週期素依賴激脢第五型(Cdk5),它對於神經系統的發育十分重要。週期素依賴激脢第五型必須和p35或p39、它的神經活化物，共同引起它在神經系統的作用。例如神經突觸生長。除此之外，週期素依賴激脢第五型在許多方面扮演重要角色。如藥物成癮、神經傳導物質釋放、NMDA接受體的磷酸化。
本論文分成三部分,分別探討給予週期素依賴激脢第五型抑制劑－roscovitine對大鼠嗎啡止痛耐受性產生、急性發炎疼痛、以及藥物引起抽筋的影響。
第一部分討論週期素依賴激脢第五型抑制劑－roscovitine對嗎啡止痛耐受性產生的影響：嗎啡耐受性和delta FosB活化有關，Cdk5是delta FosB下游標的。於大鼠椎管中以嗎啡每小時五微克連續注射五天，產生止痛耐受性。同時於椎管中以roscovitine每小時一微克連續注射，可以顯著加強嗎啡的止痛效果、減低耐受性的產生。由此可知Cdk5的活化和嗎啡止痛耐受性的產生有關。
第二部分討論週期素依賴激脢第五型抑制劑－roscovitine對急性發炎性疼痛的影響：對大鼠後腳掌注射福馬林，因疼痛產生縮腳現象。此種疼痛反應包含兩部分，第一期疼痛反應持續約數分鐘、短暫緩解後，第二期疼痛反應持續約一小時。第一期被視為是福馬林直接的疼痛作用， 第二期則和中樞加強作用有關。注射福馬林三十分鐘前，分別在大鼠椎管中注射五十、一百、二百微克roscovitine，可以顯著減少第二期的縮腳現象，而二百微克roscovitine更可以顯著減少第一期的縮腳現象。由此可知Cdk5的活化和發炎疼痛及引起中樞疼痛加強作用有關。
第三部分討論週期素依賴激脢第五型抑制劑－roscovitine對藥物引起顳葉癲癇的影響：長期癲癇發作會提高Cdk5的活性，而週期素依賴脢第五型抑制劑－roscovitine可抑制NMDA對於海馬迴所引起長期增強作用。在藥物引起癲癇前三十分鐘，於大鼠腦室內注射一百微克roscovitine，可顯著降低因抽筋引起的死亡率。經一個月追蹤，注射組比未處理組明顯縮短水迷宮的脫逃時間（和空間記憶有關）。因此，Cdk5和癲癇致病機轉有關，並且其抑制可作為癲癇之治療。

Cyclin-dependent kinase-5 (Cdk5) was identified as a serine/threonine kinase that plays an important role in neuronal development. Association with one of the neuronal activators, p35 or p39, is required for Cdk5 to elicit its diverse effects in the nervous system, such as neurite outgrowth. In addition to these, increasing evidence suggests that Cdk5 also plays an important role in cocaine addiction, neurotransmitter release, NMDA receptor phosphorylation. This thesis is divided into three parts which deals with the effects of Cdk5 inhibitor－roscovitine on the morphine tolerance development, acute inflammatory pain, and pilocarpine-induced seizure respectively.
The first part explored the effect of Cdk5 inhibitior－roscovitine on the morphine antinociceptive tolerance development. Delta FosB activation is involved in morphine tolerance. Cyclin-dependent kinase- 5 (Cdk5) is found to be the downstream target of delta FosB. We examined the effects of the potent selective Cdk5 inhibitor－roscovitine on the development of antinociceptive tolerance of morphine. Tolerance was induced by continuous infusion of morphine 5 µg/hr intrathecally (i.t.) for 5 days. The effect of co-administration of roscovitine 1 µg/hr i.t. for 5 days was also examined. Roscovitine co-administration enhanced the antinociceptive effect of morphine in morphine tolerant rats. It also shift the morphine antinociceptive dose－response curve to the left during morphine tolerance induction, and reduced the increase in the ED50 of morphine two-fold. Collectively, these findings suggest Cdk5 modulation may be involved in the development of morphine tolerance and its inhibitor will enhance antinociceptive effect.
The second part discussed the roscovitine effect on acute inflammatory pain. Formalin injected into the rat hind paw will evoke flinching (consisting of an elevation and shrinking back of the injected paw), a reliable parameter of pain behavior. The nociceptive response to formalin occurs in a biphasic pattern: there isan initial acute period (phase 1), and after a short period of remission, phase 2 begins and consists of a longer period (1 hour) of sustained activity. The initial response was initially attributed to a direct algogenic effect of formalin, whereas phase 2 was associated with the central sensitization. In this study, the Cdk5 inhibitor－roscovitine was injected intrathecally to elucidate the mechanism of Cdk5 activation during formalin-induced hyperalgesia. The 50 ul of 5% formalin solution was used as the noxious stimulant. The rats were injected with 0, 50, 100, and 200ug roscovitine intrathecally thirty minutes before hind paw formalin injection. Intrathecal 200ug roscovitine injection attenuates the phase I flinch response. And intrathecal 50, 100, and 200ug roscovitine injection suppress phase II flinch response effectively. Roscovitine administration could effectively suppress the formalin-induced flinch behavior. This implies the activation of Cdk5 plays an important role in the sensitization after nociceptive stimulation.
The third part focus on the roscovitine effect on the pilocarpine induced seizure. Pilocarpine temporal lobe epilepsy model is widely used. Chronic electroconvulsive therapy could upregulate Cdk5 activity. Cdk5 inhibitor－roscovitine could suppress NMDA induced long-term potentiation in hippocampal slice. Intracerebroventricular injection of 100μg roscovitine 30 min before pilocarpine-induced epilepsy could significantly decrease the seizure-induced mortality ( 11% in roscovitine group VS 77% in control group). The escape latency, spatial memory impairment, in the pilocarpine-induced seizure group is significant longer than the roscovitine pretreatment group in the Morris water maze test after one month (p＜0.05). It is concluded Cdk5 may play an important in the pathogenesis of epilepsy. Therefore, Cdk5 inhibition may become another way for the epilepsy treatment.

Table of contents
Acknowledgement ……………………………………………………III
中文摘要……………………………………………………………..IV
Abstract ………………………………………………………………..V
Table of contents ……………………………………………………………….VII
List of tables and figures ……………………………………………………..VIII
Introduction
Overviews of Cdk5 …………………………………………………………………… 1
The Cdk5/p35 system: structural aspects …………………………………………….. 2
Functional organization of Cdk5 in cells …………………………………………….. 4
Cdk-5 interacting proteins ……………………………………………………………. 5
Regulation of the Cdk5－p35 complex ………………………………………………. 7
The Cdk5 in Alzheimer pathology …………………………………………………… 7
Cdk5 and DARPP-32 ………………………………………………………………… 11
Cdk5: Balancing Excitation and Inhibition ………………………………………….. 12
Reference …………………………………………………………………………….. 14
Part I : The effect of intrathecal Cdk5 inhibitor－roscovitine on morphine antinociceptive tolerance Sprague–Dawley in rats
Abstract ……………………………………………………………………………… 22
Introduction ………………………………………………………………………….. 23
Materials and Methods ………………………………………………………………..24
Results ………………………………………………………………………………...25
Discussion …………………………………………………………………………….26
Reference …………………………………………………………………………….. 28
Part II : The effect of intrathecal Cdk5 inhibitor－roscovitine on formalin-induced pain behavior in Sprague–Dawley rats
Abstract ……………………………………………………………………………….34
Introduction …………………………………………………………………………...36
Materials and Methods ………………………………………………………………..37
Results ………………………………………………………………………………...39
Discussion …………………………………………………………………………….39
Reference ……………………………………………………………………………...41
Part III: The effect of intracerebroventricular Cdk5 inhibitor — roscovitine on the pilocarpine seizure death and escape latency in Morris water maze in adult Sprague–Dawley rats
Abstract ………………………………………………………………………………..45
Introduction ……………………………………………………………………………46
Materials and Methods ……………………………………………………………….. 47
Results …………………………………………………………………………………48
Discussion ……………………………………………………………………………..49
Reference ………………………………………………………………………………53

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