In the past decade, resting-state fMRI (rsfMRI) has emerged as a valuable tool for mapping long-range brain functional networks¹. These functionally coupled networks echoed many known features of anatomical organization², but interpreting rsfMRI is complicated. Functional coupling changes dynamically³, suggesting that it is constrained, but not fully dictated, by anatomical connections. Furthermore, the low frequency coherent rsfMRI signals (<0.1Hz) do not match directly to the bandwidth of established neuronal oscillations^4-6, highlighting a gap in our knowledge regarding the neuronal basis of rsfMRI underlying long-range brain functional connectivity. Our recent preliminary study indicated that low frequency optogenetic stimulation in the dorsal dentate gyrus (DG) evoked long-range cortical responses⁷. Inspired by this, optogenetic stimulation and rsfMRI were combined here to investigate the neuronal basis of rsfMRI connectivity by probing alternations of brain functional connectivity before, during and after low frequency stimulation in dorsal DG.

Animal Preparation and Optogenetic Stimulation: AAV5-CaMKIIa::ChR2(H134R)-mCherry was injected into dorsal DG of adult male rats (n=24) (Fig. 1a). After 4 weeks, an optical fiber was implanted in the injection site (Fig. 1b). rsfMRI (n=18) and local field potentials (LFP) (n=6) experiments were conducted. Continuous 1Hz blue light stimulation (100ms pulse, 40mW/mm²) was delivered during acquisition. Opaque tape was used to ensure no light leakage causing visual stimulus.

rsfMRI Protocol and Data Analysis: rsfMRI data was acquired at 7T (Fig. 2a). Standard preprocessing followed by seed-based analysis (SBA) was applied to quantify rsfMRI connectivity between bilateral primary somatosensory (S1), auditory (A1) and visual (V1) cortices. Frequency spectra were plotted. LFP Protocol and Data Analysis: Electrodes were inserted to bilateral V1 (Fig. 2b). LFP correlation was calculated in different bands, namely slow-wave (0.01-1Hz), delta (1-4Hz), theta (4-8Hz), alpha (8-14Hz), beta (14-30Hz) and gamma (30-100Hz). Frequency spectra were plotted.

Figs. 1c-1d confirm ChR2-mCherry expression in targeted CaMKIIa neurons of dorsal DG. Figs 1e-1f demonstrate that optogenetic stimulation evoked spike and LFP responses at dorsal DG. Fig. 3 shows the bilateral S1, A1, and V1 functional connectivity maps (measured as correlation coefficient from SBA) before, during and after 1Hz optogenetic stimulation. Fig. 4 compares the correlation coefficients and rsfMRI spectra before, during and after stimulation in bilateral S1, A1, and V1 networks. Fig. 5 depicts the bilateral V1 LFP correlations and spectra.

During 1Hz optogenetic stimulation in dorsal DG, bilateral S1, A1 and V1 functional connectivity strengths increased (Figs. 3-4), as well as the coherence of slow-wave and delta oscillations in bilateral V1 (Fig. 5). Previous studies postulated that evoked responses in DG could propagate to the neocortex via enthorinal cortex and low frequency stimulation might facilitate hippocampal activities⁸, but no conclusive evidence was provided. Large-scale cortical activations recruited by low frequency optogenetic stimulation in DG revealed by our recent preliminary study⁷ and the functional connectivity enhancement demonstrated by our current study (Figs. 3-4) demonstrate that the evoked responses in DG can propagate to the neocortex and alter neocortical functional networks.

After 1Hz optogenetic stimulation, bilateral S1, A1 and V1 functional connectivity strengths were higher than the baseline (Figs. 3-4), as well as the coherence of slow-wave and delta oscillations in bilateral V1 (Fig. 5). Such functional connectivity enhancement indicated reorganization of brain functional networks that might arise from plasticity. This might suggest that low frequency stimulation has functional and therapeutic implications. It also suggested that low frequency stimulation in DG could initiate long-term potentiation in the neocortex, which can be associated to previous studies that showed long-term potentiation can be elicited by low frequency electrical stimulation in DG⁹.

In addition, changes in rsfMRI connectivity and LFP correlations indicated that the coherence in slow-wave and delta oscillations can contribute to rsfMRI connectivity (Figs. 3-5). In addition, rsfMRI signals and LFP oscillations contain larger contribution from low frequency components (~0.01Hz and ~1Hz, respectively) during and after stimulation (Figs. 4-5). These results supported previous studies, which postulated that rsfMRI signals are associated with low frequency oscillations^3,4. Nevertheless, the rsfMRI signals (<0.1Hz) did not fully match the bandwidth of slow-wave (<1Hz) and delta (1-4Hz) oscillations. This may suggest that these oscillations contribute to rsfMRI signals via cross-frequency coupling⁵.

In conclusion, low frequency optogenetic stimulation of dentate gyrus enhanced brain functional connectivity. This indicated that low frequency neuronal oscillations contribute and underlie the synchronized long-range rsfMRI brain functional networks. Furthermore, the results provided functional and therapeutic implications for future application of low frequency optogenetic stimulation. This study also highlighted the possibility of utilizing optogenetic manipulation to investigate the neuronal basis of rsfMRI functional connectivity.