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Optogenetically-initiated low frequency dorsal hippocampal activity enhances resting-state fMRI connectivity and visual memory retrieval performance
Russell W. Chan1,2,3, Eddie C. Wong1,2, Alex T. L. Leong1,2, Xunda Wang1,2, Celia M. Dong1,2, Karim E. Hallaoui1,2, Lee W. Lim4, and Ed X. Wu1,2

1Laboratory of Biomedical Imaging and Signal Processing, The University of Hong Kong, Hong Kong, China, 2Department of Electrical and Electronic Engineering, The University of Hong Kong, Hong Kong, China, 3Neurology and Neurological Sciences, Stanford University, Stanford, CA, United States, 4School of Biomedical Sciences, The University of Hong Kong, Hong Kong, China

Synopsis

Our recent study demonstrated that low frequency optogenetically-initiated hippocampal activities enhances brain-wide resting-state fMRI connectivity. However, the behavioral consequence of such connectivity enhancement remains unknown. Since hippocampus is known to play a prominent role in memory, we assessed the effects of such connectivity enhancement on short-term and long-term memory. Our experimental results demonstrated that, while low frequency dorsal hippocampus stimulation enhanced interhemispheric fMRI connectivity (in hippocampus, V1, A1 and S1), it also improved the long-term visual memory by enhancing memory retrieval (in contrast to memory encoding) performance.

Purpose

Functional connectivity measured by resting-state fMRI (rsfMRI) has been suggested to be an expression of brain-wide network behavior underlying cognitive functions1,2. Our recent study demonstrated that brain-wide functional connectivity was enhanced during and after low frequency optogenetic stimulation in dorsal dentate gyrus3 (dDG), a sub-region of the dorsal hippocampus (dHP). However, the behavioral consequence of such functional connectivity enhancement remains unknown. dHP plays a prominent role in memory4,5, especially memory encoding6, consolidation7 and retrieval6,8. However, the contribution of low frequency hippocampal-cortical activity initiated in dDG to memory functions is still unclear. In this study, we utilized low frequency optogenetic stimulation of dDG to enhance brain-wide functional connectivity. We then applied novel-object recognition (NOR) tests9,10 to assess the effects of low frequency dDG/dHP activities on short-term and long-term memory, as well as memory encoding and memory retrieval.

Materials and Methods

Animal preparation: AAV5-CaMKIIα::ChR2(H134R)-mCherry was injected to dDG of adult male SD rats (Fig. 1). After 6-weeks, an optical fiber was acutely or chronically implanted to deliver optogenetic stimulation for rsfMRI experiments or NOR tests, respectively. Note that NOR tests were applied 2-weeks after chronic fiber implantation. Also note that sham animals with fiber implantation but without ChR2 injection were used as controls for NOR.

rsfMRI experiment and analysis: rsfMRI scans were performed before (PRE), during (DURING) and after (POST) optogenetic stimulation (n=18; Fig. 2). Note that DURING and POST were interleaved. Continuous 10-mins stimulation (473nm; 40mW/mm2; 1Hz; 10% duty cycle) was used to mimic low frequency hippocampal activities in dDG. rsfMRI data were acquired at 7T using GE-EPI (FOV=32×32mm2, matrix=64×64, α=50°, TE/TR=20/750ms). Standard preprocessing and seed-based analysis were applied to measure the bilateral functional connectivity in dorsal hippocampus (dHP), primary visual cortex (V1), primary auditory cortex (A1) and primary somatosensory cortex (S1).

Novel object recognition (NOR) behavioral tests and analysis: To assess low frequency dDG/dHP activities on general short-term and long-term memory, optogenetic animals (n=15) and sham controls (n=11) received low frequency (1 Hz) optogenetic stimulation for 10-mins prior to the start of acquisition, short-term memory and long-term memory phases (Paradigm-A; Fig. 3A). To further assess memory encoding and memory retrieval of long-term memory, optogenetic animals received stimulation prior to short-term memory phase (Paradigm-B, n=7; Fig. 3B) and long-term memory phase (Paradigm-C, n=9; Fig. 3C), respectively. In brief, animals were first habituated for 15-mins in the arena. The next day, animals were exposed to two identical objects for 3-mins during acquisition phase. 1-hr afterwards, animals were presented with the familiar object and a novel object for 3-mins to assess short-term memory. This assessment assumes animals would spend more time with the novel object if it could remember the familiar object. 24-hrs later, animals were again presented with one familiar and one novel object to assess long-term memory. Discrimination index11,12 (DI) was used to quantify the exploration preference for novel object. A positive DI indicates a preference for novel object.

Results and Discussion

Anatomical MRI scans and histology confirmed viral injection, fiber implantation, and ChR2-mCherry expression in dDG (Fig. 1). Interhemispheric functional connectivity of dHP, V1, A1 and S1 increased significantly DURING stimulation (Fig. 4; dHP: 37.3±7.4%, p < 0.01; V1: 55.6±11.5%, p < 0.01; A1: 44.2±6.9%, p < 0.05; S1: 43.3±9.0%, p < 0.01; one-way ANOVA followed by Bonferroni’s post-hoc test), and sustained POST stimulation (Fig. 4; dHP: 46.2±8.6%, p < 0.001; V1: 72.9±13.8%, p < 0.001; A1: 53.9±7.6%, p < 0.01; S1: 58.9±10.9%, p < 0.001; one-way ANOVA followed by Bonferroni’s post-hoc test). These results demonstrate that low frequency dDG stimulation enhanced interhemispheric functional connectivity in HP, V1, A1 and S1.

NOR results are shown in Fig. 5. Optogenetic animals under Paradigm-A and Paradigm-C spent significantly more time exploring the novel object (i.e., positive DI) during long-term memory phase, but not sham controls under Paradigm-A and optogenetic animals under Paradigm-B. During short-term memory phase, all groups displayed a preference for novel object (Fig. 5) as expected9,13. These results indicate that low frequency dDG stimulation improved long-term memory, in particular memory retrieval but not memory encoding.

In summary, low frequency dDG stimulation enhanced interhemispheric functional connectivity in HP, V1, A1 and S1, and such enhancement closely parallels the improved long-term memory, particularly the memory retrieval but not memory encoding. Our findings provide novel evidence on the “functions” of resting-state connectivity observed by rsfMRI. Furthermore, our approach here presents a new direction to modulate brain connectivity for potential behavioral improvements in normal and diseased brains.

Acknowledgements

This work was supported by the Hong Kong Research Grant Council (Grants C7048-16G and HKU17103015 to E.X.W.).

References

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Figures

Figure 1. Experimental setup for optogenetic stimulation and histological characterization of CaMKIIα::ChR2 viral expression in dorsal dentate gyrus (dDG). Schematic (Left) and T2-weighted anatomical image (Middle) show the viral injection and fiber implantation sites, respectively. Histology image (Right, green box) show respective ChR2 expression in dDG.

Figure 2. Schematic of the optogenetic stimulation setup and corresponding rsfMRI paradigms. rsfMRI scans were performed before (PRE), during (DURING) and after (POST) optogenetic stimulation. Note that DURING and POST were interleaved. Continuous 10-mins stimulation (473nm; 40mW/mm2; 1Hz; 10% duty cycle) was used to mimic low frequency hippocampal activities in dDG.

Figure 3. To assess general short-term and long-term memory, optogenetic animals and sham controls received low frequency (1 Hz) optogenetic stimulation for 10-mins prior to all phases (Paradigm-A). To further assess memory encoding and memory retrieval of long-term memory, optogenetic animals received stimulation prior to short-term memory phase (Paradigm-B) and long-term memory phase (Paradigm-C), respectively. Briefly, animals were habituated in the arena for 15-mins. The next day, animals were exposed to two identical objects for 3-mins during acquisition phase. Novel-object recognition tests were then performed 1 hour and 24 hours later to assess short- and long-term memory functions, respectively.

Figure 4. RsfMRI connectivity maps of dHP, V1, A1 and S1 (left) and corresponding quantification of interhemispheric connectivity (right) PRE, DURING and POST low frequency (1Hz) dDG optogenetic stimulation (*, ** and *** denotes p < 0.05, p < 0.01 and p < 0.001, respectively; error bars indicate ± s.e.m.). Seed location is indicated by a blue crosshair. These results demonstrate that low frequency dDG stimulation enhanced interhemispheric functional connectivity in HP, V1, A1 and S1.

Figure 5. Boxplot summary of the preference for novel object in all animals during short- and long-term memory phases. Results were quantified by discrimination index (DI; one-way ANOVA followed by Bonferroni’s post-hoc test; *** denotes p < 0.001, respectively). Optogenetic animals under Paradigm-A and Paradigm-C spent significantly more time exploring the novel object during long-term memory phase, but not sham controls under Paradigm-A and optogenetic animals under Paradigm-B. During short-term memory phase, all groups displayed a preference for novel object as expected. These results indicate that low frequency dDG stimulation improved long-term memory, in particular memory retrieval but not memory encoding.

Proc. Intl. Soc. Mag. Reson. Med. 26 (2018)
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