Objective      To establish a method of recording Ca

2+

activity at axonal terminals of neural projection and then study the activity pattern of the pathway from the entorhinal cortex t

o the hippocampus of mice under anesthetized and freely behaving conditions.       Methods      AAV-Syn-GCaMP5G was injected into the medial entorhinal cortex (MEC) superficial layer of male C57BL/6 mice at age of 8~10 weeks. In 1 month later

optical fiber was implanted onto the stratum moleculare of dentate gyrus (DG) to record Ca

2+

activity at the axonal terminals under both anesthetized and freely behaving conditions. The AAV-Syn-eGFP injected mice were recorded as the control group. Ca

2+

activity dynamics at the axon terminal sites were compared before and after tetrodotoxin (TTX) was injected into the MEC superficial layers of the anesthetized mice. The mice in the control group were injected with artificial cerebrospinal fluid (ACSF).       Results      The axonal fibers projecting from the entorhinal cortex to DG were densely labeled by GCaMP5G. Ca

2+

signals from the axonal terminals can be recorded steadily under both anesthetized and freely behaving conditions by using optical fiber photometry. However

no signals were detected in eGFP-labeled mice. After injecting TTX into the superficial layers of MEC

the maximum amplitude of Ca

2+

signals within 100 s from axonal terminals descended to 12.6% of Ca

2+

signals recorded before injection. Compared with the ACSF-injected mice

TTX significantly decreased Ca

2+

activity at the axon terminals (P0.01). The Ca

2+

activity from the axonal terminals in the hippocampus exhibited unsynchronized pattern and higher amplitude in freely behaving condition as compared with the rhythmic-synchronized pattern under anesthetized condition.       Conclusion       Utilizing specific labeling of neuronal projection with GCaMP5G and optical fiber photometry

Ca

2+

signals from the axonal terminal sites of long-range projections can be recorded reliably under both anesthetized and freely behaving conditions. The Ca

2+

activity from the axonal terminals of entorhinal-hippocampal projec

tions may exhibit higher level of dynamics in freely behaving mice.