改變應和長期記憶的形成有關；（2）凡蛋白質總量改變但其磷酸化程度不變，表示該蛋白質的去磷酸化和記憶的提取有關。在古柯鹼形成的制約場地偏好記憶中，杏仁核內CaMKⅡ的磷酸化應和該記憶的提取有關。

　Drug-associated cues can maintain drug use and contribute to relapse even after long periods of abstinence. Thus, it is of importance to understand the underlying mechanism of the drug-associated memory for developing effective treatment of drug-addiction. Methamphetamine and cocaine, two abused drugs of choice in local area, possess reinforcing properties that can be used to support the approach responses to their associated environmental cues in a drug-induced conditioned place preference paradigm with rodent models. Many lines of evidence have demonstrated that methamphetamine- and cocaine-induced conditioned place preferences (CPP) are both long-term memories. This study aimed to determine the relevance of several intracellular signaling molecules, known for their involvement in various kinds of long-term memory, to the formation and retrieval of methamphetamine- or cocaine- induced conditioned place preference. Male C57BL/6Nj mice were divided into two groups. One group (Exp group) received a 3-day drug-place conditioning protocol, while the other group (Ctl group) received a similar drug dosing protocol in their home cage. These mice were killed thirty minutes after the 15-min CPP test. Animals in the Exp group exhibiting the drug-induced CPP and animals of the Ctl group exhibiting no evident place preference were used for further data analysis. Western blotting was applied to compare the levels of phosphorylated and total proteins of PKA, CREB, and CaMKⅡ in selected brain regions, including prefrontal cortex, nucleus accumbens, striatum, amygdala, and hippocampus between the Exp and Ctl groups. 　Our results demonstrated that (1) upregulation of accumbal PKA and downregulation of amygdaloid CREB could be responsible for the storage of the methamphetamine-induced conditioned place preference; (2) dephosphorylation of accumbal phosphorylated PKA and amygdaloid phophorylated CREB could be involved in the retrieval of the methamphetamine-induced conditioned place preference; and (3) elevated pCaMKⅡ expression in amygdala may take part in the retrieval of the cocaine-induced conditioned place preference and inhibition of CaMKⅡ activation by intra-amygdala KN-62 infusion was found to attenuate the cocaine-induced CPP.

1.　Abel T, Lattal KM. Molecular mechanisms of memory acquisition, consolidation and retrieval. Curr Opin Meurobiol 11:180-187, 2001.

2.　Arnsten AF, Ramos BP, Birnbaum SG, Taylor JR. Protein kinase A as a therapeutic target for memory disorders: rationale and challenges. Trends in molecular medicine 11(3):121-128, 2005.

3.　Barnes TD, Kubota Y, Hu D, Graybiel AH. Activity of striatal neurons reflects dynamic encoding and recoding of procedural memories. Nature 437:1158-1161, 2005.

4.　Beninger, RJ, Nakonechny, PL, Savina, I. cAMP-dependent protein kinase and reward-related learning: Intra-accumbens Rp-cAMPS blocks amphetamine-produced place conditioning in rats. Psychopharmacology 170:23-32, 2003.

5.　Carelli RM, Ijames SG. Selective activation of accumbens neurons by cocaine-associated stimuli during a water/cocaine multiple schedule. Brain Res 907:256-161, 2001.

6.　Dash PK, Karl KA, Colicos MA, Prywes R, Kandel ER. cAMP response element-binding protein is activated by Ca2+/calmodulin- as well as cAMP-dependent protein kinase. PNAS 88:5061-5065, 1991.

7.　Davis HP, Squire LR. Protein synthesis and memory: a review. Psychol. Bull 96: 518-559, 1983.

8.　Delfs JM, Schreiber L, Kelley AE. Microinjection of cocaine into the nucleus accumbens elicits locomotor activation on the rat. J Neurosce 10:303-310, 1990.

9.　Everitt, BJ, Wolf ME. Psychomotor stimulant addiction: a neural systems perspective. J. Neurosci., 22:3312-3320, 2002.

10.　Gehrke BJ, Harrod SB, Cass WA, Bardo MT. The effect of neurotoxic doses of methamphetamine on methamphetamine-conditioned place preference in rats. Psychopharmacology 166:249-257, 2003.

11.　Hagiwara M, Alberts A, Brindle P. transcriptional attenuation following cAMP induction requires PP-1 mediated dephosphorylation of CREB. Cell 70:105-113, 1992.

12.　Heged AN, DiAntonio A. ubiquitin and the synapse. Nat. Rev. Neurosci. 3:854-861, 2002.

13.　Jentsh JD, Olausson P, Nestler EJ, Taylor JR. Stimulation of protein kinase A activity in the rat amygdala enhances reward-related learning. Biol Psychiatry 52(2):111-8, 2002.

14.　Josselyn SA, Nguyen PV. CREB, synapses and memory disorders: past progress and future challenges. Curr Drug Targets CNS Neurol Disord. 4(5):481-97, 2005.

15.　Kida S, Josselyn SA, de Ortiz SP, Kogan JH, Chevere I, Masushige S, Silva AJ. CREB required for the stability of new and reactivated fear memories. Nat. Neurosci 5:348-355, 2002.

16.　Koh, MT, Bernstein IL. Inhibition of protein kinase A activity during conditioned taste aversion retrieval: interference with extinction or recondolidation of a memory? Neuroreport 14:405-407, 2003.

17.　Lee JL, Everitt BJ, Thomas KL. Independent cellular processes for hippocampal memory consolidation and reconsolidation. Science 304:839-843, 2004.

18.　Lien WH, Yeh TL, Yang YK, Cherng CF, Chen HH, Chen PS, Yu L. Cycloheximide enhances maintenance of methamphetamine-induced conditioned place preference. Chin J Physiol 47:23-30, 2004.

19.　Lisman J, Malenka RC, Nicoll RA, Malinow R. Learning mechanisms: the case for CaM-KII. Science 276:2001-2002, 1997.

20.　Maren S, Fanselow MS. Electrolytic lesions of the fimbria fornix, dorsal hippocampus, or entorhinal cortex produce anterograde deficits in contextual fear conditioning in rats. Neurobiol Learn Mem 67: 142-149, 1997.

21.　Martin SJ, Grimwood PD, Morris RGM. Synaptic plasticity amd memory. Annu. Rev. Neurosci. 23:649-711, 2000.

22.　Matthies H, Reymann KG. Protein kinase A inhibitors prevent the maintenance of hippocampal long-term potentiation. NeuroReport 4: 712-714, 1993.

23.　Mattson BJ, Bossert JM, Simmons DE, Nozaki N, Nagarkar D, Kreuter JD, Hope BT. Cocaine-induced creb phosphorylation in nucleus accumbens of cocaine-sensitized rats is enabled by enhanced activation of extracellular signal-related kinase, but not kinase A. J Neurochemistry 95:1481-1494, 2005

24.　Mayford A, Bach ME, Huang YY, Wang L, Hawkins RD, Kandel ER. Control of memory formation through regulated expression of a CaMKⅡ transgene. Science 274:1678-1683, 1996.

25.　Micheau J, Riedel G. Protein kinases: which one is the memory molecule? Cell Mol Life Sci. 55(4):534-48, 1999.

26.　Miller CA, Marshall JF. Molecular substrates for retrieval and reconsolidation of cocaine-associated contextual memory. Neuron 47:873-884, 2005.

27.　Mueller D, Perdikaris D, Stewart J. Persistence and drug-induced reinstatement of a morphine-induced conditioned place preference. Behav Brain Res 136:389-397, 2002

28.　Mulkey RM, Herron CE, Malenka RC. An essential role for protein phosphatases in hippocampal long-term depression. Nature 369:486-488, 1994.

29.　Nestler, EJ. Molecular basis of long-term plasticity underlying addiction. Nat. Rev. Neurosci. 2:119-128, 2001.

30.　Pierce RC, Quick EA, Reeder DC, Morgan ZR, Kalivas PW. Calcium-mediated second messengers modulate the expression of behavioral sensitization to cocaine. J Pharmacol Exp Ther. 286(3):1171-6, 1998.

31.　Rodrigues SM, Farb CR, Bauer EP, LeDoux JE, Schafe GE. Pavlovian fear conditioning regulates Thr286 autophosphorylation of Ca2+/calmodulin-dependent protein kinase II at lateral amygdala synapses. J Neurosci. 24(13):3281-8, 2004.

32.　Sheng M, Thompson MA, Greenberg ME. CREB: a Ca(2+)-regulated transcription factor phosphorylated by calmodulin-dependent kinase. Science 252:1427-1430, 1991.

33.　Tan SE. Impairing the amphetamine conditioning in rats through the inhibition of hippocampal calcium/calmodulin-dependent protein kinase Ⅱ activity. Neuropharmacology 42:540-547, 2002

34.　Tan SE, Liang KC. Inhibitory avoidance learning alters amygdala calcium/calmodulin-dependent protein kinase Ⅱ activity in rats. Brain Res. 711:234-240, 1997.

35.　Torii N, Kamishita T, Otsu Y, Tsumoto T. an inhibitor for calcineurin, FK506, blocks induction of long-term depression in rat visual cortex. Neuroscience Letters. 185:1-4, 1995.

36.　Wadzinski BZ, Wheat WH, Jaspers S. Nuclear protein phosphatase 2 A dephosphorylates protein A-phosphorylated CREB and regulates CREB transcriptional stimulation. Mol Cell Biol. 13: 2822-2834, 1993.

37.　Wang, JH, Stelzer A. Inhibition of phosphatase 2B prevents expression of hippocampal long-term potentiation. NeuroReport. 5:2377-2380, 1994.

38.　Yasoshima Y, Morimoto T, Yamamoto T. Different disruptive effects on the acquisition and expression of conditioned taste aversion by blockades of amygdalar ionotropic and metabotropic glutamatergic receptor subtypes in rats. Brain Res. 869:15-24, 2000.

39.　Yun IA, Fields HL. Basolateral amydala lesions impair both cue- and cocaine-induced reinstatement in animals trained on a discriminative stimulus task. J Neurosci 121:747-757, 2003.