Graded recruitment of pupil-linked neuromodulation by parametric stimulation of the vagus nerve

Vagus nerve stimulation (VNS) is thought to affect neural activity by recruiting brain-wide release of neuromodulators. VNS is used in treatment-resistant epilepsy, and is increasingly being explored for other disorders, such as depression, and as a cognitive enhancer. However, the promise of VNS is only partially fulfilled due to a lack of mechanistic understanding of the transfer function between stimulation parameters and neuromodulatory response, together with a lack of biosensors for assaying stimulation efficacy in real time. We here develop an approach to VNS in head-fixed mice on a treadmill and show that pupil dilation is a reliable and convenient biosensor for VNS-evoked cortical neuromodulation. In an ‘optimal’ zone of stimulation parameters, current leakage and off-target effects are minimized and the extent of pupil dilation tracks VNS-evoked basal-forebrain cholinergic axon activity in neocortex. Thus, pupil dilation is a sensitive readout of the moment-by-moment, titratable effects of VNS on brain state.

(averaged across highest two pulse widths and all train rates). Each line is a session. (F) Relationship between observed leak fraction and measured cuff electrode impedance. Each panel is for the indicated pulse amplitude bin. Each data point is a session; red lines are fitted 1 st order polynomial; stats are Pearson correlation. Panels B-D: N=18 (total of 83 sessions). Panels E-F: N=10 (total of 45 sessions).

Supplementary Figure 2. (A)
VNS-evoked pupil response scalars plotted separately per animal, and separately for pulse amplitudes and rates (collapsed across widths). Box plots indicate the median (center line), first quartiles (box edges), minimum/maximum values (whiskers), and outliers (diamonds; based on interquartile range). (B) As A, but for pulse widths and rates (collapsed across amplitudes). Source data are provided as a Source Data file.

Supplementary Figure 4. (A)
VNS-evoked pupil time-courses, for ungrounded animals with intact nerve. Each trace is a charge/pulse bin and train rate. Grey window, VNS train; red bar, interval for VNS-evoked pupil response scalar measures (see Methods); data are presented as mean values ± s.e.m. (across VNS events). N=45 repetitions for each unique parameter combination (before artefact rejection; Methods). (B) As A, but for single cut nerve. N=14 repetitions for each unique parameter combination (before artefact rejection; Methods). (C) As A, but for double cut nerve. N=24 repetitions for each unique parameter combination (before artefact rejection; Methods). (D) VNS-evoked pupil response scalar measures separately per charge/pulse bin and train rate, for ungrounded animals with intact nerve. Colored lines, fitted log logistic function (Methods); data are presented as mean values ± 1.96 × s.e.m. (across VNS events; stats, two-sided one sample t-test (tested against 0; ***p < 0.001, **p < 0.01; *p < 0.05, corrected for false discovery rate). (E) As D, but for single cut nerve. (F) As D, but for double cut nerve. (G-L) As A-F, but for grounded animals. N=22, N=17 and N=14 repetitions for each unique parameter combination, respectively (before artefact rejection; Methods). Source data are provided as a Source Data file.

Supplementary Figure 5. (A)
Time-frequency representation of VNS-evoked eye movement responses, expressed as % signal change from baseline (Methods), for ungrounded animals with intact nerve. N=45 repetitions for each unique parameter combination (before artefact rejection; Methods). (B) As A, but for single cut nerve. N=14 repetitions for each unique parameter combination (before artefact rejection; Methods). (C) As A, but for double cut nerve. N=24 repetitions for each unique parameter combination (before artefact rejection; Methods). (D) VNS-evoked phase-locked horizontal (top) and vertical (bottom) eye movement response measures (Methods) separately per charge/pulse bin and train rate. Colored lines, fitted log logistic function (Methods); stats, two-sided one sample t-test (tested against 0; ***p < 0.001, **p < 0.01; *p < 0.05, corrected for false discovery rate); data are presented as mean values ± 1.96 × s.e.m. (across VNS events). (E) As D, but for single cut nerve. (F) As D, but for double cut nerve. (G-L) As A-F, but for grounded animals. N=22, N=17 and N=14 repetitions for each unique parameter combination, respectively (before artefact rejection; Methods). Source data are provided as a Source Data file. Correspondence between co-registered frame and the reference image time-locked to VNS separately for pulse charge and collapsed across pulse rate. Grey window, VNS train; purple bar, interval for VNS-evoked correspondence measures (Methods); data are presented as mean values ± s.e.m. (across VNS events). (C) VNS-evoked correspondence measures separately per pulse charge bin and pulse rate. Colored lines, fitted log logistic function (Methods); data are presented as mean values ± 1.96 × s.e.m. (across VNS events); stats, two-sided one sample t-test (tested against 0; ***p < 0.001, **p < 0.01; *p < 0.05 false discovery rate corrected). (D) VNS-evoked pupil responses separately for pulse amplitudes (left), widths (middle) and rates (right) collapsed across two other stimulation parameters. Grey window, VNS pulse (10 s); red bars, interval for VNS-evoked pupil response measures (Methods); data are presented as mean values ± s.e.m. (across VNS events). (E) VNS-evoked pupil response measures separately for pulse amplitudes and rates (collaped across widths, left) and separately for pulse widths and rates (collapsed across amplitudes, right). VNS-evoked calcium response scalars plotted separately per animal, and separately for pulse amplitudes and rates (collapsed across widths). Box plots indicate the median (center line), first quartiles (box edges), minimum/maximum values (whiskers), and outliers (diamonds; based on interquartile range). (K) As J, but for for pulse widths and rates (collapsed across amplitudes). All panels except G,I: N=12 repetitions for each unique parameter combination (before artefact rejection; Methods). Panel I: N=120 repetitions of the same parameter combination (before artefact rejection; Methods). Source data are provided as a Source Data file.

Supplementary Figure 7. (A)
Left: histogram of VNS-evoked walking speed in 'parameter exploration experiment' (intact nerve, ungrounded). Right: as left, but zoomed in on the large peak around zero walking speed. Dashed red line, cutoff for defining walking. N=10 (total of 45 sessions). (B) VNS-evoked walk probability (without correction for reversion to the mean; see Methods) separately for pulse amplitudes and rates (collapsed across widths, left) and separately for pulse widths and rates (collapsed across amplitudes, right). Black line, spontaneous walk probability in pre-stimulation baseline interval; colored lines, fitted log logistic function (Methods); data are presented as mean values ± 1.96 × s.e.m. (across VNS events). (C) As B, but for charge bins. (D) VNS-evoked walking speed (on all trials; without correction for reversion to the mean; see Methods) separately for pulse amplitudes and rates (collapsed across widths, left) and separately for pulse widths and rates (collapsed across amplitudes, right). Black line, spontaneous walk probability in pre-stimulation baseline interval; colored lines, fitted log logistic function (Methods); data are presented as mean values ± 1.96 × s.e.m. (across VNS events). (E) As D, but for charge bins. (F) VNSevoked walk probability in grounded animals (see Methods) separately for pulse amplitudes and rates (left) and separately for pulse widths and rates (right). Colored lines, fitted log logistic function (Methods); data are presented as mean values ± 1.96 × s.e.m. (across VNS events). (G) As A, but for charge bins. Stats, two-sided one sample t-test (tested against 0; ***p < 0.001, **p < 0.01; *p < 0.05 false discovery rate corrected). (H,I) As F,G, but for walking speed on walk trials only. Panels A-D: N=27 repetitions for each unique parameter combination (before artefact rejection; Methods). Panels F-I: N=24 repetitions for each unique parameter combination (before artefact rejection; Methods). Source data are provided as a Source Data file.