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Stimulation of basal forebrain cholinergic neurons induces decreased functional connectivity in the default mode-like network in rats
Lore M. Peeters1, Monica Van den Berg1, Rukun Hinz1, and Georgios A. Keliris1
1Bio-Imaging Lab, University of Antwerp, Antwerp, Belgium

Synopsis

Understanding the role of the basal forebrain (BFB) in controlling the dynamics of brain processes is of great interest. We aimed to investigate the influence of selective unilateral stimulation of BFB cholinergic neurons on functional connectivity (FC) in the rodent default-mode like network (DMLN). By combining resting-state functional MRI (rsfMRI) with chemogenetics, we demonstrate that stimulation of the cholinergic neurons in the right BFB significantly decreased right intra-hemispheric and inter-hemispheric FC in the DMLN in rats. These findings provide new critical insights into the interplay between attentional networks and DMLN in rodents.

INTRODUCTION

The role of the basal forebrain (BFB) in controlling the dynamics of large-scale functional networks, such as the default-mode network (DMN), are of great interest. Previous studies have shown that the cholinergic corticopetal neurons originating from the BFB are strongly involved in attentional processes [1-5]. During attention-demanding tasks, the increased activity in the attention networks has been shown to be associated with decreased activity in the DMN [6, 7]. Although this antagonistic relationship between both networks could be repeatedly replicated, the underlying mechanisms remain to be fully understood. Interestingly, recent studies proposed that the BFB plays a role in the regulation of the DMN [8]. Here, we aim to investigate the influence of selective unilateral stimulation of BFB cholinergic neurons on functional connectivity (FC) in the rodent default-mode like network (DMLN).

METHODS

Adult Long Evans rats (n=28) were subjected to stereotactic surgery targeting the right basal forebrain with an adeno-associated (AAV) virus. Rats received either (n=16), AAV8-hSyn_DIO_hM3Dq(gq)-mCherry (Addgene, #44361), for the expression of DREADDs in cholinergic neurons or a control virus (n=12), AAV8-hSyn-DIO-mCherry (Addgene, #50459). Ex vivo evaluation of the DREADDs expression showed that expression was limited to cholinergic neurons in the right basal forebrain, indicating a successful cell-type specific transfection (fig 1). All imaging procedures started at least six weeks after viral injections and were performed on a 9.4 T MRI system (Bruker Biospec, Germany). For all scans rats were anesthetized with an intravenous (i.v.) bolus of 0.05 mg/kg medetomidine, followed by a i.v. infusion of 0.1 mg/kg/h medetomidine and 0.2% isoflurane. RsfMRI scans started at 35 minutes after the medetomidine bolus injection and were acquired using a T2* weighted echo planar imaging (EPI) sequence (TE: 18 ms, TR: 2000ms, FOV: (30 x 30) mm2, matrix [128 x 96], 16 slices of 0.8 mm). First, a 5-minute baseline rsfMRI was acquired. Second, a 20 minute rsfMRI scan was acquired during which the CNO or vehicle (i.e. saline) was intravenously administered at 5 minutes after the start of the scan. Then, a final 5 minute rsfMRI scan was acquired. These three scans were acquired during one scan session. Two scan sessions were acquired for each animal in the DREADDs-expressing group, during which the effect of either CNO or vehicle was evaluated. One scan session was acquired for each animal in the control group to evaluate the off-target effects of CNO. Breathing rate, temperature and blood oxygenation were monitored during the scan sessions. All data was processed using SPM12 and REST v1.8 in Matlab 2014a. Statistical analysis was performed using SPM12, JMP and GraphPad Prism 6.

RESULTS & DISCUSSION

Regions of interest (ROIs) were drawn in brain regions of the DMLN in rats and their FC alterations were followed up over time after administration of Clozapine-N-oxide (CNO). It has been demonstrated that DREADDs-induced neural activity modulations become apparent at 5 minutes after administration of its ligand CNO and lasts for at least 1 hour [9]. Similarly, in this study, significantly decreased FC in the right DMLN could be observed in the DREADDs-expressing rats starting at 5 – 10 minutes post-CNO injection which reached a plateau after 10-15 minutes (fig 2). Next, to better understand the effect of right BFB stimulation on bilateral DMLN FC, a ROI-based analysis was performed using bilateral DMLN ROIs. Results demonstrated that CNO-induced stimulation of the right BFB cholinergic system significantly decreased the FC between various DMLN ROI pairs (fig 3A). Additionally, comparing the average intra-hemispheric and inter-hemispheric FC of the DMLN between both groups revealed significantly decreased FC in the right hemisphere and between both hemispheres (fig 3B). Furthermore, a seed based analysis on two hub regions of the DMLN showed significant decreases in FC after injection of CNO (fig 4). In contrast, CNO injection in the sham group and saline injection in the DREADDs-expressing animals, did not elicit FC alterations. Interestingly, various functional imaging studies have identified anti-correlations between the attentional networks, or “task-positive networks” and the DMN or “task-negative network” [6, 10, 11]. Additionally, a recent study in humans demonstrated that negative BOLD responses in DMN brain regions can be induced by the presentation of random flashing checkerboard visual stimuli [12]. Other studies observed significantly decreased FC in the DMN when subjects were preforming a cognitive task [13, 14]. More recently, Hinz et al. (2019) evaluated the BOLD responses in DMLN regions upon visual stimulation in sedated rats. The authors demonstrated that visual stimulation could deactivate nodes of the DMLN which was associated with decreased FC within the DMLN [15]. The similarity between the latter finding and our results might suggest involvement of the basal forebrain cholinergic system in mediating decreased FC in DMLN upon presentation of visual stimuli in sedated rats.

CONCLUSION

Our study demonstrates decreased right intra- and interhemispheric FC in the DMLN upon activation of basal forebrain cholinergic neurons in sedated rats. We conjecture that our DREADDs-induced stimulation mediates decreased DMLN FC through similar pathways as task-related DMLN suppression. To conclude, our findings provide new critical insights into the interplay between attentional networks and DMLN in rodents.

Acknowledgements

This study was supported by the Fund of Scientific Research Flanders (FWO G048917N) and the Research Fund of the University of Antwerp (BOF DOCPRO FFB150340). Johan van Audekerke provided excellent technical support. We thank Dr. Gaurav Majumdar for supervising the immunohistochemistry as well as Prof. Dr. Timmermans and Dr. Pintelon from the Laboratory of Cell Biology and Histology from the University of Antwerp for acquiring the histological images presented in Fig 1. The Leica SP 8 (Hercules grant AUHA.15.12) microscope was funded by the Hercules Foundation of the Flemish Government. The computational resources and services used in this work were provided by the HPC core facility CalcUA of the University of Antwerp, the VSC (Flemish Supercomputer Center), funded by the Hercules Foundation and the Flemish Government – department EWI.

References

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Figures

Fig. 1: (A) Cre-dependent viral vectors tagged with mCherry were injected in the right basal forebrain of ChAT-cre transgenic rats. (B) Immunohistochemistry was performed to check the expression of the DREADDs. The expression of mCherry is represented in red and cell nuclei are blue. (C) ChAT, an enzyme present in cholinergic neurons, is presented in green. (D) Co-localization of mCherry and ChAT in yellow (red + green). All cholinergic neurons in the target region were transfected and no mCherry was observed in structures other than cholinergic neurons.

Fig. 2: DREADD-induced decreased FC in the right DMLN. Quantification of the FC strength in the right DMLN (z-scores) in different time-windows demonstrated significant decreases only after CNO injection in the DREADD-expressing group (orange). Saline injections in the same group (light blue) or CNO-injections in sham animals (dark blue) demonstrated no significant changes in comparison to baseline. Linear mixed model with Student’s T correction for multiple comparisons, ** p≤0.01


Fig. 3: (A) Pairwise z-transformed FC matrix after saline injection (lower half) and after CNO injection (upper half). The colour bar represents z-scores. The stars indicate significant differences (repeated measures Two-way ANOVA with Sidak’s correction for multiple comparisons). (B) Bar-graphs of the average intra-hemispheric FC (z-scores) in the DMLN of the right hemisphere and left hemisphere, as well as the average inter-hemispheric FC. The z-scores of both groups were statistically compared using a paired Student’s T-test. * p≤0.05, ** p≤0.01, *** p≤0.001, ****p≤0.0001.

Fig. 4: (A) and (C) represent mean statistical FC maps of the right cingulate cortex and right retrosplenial cortex respectively as seed regions in the DREADDs-expressing group after injection of saline and CNO (One-sample T-test, FWE corrected p≤0.05, minimal cluster size = 10 voxels). (B) and (D) show the quantification of the FC strength as mean T-values ± SEM for both seed regions (right cingulate cortex (B) and right retrosplenial cortex (D)). Paired Student’s T-test, * p≤0.05, ** p≤0.01. Abbreviations: functional connectivity (FC).

Proc. Intl. Soc. Mag. Reson. Med. 28 (2020)
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