1 Brain Perturbation and Neuroimaging in NHPs 1 2 Combining Brain Perturbation and Neuroimaging in Non-human Primates 3 P. Christiaan Klink1,*, Jean-François Aubry2, Vincent P. Ferrera3, Andrew S. Fox4, Sean Froudist-Walsh5, 4 Bechir Jarraya6,7, Elisa Konofagou8, Richard Krauzlis9, Adam Messinger10, Anna S. Mitchell11, Michael 5 Ortiz-Rios12,13, Hiroyuki Oya14, Angela C. Roberts15, Anna Wang Roe16, Matthew F. S. Rushworth11, Jérôme 6 Sallet11,17, Michael Christoph Schmid12,18, Charles E. Schroeder19, Jordy Tasserie6, Doris Tsao20, Lynn Uhrig6, 7 Wim Vanduffel21, Melanie Wilke13,22, Igor Kagan13,* & Christopher I. Petkov12,* 8 REVISION 1 2021-03-07 9 10 Author affiliations 11 1 Department of Vision & Cognition, Netherlands Institute for Neuroscience, Amsterdam, The Netherlands 12 2 Physics for Medicine Paris, Inserm U1273, CNRS UMR 8063, ESPCI Paris, PSL University, Paris, France 13 3 Department of Neuroscience & Department of Psychiatry, Columbia University Medical Center, New York, NY, 14 USA; Zuckerman Mind Brain Behavior Institute, Columbia University, New York, NY, USA 15 4 Department of Psychology & California National Primate Research Center, University of California, Davis, CA, USA 16 5 Center for Neural Science, New York University, New York, NY, USA 17 6 NeuroSpin, Commissariat à l'Énergie Atomique et aux Énergies Alternatives (CEA), Institut National de la Santé et de 18 la Recherche Médicale (INSERM), Cognitive Neuroimaging Unit, Université Paris-Saclay, France; 19 7 Foch Hospital, UVSQ, Suresnes, France 20 8 Ultrasound and Elasticity Imaging Laboratory, Department of Biomedical Engineering, Columbia University, New 21 York, NY, USA; Department of Radiology, Columbia University, New York, NY, USA 22 9 Laboratory of Sensorimotor Research, National Eye Institute, Bethesda, Maryland, USA 23 10 Laboratory of Brain and Cognition, National Institute of Mental Health, Bethesda, Maryland, USA 24 11 Department of Experimental Psychology, Oxford University, Oxford, United Kingdom 25 12 Newcastle University Medical School, Newcastle upon Tyne, United Kingdom; 26 13 German Primate Center, Leibniz Institute for Primate Research, Göttingen, Germany 27 14 Iowa Neuroscience Institute, Carver College of Medicine, University of Iowa, Iowa City, IA, USA; Department of 28 Neurosurgery, University of Iowa, Iowa city, IA, USA 29 15 Department of Physiology, Development and Neuroscience, Cambridge University, Cambridge, United Kingdom 30 16 Interdisciplinary Institute of Neuroscience and Technology, School of Medicine, Zhejiang University, Hangzhou 31 310029, China 32 17 Univ Lyon, Université Lyon 1, Inserm, Stem Cell and Brain Research Institute U1208, Bron, France; Wellcome 33 Centre for Integrative Neuroimaging, Department of Experimental Psychology, University of Oxford, Oxford, United 34 Kingdom 35 18 Faculty of Science and Medicine, University of Fribourg, Chemin du Musée 5 CH-1700 Fribourg, Switzerland 36 19 Nathan Kline Institute, Orangeburg, NY, USA; Columbia University, New York, NY, USA 37 20 Division of Biology and Biological Engineering, Tianqiao and Chrissy Chen Institute for Neuroscience; Howard 38 Hughes Medical Institute; Computation and Neural Systems, Caltech, Pasadena, CA, USA 39 21 Laboratory for Neuro- and Psychophysiology, Neurosciences Department, KU Leuven Medical School, Leuven, 40 Belgium; Leuven Brain Institute, KU Leuven, Leuven Belgium; Harvard Medical School, Boston, Massachusetts, 41 USA; Massachusetts General Hospital, Martinos Center for Biomedical Imaging, Charlestown, Massachusetts, USA 42 22 Department of Cognitive Neurology, University Medicine Göttingen, Göttingen, Germany 43 44 45 * Correspondence: 46 P.C.K.: [email protected], Netherlands Institute for Neuroscience, Meibergdreef 47, 1105 BA Amsterdam, The 47 Netherlands. 48 I.K.: [email protected], German Primate Center, Kellnerweg 4, 37077 Göttingen, Germany. 49 C.I.P.: [email protected], Newcastle University, Newcastle upon Tyne NE1 7RU, UK. 50 2 1 3 1 Brain Perturbation and Neuroimaging in NHPs 1 Abstract 2 Brain perturbation studies allow detailed causal inferences of behavioral and neural processes. Because the 3 combination of brain perturbation methods and neural measurement techniques is inherently challenging, 4 research in humans has predominantly focused on non-invasive, indirect brain perturbations, or neurological 5 lesion studies. Non-human primates have been indispensable as a neurobiological system that is highly 6 similar to humans while simultaneously being more experimentally tractable, allowing visualization of the 7 functional and structural impact of systematic brain perturbation. This review considers the state of the art in 8 non-human primate brain perturbation with a focus on approaches that can be combined with neuroimaging. 9 We consider both non-reversible (lesions) and reversible or temporary perturbations such as electrical, 10 pharmacological, optical, optogenetic, chemogenetic, pathway-selective, and ultrasound based interference 11 methods. Method-specific considerations from the research and development community are offered to 12 facilitate research in this field and support further innovations. We conclude by identifying novel avenues 13 for further research and innovation and by highlighting the clinical translational potential of the methods. 14 15 Keywords 16 Microstimulation, Optogenetics, Chemogenetics, Ultrasound, Lesion, Infrared, Causality, fMRI, primates 17 18 Highlights 19 Combined brain perturbation and neuroimaging can reveal causal brain mechanisms. 20 Overview of perturbation methods used with non-human primate neuroimaging. 21 Methodological considerations of the different techniques are discussed. 22 Translational potential and future directions are laid out and critically assessed. 23 2 2 3 1 Brain Perturbation and Neuroimaging in NHPs 1 1. Introduction 2 The brain is a complex dynamical network and an advanced understanding of its functional mechanisms 3 requires both observational and perturbation studies. Observational studies aim to understand neural systems 4 within a particular context. They are typically descriptive and correlative. Brain perturbation studies on the 5 other hand can provide insights on cause and effect. By manipulating a neural substrate and observing the 6 consequences of perturbation on relevant output measures (e.g., behavior, neural activity patterns, etc.) one 7 can demonstrate the substrate’s necessity and sufficiency for particular behaviors or cognitive functions 8 (Krakauer et al., 2017; Marinescu et al., 2018). Neuroscience is rich with examples of observational studies 9 leading to later experimental perturbations, from deducing the neurochemical properties of the giant squid 10 action potential (Hodgkin and Huxley, 1952) to the recent successes of techniques like optogenetics, 11 whereby neurons are transfected with light-sensitive channels that allow experimental control over action 12 potentials by shining light of specific wavelengths onto the neurons (Deisseroth, 2015). 13 The field of neuroimaging is often criticized for its descriptive or correlational nature (Ramsey et al., 2010). 14 In response, a range of analytical techniques have been developed as a proxy to derive causal inferences 15 from otherwise correlational neuroimaging data. Examples of such approaches are dynamic causal modeling 16 (DCM) for functional Magnetic Resonance Imaging studies (fMRI) (Friston et al., 2019, 2003) or Granger 17 causality for electroencephalography (EEG) and magnetoencephalography (MEG) (Friston et al., 2012; Seth 18 et al., 2015; Zhou et al., 2009). Despite an increase in the use of such analytical methods, they cannot 19 replace the need for causal perturbation, and often require additional validation with empirical perturbation 20 approaches. In humans, the combination of neuroimaging and transcranial magnetic stimulation (TMS) 21 (Driver et al., 2009; Ruff et al., 2009), transcranial direct/alternating current stimulation (tDCS/tACS) 22 (Cabral‐Calderin et al., 2015; Saiote et al., 2013), or more recently transcranial focussed ultrasound 23 stimulation (tFUS) (Lee et al., 2016a; Legon et al., 2018; Verhagen et al., 2019), has allowed non-invasive 24 disruption or potentiation of neural processes with simultaneous monitoring of the effects of these 25 perturbations on local and global brain activity patterns and behavior. 26 Brain perturbation techniques in both humans and animal models vary greatly in their scale and precision of 27 effects (Figure 1) with a tendency for non-invasive approaches to broadly affect many areas. All techniques 28 furthermore come with their own advantages and limitations. On one side of this spectrum, inferences from 29 neurological patient studies remain important, but such studies often have to deal with substantial lesion size 30 and large variability in lesion location across patients. Pre-lesion experimental controls in the same 31 individuals are almost always absent necessitating between-subjects comparisons with unaffected control 32 participants. Some newer non-invasive approaches, like ultrasound stimulation, can provide more focal deep 33 brain stimulation, and efforts are underway to extend their use from animal models to humans (Legon et al., 34 2020, 2014; Szablowski et al., 2018). All of the more precise perturbation methods, however, are invasive 35 and as a consequence their application is restricted to animal models and, under restricted circumstances, 36 human neurosurgery patients. The development