Optogenetics seminars

Genetic targeting of neuronal cells with activity reporters (calcium or voltage indicators) has initiated the paradigmatic transition whereby photons have replaced electrons for reading large-scale brain activities at cellular resolution. This has alleviated the limitations of single cell or extracellular electrophysiological probing, which only give access to the activity of at best a few neurons simultaneously and to population activity of unresolved cellular origin, respectively. In parallel, optogenetics has demonstrated that targeting neuronal cells with photosensitive microbial opsins, enables the transduction of photons into electrical currents of opposite polarities thus writing, through activation or inhibition, neuronal signals in a non-invasive way. These progresses have in turn stimulated the development of sophisticated optical methods to increase spatial and temporal resolution, light penetration depth and imaging volume. Today, nonlinear microscopy, combined with spatio-temporal wave front shaping, endoscopic probes engineering or multi scan heads design, enable in vivo in depth, simultaneous recording of thousands of cells in mm 3 volumes at single-spike precision and single-cell resolution. Joint progress in opsin engineering, wave front shaping and laser development have provided the methodology, that we named circuits optogenetics, to control single or multiple target activity independently in space and time with single- neuron and single-spike precision, at large depths. Here, we will review the most significant breakthroughs of the past years, which enable reading and writing neuronal activity at the relevant spatiotemporal scale for brain circuits manipulation, with particular emphasis on the most recent advances in circuit optogenetics.

To enable the understanding and repair of complex biological systems, such as the brain, we are creating novel optical tools that enable molecular-resolution maps of such systems, as well as technologies for observing and controlling high-speed physiological dynamics in such systems. First, we have developed a method for imaging specimens with nanoscale precision, by embedding them in a swellable polymer, homogenizing their mechanical properties, and exposing them to water – which causes them to expand manyfold isotropically. This method, which we call expansion microscopy (ExM), enables ordinary microscopes to do nanoscale imaging, in a multiplexed fashion – important, for example, for brain mapping. Second, we have developed a set of genetically-encoded reagents, known as optogenetic tools, that when expressed in specific neurons, enable their electrical activities to be precisely driven or silenced in response to millisecond timescale pulses of light. Finally, we are designing, and evolving, novel reagents, such as fluorescent voltage indicators and somatically targeted calcium indicators, to enable the imaging of fast physiological processes in 3-D with millisecond precision. In this way we aim to enable the systematic mapping, control, and dynamical observation of complex biological systems like the brain. The talk will be simultaneously interpreted English-Spanish) by the Interpreter, Mg. Lourdes Martino. Para permitir la comprensión y reparación de sistemas biológicos complejos, como el cerebro, estamos creando herramientas ópticas novedosas que permiten crear mapas de resolución molecular de dichos sistemas, así como tecnologías para observar y controlar la dinámica fisiológica de alta velocidad en dichos sistemas. Primero, hemos desarrollado un método para obtener imágenes de muestras con precisión a nanoescala, incrustándolas en un polímero hinchable, homogeneizando sus propiedades mecánicas y exponiéndolas al agua, lo que hace que se expandan muchas veces isotrópicamente. Este método, que llamamos microscopía de expansión (ExM), permite que los microscopios ordinarios obtengan imágenes a nanoescala, de forma multiplexada, lo que es importante, por ejemplo, para el mapeo cerebral. En segundo lugar, hemos desarrollado un conjunto de reactivos codificados genéticamente, conocidos como herramientas optogenéticas, que cuando se expresan en neuronas específicas, permiten que sus actividades eléctricas sean activadas o silenciadas con precisión en respuesta a pulsos de luz en una escala de tiempo de milisegundos. Finalmente, estamos diseñando y desarrollando reactivos novedosos, como indicadores de voltaje fluorescentes e indicadores de calcio dirigidos somáticamente, para permitir la obtención de imágenes de procesos fisiológicos rápidos en 3-D con precisión de milisegundos. De esta manera, nuestro objetivo es permitir el mapeo sistemático, el control y la observación dinámica de sistemas biológicos complejos como el cerebro. La conferencia será traducida simultáneamente al español por la intérprete Mg. Lourdes Martino.

My lab studies the design principles of cytoskeletal materials the drive cellular morphogenesis, with a focus on contractile machinery in adherent cells. In addition to force generation, a key feature of these materials are distributed force sensors which allow for rapid assembly, adaptation, repair and disintegration. Here I will describe how optogenetic control of RhoA GTPase is a powerful and versatile force spectroscopy approach of cytoskeletal assemblies and its recent use to probe repair response in actomyosin stress fibers. I will also describe our recent identification of 18 proteins from the zyxin, paxillin, Tes and Enigma families with mechanosensitive LIM (Lin11, Isl- 1 & Mec-3) domains that bind exclusively to mechanically stressed actin filaments. Our results suggest that the evolutionary emergence of contractile F-actin machinery coincided with, or required, proteins that could report on the stresses present there to maintain homeostasis of actively stressed networks.

Seizure onset is a critically important brain state transition that has proved very difficult to predict accurately from recordings of brain activity. I will present new data acquired using a range of optogenetic and imaging tools to characterize exactly how cortical networks change in the build-up to a seizure. I will show how intermittent optogenetic stimulation ("active probing") reveals a latent change in dendritic excitability that is tightly correlated to the onset of seizure activity. This data relates back to old work from the 1980s suggesting a critical role in epileptic pathophysiology for dendritic plateau potentials. Our data show how the precipitous nature of the transition can be understood in terms of multiple, synergistic positive feedback mechanisms.

The vagus nerve contains a diversity of sensory neurons that detect peripheral stimuli such as blood pressure changes at the aortic arch, lung expansion during breathing, meal-induced stomach distension, and chemotherapeutics that induce nausea. Underlying vagal sensory mechanisms are largely unresolved at a molecular level, presenting tremendously important problems in sensory biology. We charted vagal sensory neurons by single cell RNA sequencing, identifying novel cell surface receptors and classifying a staggering diversity of sensory neuron types. We then generated a collection of ires-Cre knock-in mice to target each neuron type, and adapted genetic tools for Cre-based anatomical mapping, in vivo imaging, targeted ablation, and optogenetic control of vagal neuron activity. We found different sensory neuron types that innervate the lung and exert powerful effects on breathing, others that monitor and control the digestive system, and yet others that innervate that innervate the larynx and protect the airways. Together with Ardem Patapoutian, we also identified a critical role for Piezo mechanoreceptors in the sensation of airway stretch, which underlies a classical respiratory reflex termed the Hering-Breuer inspiratory reflex, as well as in the neuronal sensation of blood pressure and the baroreceptor reflex.

The design of neural circuits, with large numbers of neurons interconnected in vast networks, strongly suggest that they are specifically build to generate emergent functional properties (1). To explore this hypothesis, we have developed two-photon holographic methods to selective image and manipulate the activity of neuronal populations in 3D in vivo (2). Using them we find that groups of synchronous neurons (neuronal ensembles) dominate the evoked and spontaneous activity of mouse primary visual cortex (3). Ensembles can be optogenetically imprinted for several days and some of their neurons trigger the entire ensemble (4). By activating these pattern completion cells in ensembles involved in visual discrimination paradigms, we can bi-directionally alter behavioural choices (5). Our results demonstrate that ensembles are necessary and sufficient for visual perception and are consistent with the possibility that neuronal ensembles are the functional building blocks of cortical circuits. 1. R. Yuste, From the neuron doctrine to neural networks. Nat Rev Neurosci 16, 487-497 (2015). 2. L. Carrillo-Reid, W. Yang, J. E. Kang Miller, D. S. Peterka, R. Yuste, Imaging and Optically Manipulating Neuronal Ensembles. Annu Rev Biophys, 46: 271-293 (2017). 3. J. E. Miller, I. Ayzenshtat, L. Carrillo-Reid, R. Yuste, Visual stimuli recruit intrinsically generated cortical ensembles. Proceedings of the National Academy of Sciences of the United States of America 111, E4053-4061 (2014). 4. L. Carrillo-Reid, W. Yang, Y. Bando, D. S. Peterka, R. Yuste, Imprinting and recalling cortical ensembles. Science 353, 691-694 (2016). 5. L. Carrillo-Reid, S. Han, W. Yang, A. Akrouh, R. Yuste, (2019). Controlling visually-guided behaviour by holographic recalling of cortical ensembles. Cell 178, 447-457. DOI:https://doi.org/10.1016/j.cell.2019.05.045.

Understanding how neural circuits in the brain compute information not only requires determining how individual inhibitory and excitatory elements of circuits are wired together, but also a detailed knowledge of their functional interactions. Recent advances in optogenetic techniques and mouse genetics now offer ways to specifically probe the functional properties of neural circuits with unprecedented specificity. Perhaps one of the most heavily interrogated circuits in the mouse brain is one in the retina that is involved in coding direction (reviewed by Mauss et al., 2017; Vaney et al., 2012). In this circuit, direction is encoded by specialized direction-selective (DS) ganglion cells (DSGCs), which respond robustly to objects moving in a ‘preferred’ direction but not in the opposite or ‘null’ direction (Barlow and Levick, 1965). We now know this computation relies on the coordination of three transmitter systems: glutamate, GABA and acetylcholine (ACh). In this talk, I will discuss the synaptic mechanisms that produce the spatiotemporal patterns of inhibition and excitation that are crucial for shaping directional selectivity. Special emphasis will be placed on the role of ACh, as it is unclear whether it is mediated by synaptic or non-synaptic mechanisms, which is in fact a central issue in the CNS. Barlow, H.B., and Levick, W.R. (1965). The mechanism of directionally selective units in rabbit's retina. J Physiol 178, 477-504. Mauss, A.S., Vlasits, A., Borst, A., and Feller, M. (2017). Visual Circuits for Direction Selectivity. Annu Rev Neurosci 40, 211-230. Vaney, D.I., Sivyer, B., and Taylor, W.R. (2012). Direction selectivity in the retina: symmetry and asymmetry in structure and function. Nat Rev Neurosci 13, 194-208

The dynamic nature of sleep in most animals suggests distinct stages which serve different functions. Genetic sleep induction methods in animal models provide a powerful way to disambiguate these stages and functions, although behavioural methods alone are insufficient to accurately identify what kind of sleep is being engaged. In Drosophila, activation of the dorsal fan-shaped body (dFB) promotes sleep, but it remains unclear what kind of sleep this is, how the rest of the fly brain is behaving, or if any specific sleep functions are being achieved. Here, we developed a method to record calcium activity from thousands of neurons across a volume of the fly brain during dFB-induced sleep, and we compared this to the effects of a sleep-promoting drug. We found that drug-induced spontaneous sleep decreased brain activity and connectivity, whereas dFB sleep was not different from wakefulness. Paradoxically, dFB-induced sleep was found to be even deeper than drug- induced sleep. When we probed the sleeping fly brain with salient visual stimuli, we found that the activity of visually-responsive neurons was blocked by dFB activation, confirming a disconnect from the external environment. Prolonged optogenetic dFB activation nevertheless achieved a significant sleep function, by correcting visual attention defects brought on by sleep deprivation. These results suggest that dFB activation promotes a distinct form of sleep in Drosophila, where brain activity and connectivity remain similar to wakefulness, but responsiveness to external sensory stimuli is profoundly suppressed.