Instituto de Biología Celular y Neurociencia "Prof. E. De Robertis" (IBCN), Facultad de Medicina, CONICET - Universidad de Buenos Aires, Calle Paraguay 2155 3er piso, 1121, Ciudad de Buenos Aires, Argentina.
Cátedra de Farmacología, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Buenos Aires, Argentina.
Mol Autism. 2021 Mar 6;12(1):23. doi: 10.1186/s13229-021-00428-8.
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.
自闭症谱系障碍（ASD）是一种突触病，其特征是特定区域的突触改变和神经炎症。在丙戊酸（VPA）模型中已经描述了海马突触的结构和粘附特征。然而，神经元和小胶质细胞对海马突触模式及其出现的时间过程的贡献仍然未知。
使用在胚胎第 10.5 天经腹腔注射 VPA（450mg/kg）或生理盐水（对照）的怀孕大鼠所生的雄性幼仔。作为自闭症样特征，评估了成熟度、探索性活动和社交互动。在出生后第 3 天和 35 天评估海马 CA3 区的突触、细胞粘附和小胶质细胞标志物。使用来自对照和 VPA 动物的海马神经元原代培养物研究突触特征和谷氨酸诱导的结构重塑。从小鼠原代培养的小胶质细胞中评估基础和刺激介导的反应性，这些小胶质细胞来自对照和 VPA 动物。
在 PND3，在 VPA 行为缺陷明显之前，VPA 动物的海马突触小泡蛋白免疫反应性和神经元细胞粘附分子（NCAM）与其多涎酸化形式（PSA-NCAM）之间的平衡得以保留，同时没有小胶质细胞增生。在 PND35，与行为缺陷的建立同时，VPA 大鼠的海马显示兴奋性突触较少，NCAM/PSA-NCAM 平衡增加，而没有小胶质细胞增生。体外突触发生初期，来自 VPA 动物的海马神经元培养物的突触突点数保持不变，但兴奋性突触较少，NCAM/PSA-NCAM 平衡增加，对谷氨酸诱导的结构突触重塑的抵抗力增强。在没有神经元的情况下分离自 VPA 动物并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反应性。结果表明，在没有胶质细胞的情况下，来自 VPA 动物的海马神经元反映了体内的突触模式，并表明在产前期间神经元被启动，而海马小胶质细胞没有内在改变。
我们的研究表明小胶质细胞的作用对于发展神经元改变或对抗海马中的神经元结果不是决定性的，并强调了海马神经元和结构可塑性在 VPA 大鼠模型中建立突触改变的关键作用。
