Intracellular chloride regulation in olfactory neurons

描述（由申请人提供）：缺乏检测气味的能力是一个非常真实的障碍。理解嗅觉系统的核心是确定输入，初级嗅觉感觉神经元（OSN）的功能。我们的长期目标是了解嗅觉神经元的功能。在环核苷酸门控通道（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 et al. 1996; Delay and Restrepo 1999）。由于Ca激活的Cl-通道是cAMP/CNG通道途径中唯一已知的下游元件，这表明Cl电流可以介导两种类型的反应。如果氯离子（Ecl）的逆转电位在各个OSN中不同，则这是可能的。因此，确定细胞内CI的水平，[CI]i，和[CI]i与气味刺激的动态变化是理解CI在气味转导中发挥的作用的关键。我们的一般假设是，细胞内CI-调节嗅觉神经元的功能是必不可少的。 目标1.在静止条件下和用气味刺激表征[Cl]i。我们的假设预测，氯离子水平将在个体OSN之间变化，并且细胞内氯离子的水平将决定OSN对cAMP介导的气味的响应。我们将使用CI敏感染料通过光学成像来表征分离的OSN中的静息[CI]i水平。 目标二。确定两种氯离子转运蛋白，Na/K/2CI协同转运蛋白的亚型NKCC 1和K/CI协同转运蛋白的亚型KCC 2是否在OSN中表达，以及是否存在局部表达。确定改变NKCC 1和KCC 2的活性对静息和气味刺激神经元中[CI]i的影响。第二个预测是，特定的Cl-协同转运蛋白在OSN中表达，改变这些转运蛋白的功能将改变[Cl]i的水平。我们将专注于NKCC 1和KCC 2，因为这些CI共转运蛋白已被证明对发育中的神经元中的[CI]i至关重要，并且如我们的初步数据所示，似乎存在于嗅觉组织中。 目标3：确定[CI]i变化与电生理反应之间的相关性。第三个有待检验的预测是，不同水平的细胞内氯化物将直接影响OSN对气味的电生理反应。我们将结合联合收割机光学测量[CI]i与膜片钳记录，以确定是否CI-的流量与OSN的电响应。

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.