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Thalamic low frequency activity contributes to resting-state cortical interhemispheric MRI functional connectivity

Xunda Wang^1,2, Alex T. L. Leong^1,2, Russell W Chan^1,2, and Ed X. Wu^1,2

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

Synopsis

The brain consists of numerous interconnected parallel and hierarchical networks subserving sensory, behavioral and cognitive functions. Resting-state functional MRI (rsfMRI) connectivity has helped study the complex brain-wide functional networks. Yet, less is known about the role of thalamus in rsfMRI connectivity. Utilizing optogenetic excitation and pharmacological inactivation to manipulate the neural activity of somatosensory thalamocortical neurons, we demonstrate that thalamus contributes to rsfMRI connectivity within and beyond its sensory modality, likely through the recruitment of interhemispheric low frequency neural oscillations at all cortical layers. Our work highlights the thalamus as a pivotal structure underpinning rsfMRI connectivity observations.

Purpose

Resting-state functional MRI (rsfMRI) connectivity is a robust measurement tool for interrogating complex brain-wide functional networks^1-3. Intrinsic rsfMRI connectivity networks and their changes in normal and diseased brains have been increasingly recognized and utilized by the scientific and clinical community as important cognitive performance predictors and disease biomarkers^4-7. However, the interpretation of these findings is challenging as the neural bases of rsfMRI connectivity remain unclear. By characterizing changes in cortical interhemispheric rsfMRI connectivity due to corpus callosum congenital defects or damage^8-10, several human and animal studies strongly indicate the involvement of subcortical structures (e.g., thalamus) and polysynaptic connections in maintaining rsfMRI connectivity. Thalamus is known to modulate diverse long-range functional neural integration involved in cortico-cortical processing^11,12. Such functional role is structurally supported by the massive polysynaptic connections¹³ and the long-range propagation/coupling of low frequency (<10Hz) spontaneous oscillatory neuronal activities in the brain-wide thalamo-cortical networks^14-16. However, few studies directly interrogate the role of thalamus in rsfMRI connectivity. To examine whether and how thalamus contributes to rsfMRI connectivity, here we directly perturb ventral posteromedial thalamus (VPM) and monitor brain-wide rsfMRI connectivity with a multi-modal approach of rsfMRI, neuromodulation (i.e., optogenetic stimulation and pharmacological inhibition) and electrophysiology recordings.

Method

Animal preparation: AAV5-CaMKIIα::ChR2(H134R)-mCherry (Figure 1a) was injected to VPM of adult male SD rats (rsfMRI: n=8; electrophysiology: n=9; 200-250g). Four weeks after injection, an opaque optical fiber cannula (d=450μm; for optogenetic stimulation) or a glass capillary tube (d=300μm; for tetrodotoxin, TTX, infusion; n=10) was implanted at VPM. All experiments were performed under 1.0% isoflurane.

rsfMRI and electrophysiology experiments: rsfMRI data was acquired at 7T using GE-EPI. For optogenetic stimulation, 1Hz blue (473nm) light was presented continuously (10% duty cycle, 40mW/mm2; Figure 1b). For TTX infusion, 5μL (5ng/μL) was used (Figure 1c). Standard preprocessing followed by seed-based analysis was applied to map and quantify interhemispheric rsfMRI functional connectivity of primary somatosensory barrel field (S1BF), secondary somatosensory (S2), primary visual (V1) and auditory (A1) cortices. Local field potential (LFP) data was acquired from bilateral S1BF using multi-depth electrodes (16 channels). Interhemispheric connectivity and intrahemispheric/local power spectra were computed for both rsfMRI and LFP data.

Results

Optogenetic stimulation of VPM thalamocortical excitatory neurons increases brain-wide cortical interhemispheric rsfMRI connectivity: 1Hz VPM stimulation enhanced the spatial extent and the strength of interhemispheric rsfMRI connectivity in S1BF, S2, V1 and A1 (Figures 2a, b). Further analyses of the interhemispheric connectivity and intrahemispheric power spectra showed enhancements at the infraslow (0.01-0.1Hz) frequency in both interhemispheric connectivity (Figure 2c) and intrahemispheric rsfMRI BOLD activity (Figure 2d).

Optogenetic stimulation of VPM thalamocortical excitatory neurons increases cortical interhemispheric LFP connectivity of low frequency neural oscillations at all cortical layers: Layer estimation and channel realignment were performed using high-resolution current source density analysis (Figure 3c). Although the deeper cortical layers were observed to have stronger interhemispheric LFP connectivity than the upper layers pre-stimulation (Figures 4a, b), interhemispheric connectivity strengthened significantly at all layers post stimulation, particularly the correlation of slow, delta and theta oscillations (Figures 4a, b). We also observed an increase in the intrahemispheric LFP power of slow, delta and theta oscillations at all layers of bilateral S1BF post-stimulation.

Pharmacological inhibition of VPM thalamocortical neurons decreases brain-wide cortical interhemispheric rsfMRI connectivity: Pharmacological inhibition weakened the spatial extent and the strength of interhemispheric rsfMRI connectivity in S1BF, S2, V1, and A1 (Figures 5a, b). For the interhemispheric connectivity and intrahemispheric/local power spectra, we detected significant decreases in the interhemispheric connectivity (Figure 5c) and the strength of intrahemispheric rsfMRI BOLD activity (Figure 5d) at infraslow (0.01-0.1Hz) frequency post stimulation.

Discussion and Conclusion

Our findings demonstrated that thalamically-evoked low frequency activity by optogenetic stimulation of VPM thalamocortical excitatory neurons increased brain-wide cortical interhemispheric rsfMRI connectivity. Meanwhile, the pharmacological inhibition of VPM thalamocortical neurons decreased interhemispheric rsfMRI connectivity. Together, these results support the role of thalamo-cortical low frequency activities in modulating brain-wide cortical interhemispheric rsfMRI connectivity, highlighting the key contributions of thalamus to rsfMRI connectivity. Previous studies suggest that polysynaptic connections and long-range coupling of low frequency neural oscillations (mainly <10Hz) are related to the thalamic influence on brain-wide rsfMRI connectivity^17-19. Indeed, in our previous study, 1Hz VPM stimulation showed downstream neural activity propagation to numerous non-somatosensory regions²⁰. Hence, it is highly likely that neural activity propagates to the visual and auditory cortices, leading to subsequent increase in their respective interhemispheric rsfMRI connectivity²¹. Taken together, our study presents compelling evidence on the contributions of low frequency neural oscillations in thalamo-cortical networks to brain-wide rsfMRI connectivity.

Acknowledgements

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

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Figures

Histological characterization of ChR2::CaMKIIα viral expression in VPM thalamocortical excitatory neurons, optogenetic/TTX experiment setup and paradigm. (a) Confocal images of ChR2-mCherry expression in VPM; Lower magnification (Left) and higher magnification (Right). Overlay of images costained for the nuclear marker DAPI, excitatory marker CaMKIIα, and mCherry revealed colocalization of mCherry and CaMKIIα in the cell body of thalamo-cortical neurons (indicated by white arrows). (b) Illustration of optogenetic stimulation setup and experimental timeline (left), and 2mins-on-2mins-off 1 Hz optogenetic stimulation for an OG-On scan (right). (c) Illustration of TTX infusion setup (left), and experimental timeline and a typical rsfMRI scan (right).

rsfMRI connectivity in all sensory cortices are enhanced by low frequency (1 Hz) optogenetic excitation of thalamocortical excitatory neurons (n = 8) (a) Functional connectivity maps of S1BF, S2, V1 and A1 pre- and post- optogenetic stimulation (asterisk, stimulation site; blue crosshair, seed and ROI location). (b) Quantification for averaged interhemispheric correlation coefficient values. (c) and (d) Interhemispheric rsfMRI connectivity and intrahemispheric rsfMRI power spectra with quantifications in 0.01-0.1 Hz frequency range (error bar indicates ± sem., two-tail sample t tests; *, **, *** and **** denote P < 0.05, P < 0.01, P < 0.001 and P < 0.0001).

Electrophysiology recording locations, representative traces during optogenetic stimulation, and schematic diagram for cortical layer identification/realignment in S1BF. (a) Illustration of multisite recording electrodes locations for the electrophysiology experiment. (b) Representative LFP traces during optogenetic stimulation (redline represent the stimulation onset) overlaid on the high-resolution current source density (CSD) maps showing the layer estimation and method for realignment of electrodes. Note that CSD analysis was performed here to facilitate the identification and realignment of bilateral somatosensory cortical layers for subsequent interhemispheric and intrahemispheric LFP analyses.

Interhemispheric LFP connectivity and intrahemispheric LFP power of low frequency neural oscillations at all cortical layers were elevated by low frequency (1 Hz) optogenetic stimulation of VPM (n = 9). (a) Interhemispheric connectivity spectra between bilateral S1BF. (b) Quantifications of interhemispheric connectivity values. One-way ANOVAs with post hoc corrections for baseline comparison. Paired two-tail sample t tests for the comparisons between PRE and POST optogenetic stimulation. Error bar indicates ±SEM (*, **, ***, and **** denote P < 0.05, P < 0.01, P < 0.001, and P < 0.0001) (c) Intrahemispheric resting-state LFP power spectra for bilateral S1BF.

TTX inhibition of VPM thalamocortical excitatory neurons decreases interhemispheric rsfMRI connectivity in sensory cortices. (a) Functional connectivity maps of S1BF, S2, V1 and A1 before and after TTX infusion (n = 10; asterisk, infusion site; blue crosshair, seed and ROI location). (b) Statistical analysis for averaged interhemispheric correlation coefficient values. (c) and (d) Interhemispheric rsfMRI connectivity spectrum and intrahemispheric rsfMRI power spectrum with quantifications in 0.01-0.1 Hz frequency range (n = 10; error bar indicates ± sem., two-tail sample t tests; *, ** and *** denote P < 0.05, P < 0.01 and P < 0.001).

Proc. Intl. Soc. Mag. Reson. Med. 27 (2019)

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