Optogenetic stimulation of cell bodies versus axonal terminals generate comparable activity and functional connectivity patterns in the brain

Abstract

Optogenetic techniques are often employed to dissect neural pathways with presumed specificity for targeted projections. In this study, we used optogenetic fMRI to investigate the effective landscape of stimulating the cell bodies versus one of its projection terminals. Specifically, we selected a long-range unidirectional projection from the ventral subiculum (vSUB) to the nucleus accumbens shell (NAcSh) and placed two stimulating fibers-one at the vSUB cell bodies and the other at the vSUB terminals in the NAcSh. Contrary to the conventional view that terminal stimulation confines activity to the feedforward stimulated pathway, our findings reveal that terminal stimulation induces brain activity and connectivity patterns remarkably similar to those of vSUB cell body stimulation. This observation suggests that the specificity of optogenetic terminal stimulation may induce antidromic activation, leading to broader network involvement than previously acknowledged.

Keywords: Antidromic activation; Functional connectivity; Nucleus accumbens shell (NAcSh); Optogenetics; Ventral subiculum (vSUB); fMRI.

Fig. 1.

Optogenetic stimulation of vSUB cell bodies and their terminals in NAcSh induced similar brain-wide activity patterns. (A) Schematic representation of optogenetic stimulation sites, including the vSUB cell bodies and the NAcSh terminals. (B) Expression of eYFP in vSUB cell bodies and NAcSh terminals (green) against DAPI staining (blue), with corresponding optical fiber placements. (C) Overview of eYFP expression in vSUB-NAcSh terminals. (D) Position of optical fibers (indicated by arrows). (E) Voxel-wise activation maps for both vSUB and NAcSh stimulations across frequencies, showing overlapping voxβel distributions. (one sample t-test, corrected p < 0.05 using 3dClustSim) Correlation analysis based on the responsive voxels indicated a strong spatial relationship between NAcSh and vSUB stimulation. The voxels were categorized into three groups: voxels in vSUB Stim only, voxels in NAcSh Stim only, and voxels present in both vSUB Stim and NAcSh Stim (coactivation). (F) Average time series derived from the anatomical vSUB and NAc ROIs. (G) One-sample t-test of vSUB and NAcSh Stim in eYFP expression group (one sample t-test, corrected p < 0.05 using 3dClustSim). Analysis of (H) FWHM and (I) maximum CBV changes for each voxel across frequencies, showing a strong relationship between NAcSh and vSUB using the overlapping voxels identified in (E), specifically those present in both vSUB and NAcSh stimulation. (***p < 0.001).

Fig. 2.

Optogenetic stimulation of vSUB cell bodies and their terminals in NAcSh induced similar brain-wide functional connectivity (FC) patterns. (A) Seed-based analysis of vSUB and NAc FC in the ChR2 group showed similar FC patterns with overlapping voxel distributions (one sample t-test, corrected p < 0.05 using 3dClustSim). Pie chart showing the proportion of voxels categorized as being in vSUB seed only, NAc seed only, or in both vSUB and NAcSh seed (co-connectivity) for the ChR2 group with corresponding correlation analysis between vSUB and NAcSh stimulation for their respective voxels. Correlation analysis based on identified voxels indicated a strong spatial relationship (all p-value <0.001) between NAc and vSUB FC. (B) Identical seed-based FC analysis in the eYFP group does not identify stimulation effects. (C) ICA with 50 components derived from the baseline (pre-stimulation) period, and the FC matrix derived from the vSUB (lower left, purple scale heatmap) and NAcSh (upper right, green scale heatmap) stimulation. (D) Correlation analysis revealed a significantly higher correlation of stimulation-induced FC pattern changes (stimulation - baseline) in the ChR2 group compared to the eYFP group (z-value = 5.72, p < 0.001). (E) LME analysis revealed no significant effect of stimulation site (vSUB vs. NAcSh) on behavior, while optogenetic expression significantly increased nosepokes in self-stimulation and decreased mean stimulation in aversive behavior at 20 mW (*p < 0.05).