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: 10490262
• 负责人: Chelsie L Brewer
• 金额: $ 4.18万
• 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 2023-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10490262
• 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.

项目摘要 炎症性损伤可能会对伤害性神经循环产生巨大的影响，并具有潜在的损伤。 外围感觉神经元突触到投射到中脑周围灰色（PAG）的脊柱神经元上。 尽管这些发现表明注射强度对PAG的激发脊柱输入，但 PAG中的异质神经通路对疼痛传播的影响不同。啮齿动物研究表明GABA- 和谷氨酸能PAG神经元分别喜欢伤害性伤害感和镇痛。我建议隔离脊柱 使用光遗传学输入PAG，并确定上升输入到GABA能PAG神经元是否为 加强，因此减少了谷氨酸能PAG人群的兴奋。此外， 大麻素可以通过PAG中的突触前抑制来促进镇痛，这可以通过 炎症 - 在此提案中测试的可能性。而且，由于炎症损伤会导致长期 周围突触的增强（LTP）在脊柱投射神经元上，该提案将测试LTP Spino-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 PAG中的脊柱传入后会分别缓解炎症性损伤后的疼痛或更严重的疼痛。 目标1：确定炎症性损伤强度是否将脊柱输入到GABA能神经元中，从而减少 输入谷氨酸能PAG神经元。我将使用PAG中的体外补丁钳记录来解决这一假设 从福尔马林注射（炎症性损伤）和对照动物中切成薄片。表达转基因的小鼠 升级脊柱项目中的ChannelRhopopsin（CHR2）将允许选择性和临时精确 刺激PAG中的脊柱传入。我将使用启动子 - 特定病毒。 a）我将确定炎症是否影响突触前强度（释放位点的数量 和释放的概率），进料抑制的大小以及脊柱输入对大麻素的敏感性 GABA和谷氨酸能PAG神经元。 b）我将确定炎症损伤是否向脊柱输入 使用刺激方案和腺苷酸环化酶激活剂（Forskolin）的PAG来控制LTP 动物和评估是否会因炎症性损伤阻塞。 目标2：确定抑制脊柱输入对PAG是否减少炎症性疼痛，以及是否激活这些输入 会加剧和/或复制炎症性疼痛。此目标将在PAG区域的PAG区域使用 在测量疼痛行为的同时，在上升的脊柱传入中表达Opsin的转基因小鼠。一个） 表达卤代紫红质的小鼠将用于确定抑制脊柱传入是否会减少福尔马林诱发的 炎症性疼痛。 b）表达CHR2的高保真CHETA变体的小鼠（模仿爆发） Spino-Pag投影神经元的模式将用于确定是否激活上升的脊柱末端 PAG注射后炎症性疼痛行为恶化。最后，受伤后 解决了，我将通过激活PAG中的脊柱末端来确定是否可以恢复核心行为。