Control of Fear/Defensive Behavior by Brain Derived Neurotrophic Factor

该项目的主要目标是了解海马体的变化如何导致病理性攻击。我们以前发现，限制在海马CA3区的脑源性神经营养因子（BDNF）的条件性敲除（KO）小鼠比野生型（WT）小鼠更具攻击性。这些动物的血清素（5-HT）水平降低，血清素受体3（5-HTr3）的表达和活性增加，5-HTr3的过度激活增加了野生型小鼠的攻击性。5-HT水平的降低似乎引起BDNF KO小鼠中5-HTr3的上调，因为长期给予氟西汀，其升高细胞外5-HT的浓度，使5-HTr3的表达和活性正常化，其还降低攻击性。在寻找由5-HTr3调节并可能与攻击性相关的海马的生理特性时，我们发现5-HTr3激动剂抑制卡巴胆碱对海马γ振荡的诱导。 在上一个财政年度，我们关注了两个问题：1）海马中BDNF的丢失如何降低5-HT的水平？2)通过5-HTr3抑制伽马振荡的机制是什么？ 为了解决第一个问题，我们首先检查了5-HT再摄取，这被认为是BDNF的影响，并发现KO和对照WT小鼠之间没有差异。这一结果表明，较低的5-HT释放可能是造成KO小鼠中5-HT浓度降低的原因。在真实的时间内精确测量5-HT释放仍然是一个技术挑战，因为释放部位是分隔的，并且不能容易地通过人造测量工具进入。为了避免这个问题，我们使用由5-HTr 3介导的快速5-HT传递作为5-HT释放的量度。5-HTr 3是唯一的离子型5-HT受体，它是一种阳离子通道，在5-HT释放后几毫秒内打开。通过通道的电流反映了由电刺激释放的5-HT的量。该通道在海马抑制性神经元（5-HT3细胞）的亚群中表达，所述亚群在含有GFP基因的转基因小鼠中容易鉴定，所述GFP基因在5-HTr 3a基因启动子的控制下。我们从海马5-HT 3细胞开始进行全细胞记录，初步数据表明BDNF KO小鼠的5-HT末端释放的5-HT少于WT对照组的末端。 为了解决关于5-HTr 3激活抑制γ振荡的机制的第二个问题，我们检查了5-HTr 3激动剂m-CPBG对5-HT3神经元的细胞特性和表达小清蛋白（PV）的中间神经元的振荡特性的影响，这是γ振荡的主要驱动力。我们发现m-CPBG通过增加后超极化（AHP）降低5-HT 3细胞的兴奋性和放电频率。这种增加AHP介导的钙激活的小电导钾（SK）通道，这是激活的5-HTr3介导的去极化和钙离子内流的结果。与此同时，m-CPBG衰减PV-中间神经元的抑制电流，并增加其放电，这变得更加随机，与振荡相位的耦合更小。基于这些观察结果，我们提出，激活5-HTr 3干扰γ振荡衰减抑制输入从5-HT3细胞PV中间神经元和增加随机发射的PV细胞。 我们未来的目标是了解海马的振荡活动可能与攻击性，以及5-HTr 3是否通过改变海马的振荡来调节攻击性。

The main goal of this project is to understand how changes in the hippocampus may cause pathological aggression. We have previously found that mice with the conditional knockout (KO) of Brain Derived Neurotrophic Factor (BDNF) restricted to the hippocampal area CA3 are more aggressive than their wild type (WT) counterparts. These animals had reduced levels of serotonin (5-HT) and increased expression and activity of the serotonin receptor 3 (5-HTr3), and over-activation of 5-HTr3 increases aggression in wild type mice. The decrease in the levels of 5-HT appeared to cause the up-regulation of 5-HTr3 in BDNF KO mice, because chronic administration of fluoxetine, which elevates concentration of extracellular 5-HT, normalized expression and activity of 5-HTr3, it also reduced aggression. In search for physiological properties of the hippocampus that are regulated by 5-HTr3 and possibly correlate with aggression, we found that 5-HTr3 agonist suppresses induction of hippocampal gamma oscillations by carbachol. During the last fiscal year, we focused on two questions: 1) How loss of BDNF in the hippocampus reduces levels of 5-HT? 2) What mechanisms underlie suppression of gamma oscillations through 5-HTr3? To address the first question, we first examined 5-HT reuptake, which is thought to be influenced by BDNF, and found no difference between KO and control WT mice. This result suggested that lower 5-HT release could be responsible for the reduced 5-HT concentration in KO mice. Accurate measurement of 5-HT release in real time remains a technical challenge, because the release sites are compartmentalized and cannot be readily accessed by a human-made measuring tool. To circumvent this problem, we used fast 5-HT transmission mediated by 5-HTr3 as a measure of 5-HT release. 5-HTr3 is the only ionotrophic 5-HT receptor, which it is a cation channel that opens within milliseconds upon 5-HT release. The current across the channel reflects the amounts of 5-HT released by an electrical stimulus. The channel is expressed in a subpopulation of hippocampal inhibitory neurons (5-HT3 cells), which are readily identifiable in transgenic mice containing GFP gene under the control of 5-HTr3a gene promoter. We began whole cell recordings from hippocampal 5-HT3 cells and preliminary data suggest that 5-HT terminals in BDNF KO mice release less 5-HT than the terminals in WT controls. To address the second question about mechanisms whereby 5-HTr3 activation suppresses gamma oscillations, we examined effects of 5-HTr3 agonist m-CPBG on the cellular properties of 5-HT3 neurons and oscillatory properties of parvalbumine (PV) expressing interneurons, which are the main driving force for gamma oscillations. We found that m-CPBG reduced excitability and firing frequency of 5-HT3 cells by increasing afterhyperpolarization (AHP). This increase in AHP was mediated by calcium-activated small conductance potassium (SK) channels, which were activated as a result of 5-HTr3-mediated depolarization and calcium influx. At the same time, m-CPBG attenuated inhibitory currents in PV-interneurons and increased their firing, which became more random and less coupled to the oscillation phase. Based on these observations, we propose that activation of 5-HTr3 interferes with gamma oscillations by attenuating inhibitory input from 5-HT3 cells to PV interneurons and by increasing random firing of PV cells. Our future goals are to understand how oscillatory activity of hippocampus may relate to aggression and whether 5-HTr3 modulates aggression by changing hippocampal oscillations.