Functional Mapping Of The Entorhinal–Hippocampal Projection For Spatial Exploration

Objective: Entorhinal cortex?EC?and hippocampus are crucial for spatial learning and memory,exploration and navigation.The place cells in hippocampus and grid cells,head direction cells,border cells and speed cells in medial entorhinal cortex contribute to forming idiothetic location sensation that is the basis of spatial exploration and navigation.The connections between entorhinal cortex and hippocampus include the projection from EC layer ? neurons to granule cells in detate gyrus?DG?,from EC layer ? neurons to pyramidal cells in CA1 and from granule cells in DG to neurons in CA3 and then to CA1.The information synthesizes in CA1 and is subsequently transferred to the deep layer ? and ? of entorhinal cortex.Since entorhinal cortex has clear functional and morphological associations with hippocampus in spatial exploration and navigation,we aimed to identify the spatial exploration coding pattern by recording calcium signals of the axons projected from the layer ? of medial entorhinal cortex to DG.Methods:?1?AAV-Syn-GCaMP5 G was injected into the layer ? of MEC using stereotaxic location to infect the neurons for expressing Ca²⁺ indicator protein in axon projection from MEC? to DG.?2?After that,the optical fiber was implanted into the molecular stratum of DG to detect the calcium signals from axonal terminals of MEC? neurons.We recorded mice's freely exploring behavior and conducted contrast experiments in AAV-Syn-eGFP injected mice,and checked the blocking effects at the axonal terminals by injecting muscimol or TTX into layer ? of MEC.We also checked the reliability of the method of Ca²⁺ signal recording by optical fiber photometry system.?3?We identified the correlation between axonal terminals' Ca²⁺ signals and freely exploring by combining analysis of the recorded Ca²⁺ signals and exploring behaviors,comparing the Ca²⁺ signals of exploration and non-exploration such as running on treadmill,grooming and eating.We also tested the changes of exploring Ca²⁺ signals after blocking the visual input.?4?The exploring Ca²⁺ signals from four different sites at MEC?-DG axonal terminals were recorded using 4-channel optical fibers photometry system with a mouse and different axonal terminals areas were recorded with six mice.Finally,the specific f unctional map of MEC?-DG axonal terminals associated with spatial exploration encoding was drawn according to 24 sites of six mice which cover a large area of axonal terminals.Results:?1?The AAV mainly infected layer ? neurons of MEC and made the neuro n express a great deal of Ca²⁺ signals indicator protein?GCaMP5G?.Although layer ? neurons were also labeled,the number of labeled neurons by GCaMP5 G was much smaller than that in layer ?.The labeled axons also mainly projected to the molecular stri atum of DG but not to CA1.?2?The Ca²⁺ signals of MEC?-DG axonal terminals fired simultaneously with a rhythm when the animal was anesthetized but not when freely exploring.No Ca²⁺ signals like dynamics were detected on eGFP labeled mice.The axonal terminals Ca²⁺ signals were blocked after injecting TTX or muscimol into MEC superficial layers.TTX's blocking effect could not be removed.But when the muscimol was washed out,the Ca²⁺ signals can recover completely.The axonal terminals Ca²⁺ signals could be detected stably up to 14 days.?3?MEC?-DG axonal terminals Ca²⁺ signals and freely spatial exploration were highly correlated,and the latency between their onsets was about 318 ms.The amplitudes of Ca²⁺ signals during running on treadmill,grooming and eating were much lower than during spatial exploration.After the visual input was blocked,the amplitude of Ca²⁺ signals during exploration decreased significantly.The Ca²⁺ signals during exploration were much stronger for novel object than familiar object.This phenomenon also occurred when exploring with hunger physical condition as compared with satiety condition.?4?The 4-channel optical fibers photometry system could simultaneously detect Ca²⁺ signals from four different sites of axonal terminals of MEC?-DG projection.The Ca²⁺ signals from four sites activated with high synchronization.The functional encoding of MEC?-DG axonal terminals for spatial exploration had good space selectivity.And the ratio of hot axon terminals sites is around 50%.Conclusions:?1?We built an excellent functional labeling method of MEC?-DG pathway by injecting AAV-Syn-GCaMP5 G into MEC?.?2?A reliable method of detecting Ca²⁺ signals from MEC?-DG axonal terminals in freely exploring mice was built successfully.?3?MEC?-DG axonal terminals' Ca²⁺ signals that could be influenced by visual input were closely related to spatial exploration encoding.The spatial exploring Ca²⁺ signals could also be fluctuated by environmental factors and physical conditions.?4?Different sites of MEC?-DG axonal terminals encoded spatial exploration with highly synchronization.Around 50% of axonal terminals sites had obvious space selectivity on spatial exploration encoding and were distributed dispersedly.