Inflammatory injury-mediated synaptic plasticity in the periaqueductal gray

导水管周围灰质炎症损伤介导的突触可塑性

• 批准号: 10285503
• 负责人: Chelsie L Brewer
• 金额: $ 6.86万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-30 至 2023-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10285503
• 关键词: Absence of pain sensation; Acute; Address; Adenylate Cyclase; Affect; Afferent Neurons; Agonist; Analgesics; Area; Behavior; Cannabinoids; Cells; Chemosensitization; Chronic; Complex; Control Animal; Development; Formalin; Forskolin; Frequencies; Future; Glutamates; Halorhodopsins; Hyperalgesia; Immune; Implant; In Vitro; Inflammation; Inflammatory; Injections; Injury; Intercept; Knowledge; Label; Left; Long-Term Potentiation; Measures; Mediating; Methods; Midbrain structure; Mus; Nerve; Neurons; Nociception; Opsin; Optics; Pain; Pain management; Pathology; Pathway interactions; Pattern; Peripheral; Phase; Play; Population; Probability; Process; Property; Protocols documentation; Public Health; Rodent; Role; Signal Transduction; Site; Slice; Spinal; Spinal Cord; Spinal cord posterior horn; Spinal nerve structure; Synapses; Synaptic plasticity; Testing; Variant; Virus; afferent nerve; anandamide; cannabinoid receptor; chronic pain; dorsal horn; gamma-Aminobutyric Acid; glutamatergic signaling; improved; in vivo; inflammatory pain; insight; midbrain central gray substance; neural circuit; neuronal circuitry; novel; optogenetics; pain behavior; pain processing; patch clamp; presynaptic; promoter; somatosensory; transmission process

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.

项目总结 炎症损伤可以对伤害性神经回路产生实质性的、持久的影响，并增强 外周感觉神经元与投射到中脑导水管周围灰质(PAG)的脊髓神经元突触。 尽管这些发现表明炎症增强了兴奋性脊髓对PAG的传入，但复杂的和 PAG中的异质性神经回路对疼痛传递有不同的影响。啮齿动物研究表明，GABA- 谷氨酸能PAG神经元分别促进伤害性感受和镇痛作用。我建议分离出脊椎 使用光遗传学对PAG的输入，并确定GABA能PAG神经元的上行输入是否 增强，从而降低谷氨酸能PAG群体的兴奋。此外， 大麻素可能通过抑制PAG中的突触前抑制来促进镇痛，这种抑制可以通过 炎症--在这项提案中测试了一种可能性。此外，由于炎症损伤会导致长期的 外周突触对脊髓投射神经元的增强(LTP)，这一提议将在 Spino-PAG电路。最后，我将在体内使用这种光遗传方法来确定抑制和激活 炎性损伤后PAG内的脊髓传入神经分别可减轻或加重疼痛。 目的1：确定炎症损伤是否增强了脊髓对GABA能PAG神经元的输入，从而减少了 谷氨酸能PAG神经元的输入。我将使用PAG的体外膜片钳记录来解决这一假说 福尔马林注射(炎性损伤)和对照动物的切片。转基因小鼠表达 上升脊髓投射中的通道视紫红质(ChR2)将允许选择性和时间上的精确 刺激PAG内的脊髓传入。我将用启动子标记GABA和谷氨酸能PAG神经元- 特定的病毒。A)我将确定炎症是否影响突触前强度(释放部位的数量 和释放的概率)、前馈抑制的大小和脊髓输入对大麻素的敏感性 GABA能和谷氨酸能的PAG神经元。B)我将确定炎症性损伤是否加强了脊髓对 使用刺激方案和腺苷环化酶激活剂(Forskolin)产生LTP的PAG作为对照 并评估这种影响是否被炎性损伤所阻断。 目的2：确定抑制脊髓对PAG的输入是否减少炎性疼痛，以及是否激活这些输入 可加重和/或复制炎症性疼痛。这个目标将在PAG区域使用光学植入物 转基因小鼠在上升的脊髓传入中表达视蛋白，同时测量疼痛行为。a) 表达卤视紫红质的小鼠将被用来确定抑制脊髓传入是否减少福尔马林诱发的 炎症性疼痛。B)表达高保真Cheta变体ChR2的小鼠(以模拟爆发发射 Spino-PAG投射神经元的模式)将被用来确定是否激活在 后爪注射福尔马林后，PAG加重炎性疼痛行为。最后，在受伤之后 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明示行为。