Functional role of the neuron-satellite glial cell unit in the autonomic nervous system

Abstract

The sympathetic and parasympathetic nervous system, constituting the autonomic nervous system, play a central role in maintaining homeostasis in our body by coordinating the functions of internal organs. While autonomic ganglia have traditionally been viewed as relays for neural signals from the brain stem and spinal cord to the effector tissues, emerging evidences suggests that ganglia internally process and integrate a variety of signals to modulate the functions of terminal effector organs. This highlights the importance of an interplay between individual cells within the ganglia for balanced autonomic function. Autonomic ganglia are composed of neurons and satellite glial cells (SGCs) that surround xiii them. The anatomically close proximity between these neurons and SGCs is believed to contribute to the regulation of homeostasis in autonomic ganglia through mutual neuroglial communication. While most of the research on autonomic ganglia has mainly focused on the structure and function of neurons, recently, many scientists have sought to elucidate the roles of SGCs. SGC is a major type of glial cell in the autonomic ganglia, found to wrap around the neuronal cell body. This unique organization of neuron-glial unit is well-noted. However, little is known about the functional roles of the autonomic neuron-SGC units. In this study, I established a culture system for partially dissociated neuron-SGC units from the superior cervical ganglia (SCG). The first part of the project involves identifying calcium (Ca2+) signaling machinery of store-operated Ca2+ entry (SOCE) and receptoroperated Ca2+ entry (ROCE) in sympathetic neurons and SGCs in responding to the various extracellular stimuli. The second part of the project aims to investigate purinergic communications between sympathetic neurons and SGCs, where a somatic ATP release may have a potential role. The rat SCG was isolated, and sympathetic neurons attached to SGCs were dissociated using a partial enzymatic digestion method. Immunohistochemical and immunofluorescent experiments confirmed the presence of sympathetic SGCs attached to the neurons. Subsequently, I investigated the components of SOCE and ROCE, critical for cellular Ca2+ homeostasis in rat sympathetic ganglia under normal and pathological states. Quantitative RT-PCR was employed to detect the expressions of Orai 1/2/3 channels, stromal interaction molecules 1/2 (STIM1/2), and the transient receptor potential cation channels 1/3/6 xiv (TRPCs) in sympathetic ganglia. Functional studies were performed using Ca2+ imaging technique with Fura-2/AM. Both neurons and SGCs exhibited SOCE when the internal storage of Ca2+ was depleted by cyclopiazonic acid (CPA) along with the removal of extracellular Ca2+. The subsequent recovery from the ER depletion upon the restoration of extracellular Ca2+ via SOCE was rapid. Intracellular Ca2+ imaging revealed that the magnitude of SOCE was significantly larger in the SGCs than in the neurons. Unlike neurons, the SOCE in the SGCs were accompanied with substantial Ca2+ oscillation. Orai1 and STIM1 were identified as the major components of SOCE machinery in sympathetic ganglia. SOCE was significantly suppressed by GSK7975A (1 μM), a selective Orai1 blocker, and Pyr6 (3 μM), a SOCE blocker. Acute inflammation was induced with lipopolysaccharide (LPS) to assess changes in the function of neuron-SGC unit. Glial fibrillary acidic protein (GFAP) and Toll-like receptor 4 (TLR4) were upregulated to indicate neuro-glial inflammation in sympathetic ganglia. Importantly, LPS attenuated SOCE by downregulating Orai1 and STIM1 expressions. This suggests that SOCE is highly susceptible to inflammation, potentially influencing sympathetic neuronal activity and thereby autonomic output. In an effort to characterize the ROCE components in sympathetic ganglia, TRPC3 and TRPC6 were further investigated. In this regard, carbachol and selective TRPC inhibitors, Pyr3 (TRPC 3 inhibitor, 10 μM), and SAR7334 (TRPC 6 inhibitor, 10 μM), were employed to identify the presence of TRPCs in SCG neurons and SGCs. Additionally, TRPCs, especially TRPC3, may also be a partial component of SOCE machinery as CPA-induced xv SOCE activity was reduced by lanthanum chloride (non-selective TRPC inhibitor, 50 μM) and Pyr3. In contrast to sympathetic SOCE profiles, Ca2+ signaling through TRPCs was significantly larger in sympathetic neurons compared to the SGCs. Collectively, these findings suggested that Ca2+ is mediated by Orai, STIM, and TRPC channels in sympathetic ganglia, potentially contributing to functions of sympathetic neuron-SGC units. Neuronal and glial Ca2+ signaling is critical for controlling sympathetic neural excitability and communications in neuron-SGC units. Adenosine triphosphate (ATP) is co-released with norepinephrine from postganglionic sympathetic nerve terminals, mediating fast excitatory synaptic transmission to various visceral tissues. In addition, ATP is also extrasynaptically released from autonomic neurons. To date, however, the functional significance of somatic ATP release in the sympathetic ganglia remains elusive. Upon depolarization of the SCG neurons by application of high potassium (K+ ), intracellular Ca2+ is significantly increased in the SGCs attached to the SCG neurons, but not in singly isolated glial cells. This strongly suggests that neuronal excitation causes a local release of mediator that selectively affects the attached SGCs. Furthermore, the extracellular application of high K+ significantly diminished the FM1-43-stained and quinacrine-stained vesicular puncta in the soma of SCG neuron, suggesting somatic ATP release. ATP luminescence assay was conducted to quantify ATP released from cultured sympathetic neurons. Upon high K+ stimulation, the extracellular ATP concentration was significantly increased to confirm the somatic ATP released from sympathetic neurons. The pretreatment of neurons with the voltage-gated calcium channel (VGCC) inhibitor, xvi cadmium chloride (Cd 2+ , 100 μM), and Ca2+ ion chelator, BAPTA (30 μM), hindered the Ca2+ influx from the external buffer upon neuronal depolarization, and no significant ATP release was observed. Next, the purinergic agonist, ATP (100 μM), and pan-purinergic antagonists such as suramin (P2 inhibitor, 30 μM) and PPADS (P2X blocker, 30 μM) were applied to characterize the purinergic Ca2+ signaling in sympathetic neurons and SGCs. Both inhibitors almost abolished the ATP-induced Ca2+ responses in sympathetic neuronSGC units. The screening of transcripts encoding purinergic receptors confirmed the presence of P2X4, P2X7, and P2Y1 receptors in rat SCG. Intracellular Ca2+ was increased in both neurons and SGCs after ATP (50 μM) stimulation. BzATP (selective P2X7 agonist, partial agonist for P2X4/P2Y1, 50 μM) induced activation of purinergic Ca2+ signaling which was differentially inhibited by A438079 (potent P2X7 inhibitor, 30 μM) in SGCs, and by MRS2179 (selective P2Y1 inhibitor, 30 μM) in sympathetic neurons. Based on the provided context, the allosteric modulators of P2X4 and P2X7 receptors, zinc chloride (Zn2+, 5 μM) and cadmium chloride (Cd2+, 20 μM), exhibited variable Ca2+ responses to ATP and BzATP in sympathetic SGCs. This suggests the potential presence of both heteromeric or homomeric P2X4/7 receptors in sympathetic SGCs. Consequently, this diversity in receptor composition contributes to the provision of a wide range of Ca2+ responses in SGCs when exposed to external stimuli. Taken together, these data suggested that the vesicular ATP released from the sympathetic neurons may trigger the activation of multiple candidate purinergic receptors in the SGCs through Ca2+ signaling. In conclusion, the unique anatomical arrangement suggests signal exchanges between xvii sympathetic neurons and SGCs. The sympathetic neurons and SGCs functionally express multiple Ca2+ signaling machineries, which are essential for mediating sympathetic neuroglial communication. 자율신경계를 구성하는 교감신경계와 부교감신경계는 내부 장기의 기능을 균 형있게 조절하여 우리 몸의 항상성을 유지하는데 중심적인 역학을 한다. 이러 한 단서들은 균형 잡힌 자율신경 기능을 수행하기 위해 신경절 내 개별 세포 간의 상호 작용의 중요성을 강조한다. 자율신경절은 뉴런과 이를 둘러싸고 있 는 위성교세포 (SGC)로 구성된다. 이들 뉴런과 위성교세포 사이의 해부학적 독 특한 구조는 상호 교신을 용이하게 하여 자율신경절의 항상성 조절에 기여하는 것으로 여겨진다. 본 연구에서는 교감신경절의 뉴런-위성교세포 단위를 배양할 수 있는 세포 배양 시스템을 개발하고 다양한 외부 자극에 반응하는 교감 신경 절 뉴런과 위성교세포의 칼슘 항상성에 기반하는 기능적 메커니즘을 연구했다. 쥐의 상경신경절(SCG)을 적출하여 SGC가 부착되어 있는 뉴런-위성교세포 유닛 을 분리했다. 면역조직화학 및 면역형광 실험을 통해 위성교세포를 확인 후 정 - 156 - 량적 RT-PCR을 사용하여 교감신경절에서 Orai1 채널, STIM1 및 TRPC 1/3/6의 발 현을 확인했다. 기능적 연구는 Fura-2/AM을 이용한 칼슘 이미징 기술을 응용하 여 수행했다. CPA에 의해 ER내 Ca2+이 고갈될 때 뉴런과 유선교세포에서 모두 SOCE을 확인했으나 세포내 Ca2+ 유입은 뉴런보다 SGC에서 훨씬 더 크다는 것을 확인하였다. 또한, 뉴런과 달리 위성교세포의 SOCE는 큰 Ca2+ 진동을 동반했다. 뉴런-SGC 단위의 기능 변화를 확인하기 위해 동물 모델에서 LPS로 급성 염증을 유도했다. 염증 동물 모델의 상경신경절에서 GFAP와 TLR4가 많이 발현되어 있 음을 확인하였고는 반대로 Orai1 및 STIM1 발현이 감소됨을 확인하였다. 따라 서 SOCE는 염증에 매우 취약하며, 이는 교감 신경 활동에 영향을 미칠 수 있다. 종합하면, 위성교세포에서의 Ca2+은 교감신경절의 다양한 SOCE과 TRPC 채널들에 의해 매개되며 이는 자율신경 기능조절에 기여하는 것으로 추정된다. 뉴런과 위성성교세포의 Ca2+ 신호전달은 교감신경 흥분성을 제어하고 자율신경 미세환 경에서의 변화를 중재하는 데 중요하다. 80mM KCl을 사용하여 상경신경절 뉴런 을 탈분극시키면 세포내 Ca2+은 상경신경절 뉴런뿐만 아니라 뉴런 옆에 부착된 비흥분성 위성교세포에서도 유의하게 증가한다. 이는 신경 흥분성이 뉴런에 부 착된 위성교세포에만 영향을 미칠 수 있는 중간 매개체의 국소 방출을 유발한 다는 것을 강력히 시사하며 본 연구에서는 교감 신경 뉴런에서 방출된 소포성 ATP가 Ca2+ 신호 전달을 통해 뉴런에 단단히 부착된 위성교세포의 퓨린성 수용 체 P2X4와 P2X7의 활성화를 유발할 수 있음을 확인했다.