Inflammatory injury-mediated synaptic plasticity in the periaqueductal gray

项目摘要 炎性损伤可对伤害性神经回路具有实质性的、持久的影响，并增强伤害性神经回路的功能。 外周感觉神经元突触到投射到中脑导水管周围灰质（PAG）的脊髓神经元上。 尽管这些发现表明炎症增强了PAG的兴奋性脊髓输入，但复合物和 PAG中的异质性神经回路差异性地影响疼痛传递。啮齿动物研究表明GABA- 和多巴胺能PAG神经元分别促进伤害感受和镇痛。我建议将脊髓 使用光遗传学输入到PAG，并确定对GABA能PAG神经元的上行输入是否 加强，从而减少兴奋的多巴胺能PAG的人口。此外，委员会认为， 大麻素可能通过PAG中的突触前抑制来促进镇痛， 炎症-在本提案中测试的可能性。此外，由于炎症损伤导致长期的 增强（LTP）的外周突触到脊髓投射神经元，这一建议将测试LTP在 自旋PAG电路最后，我将在体内使用这种光遗传学方法来确定是否抑制和激活 脊髓传入纤维在PAG后可分别减轻或加重炎性损伤后的疼痛。 目的1：确定炎性损伤是否加强了对GABA能PAG神经元的脊髓输入，从而减少了对GABA能PAG神经元的刺激。 输入到多巴胺能PAG神经元。我将在PAG中使用体外膜片钳记录来解决这个假设 来自福尔马林注射（炎性损伤）和对照动物的切片。基因修饰小鼠表达 通道视紫红质（Chr 2）在上行脊髓投影将允许选择性和时间精确 刺激PAG中的脊髓传入神经。我将标记GABA和多巴胺能PAG神经元，使用启动子- 特定病毒A）我将确定炎症是否影响突触前强度（释放部位的数量 和释放的可能性）、前馈抑制的幅度和脊髓输入对大麻素的敏感性， GABA能和GABA能PAG神经元。B）我将确定炎性损伤是否增强了脊髓对脊髓的输入。 PAG使用刺激方案和腺苷酸环化酶激活剂（毛喉素）产生LTP， 动物，并评估这种作用是否被炎性损伤所阻断。 目的2：确定抑制PAG的脊髓输入是否能减轻炎性疼痛，以及激活这些输入是否能减轻炎性疼痛。 可加重和/或复制炎性疼痛。这一目标将使用光学植入物在PAG区域， 基因修饰小鼠在上行脊髓传入中表达视蛋白，同时测量疼痛行为。A）、 表达盐视紫红质的小鼠将被用于确定抑制脊髓传入是否减少福尔马林诱发的脊髓损伤。 炎性疼痛。B）表达Chr 2的高保真ChETA变体的小鼠（以模拟爆发放电 脊髓-PAG投射神经元的模式）将用于确定是否激活脊髓中的上行终末。 PAG对福尔马林注射后的炎性痛行为有明显的抑制作用。最后，在受伤后， 解决后，我将确定是否可以通过激活PAG中的脊髓终末来恢复伤害性行为。

PROJECT SUMMARY Inflammatory injury can have substantial, lasting effects on nociceptive neurocircuits, and potentiates peripheral sensory neuron synapses onto spinal neurons projecting to the midbrain periaqueductal gray (PAG). Although these findings suggest that inflammation strengthens excitatory spinal input to the PAG, complex and heterogenous neurocircuitry in the PAG differentially affects pain transmission. Rodent studies suggest GABA- and glutamatergic PAG neurons facilitate nociception and analgesia, respectively. I propose to isolate spinal input to the PAG using optogenetics and determine if ascending inputs to GABAergic PAG neurons are strengthened, consequently reducing excitation of the glutamatergic PAG population. Furthermore, cannabinoids may promote analgesia via presynaptic inhibition in the PAG, which could be reduced by inflammation—a possibility tested in this proposal. Moreover, since inflammatory injury results in long-term potentiation (LTP) of peripheral synapses onto spinal projection neurons, this proposal will test for LTP in the spino-PAG circuit. Finally, I will use this optogenetic approach in vivo to determine if inhibiting and activating spinal afferents in the PAG after can alleviate or worsen pain after inflammatory injury, respectively. Aim 1: Ascertain if inflammatory injury strengthens spinal input to GABAergic PAG neurons, thereby reducing input to glutamatergic PAG neurons. I will address this hypothesis using in vitro patch-clamp recordings in PAG slices from formalin-injected (inflammatory injury) and control animals. Genetically modified mice expressing channelrhodopsin (Chr2) in ascending spinal projections will allow for selective and temporally precise stimulation of spinal afferents in the PAG. I will label GABA- and glutamatergic PAG neurons using promoter- specific viruses. A) I will determine if inflammation affects the presynaptic strength (number of release sites and probability of release), magnitude of feedforward inhibition, and cannabinoid sensitivity of spinal input to GABA- and glutamatergic PAG neurons. B) I will determine if inflammatory injury potentiates spinal input to the PAG using stimulation protocols and an adenylyl cyclase activator (forskolin) to generate LTP in control animals and assess if this effect is occluded by inflammatory injury. Aim 2: Determine if inhibiting spinal input to the PAG reduces inflammatory pain, and if activating these inputs can exacerbate and/or replicate inflammatory pain. This aim will use optical implants over the PAG region of genetically modified mice expressing opsins in ascending spinal afferents, while measuring pain behaviors. A) Mice expressing halorhodopsin will be used to determine if inhibiting spinal afferents reduces formalin-evoked inflammatory pain. B) Mice expressing the high-fidelity ChETA variant of Chr2 (to mimic the burst firing patterns of spino-PAG projection neurons) will be used to determine if activating ascending spinal terminals in the PAG worsens inflammatory pain behaviors after hindpaw formalin injection. Finally, after the injury has resolved, I will determine if nocifensive behaviors can be reinstated by activating spinal terminals in the PAG.