Inflammation-induced changes in spinal GABA-A receptor signaling

描述(由申请人提供)：GABA-A受体(GAR)介导的对脊髓传入的突触前抑制对于建立伤害性阈值至关重要。在没有损伤的情况下，脊髓注射GAR拮抗剂会产生痛觉过敏和异位痛觉，而GAR激动剂是止痛剂。然而，我们最近证明，在存在持续性炎症的情况下，GAR信号发生了变化，以至于脊髓GAR的激活实际上有助于炎性痛敏：脊髓给药GAR拮抗剂逆转炎性痛敏，而GAR激动剂则加剧炎性痛敏。我们的初步数据表明，这种变化反映了GAR信号的突触前变化，包括不同的GAR受体亚基的出现和阴离子平衡电位(Eanion)的去极化变化。Eanion中的去极化转变可能使GAR的激活变得兴奋。然而，我们最近的数据也表明，咪达唑仑是一种苯二氮卓类受体激动剂，在炎症存在的情况下仍保持镇痛效果，这表明仅靠Eanion的去极化转变不足以解释炎症诱导的GAR信号转变。脊髓GAR信号的明显变化对1)我们对持续性疼痛的潜在机制的理解，2)一系列全身麻醉药的临床应用，如异氟醚、异丙酚和依托咪酯的临床应用，以及3)开发治疗疼痛的新的治疗干预措施具有深远的意义。因此，我们提出了在4个特定目标下描述的一系列实验，旨在确定在存在和不存在炎症的情况下脊髓GAR信号转导的机制。在特定的目标1中，我们将描述炎症引起的GAR介导的电流的生物物理性质和药理学的变化以及皮肤感觉神经元阴离子稳态的调节。在特定的目标2中，我们将描述炎症诱导的皮肤感觉神经元中GAR亚单位表达和分布的变化。在具体目标3中，我们将评估炎症诱导的GAR亚单位表达和细胞内阴离子稳态变化的功能后果。在特定的目标4中，我们将确定皮肤感觉神经元中特定的GAR亚单位在多大程度上介导炎症情况下GAR信号的前伤害反应的出现。在这4个目标下描述的实验，涉及从行为药理学到病毒载体介导的基因表达操纵的一系列方法，旨在测试持续炎症导致GAR信号变化的中心假设，该变化反映了初级传入神经元Eanion和GAR亚单位表达的组合变化。公共卫生相关性：初级传入神经元中央终末上的3-氨基丁酸-A(GABA-A)受体似乎在抑制急性疼痛和在有损伤的情况下维持持续性疼痛方面发挥关键作用。GABA-A受体信号转导变化的机制尚不清楚，但它对1)我们理解持续性疼痛的潜在机制，2)一系列全麻药的临床应用，如异氟醚、异丙酚和依托咪酯，以及3)开发治疗疼痛的新的治疗干预措施具有深远的意义。因此，以最小化全身麻醉药的有害后果和确定治疗干预的新靶点为最终目标，我们提出了一系列实验，旨在确定炎症存在时脊髓GABA-A受体信号转移的潜在机制。

DESCRIPTION (provided by applicant): GABA-A receptor (GAR) mediated presynaptic inhibition of afferent input to the spinal cord is critical for the establishment of nociceptive threshold. In the absence of injury, spinal administration of GAR antagonists produce hyperalgesia and allodynia whereas GAR agonists are analgesic. We recently demonstrated, however, that in the presence of persistent inflammation, there is a shift in GAR signaling such that activation of spinal GARs actually contributes to inflammatory hyperalgesia: spinal administration GAR antagonists reverse inflammatory hyperalgesia while GAR agonists exacerbate it. Our preliminary data suggest this shift reflects a presynaptic change in GAR signaling which includes the emergence of distinct GAR receptor subunits and a depolarizing shift in the anion equilibrium potential (Eanion). A depolarizing shift in Eanion may enable GAR activation to become excitatory. However, our recent data also indicate that midazolam, a benzodiazepine receptor agonist, retains analgesic efficacy in the presence of inflammation, indicating that a depolarizing shift in Eanion alone is insufficient to account for the inflammation-induced shift in GAR signaling. The apparent shift in spinal GAR signaling has profound implications for 1) our understanding of the underlying mechanisms of persistent pain, 2) the clinical use of an array of general anesthetics such a isoflurane, propofol and etomidate, and 3) the development of novel therapeutic interventions for the treatment of pain. Therefore, we have proposed a series of experiments described under 4 specific aims, designed to identify mechanisms underlying spinal GAR signaling in the presence and absence of inflammation. In Specific Aim 1, we will characterize inflammation-induced changes in the biophysical properties and pharmacology of GAR mediated currents and the regulation of anion homeostasis in cutaneous sensory neurons. In Specific Aim 2, we will characterize inflammation-induced changes in the expression and distribution of GAR subunits in cutaneous sensory neurons. In Specific Aim 3, we will assess the functional consequences of inflammation-induced changes in GAR subunit expression and intracellular anion homeostasis. And in Specific Aim 4, we will determine the extent to which specific GAR subunits in cutaneous sensory neurons mediate the emergence of pronociceptive actions of GAR signaling in the presence of inflammation. The experiments described under these 4 aims, involving an array of approaches ranging from behavioral pharmacology to viral vector mediated manipulation of gene expression, are designed to test the central hypothesis that persistent inflammation results in changes in GAR signaling that reflect a combination of changes in Eanion and GAR subunit expression in primary afferent neurons. PUBLIC HEALTH RELEVANCE: 3-aminobutyric acid-A (GABA-A) receptors on the central terminals of primary afferent neurons appear to play critical role both in the inhibition of acute pain and the maintenance of persistent pain in the presence of injury. The mechanisms underlying this shift in GABA-A receptor signaling remain to be identified, but it has profound implications for 1) our understanding of the underlying mechanisms of persistent pain, 2) the clinical use of an array of general anesthetics such a isoflurane, propofol and etomidate, and 3) the development of novel therapeutic interventions for the treatment of pain. Therefore, with the ultimate goals of both minimizing deleterious consequences of general anesthetics and identifying novel targets for therapeutic interventions, we have proposed a series of experiments designed to identify mechanisms underlying the shift in spinal GABA-A receptor signaling in the presence of inflammation.