In Vitro Synapse Development in the Cultured Neuronal Network Formed from Dissociated Chick Forebrain Neurons

The formation of in vitro synapses and the confirmation of their function in the cultured neuronal network formed from dissociated chick forebrain neurons (FBNs) were first studied in 1981 and 1993 by two different research groups. Since then, no more progress has been seen in the area, possibly due to the difficulty in maintaining a lifespan of the culture longer than 14 days. We have reported previously our successful establishment of the culture using high plating density of 2,000 cells per mm 2 on the microelectrode array (MEA) platform that can achieve a lifespan of several months in culture. This abstract presents our findings on the early development of the structure and the function of the synapses during the first three weeks in culture. Chick FBNs were cultured on costar plates and MEA chips at the same time and with the same plating densities. Immunocytochemistry was used to examine the density/distribution of the synapses of the neuronal network on costar plates over time. MEA was used to record spontaneous spiking activity (SSA) from the neuronal network on its surface, which demonstrates successful network‐wide synaptic transmission. Results show that 1) the development of synaptic structure and function in vitro is faster than reported in vivo; 2) synapses function right after their formation as indicated by the rapid increase in the number of active channels net‐wide, with the peak of the active channel count appearing at 12 days in vitro (DIV) at our plating density; 3) SSA recorded in active channels shows a well‐coordinated firing pattern shortly after these channels became active; and 4) active channel development is faster in the presence of 2% fetal bovine serum before peak active channel count was reached. These findings suggest that the early synapse development characterized in this study can be used as a convenient non‐mammalian model for a wide spectrum of studies with various research purposes, such as assessing the effects of pharmacological agents or neurotoxic chemicals on the structure and function of the synapses. Support or Funding Information This work was partially supported by funding from the National Institutes of Health through SC COBRE (P20RR021949), the National Natural Science Foundation of China (No. 31070847 and 31370956), the Strategic New Industry Development Special Foundation of Shenzhen (No. JCYJ20130402172114948), and Guangdong Provincial Department of Science and Technology, China (2011B050400011). 1Confocal images showing positive staining of synapses in a developing E8 chick‐FBN‐culture at 16 days in vitro (DIV). A: positive staining of the post‐synaptic marker protein MAP2 (green) distributed in neuronal soma and dendrites; B: positive staining of the pre‐synaptic marker Synapsin I (red) in a dot pattern; C: a merged image of A and B where yellow dots represent synapses.2Phase‐contrast images confirming the synapse identities in the C‐FBN culture by co‐staining two pre‐synaptic markers. A: positive staining of Synapsin I (red); B: positive staining of Synaptophysin (green); and C: co‐staining of Synapsin I and Synaptophysin (yellow).3Synaptogenesis illustrated by an increase in Synapsin I positive staining. A: at 4 DIV, see positively stained soma and some neurites where dots of Synapsin I are loose; B: at 7 DIV, see much more neurites and denser dots of Synapsin I; C: at 16 DIV, see densest dots of Synapsin I in soma and neurites that are merged.ACC development in NB and NB + during the first 3 weeks in vitro. ACC: active channel count; TCC: total channel count. N = 3 batches of dissections; n = 3 MEAs per batch for both NB group and NB + group. So, the maximum TCC for a particular DIV = (59 channels/MEA)*(3 MEAs)*(3 batches) = 531 channels. One‐tail Z test approximation was used to test whether the portion of ACC/TCC in NB + was significantly higher than in NB. (* p < 0.05; ** p < 0.01).