Delayed crosstalk between astrocytes and neurons leads to depressive-like behaviors

The proper function establishment of nervous system is characterized by closely crosstalk among multiple cell types during the critical periods. Impeding or delaying the crosstalk results in abnormal neural functions and neurodevelopmental disorders. However, the lack of robust mouse models to study the crosstalk between astrocytes and neurons thus renders unclear the implications of impeding such interactions. Here we found that Egfr knockout led to the absence of astrocytes during the critical period of neuronal maturation, with recovery observed in adult mice, providing an exceptional opportunity to investigate the consequences of delaying crosstalk between astrocytes and neurons. We show that glial progenitor cells exhibit a rounded and smooth morphology, displaying few or no protrusions, in the absence of Egfr, which stands in stark contrast to the multi-protrusion morphology observed in the control cells. This phenomenon arises as a direct consequence of the impaired Egfr-pERK-Epb41l2 signaling axis, leading to delayed crosstalk between astrocytes and neurons. This delay results in reduced neuronal dendritic complexity and decreased neuronal excitability (due to damage to the Sema6a-Plxna2/4 receptor pair between astrocytes and neurons during the critical period), ultimately leading to the manifestation of depressive-like behaviors in adult mice. scRNA-Seq: FlashTag (CellTrace Yellow, Life Technologies, #C34567, 0.5 μl of 10 mM) was injected into the cortical lateral ventricle of control mice, hGFAP-Cre; Egfr-cko and hGFAP-Cre; MAP2k1/2-dcko mice at E18 and E16, respectively. P2 and E17 brains were harvested respectively and submerged in fresh ice-cold Hanks’ balanced salt solution (Gibco 14175-095). The dorsal cortices were cut into small pieces and dissociated into a single-cell suspension using a Papain Cell Dissociation Kit by following the manufacturer’s instruction. The tdTomato+ (tdT+) cells were captured and harvested through fluorescence-activated cell sorting (FACS). The Chromium droplet-based sequencing platform (10X Genomics) was used to generate scRNA-Seq libraries, following the manufacturer’s instructions (manual document part number: CG00052 Rev C). The cDNA libraries were purified, quantified using an Agilent 2100 Bioanalyzer, and sequenced on an Illumina Novaseq 6000.RNA-Seq: The P50 PFC tissue was collected from hGFAP-Cre; Egfr-cko and same-litter homozygotes . Direct-zol RNA Miniprep kit (Zymo, catalog #R2050) was used to isolate total RNA following the manufacturer’s instructions. The quality of RNA was assessed by measuring RNA concentration and purity, as well as evaluating RNA integrity (RIN > 7). After removing rRNA from the sample, purified mRNA was obtained. Following cDNA synthesis, a sample library is constructed and subjected to sequencing analysis. Gene expression levels were reported as fragments per kilobase of exon model per million mapped reads (FPKM). P value <0.05 grip was defined as differentially expressed genes.