Research On Morphological And Functional Integration Of Engrafted Astrocytes In Adult Mouse Cortex

Object: Astrocytes, the most widely distributed glial cells in the brain, make extensive contacts with both neurons and cerebral blood vessels and regulate blood flow and brain functions. The previous studies have proved that sensory stimulation is able to cause the responses in both neurons and astrocytes. On the other hand, astrocytes can regulate the neuronal activity by releasing transmitters, e.g. glutamate. Dysfunctions of astrocytes cause many neurological and psychiatric disorders in both humans and animals. Therefore, glia transplantation may be a potential therapy method to cure nervous system diseases. Indeed, previous studies have reported that transplantation of glial progenitor cells or astrocytes can effectively improve brain functions and behaviors in diseased animals. However, there is still no in vivo evidence to confirm whether transplanted astrocytes can synaptically integrate into the host neural circuits and fulfill basic brain functions. In our experiments, we studied the functions of the engrafted glial cells in adult mouse cortex in vivo at the single cellular resolution by two-photon imaging. Finally, using correlated light and electron microscopy(CLEM) technique, we also studied the ultrastructure of engrafted astrocytes integrated into host neural network.Method: ①Neural stem cells(NSCs) were isolated from the 14.5 d~15.5 d embryonic cortex, and were induced into glial progenitors and astrocytes. NSCs, glial progenitors and astrocytes derived from NSCs were observed by immunofluorescence and confocal calcium imaging approach, in order to detect their morphology and in vitro functions. ②Glial progenitors were transplanted into the cerebral cortex of adult mice. The morphology of the engrafted cells was detected by immunostaining and two-photon imaging at different weeks(4W, 8W, 12 W, 25 W, 45W) after glial progenitor transplantation. ③The Ca2+ responses of engrafted astrocytes to hindlimb stimulation were observed by in vivo two-photon imaging at different time points after transplantation. ④Using the correlated light and electron microscopy(CLEM) technique, we studied the subcellular structure between engrafted astrocytes and axon projections from cholinergic neurons of NBM.Result: ①NSCs were induced into glial progenitors by the differential medium(DMEM/F12+B27+10 ng/mL CNTF+30% FBS). Percentage of glial progenitors derived from NSCs was 87%. Percentage of astrocytes derived from glial progenitors was 83%. In addition, astrocytes derived from NSCs were able to respond to ATP or thapsigargin in vitro. ②Engrafted glial progenitors could differentiate into astrocytes in vivo 12 weeks after transplantation, and could survive for at least 45 W. ③ The spontaneous and stimulation- induced Ca2+transients of engrafted astrocytes could be detected at 12 W after transplantation. The response latencies and amplitudes were similar to those of host astrocytes. ④ Light microscopy results showed that α7-nACh Rs expression on the engrafted astrocytic processes were closed to the presynaptic cholinergic terminals in cortex projected from the cholinergic neuron in NBM. ⑤The electron microscopy results showed both “tripartite synapses” or “synapse-like” structures could be found between engrafted astrocytic processes and axon terminals projected from the neurons in NBM.Conclusions: ①The embryonic NSCs can be induced into mature astrocytes in vitro by CNTF and FBS. These astrocytes derived from NSCs can express ATP receptors and exhibit normal intracellular calcium stores. ② After transplantation of astrocytic progenitors in the cortex of adult mice, they can differentiate into mature astrocytes at 12 weeks after the transplantation. ③Engrafted astrocytes show spontaneous Ca2+ responses and can also respond to hindlimb stimulation, implying that engrafted astrocytes are functionally integrated into the host neural network. ④ “Tripartite synapses” and “synapse-like” structures can be formed between engrafted astrocytic processes and axon terminal projections from the cholinergic neurons of NBM. In summary, glial progenitors derived from NSCs can develope into mature astrocytes in vivo, and are fully integrated into the host neural network at structural and functional levels after transplantation.