Abstract
The complex connectivity of the mammalian brain underlies its function, but understanding how interconnected brain regions interact in neural processing remains a formidable challenge. Here we address this problem by introducing a genetic probe that permits selective functional imaging of distributed neural populations defined by viral labeling techniques. The probe is an engineered enzyme that transduces cytosolic calcium dynamics of probe-expressing cells into localized hemodynamic responses that can be specifically visualized by functional magnetic resonance imaging. Using a viral vector that undergoes retrograde transport, we apply the probe to characterize a brain-wide network of presynaptic inputs to the striatum activated in a deep brain stimulation paradigm in rats. The results reveal engagement of surprisingly diverse projection sources and inform an integrated model of striatal function relevant to reward behavior and therapeutic neurostimulation approaches. Our work thus establishes a strategy for mechanistic analysis of multiregional neural systems in the mammalian brain.
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Data availability
Source data for Figs. 1–5 and the associated Extended Data figures are provided with this paper.
Code availability
Processing scripts used in the data analysis are available from the corresponding author on request.
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Acknowledgements
This research was funded by NIH grant numbers R01 DA038642, R24 MH109081, UF1 NS107712 and U01 NS103470 and a grant from the MIT Simons Center for the Social Brain to A.J. S.G. was supported by an HHMI International Student Research Fellowship and Sheldon Razin Fellowship from the McGovern Institute for Brain Research. N.L. was supported by a Stanley Fahn Research Fellowship from the Parkinson’s Disease Foundation. M.S. was the recipient of a Marie Curie Individual Fellowship from the European Commission. T.X. was a visiting student from the Beijing University of Chinese Medicine, funded by a scholarship from the China Scholarship Council. J.I.D. was supported by the Johnson & Johnson UROP Scholars Program at MIT. We are grateful to S. Lall and B. Sabatini for comments on the manuscript, and to A. Devor, I. Wickersham and H. Sullivan for conversations. We also thank L. McLain for providing CAD cells and T. Poulos of the University of California, Irvine and P. Ortiz de Montellano at the University of California, San Francisco for providing NOS constructs. R. Neve of the Massachusetts General Hospital is acknowledged for production of HSV vectors.
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S.G., N.L., M.S., B.B.B. and A.J. designed the research. S.G., N.L., M.S., B.B.B., T.X., J.I.D. and U.D.S. performed the in vitro and in vivo experiments. K.X., N.D. and N.B.E. implemented the brain-clearing histology procedures under the direction of K.C. S.G., N.L., M.S. and A.J. analyzed the results. S.G., N.L. and A.J. wrote the paper.
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Extended data
Extended Data Fig. 1 NOSTIC functionality in a neuronal cell line.
a, NOSTIC-1 fused to mCherry (cyan), as well as mCherry alone (gray), were expressed in CAD cells and stimulated with A23187 in the absence (–) or presence (+) of 100 µM 1400W; nitrite formation following NO release from the stimulated cells was measured for each condition. The effect of 1400W inhibition is significant for NOSTIC-expressing cells (paired t-test p = 0.013, n = 4) but not for control cells (p = 0.59, n = 4). b, NO production by A23187-stimulated CAD cells was visualized by staining with the fluorescent NO probe diaminofluorescein (DAF). NOSTIC-expressing cells indicated by red mCherry staining show higher levels of green DAF staining in the absence (top) than in the presence (bottom) of NOSTIC inhibition with 1400W. Scale bar = 200 µm. This experiment was performed twice.
Extended Data Fig. 2 Time courses of fMRI responses to forepaw stimulation.
Mean fMRI signal time courses (% signal change) observed in the S1 forelimb region in response to stimulation of the contralateral paw (pink shaded region) in the presence vs. absence of L-NAME (left) or 1400W (right), corresponding to maps and mean response amplitudes reported in Fig. 1e. For arbitrary reasons, experiments testing effects of L-NAME and 1400W were performed with stimulation blocks of 20 s and 10 s, respectively, and differences in the response durations observed left vs. right arise from this fact.
Extended Data Fig. 3 Spatial extent of xenograft-induced hemogenetic responses.
a, Left: Anatomical MRI showing T2-weighted (T2w) contrast in the neighborhood of cell implantation for a represenative animal from the experiments of main text Fig. 2. Right: Average BOLD contrast map (color) induced by A23187 stimulation in units of percent signal change (%SC), overlayed on a T2-weighted scan. b, Cross sections of the contrast patterns through the dotted line in panel a, with solid lines denoting means and shading denoting SEM across six animals’ BOLD (red) and T2w (black) signal changes. Full width at half maximum is 1.4 ± 0.1 mm for the hemogenetic BOLD responses, indicating point-spread function comparable to or less than conventional hemodynamic fMRI.
Extended Data Fig. 4 Evaluation of HSV vectors in cell culture.
a, Western blotting was used to analyze cells transfected (Transfec.) with GFP or NOSTIC-1 (N-1), along with cells infected with HSV encoding mCherry (mC) or NOSTIC-1-IRES-mCherry (N-1/mC). Expression and size of NOSTIC-1 was confirmed using blotting with an anti-FLAG tag (α-FLAG) antibody (top). Expression and size of mCherry in the HSV infected cells was assessed using an anti-mCherry antibody (α-mCherry, middle), and anti-glyceraldehyde 3-phosphate dehydrogenase (α-GAPDH) was used as a loading control (bottom). Relevant molecular weight (MW) markers labeled at left. Expected sizes: NOSTIC-1 140 kD, mCherry 29 kD, GAPDH 37 kD. b, Griess test measurement of nitrite production in the presence of cells transfected with GFP (gray) or NOSTIC-1 (cyan) or virally transduced with mCherry or NOSTIC-1, in the presence or absence of A23187 stimulation, as indicated. Only stimulated cells expressing NOSTIC-1 show strong evidence of stimulus-dependent NO production, as indicated by the Griess test results.
Extended Data Fig. 5 LH stimulus responses in NOSTIC-expressing rats after 1400W treatment.
Average fMRI responses to LH stimulation in the +1400W condition, among 6 animals infected with NOSTIC-encoding HSV, analogous to –1400W condition data in main text Fig. 3b. Significant responses with F-test P ≤ 0.01 shown.
Extended Data Fig. 6 Time courses of responses to LH stimulation in multiple ROIs.
Average time courses of fMRI signals observed in eight ROIs in the presence and absence of 1400W, analogous to data of Fig. 3d. Pink boxes denote stimulation period. Shading denotes SEM over six animals each.
Extended Data Fig. 7 LH stimulus responses in rats treated with control HSV vectors.
Average fMRI responses to LH stimulation in the –1400W (left) and +1400W (right) conditions, among 5 animals infected with mCherry-encoding HSV, corresponding to difference maps shown in main text Fig. 3c. Significant responses with F-test P ≤ 0.01 shown.
Extended Data Fig. 8 1400W-dependent fMRI amplitudes observed in multiple ROIs.
Absolute fMRI response amplitudes observed in each of eight ROIs examined in NOSTIC and control mCherry HSV-treated animals, analogous to data presented in Fig. 3f. 1400W-dependent effects in CPu, ECx, MCx, and SN are all significant with paired t-test *P ≤ 0.05 (n = 6).
Extended Data Fig. 9 Relative changes in fMRI amplitudes upon 1400W treatment.
ROI-averaged fMRI amplitudes from the experiments of Fig. 3 were used to compute relative signal differences as 100 x (pre – post)/pre, where pre and post are the fMRI amplitudes observed before and after 1400W application in each rat. Results are shown as box plots for NOSTIC-1-treated (left, n = 6) and control mCherry-treated (right, n = 5) animals. Box plots denote median (center line), first quartiles (boxes), and full range (whiskers) over animals. NOSTIC results are significant in CPu, ECx, MCx, and SN (t-test P ≤ 0.008); control results are significant in CCx (t-test P = 0.037).
Extended Data Fig. 10 Resting-state functional connectivity to CPu.
A map of Z-transformed correlation coefficients relating resting state fMRI signal in a striatal seed region to voxels across the rest of the brain.
Supplementary information
Supplementary Information
Supplementary Table 1 and Figs. 1–6.
Source data
Source Data Fig. 1
Source data for graphs.
Source Data Fig. 2
Source data for graphs.
Source Data Fig. 3
Signal change amplitudes.
Source Data Fig. 4
Cell counting data.
Source Data Fig. 5
Source data for graphs.
Source Data Extended Data Fig. 2
Source data for time courses.
Source Data Extended Data Fig. 6
Source data for time courses.
Source Data Extended Data Fig. 8
Signal change amplitudes.
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Ghosh, S., Li, N., Schwalm, M. et al. Functional dissection of neural circuitry using a genetic reporter for fMRI. Nat Neurosci 25, 390–398 (2022). https://doi.org/10.1038/s41593-022-01014-8
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DOI: https://doi.org/10.1038/s41593-022-01014-8
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