Autism spectrum disorders (ASD) are synaptopathies characterized by area-specific synaptic alterations and neuroinflammation. Structural and adhesive features of hippocampal synapses have been described in the valproic acid (VPA) model. However, neuronal and microglial contribution to hippocampal synaptic pattern and its time-course of appearance is still unknown. Male pups born from pregnant rats injected at embryonic day 10.5 with VPA (450 mg/kg, i.p.) or saline (control) were used. Maturation, exploratory activity and social interaction were assessed as autistic-like traits. Synaptic, cell adhesion and microglial markers were evaluated in the CA3 hippocampal region at postnatal day (PND) 3 and 35. Primary cultures of hippocampal neurons from control and VPA animals were used to study synaptic features and glutamate-induced structural remodeling. Basal and stimuli-mediated reactivity was assessed on microglia primary cultures isolated from control and VPA animals. At PND3, before VPA behavioral deficits were evident, synaptophysin immunoreactivity and the balance between the neuronal cell adhesion molecule (NCAM) and its polysialylated form (PSA-NCAM) were preserved in the hippocampus of VPA animals along with the absence of microgliosis. At PND35, concomitantly with the establishment of behavioral deficits, the hippocampus of VPA rats showed fewer excitatory synapses and increased NCAM/PSA-NCAM balance without microgliosis. Hippocampal neurons from VPA animals in culture exhibited a preserved synaptic puncta number at the beginning of the synaptogenic period in vitro but showed fewer excitatory synapses as well as increased NCAM/PSA-NCAM balance and resistance to glutamate-induced structural synaptic remodeling after active synaptogenesis. Microglial cells isolated from VPA animals and cultured in the absence of neurons showed similar basal and stimuli-induced reactivity to the control group. Results indicate that in the absence of glia, hippocampal neurons from VPA animals mirrored the in vivo synaptic pattern and suggest that while neurons are primed during the prenatal period, hippocampal microglia are not intrinsically altered. Our study suggests microglial role is not determinant for developing neuronal alterations or counteracting neuronal outcome in the hippocampus and highlights the crucial role of hippocampal neurons and structural plasticity in the establishment of the synaptic alterations in the VPA rat model.

中文翻译：

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从接受丙戊酸模型模拟体内突触模式的大鼠身上分离的海马神经元：自闭症谱系障碍早期发育过程中神经元启动的证据

自闭症谱系障碍 (ASD) 是突触病，其特征是区域特异性突触改变和神经炎症。海马突触的结构和粘附特征已在丙戊酸 (VPA) 模型中进行了描述。然而，神经元和小胶质细胞对海马突触模式的贡献及其出现的时间过程仍然未知。使用在胚胎第 10.5 天注射 VPA（450 毫克/公斤，腹腔注射）或生理盐水（对照）的怀孕大鼠出生的雄性幼崽。成熟、探索活动和社交互动被评估为类似自闭症的特征。在出生后第 3 天和第 35 天评估 CA3 海马区的突触、细胞粘附和小胶质细胞标记物。来自对照和 VPA 动物的海马神经元的原代培养物用于研究突触特征和谷氨酸诱导的结构重塑。对从对照和 VPA 动物分离的小胶质细胞原代培养物评估基础和刺激介导的反应性。在 PND3，在 VPA 行为缺陷明显之前，突触素免疫反应性和神经元细胞粘附分子 (NCAM) 与其多唾液酸化形式 (PSA-NCAM) 之间的平衡在 VPA 动物的海马体中保持不变，同时没有小胶质细胞增生。在 PND35，伴随着行为缺陷的建立，VPA 大鼠的海马体表现出较少的兴奋性突触和增加的 NCAM/PSA-NCAM 平衡，而没有小胶质细胞增生。来自培养的 VPA 动物的海马神经元在体外突触形成期开始时表现出保留的突触点数，但表现出较少的兴奋性突触以及增加的 NCAM/PSA-NCAM 平衡和对主动突触发生后谷氨酸诱导的结构性突触重塑的抵抗力。从 VPA 动物中分离并在没有神经元的情况下培养的小胶质细胞显示出与对照组相似的基础和刺激诱导的反应性。结果表明，在没有神经胶质细胞的情况下，来自 VPA 动物的海马神经元反映了体内突触模式，并表明虽然神经元在产前时期已准备好，但海马小胶质细胞并没有本质上的改变。