In-vivo detection of neuronal current remains a challenging and promising goal in fMRI. Up to present, progress has been made in phantom¹ and cell culture² studies, but no conclusive reports have been made for in-vivo studies^3,4. As neuronal current is usually comprised of a series of oscillatory waveforms rather than being a direct current, it is most likely to be detected using oscillatory current sensitive sequences. Spin-lock oscillatory excitation (SLOE) has been reported to be the most sensitive method up to date and showed sub-nanotesla level detection of oscillatory current in phantom experiment⁵. In this study, we explored the potential of using SLOE sequence to directly detect oscillatory neuronal current in vivo for the first time.

SLOE was implemented with a spiral readout for minimal TE (Fig. 1a). Phantom experiment was first performed with a single copper loop (diameter, 1.1 cm) immersed in a 3.0 mM NiCl₂ solution. A function generator was connected to the loop to generate a 100 Hz sinusoidal current with an amplitude of 0.5 nT at the center of the loop according to the Biot-Savart law. Optogenetic animal experiment was performed by first injecting 1 µl virus of AAV5-Syn-ChR2(ET/TC)-EYFP into V1 cortex of Sprague-Dawley rats (200-250 g) and letting it express for more than 7 weeks. Then optogenetically evoked oscillatory neuronal current could be evoked by delivering a series of 473 nm laser pulses at less than 80 mW/mm² irradiance above the cortex of viral injection (Fig. 3). Such an oscillatory neuronal current was expected to induce spin-lock excitation only if the oscillatory frequency equals the spin-lock intensity.

MR data acquisition was performed using a 7 T Bruker Biospec animal scanner. SLOE was used for phantom and in-vivo experiments with the same parameters: spin-lock time (T_SL) = 70 ms, spin-lock intensity (B_SL) = 100 Hz, TR/TE = 1000/5 ms, resolution = 0.47×0.47 mm², slice thickness = 1 mm. In in-vivo experiments, BOLD signal was acquired before and after the whole ncMRI acquisitions to confirm the existence of neuronal response, using a GE-EPI sequence with TR/TE = 1000/25 ms, FA = 55⁰ (Ernst angle) and identical geometry parameters with SLOE. The detailed stimuli pattern of ncMRI experiment is illustrated in Fig. 5a: light stimulation (4 ms, 100 Hz) is ON only during the spin-lock time in the first TR out of every 15 consecutive TRs, and polarity of the stimulation changes every 15 TRs. Acquisition for ncMRI is made in every first TR. After the data was 2D motion corrected and high-pass filtered at 0.006 Hz, GLM fitted analysis was performed to determine the activation map.

SLOE acquisitions in phantom are summarized in Fig. 1: with oscillatory currents of opposite polarities (Fig. 1a), the detected signal also showed opposite polarities inside and outside the loop (Fig. 1b). The expressions of ChR2-EYFP were verified with confocal images (Fig. 2a); their overlaying with NeuN (red marker) and DAPI (blue marker) demonstrates the specific expression in neuron cells (Fig. 2b). Fig. 3 shows the recorded optogenetically evoked oscillatory LFP signals at different stimulation frequencies (20 Hz, 50 Hz, 80 Hz), along with their spectrograms. BOLD activation is shown in Fig. 4, and such activation area was consistent among different rats. However, no consistent activation pattern was observed in SLOE detection with either polarity of stimulation as displayed in Fig. 5. In this work, SLOE, the most sensitive ncMRI method reported up to date was implemented on a high field 7 T scanner for in-vivo experiment. Thorough experiment design was performed including optimizations of imaging protocol and stimulation pattern, phantom verification of hypothesis, LFP verification of optogenetical stimulation, and BOLD verification of brain activation. However, SLOE detection in vivo did not lead to statistically significant results. Given the shown sub-nanotesla level of sensitivity in phantom, the main hurdle of SLOE in vivo is the vulnerability to field inhomogeneity, which should be the focus of technical refinement in the next stage.