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Optogenetic resting-state fMRI reveals thalamic modulation of long-range sensory networks

Alex T. L. Leong^1,2, Xunda Wang^1,2, Russell W. Chan^1,2, Leon C. Ho^1,2, Yongrong Qiu^1,2, Celia M. Dong^1,2, and Ed X. Wu^1,2

¹Laboratory of Biomedical Imaging and Signal Processing, The University of Hong Kong, Hong Kong, Hong Kong, ²Department of Electrical and Electronic Engineering, The University of Hong Kong, Hong Kong, Hong Kong

Synopsis

One grand challenge in contemporary neuroscience is to achieve an integrated understanding of large-scale brain-wide networks. Resting-state functional MRI (rsfMRI) has helped reveal such brain-wide networks, yet, the neural bases underlying them remain unclear. Utilizing optogenetic excitation and pharmacological inactivation to manipulate the neural activity of somatosensory thalamocortical neurons, the present study demonstrated that rsfMRI connectivity is enhanced and decreased respectively. Furthermore, our findings suggest that optogenetically-evoked propagating low frequency activity (~1Hz) within the thalamo-cortico-thalamic network facilitate the enhancement of rsfMRI connectivity. Our work offers an exciting avenue to dissect the underlying neural bases of brain-wide functional connectivity.

Purpose

The brain is a highly complex, interconnected structure with parallel and hierarchical networks distributed within and between neural systems^1,2. Functional connectivity mapping using resting-state functional MRI (rsfMRI) enables non-invasive visualization of brain-wide networks in humans³ and animals⁴ through coherent infra-slow (<0.1Hz) hemodynamic/BOLD activity. However, the exact underlying neural bases and functional significance remain unclear. Previous studies integrating large-scale electrical recordings, voltage-sensitive-dye (VSD) and Ca²⁺ imaging techniques^5-7 imply that slow, oscillating neural activity (<1Hz) constrains and elicits these rsfMRI BOLD activity. This indicate that interrogating the brain’s underlying electrical activity can provide mechanistic insights into brain-wide rsfMRI connectivity. Numerous long-range networks exist, subserving the functions of particular systems and their interactions. One classical example is the thalamo-cortical-thalamic network, which interconnects the thalamus and neocortex via thalamo-cortical and cortico-thalamic projections⁸. In this study, we optogenetically stimulate ventral posteromedial thalamus (VPM) thalamocortical excitatory neurons and pharmacologically inactivate VPM thalamocortical neurons, to investigate whether modulating and/or disrupting the neural activity of such an integral brain network could uncover the neural bases of rsfMRI.

Methods

Animal Preparation: AAV5-CaMKIIα::ChR2(H134R)-mCherry was injected to VPM of adult rats (n=8, 200-250g, male, SD strain). Four weeks after injection, an opaqued optical fiber cannula (d=450μm) was implanted at the injection site. In a separate group of rats (n=10, 400-450g, male, SD strain), a glass capillary tube (d=300μm) was implanted in VPM. All experiments were performed under 1.0% isoflurane.

Optogenetic and Tetrodotoxin (TTX) rsfMRI Experiment: For optogenetic stimulation (OG-On), blue (473nm) light was presented continuously at 1Hz (10% duty cycle, 40mW/mm²; Figure 1A). RsfMRI scans were performed before (baseline), during (OG-On) and after (OG-Off) optogenetic stimulation. Typically, five baseline rsfMRI scans were acquired before five OG-On scans were interleaved with five OG-Off scans. For TTX infusion, 5μL (5ng/μL) was used. Typically, five rsfMRI scans were acquired before (baseline) TTX infusion and five after (Post). TTX is a neurotoxin that blocks neuronal action potentials.

rsfMRI Acquisition and Analysis: RsfMRI data was acquired at 7T using GE-EPI (FOV=32×32mm², matrix=64×64, α=56°, TE/TR=20/750ms, 16 contiguous slices with 1mm thickness). Standard preprocessing followed by seed-based correlation analysis was applied to map and quantify interhemispheric/bilateral rsfMRI functional connectivity of primary somatosensory barrel field (S1BF), secondary somatosensory (S2), primary visual (V1) and auditory (A1) cortices. Interhemispheric rsfMRI connectivity spectra and intrahemispheric power spectra were then computed.

Results

Figure 1 demonstrates that interhemispheric S1BF, S2, V1 and A1 functional connectivities were strengthened significantly during OG-On period, which remained elevated during OG-Off period.

Figure 2 shows the significant decreased interhemispheric S1BF, S2, V1 and A1 functional connectivities after pharmacological blockade of VPM thalamocortical neurons by TTX.

Figure 3 presents the interhemispheric rsfMRI connectivity spectrum of S1BF, S2, V1 and A1. Coherent bilateral infra-slow (<0.1Hz) rsfMRI BOLD activity were increased and remained elevated following 1Hz optogenetic VPM stimulation, and decreased after TTX infusion.

Figure 4 shows the increased local infra-slow (<0.1Hz) rsfMRI BOLD activity of S1BF, S2, V1 and A1, which remained elevated following 1Hz VPM stimulation. TTX infusion at VPM led to a decrease in such activity.

Figure 5 demonstrates the downstream propagation of low frequency neuronal activity as measured by LFP, initiated by optogenetic excitation of VPM thalamocortical excitatory neurons at 1Hz.

Discussion and Conclusion

Our results demonstrated that optogenetic excitation of the VPM thalamocortical excitatory neurons at 1Hz enhanced brain-wide rsfMRI functional connectivity. On the other hand, TTX inactivation of thalamocortical neurons decreased rsfMRI connectivity. Spectral analysis (Figure 3) indicated that such changes were underpinned by neuromodulatory effects on the infra-slow rsfMRI BOLD activity, suggesting that propagation of low frequency activity in the thalamo-cortical circuit facilitate certain neuromodulatory mechanisms. The thalamo-cortical-thalamic network has been shown to play a pivotal role in setting the global brain state and controlling the flow of sensory information⁸. Indeed, its importance has been echoed in our recent optogenetic fMRI investigation, showing that low frequency stimulation of VPM thalamocortical excitatory neurons initiates brain-wide propagation of low frequency activities⁹ (Figure 5). Low frequency activities such as slow (<1Hz) and infra-slow (<0.1Hz) oscillations are spontaneously initiated in^10,11 or triggered by stimulation of the thalamus and cortex^7,12. Moreover, numerous evidence indicate that slow oscillations are capable of modulating neuronal excitability brain-wide¹³. These studies by others suggest that our 1Hz optogenetic stimulation of VPM initiates slow oscillations that may couple with infra-slow oscillations^14,15, leading to enhanced rsfMRI connectivity. Whereas, TTX inactivation of VPM thalamocortical neurons disrupts the initiation or propagation of these oscillations, decreasing rsfMRI connectivity. By manipulating the specific activities within the thalamo-cortical-thalamic network, optogenetic rsfMRI presents a powerful approach to dissect the neural bases of rsfMRI connectivity.

Acknowledgements

No acknowledgement found.

References

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Figures

Figure 1 (A) Illustration of optogenetic stimulation setup and experimental timeline (left), and continuous 1Hz optogenetic stimulation during a rsfMRI scan (right). (B) Functional connectivity maps of S1BF, S2, V1 and A1 before, during and after low frequency (1Hz) optogenetic stimulation of VPM thalamocortical excitatory neurons (n=8; asterisk, stimulation site; blue crosshair, seed location). (C) Analysis of averaged interhemispheric connectivity correlation coefficient values (n=8; error bar indicates ± s.e.m., one-way ANOVA with post-hoc Bonferroni correction; *, **, *** and n.s denotes P<0.05, P<0.01, P<0.001 and not significant).

Figure 2 (A) Illustration of TTX infusion setup (left), and experimental timeline and a typical rsfMRI scan (right). (B) Functional connectivity maps of S1BF, S2, V1 and A1 before and after TTX infusion into VPM (n=10; asterisk, infusion site; blue crosshair, seed location). (C) Analysis of averaged interhemispheric connectivity correlation coefficient values (n=8; error bar indicates ± s.e.m., paired Student’s t-test with post-hoc Bonferroni correction; ** and *** denotes P<0.01 and P<0.001).

Figure 3 (A) Schematic diagram of the computation steps involved in generating an interhemispheric rsfMRI connectivity spectrum. (B) Interhemispheric rsfMRI connectivity spectrum of S1BF, S2, V1 and A1 before (Baseline), during (OG-On) and after (OG-Off) low frequency (1Hz) optogenetic stimulation of VPM thalamocortical excitatory neurons. (C) Interhemispheric rsfMRI connectivity spectrum of S1BF, S2, V1 and A1 before (Baseline) and after (Post) TTX infusion into VPM.

Figure 4 (A) Intrahemispheric rsfMRI power spectrum of ipsilateral and contralateral S1BF, S2, V1 and A1 before (Baseline), during (OG-On) and after (OG-Off) low frequency (1Hz) optogenetic stimulation of VPM thalamocortical excitatory neurons. (B) Intrahemispheric rsfMRI power spectrum of ipsilateral and contralateral S1BF, S2, V1 and A1 before (Baseline) and after (Post) TTX infusion into VPM thalamocortical neurons.

Figure 5 (A) Illustration of the location of recording electrodes in bilateral S1BF and V1, and optrode in VPM. (B) Averaged evoked LFPs during low frequency (1Hz) VPM optogenetic stimulation (n=3; 10% duty cycle). (C) Summary of propagation delays from the onset of stimulation (magnified from B). (D) Schematic diagram of brain-wide downstream signal propagation at 1Hz.

Proc. Intl. Soc. Mag. Reson. Med. 25 (2017)

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