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现实的障碍。了解嗅觉系统的核心是确定输入，即初级嗅觉感觉神经元（OSNs）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电流可能介导这两种类型的反应。如果氯离子（Ecl）的逆转电位在单个osn中不同，这是可能的。因此，确定细胞内CI， [CI]i的水平以及[CI]i在气味刺激下的动态变化对于理解CI在气味转导中的作用至关重要。我们的一般假设是，细胞内CI调节对嗅觉神经元的功能至关重要。

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.