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要旨:
Introduction
Here, we expressed genetically encoded fluorescent reporter iGluSnFR1 for extracellular glutamate (Glu) sensing and genetically encoded calcium indicator GCaMP6f for calcium sensing in both neurons and astrocytes, and applied two channel fiber optic recording system in combination with blood oxygenation level-dependent signal (BOLD) fMRI. This platform offers us a more direct interpretation of neuronal transient with fMRI, thus, would expand our understanding of the signal propagation through the neuron-glia-vessel network couple to BOLD fMRI signals.
Methods
All images were acquired with a 14.1 T/26cm horizontal bore magnet (Magnex), interfaced to an AVANCE III console (Bruker) and equipped with a 12 cm gradient set, capable of providing 100 G/cm with a rise time of 150 us (Resonance Research). A transreceiver surface coil was used to acquire fMRI images. fMRI scans with block design were performed using 3D Echo planar imaging sequence: TR, 1.5 s, TE,11.5 ms, 1.92X1.92X1.92 cm3, FOV, 48X48X48 matrix, 400X400X400 um3 spatial resolution. The reporter iGluSnFR and GCaMP6f were expressed by AAV5 virus in the two hemisphere forepaw somatosensory cortex (FP-S1) with Syn or GFAP promoter. Fiber optic (200 mm) was inserted into the area which expressed the cortex for fluorescent signal recording.
Results/Discussion
Neuronal calcium and Glu signals with simultaneous fMRI from the FP-S1 of two hemispheres were acquired, respectively. Evoked neuronal calcium and Glu spikes were shown to follow each electrical pulses (Fig 1A), while the Glu spikes have earlier onset time and faster time to peak response in comparison with neuronal calcium. Also, amplitude of the evoked Glu spike increased proportionally to the amplitude of BOLD signals as the function of the stimulation intensity (Fig. 1B). The simultaneous fMRI BOLD maps and the time course of BOLD signal were shown (Fig. 1C).
Similar to previous study2, the astrocytic calcium signal is an integrated unitary spike, which has slower onset than the Glu spikes(Fig. 2A). Interestingly, we also observed the baseline drop of the Glu signal during the stimulation, which shows earlier onset with extended longer tail than the astrocytic signal. Also noteworthy is that the BOLD signals detected from both hemispheres are similar to each other(Fig.2B).
Conclusions
Concurrent glutamate and calcium recording was established with the BOLD fMRI brain mapping in anesthetized rats. This platform would lead to a better understanding of neurovascular coupling through the neuro-glial-vascular network in the animal brain. Future study will further clarify the neurovascular coupling events in the neuro-glial-vascular network and specify the source for the Glu baseline drop of during stimulation.
