组胺及作用于GABAA受体的静脉麻醉药对腹外侧视前区神经元的调节作用研究

The ascending arousal system (AAS) is responsible for the maintain of wakefulness, which is largely originates from a series of well-defined cell groups with identified neurotransmitters, while the neurons control the onset of sleep are thought to be located in the ventrolateral preoptic nucleus (VLPO) in hypothalamus. The relationship between the VLPO and the major monoamine groups of AAS appears to be reciprocal. When VLPO neurons fire rapidly during sleep, they would inhibit the monoaminergic cell groups, thus disinhibiting and reinforcing their own firing. Similarly, when monoamine neurons fire at a high rate during wakefulness, they would inhibit the VLPO, thereby disinhibiting their own firing. This reciprocal relationship is similar to a type of circuit that electrical engineers call a "flip-flop". Among, the reciprocal inhibition between tuberomammillary nucleus (TMN) and VLPO is the most important one. Interestingly, previous studies found that the majority of VLPO neurons, which are also inhibited by noradrenaline (NA), are unaffected by histamine (Gallopin et al. 2000,2004), but our eletrophysiological experiments show that a bath application of histamine (100 μM, 6 min) to acute brain slices suppressed the NA-inhibited neurons, thus disinhibiting TMN neurons, this might be one of the most important mechanism of maintainning wakefulness by histamine. Our discovery made a further recruitment to the theory of "flip-flop", it was highly valued by Prof. Clifford B. Saper, and Dr. Anna Zecharia in her Perspective (Anna Zecharia. J Physiol 588: 4057,2010). By using techniques including measuring rat EEG/EMG, realtime PCR, western blot and brain slice whole cell patch-clamp methods, our current proposal is going to further study the influence of histamine and H1, H2 agonists to rat sleep-wake cycle, the affecting on action potential, spontaneous and evoked inhibitory postsynaptic currents (IPSCs) of NA-inhibited and NA-excited neurons in VLPO, and the most important of all, the reciprocal functions between these two types of neurons or within nearby NA-excited neurons. We are interested in researching the changes of extrasynaptic GABAergic tonic release in different phases of sleep-wake cycle, too. We also focus on the mechanism of intravenous anesthetics which have sites of action on the GABAA receptor to VLPO neurons. To characterize the functions of histamine and intravenous antsthetics in VLPO to regulate wake-sleep cycle, we try to explicit the regulation of NA-inhibited and NA-excited neurons and their interrelationship, to find out the different regulatory mechanism to VLPO in sleep or wake phase. We hope this can afford new insight to treat sleep disorder and developing further theoritical basis for anaesthetics using.

"flip-flop"理论被公认是睡眠-觉醒调控的核心理论，其中下丘脑乳头结节核（TMN）神经元和腹外侧视前区（VLPO）神经元之间的交互抑制最为关键。以往研究认为组胺对VLPO神经元无影响。我们的脑片膜片钳实验表明，100 μM组胺对VLPO有明显抑制作用，这可能是组胺维持大脑觉醒状态的重要机制之一。这一发现对上述理论作了补充，受到国际同行的高度肯定和大力推荐。本课题拟深入研究组胺及H1、H2受体激动剂对大鼠睡眠-觉醒周期影响，对VLPO两类主要神经元（局部和长投射至TMN）动作电位、自发和诱发抑制性突触后电流的作用及其相互影响，对突触外GABA扩布性（tonic）释放的改变，进而研究GABA受体类静脉麻醉药对VLPO神经元的作用，明确组胺及静脉麻醉药对VLPO两类神经元各自的调控作用与相互影响，睡眠或觉醒阶段对VLPO不同的调节机制，以期为治疗睡眠障碍和临床手术麻醉用药提供借鉴。

我们研究了组胺作用于腹外侧视前区对大鼠睡眠-觉醒周期的影响表明。通过脑立体定位、腹外侧视前区微量注射组胺，脑电图仪记录及分析睡眠、觉醒时相。结果表明组胺 (100 µM/0.2 µl/侧)显著减少大鼠睡眠，增加觉醒。表明组胺在腹外侧视前区对睡眠-觉醒周期发挥重要的调节作用，具有明显的促觉醒作用。文章已被《江汉大学学报（自然科学版）》所接收，拟于2015年第1期发表。.GABAA受体是目前公认的脊椎动物脑内抑制性神经递质的作用部位。许多静脉麻醉药的作用主要是通过GABAA受体实现的，但其具体的作用机制还不明了。在麻醉过程中，这些药物对VLPO的两类GABA能神经元会发挥何种影响？其作用机制如何呢？通过脑片膜片钳实验，我们的研究表明，丙泊酚（propofol）可兴奋VLPO的NA(-)神经元而减少GABA释放，其作用至少部分是由于其对NA(+)神经元的抑制作用而引起的。文章已经发表在SCI杂志《Anesthesia & Analgesia》, 117(2): 358-363。.另外，通过脑片膜片钳实验，我们还在大鼠内侧前额叶皮质脑片上记录了甘氨酸介导电流。其电流随甘氨酸浓度增加而增大。甘氨酸可逆性地抑制第3和第6层细胞动作电位，对后者的抑制作用更强。电流钳条件下，甘氨酸可产生超级化作用。我们的研究表明甘氨酸受体在此处参与抑制活动，以调节神经元活性。论文已经被SCI杂志《Biochemical and Biophysical Research Communications》所接收。.通过一年的课题研究，我们发现组胺及作用于GABAA受体的静脉麻醉药在睡眠－觉醒的各个环节均对VLPO有非常重要的作用，通过全方位观察其局部反应，及通过行为学、分子生物学及脑片膜片钳电生理各个层次的研究，对其调节机制有了更进一步的认识。本研究对睡眠障碍的理论机制有了进一步的补充，对治疗睡眠障碍的药物开发有一定的理论指导意义，同时也对麻醉用药的药物设计及新药开发可以提供一定的借鉴作用。.到目前为止，已公开发表SCI论文1篇；另有一篇SCI论文及一篇中文文章已被接收，即将于2015年发表。