在出生后的前两周,啮齿动物的神经内氯化物浓度逐渐降低,引起从去极化到超极化γ-氨基丁酸(GABA)反应的转变。出生后的GABA变化在啮齿动物模型中的神经发育障碍和人类患者中延迟。但是延迟的GABA转移对发育中的大脑的影响仍然不清楚。在这里,我们通过用VU0463271(氯化物出口商KCC2的特异性抑制剂)处理培养物一周,研究了出生后GABA移位延迟对6至7天大小鼠器官型海马培养物中网络发育的直接和间接后果。我们验证了VU处理延迟了GABA移位并保持GABA信号去极化直到体外第9天(DIV)。我们发现,在VU治疗后,DIV9兴奋性和抑制性突触的结构和功能发育未受到影响。根据以前的研究,我们观察到,GABA信号在对照和VU处理的产后切片中已经被抑制。令人惊讶的是,VU治疗结束后14天(DIV21),我们观察到CA1锥体细胞中自发抑制性突触后电流的频率增加,而激发电流没有改变。突触数量和释放概率不受影响。我们发现放射层中的树突靶向中间神经元具有升高的静息膜电位,与对照切片相比,锥体细胞的兴奋性较低。我们的结果表明,去极化GABA信号传导不会促进P7后的突触形成,并表明出生后细胞内氯化物水平以细胞特异性方式间接影响膜特性。意义陈述:在大脑发育过程中,神经递质GABA的作用从去极化转变为超极化。这种转变被认为在突触形成中起关键作用。在用于神经发育障碍的啮齿动物模型和人类患者中,延迟移位是常见的。但它对突触发育的影响仍然不清楚。这里,在器官型海马培养物中,我们将GABA移位延迟了一周,并仔细检查了回路发育的后果。我们发现延迟转移对突触发育没有直接影响,而是导致间接的,细胞类型特异性膜特性的变化。我们的数据要求仔细评估神经发育障碍中细胞兴奋性的改变。
During the first two postnatal weeks, intraneuronal chloride concentrations in rodents gradually decrease, causing a shift from depolarizing to hyperpolarizing GABA responses. The postnatal GABA shift is delayed in rodent models for neurodevelopmental disorders and in human patients, but the impact of a delayed GABA shift on the developing brain remains obscure. Here we examine the direct and indirect consequences of a delayed postnatal GABA shift on network development in organotypic hippocampal cultures made from 6- to 7-d-old mice by treating the cultures for 1 week with VU0463271, a specific inhibitor of the chloride exporter KCC2. We verified that VU treatment delayed the GABA shift and kept GABA signaling depolarizing until DIV9. We found that the structural and functional development of excitatory and inhibitory synapses at DIV9 was not affected after VU treatment. In line with previous studies, we observed that GABA signaling was already inhibitory in control and VU-treated postnatal slices. Surprisingly, 14 d after the VU treatment had ended (DIV21), we observed an increased frequency of spontaneous inhibitory postsynaptic currents in CA1 pyramidal cells, while excitatory currents were not changed. Synapse numbers and release probability were unaffected. We found that dendrite-targeting interneurons in the stratum radiatum had an elevated resting membrane potential, while pyramidal cells were less excitable compared with control slices. Our results show that depolarizing GABA signaling does not promote synapse formation after P7, and suggest that postnatal intracellular chloride levels indirectly affect membrane properties in a cell-specific manner.SIGNIFICANCE STATEMENT During brain development, the action of neurotransmitter GABA shifts from depolarizing to hyperpolarizing. This shift is a thought to play a critical role in synapse formation. A delayed shift is common in rodent models for neurodevelopmental disorders and in human patients, but its consequences for synaptic development remain obscure. Here, we delayed the GABA shift by 1 week in organotypic hippocampal cultures and carefully examined the consequences for circuit development. We find that delaying the shift has no direct effects on synaptic development, but instead leads to indirect, cell type-specific changes in membrane properties. Our data call for careful assessment of alterations in cellular excitability in neurodevelopmental disorders.