Simultaneous GCaMP6-based fiber photometry and fMRI in rats

Abstract

Background: Understanding the relationship between neural and vascular signals is essential for interpretation of functional MRI (fMRI) results with respect to underlying neuronal activity. Simultaneously measuring neural activity using electrophysiology with fMRI has been highly valuable in elucidating the neural basis of the blood oxygenation-level dependent (BOLD) signal. However, this approach is also technically challenging due to the electromagnetic interference that is observed in electrophysiological recordings during MRI scanning.

New method: Recording optical correlates of neural activity, such as calcium signals, avoids this issue, and has opened a new avenue to simultaneously acquire neural and BOLD signals.

Results: The present study is the first to demonstrate the feasibility of simultaneously and repeatedly acquiring calcium and BOLD signals in animals using a genetically encoded calcium indicator, GCaMP6. This approach was validated with a visual stimulation experiment, during which robust increases of both calcium and BOLD signals in the superior colliculus were observed. In addition, repeated measurement in the same animal demonstrated reproducible calcium and BOLD responses to the same stimuli.

Comparison with existing method(s): Taken together, simultaneous GCaMP6-based fiber photometry and fMRI recording presents a novel, artifact-free approach to simultaneously measuring neural and fMRI signals. Furthermore, given the cell-type specificity of GCaMP6, this approach has the potential to mechanistically dissect the contributions of individual neuron populations to BOLD signal, and ultimately reveal its underlying neural mechanisms.

Conclusions: The current study established the method for simultaneous GCaMP6-based fiber photometry and fMRI in rats.

Keywords: Fiber photometry; GCaMP6; Rats; fMRI.

Figure 1

Schematic illustration of the setup of simultaneous calcium-based fiber photometry and fMRI. Center: Anesthetized rodent in bore of MRI tethered to optic patch cable. Right: Fiber photometry set-up. GCaMP is excited by a 473 nm laser chopped with an optical chopper reflected off a dichroic mirror (blue line). This signal is coupled into an optical patch cable using a microscope objective and a fiber launch. The emitted fluorescent GCaMP signal is then collected through the same optical apparatus, filtered by a GFP bandpass filter, and collected by a silicon photomultiplier (SiPMT) (green line). Top: The output of the SiPMT is amplified by a lock-in amplifier and digitized using a DAQ device. Visual stimulation is controlled by the same DAQ device to drive an LED light source. Left: Acquired data from simultaneous calcium-based fiber photometry and fMRI. Both calcium and BOLD time courses are obtained from a region of interest under the optic fiber. T1 weighted fMRI image shows implanted optic fiber with respect to a group functional activation map. Red line denotes visual stimulation of the contralateral eye.

Figure 2

Summary of experimental design.

Figure 3

Injection of AAV5.Syn.GCaMP6s into visually responsive superior colliculus. A, PSTH shows neural responses in medial superior colliculus during a 25ms light flash. Dashed line indicates 99% confidence limit. Mean waveform scale: 1ms, 500 mV. PSTH: 100 trials, 10 ms bin widths. B, Cytochrome oxidase processed coronal section through the superior colliculus. Inset shown in C. C, Fluorescent image of transfected neurons in the deep layers of the medial superior colliculus after injection of AAV5.Syn.GCaMP6s (Penn Vector Core). White arrow denotes injection area where visually responsive neurons were recorded in A.

Figure 4

A, Group functional activation map during visual stimulation (N=7). T values were thresholded at p<0.05 (FDR corrected). White arrow indicates implanted optical fiber. B, Averaged time courses of the BOLD and calcium signals around the tip of the optical fiber (5 rats, 2-sec stimulation).

Figure 5

Simultaneously acquired BOLD and calcium time courses in each individual rat. A, BOLD and calcium responses to single trials of visual stimulation. B–H, Averaged BOLD and calcium time courses for Rat 1 to 7, respectively. Green traces: calcium signals. Blue traces: BOLD signals. Shaded regions denote standard error of the mean. Red bars indicate visual stimulation periods. X axis: time in 0.1s. Left y axis: % change of BOLD signal. Right y axis: % change of calcium signal.

Figure 6

Repeated simultaneous measurement of BOLD and calcium signals. Green traces: calcium signals. Blue traces: BOLD signals. Shaded regions denote standard error of the mean. Red bars indicate visual stimulation periods. X axis: time in 0.1s. Y axis: % signal change.