Development Of Layer 1 Neurons In The Mouse Neocortex And The Function Of GABAergic Pioneer Neurons In The Developing Neocortex

Layer 1 is the outermost layer of the cerebral cortex and among the earliest formed layers of the neocortex. Layer 1 neurons play crucial roles in neocortical development, synaptic integration and information processing. Although extensive studies have characterized the properties of layer 1 neurons in the mature neocortex, it remains unclear the postnatal development of layer 1 neurons. In this study, we systematically investigated the developmental profile of layer 1 neurons during the first 2 postnatal weeks using immunohistochemistry, whole-cell patch-clamp recording, single-cell reverse transcription-polymerase chain reaction (PCR) and cell three-dimensional reconstruction. There are only two basic neuronal cell types in layer 1:Cajal-Retzius cells and y-aminobutyric acid (GABA)-ergic interneurons. The innovation findings of this project included:(1) The population of layer 1 neurons underwent dynamic developmental changes. About 96% of Cajal-Retzius cells died during the first two weeks after birth. The density of interneurons only decreased in the first postnatal week, and then remained stable. (2) Cajal-Retzius cells underwent two different morphological transformation stages. The rapid morphological maturation completed before the postnatal day 5 (P5), then degeneration started after P5. (3) The majority of GABAergic interneurons showed clear expression of at least 1 of the 6 distinct neurochemical markers, including Reelin, GABA-A receptor subunit delta (GABAaRδ). neuropeptide Y, vasoactive intestinal peptide (VIP), calretinin, and somatostatin after the first postnatal week. According to the expression of neurochemical markers, we divided the interneuron into six subtypes. In this study, we also examined the dynamic development of densities, collocation and location of these subtypes. (4) According to firing pattern, layer 1 interneurons can be divided into 2 groups:late-spiking (LS) and burst-spiking (BS) neurons. Compared with the frequency of mRNA of neuro-markers between the two groups, LS neurons preferentially expressed GABAARδ, whereas BS neurons preferentially expressed VIP. (5) We simultaneously analyzed the development of morphological and physiological properties of LS and BS neurons. Both LS and BS neurons exhibited a rapid active electrophysiological development during the first postnatal week, and passive electrophysiological and morphological development sustained to the end of the second postnatal week. In addition, despite the fine difference, there were no significant difference between the contemporary general morphological properties of LS and BS neurons. Therefore, the electrophysiological properties of an interneuron is not simply defined by its morphology.In summary, we systematically and quantitatively examined the early postnatal development of layer 1 neurons in this study. Cajal-Retzius cells gradually disappeared and underwent morphological degeneration after birth. The anatomical-electrophysiological-molecular properties of layer 1 interneurons exhibited a rapid development during the first two postnatal weeks. Our results not only enrich the knowledge of layer 1 neurons, but also reveal the potential role of layer 1 neurons in the establishment, maturation and regulation of neuronal circuits.Brain function executes through the coordinated activation of neuronal assemblies.During early postnatal development, neuronal networks produce various forms of spontaneous patterned activity that provide key signals for synaptic and circuit maturation. In both hippocampus and neocortex, coordinated activity can emerges by chemical synapse-driven. Extensive studies have revealed the graph theory of developing hippocampal networks, which follow a scale-free topology and include functional hub neurons. Hub neurons are shown to be exclusively early-bom GABAergic(pioneer intemeuron) and orchestrate synchronization in developing hippocampal networks. However, little is known about the graph theory of developing neocortex network and the role of pioneer intemeurons in the network. Tg circumvent this conundrum, we used "genetic fate mapping" that allows for the selective labeling of pioneer GABAergic neurons based on their place and time of origin. We found that cortical pioneer neurons preferred to locate in deeper layers. 71.3% and 18.8% of the cortical pioneer neurons expressed somatostatin(SOM) and parvalbumin(PV),-v respectively. Using functional multi-neuron calcium imaging(fMCI),we found that developing neocortex networks followed a scale-free topology and were existence of functional hub neurons. In addition,combining fMCI with whole cell recording,we demonstrated that pioneer intemeuron form a subpopulation of hub neurons.Perturbation of a single hub neuron influenced the entire network dynamics. These findings establish a centol role for GABAergic neurons in shaping developing neocortex networks and help provide a conceptual framework for studying neuronal synchrony.