Abstract
Electrical neurostimulation is effective in the treatment of neurological disorders, but associated recording artefacts generally limit its applications to open-loop stimuli. Real-time and continuous closed-loop control of brain activity can, however, be achieved by pairing concurrent electrical recordings and optogenetics. Here we show that closed-loop optogenetic stimulation with excitatory opsins enables the precise manipulation of neural dynamics in brain slices from transgenic mice and in anaesthetized non-human primates. The approach generates oscillations in quiescent tissue, enhances or suppresses endogenous patterns in active tissue and modulates seizure-like bursts elicited by the convulsant 4-aminopyridine. A nonlinear model of the phase-dependent effects of optical stimulation reproduced the modulation of cycles of local-field potentials associated with seizure oscillations, as evidenced by the systematic changes in the variability and entropy of the phase-space trajectories of seizures, which correlated with changes in their duration and intensity. We also show that closed-loop optogenetic neurostimulation could be delivered using intracortical optrodes incorporating light-emitting diodes. Closed-loop optogenetic approaches may be translatable to therapeutic applications in humans.
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Data availability
The source data supporting the results in this study are available from the Newcastle University research repository at https://doi.org/10.25405/data.ncl.19519630. All data generated during the study are available from the corresponding author on reasonable request.
Code availability
Analysis and modelling code are available from the Newcastle University research repository at https://doi.org/10.25405/data.ncl.19519630.
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Acknowledgements
The project CANDO (Controlling Abnormal Network Dynamics with Optogenetics) is co-funded by the Wellcome Trust (grant no. 102037; to A.J., A.O., S.N.B., G.J.C., M.O.C., P.D., M.K., F.E.N.L. and A.J.T.) and the Engineering and Physical Sciences Research Council (grant no. A000026; to A.J., A.O., S.N.B., G.J.C., M.O.C., P.D., M.K., F.E.N.L. and A.J.T.). We thank all members of the CANDO consortium (www.cando.ac.uk). A.J. is a Wellcome Trust Senior Research Fellow (grant no. 106149; to A.J.).
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The in vitro experiments were performed and analysed by M.T., A.H., D.W., F.E.N.L., M.O.C., A.J.T. and A.J. The in vivo experiments were performed and analysed by B.Z., M.T., S.N.B. and A.J. Virology and post-mortem histology were performed by C.G., F.M., E.E.M. and G.J.C. The simulations were performed by Y.W., F.H., M.K. and A.J. The closed-loop algorithm was developed by S.N.B. and A.J. The optrode was designed and manufactured by E.E.-C., A.S.I., R.G.B., S.T., A.P., N.P., J.G., N.D., T.G.C., P.D. and A.O. A.J. wrote the manuscript, with input from all authors.
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A.J., M.O.C., S.N.B. and P.D. are named as inventors in patent numbers EP3490669A1 (granted) and US20190168021 (pending), filed by Newcastle University, which cover aspects of the closed-loop stimulation method reported in this work. The other authors declare no competing interests.
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Extended data
Extended Data Fig. 1 Realigning phase shifts relative to high gamma envelope.
a LFP spectra from three different Emx1-ChR2 mouse brain slice sessions with different closed-loop filter frequencies. Bottom plot shows that the phase shifts and frequencies of driven oscillations varied across sessions. b Cross-correlation (over all phase conditions) reveals a varying phase relationship between the LFP and high gamma envelope for different sessions, while the stimulation to high gamma phase was relatively constant. c Schematic of the phase adjustment procedure. (1) During the experiment, the FIR filter generated optical stimulation that was phase shifted relative to the LFP (by an amount set by the experimenter). This stimulation drove neural activity, for which we used the high gamma envelope as a surrogate measure, which then produced LFP. (2) To assess the phase relationship between neural activity and resultant LFP, at the end of the experiment we measured the zero-lag phase of the overall LFP-gamma cross-correlation. (3) We added these two phase shifts to produce an adjusted phase shift of stimulation relative to high gamma envelope. We hypothesized that this overall phase-shift would determine whether the closed-loop dynamics was characterised by positive or negative feedback. d LFP spectra replotted after adjusting phase-shift relative to high gamma envelope. In addition, the frequency axis has been normalised by the filter frequency. As a result, the frequencies of oscillations driven by different phase-shifts are closely aligned. d Phase-shifts (relative to LFP) that drove maximal oscillation plotted against relative phase between LFP and high gamma envelope. Solid line indicates stimulation in phase with high gamma. Maximal modulation was typically obtained for phase-shifts advanced by 0–90° relative to this (circular mean 38°, dashed line).
Extended Data Fig. 2 Implantable LED array used in NHP experiments.
a Images of the silicon fork optrode comprising four shanks with 2 CREE DA2432 LEDs per shank encapsulated with silicone elastomer (NuSil MED-6015). Scale bar: 1 mm. b Measured light power output of each LED for different supply currents (blue). Also shown is the light power output for the commercial light source used for the other datasets. The horizontal axis encompasses the full range of stimulation intensities in both cases. Note that the datasets analysed here used a single LED to activate the tissue.
Extended Data Fig. 3 Replication of in vitro results using viral delivery of opsins.
a Example oscillations driven by CLOSe in quiescent brain slices taken from mice injected with AAV8-hSyn-Chronos-GFP, one of the viruses used in the NHP experiments. b Modulation of LFP power relative to no stimulation for different CLOSe phase-shifts (10 Hz filter). c Phase-dependent modulation of LFP for four different filter frequencies in slices taken from three animals. Colour indicates adjusted phase-shift associated with maximal oscillation. Dashed line shows log-linear fit to data. d 4-AP was then applied to induce seizure-like events. CLOSe modulated the duration of oscillatory bursts. Left: Max/min modulation of burst duration across 3 animals for raw data and sinusoidal fit. Colour shows phase-shift associated with max/min for individual animals. Right: Average modulation of burst duration averaged across three animals. e Same but for LFP power at seizure frequency. Error bars indicate s.e.m. over sessions. R and P values from circular-linear correlation over datasets.
Extended Data Fig. 4 CLOSe modulates the frequency and duration of high-amplitude seizure states in vivo in NHPs.
a Percentage of time spent in seizure state under no stimulation (black) and different CLOSe phase-shifts (coloured) for an example dataset. Seizure state was defined as smoothed, rectified LFP amplitude exceeding a particular threshold, ranging from zero up to the maximum amplitude observed in the no stimulation condition. b Change in time spent in seizure states under CLOSe+ (45° phase-shift) and CLOSe− (270° phase-shift) relative to no stimulation for the four datasets. c Average duration of seizure states exceeding each threshold level. d Relative change in seizure duration under CLOSe+ and CLOSe−. e Frequency of seizure states exceeding the threshold level. f Relative change in seizure frequency under CLOSe+ and CLOSe−. Thick bars in panels b,d,f indicate significant thresholds with significant phase-dependent modulation (P < 0.05, circular-linear correlation over datasets).
Extended Data Fig. 5 Seizure trajectories under different CLOSe phase-shifts in silico and in vitro.
a 2-D delay-embedded LFP trajectories during seizure bursts for the in silico model under all CLOSe phase-shifts. Colour indicates when in the cycle stimulation was delivered. b 3-D trajectories viewed from angle indicated in panel a. c,d Same but for example mouse in vitro session.
Extended Data Fig. 6 Comparison of closed-loop and open-loop stimulation strategies.
a Model simulations comparing CLOSe- versus open-loop delivery of the same optical pattern. Left: Example simulations of LFP under CLOSe- (red) and associated stimulation pattern (cyan). This light pattern was then delivered during a second run of the model (open-loop aligned, yellow). In this case, the only difference was the use of a new noise seed, leading to subtle differences in the seizure dynamics following the onset of stimulation (dashed line). We also delivered the same stimulation pattern during a third run of the model (open-loop random, blue). In this case, the model used the new noise seed and the onset of stimulation occurred at a different phase of the seizure cycle. Right: Summary of 100 model simulations. Despite using the same stimulation pattern, open-loop stimulation performed significantly worse than CLOSe-, and on average extended seizure durations relative to no stimulation. Boxes show median and interquartile range, whiskers show max/min. P values from two-tailed, paired t-test over simulations. b Model simulations comparing no stimulation versus open-loop stimulation at constant frequency. Left: Example simulations of LFP under no stimulation (black) and open-loop stimulation at 20 Hz (grey). Note that open-loop stimulation entrained the seizure cycles and extended the duration of seizures. Right: Relative duration of seizure bursts for increasing intensity of stimulation at different frequencies (average of 100 model stimulations per intensity). Burst duration increases monotonically for all frequencies. Note also that 20 Hz (the frequency of seizure cycles under no stimulation) is most effective at extending seizure duration. c Left: Example seizure bursts elicited by bath application of 4-AP to Emx1-ChR2 mouse brain slices with no stimulation (black) and with open-loop stimulation at constant frequency (grey) triggered by burst onset. Note that open-loop stimulation entrains the seizure cycles and extends the burst duration. Also shown are example seizure bursts under CLOSe with different phase-shifts (colours). Right: Burst duration and LFP power at seizure frequency relative to no stimulation under CLOSe+ (blue), CLOSe− (red) and open-loop stimulation (grey). Mean of 4 sessions from 3 animals. Error bars indicate s.e.m. over sessions. P values from two-tailed, paired t-test over sessions.
Extended Data Fig. 7 Model predictions for different closed-loop optogenetic seizure suppression strategies.
Optogenetic activation/suppression of excitatory/inhibitory neurons can be considered as weak perturbations of the seizure trajectory in different directions in the model state-space. Peripheral plots show predicted impact on seizure burst duration of closed-loop optogenetic strategies that deliver each types of stimulation in a phase-dependent manner. Strategies that suppress excitatory neurons and/or activate inhibitory neurons are generally more effective than activation of excitatory neurons and/or suppression of inhibitory neurons. Nevertheless, most strategies can result in either lengthening or shortening of seizures depending on the phase at which stimulation is delivered, and in all cases, there are particular phases which are more effective at suppressing seizures. Thus optogenetic seizure suppression strategies using a range of different opsins/cell-targets may be improved by delivering stimulation in a closed-loop manner.
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Supplementary Tables 1 and 2 and caption for Supplementary Video 1.
Supplementary video 1
Model trajectories with different CLOSe phase shifts.
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Zaaimi, B., Turnbull, M., Hazra, A. et al. Closed-loop optogenetic control of the dynamics of neural activity in non-human primates. Nat. Biomed. Eng 7, 559–575 (2023). https://doi.org/10.1038/s41551-022-00945-8
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DOI: https://doi.org/10.1038/s41551-022-00945-8
