Intracellular chloride regulation in olfactory neurons.

描述(由申请人提供)：缺乏嗅觉能力是一个非常现实的障碍。了解嗅觉系统的核心是确定输入，即初级嗅觉神经元(OSN)的功能。我们的长期目标是了解嗅觉神经元的功能。在循环核苷酸编码通道(CNG)敲除小鼠模型中进行的研究表明，CNG通道参与兴奋性和抑制性反应(Brunet，Gold等人)。1996年；Delay和Restrepo，1999年)。由于钙激活的CI通道是cAMP/CNG通道通路中唯一已知的下游元件，这表明CI电流可能介导了这两种类型的反应。如果在各个OSN中氯的反转电位(ECL)不同，这是可能的。因此，确定细胞内CI水平，[CI]i，以及气味刺激下[CI]i的动态变化对于理解CI在气味转导中所起的作用至关重要。我们的一般假设是，细胞内CI调节对嗅神经元的功能是必不可少的。 目的1.研究[CI]I在静息和气味刺激条件下的特性。我们的假设预测，氯离子水平将在不同的OSN中有所不同，细胞内氯的水平将决定OSN对cAMP介导的气味的反应。我们将通过使用CI敏感染料的光学成像来表征孤立OSN中的静息[CI]i水平。 目的2.确定两种氯离子转运体--Na/K/2CI共转运体NKCC1和K/CI共转运体KCC2在OSN中是否表达，以及是否有定位表达。确定改变NKCC1和KCC2活性对静息和气味刺激神经元[CI]i的影响。第二个有待检验的预测是，特定的氯转运体在OSN中表达，改变这些转运体的功能将改变[CI]i的水平。我们将重点关注NKCC1和KCC2，因为这些CI共转运体在神经元发育过程中对[CI]i至关重要，而且，正如我们的初步数据所示，似乎存在于嗅觉组织中。 目的3.确定[CI]i变化与电生理反应之间的相关性。有待检验的第三个预测是，不同水平的细胞内氯将直接影响OSN对气味的电生理反应。我们将结合[CI]i的光学测量和膜片钳记录来确定CI-的流量是否与OSN的电响应相关。

DESCRIPTION (provided by applicant): The lack of the ability to detect odors is a very real handicap. Central to the understanding of the olfactory system is determining how the inputs, the primary olfactory sensory neurons (OSNs) function. Our long-term goal is to understand how olfactory neurons function. Studies conducted in a cyclic nucleotidegated channel (CNG) knockout mouse model, suggest that the CNG channel is involved in both the excitatory and inhibitory responses (Brunet, Gold et al. 1996; Delay and Restrepo 1999). Since the Ca-activated CI- channel is the only known downstream element in the cAMP/CNG channel pathway, this suggests that the CI current may mediate both types of responses. This would be possible if the reversal potential of chloride (Ecl) was different in individual OSNs. Thus determining the level of intracellular CI, [CI]i, and the dynamic changes in [CI]i with odor stimulation is critical to understanding the role CI plays in odor transduction. Our general hypothesis is that intracellular CI- regulation is essential to the function of olfactory neurons. Aim 1. Characterize [CI]i under resting conditions and with odor stimulation. Our hypothesis predicts that chloride levels will vary among individual OSNs and that the level of intracellular chloride will determine the response of OSNs to cAMP mediated odors. We will characterize resting [CI]i levels in isolated OSNs with optical imaging using a CI sensitive dye. Aim 2. Establish whether two chloride ion transporters, an isoform of the Na/K/2CI cotransporter, NKCC1, and an isoform of the K/CI cotransporter, KCC2, are expressed in OSNs and if there is localized expression. Determine the effects of altering the activity of NKCC1 and KCC2 on [CI]i in resting and odor-stimulated neurons. The second prediction to be tested is that specific Cl- cotransporters are expressed in OSNs and altering the function of these transporters will change the levels of [CI]i. We will focus on NKCC1 and KCC2 since these CI co-transporters have been shown to be critical to [CI]i in developing neurons and, as shown in our preliminary data, appear to be present in olfactory tissue. Aim 3. Determine the correlation between changes in [CI]i and electrophysiological responses. The third prediction to be tested is that different levels of intracellular chloride will directly affect the electrophysiological response of OSNs to odorants. We will combine optical measurements of [CI]i with patch clamp recording to determine whether the flux of CI- is correlated with the electrical responses of the OSN.