Optogenetics seminars

Neural responses in the visual system are usually not purely visual but depend on behavioural and internal states such as arousal. This dependence is seen both in primary visual cortex (V1) and in subcortical brain structures receiving direct retinal input. In this talk, I will show that modulation by behavioural state arises as early as in the output of the retina.To measure retinal activity in the awake, intact brain, we imaged the synaptic boutons of retinal axons in the superficial superior colliculus (sSC) of mice. The activity of about half of the boutons depended not only on vision but also on running speed and pupil size, regardless of retinal illumination. Arousal typically reduced the boutons’ visual responses to preferred direction and their selectivity for direction and orientation.Arousal may affect activity in retinal boutons by presynaptic neuromodulation. To test whether the effects of arousal occur already in the retina, we recorded from retinal axons in the optic tract. We found that, in darkness, more than one third of the recorded axons was significantly correlated with running speed. Arousal had similar effects postsynaptically, in sSC neurons, independent of activity in V1, the other main source of visual inputs to colliculus. Optogenetic inactivation of V1 generally decreased activity in collicular neurons but did not diminish the effects of arousal. These results indicate that arousal modulates activity at every stage of the visual system. In the future, we will study the purpose and the underlying mechanisms of behavioural modulation in the early visual system

My lab seeks to understand how the mammalian brain performs the computations that underlie cognitive functions, including decision-making, short-term memory, and spatial navigation, at the level of the building blocks of the nervous system, cell types and neural populations organized into circuits. We have developed methods to measure, manipulate, and analyze neural circuits across various spatial and temporal scales, including technology for virtual reality, optical imaging, optogenetics, intracellular electrophysiology, molecular sensors, and computational modeling. I will present recent work that uses large scale calcium imaging to reveal the functional organization of the mouse posterior cortex for flexible decision-making during spatial navigation in virtual reality. I will also discuss work that uses optogenetics and calcium imaging during a variety of decision-making tasks to highlight how cognitive experience and context greatly alter the cortical circuits necessary for navigation decisions.

Coping with threatening situations requires both identifying stimuli predicting danger and selecting adaptive behavioral responses in order to survive. The dorso medial prefrontal cortex (dmPFC) is a critical structure involved in the regulation of threat-related behaviour, yet it is still largely unclear how threat-predicting stimuli and defensive behaviours are associated within prefrontal networks in order to successfully drive adaptive responses. To address these questions, we used a combination of extracellular recordings, neuronal decoding approaches, and optogenetic manipulations to show that threat representations and the initiation of avoidance behaviour are dynamically encoded in the overall population activity of dmPFC neurons. These data indicate that although dmPFC population activity at stimulus onset encodes sustained threat representations and discriminates threat- from non-threat cues, it does not predict action outcome. In contrast, transient dmPFC population activity prior to action initiation reliably predicts avoided from non-avoided trials. Accordingly, optogenetic inhibition of prefrontal activity critically constrained the selection of adaptive defensive responses in a time-dependent manner. These results reveal that the adaptive selection of active fear responses relies on a dynamic process of information linking threats with defensive actions unfolding within prefrontal networks.

Higher-order thalamic nuclei have extensive connections with various cortical areas. Yet their functionals roles remain not well understood. In our recent studies, using optogenetic and chemogenetic tools we manipulated the activity of a higher-order thalamic nucleus, the lateral posterior nucleus (LP, analogous to the primate pulvinar nucleus) and its projections and examined the effects on sensory discrimination and information processing functions in the cortex. We found an overall suppressive effect on layer 2/3 pyramidal neurons in the cortex, resulting in enhancements of sensory feature selectivities. These mechanisms are in place in contextual modulation of cortical processing, as well as in cross-modality modulation of sensory processing.

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.

Mammalian babies are “sensory traps” for parents. Various sensory cues from the newborn are tremendously efficient in triggering parental responses in caregivers. We recently showed that core aspects of maternal behavior such as pup retrieval in response to infant vocalizations rely on active learning of auditory cues from pups facilitated by the neurohormone oxytocin (OT). Release of OT from the hypothalamus might thus help induce recognition of different infant cues but it is unknown what sensory stimuli can activate OT neurons. I performed unprecedented in vivo whole-cell and cell-attached recordings from optically-identified OT neurons in awake dams. I found that OT neurons, but not other hypothalamic cells, increased their firing rate after playback of pup distress vocalizations. Using anatomical tracing approaches and channelrhodopsin-assisted circuit mapping, I identified the projections and brain areas (including inferior colliculus, auditory cortex, and posterior intralaminar thalamus) relaying auditory information about social sounds to OT neurons. In hypothalamic brain slices, when optogenetically stimulating thalamic afferences to mimic high-frequency thalamic discharge, observed in vivo during pup calls playback, I found that thalamic activity led to long-term depression of synaptic inhibition in OT neurons. This was mediated by postsynaptic NMDARs-induced internalization of GABAARs. Therefore, persistent activation of OT neurons following pup calls in vivo is likely mediated by disinhibition. This gain modulation of OT neurons by infant cries, may be important for sustaining motivation. Using a genetically-encoded OT sensor, I demonstrated that pup calls were efficient in triggering OT release in downstream motivational areas. When thalamus projections to hypothalamus were inhibited with chemogenetics, dams exhibited longer latencies to retrieve crying pups, suggesting that the thalamus-hypothalamus noncanonical auditory pathway may be a specific circuit for the detection of social sounds, important for disinhibiting OT neurons, gating OT release in downstream brain areas, and speeding up maternal behavior.

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.

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.

Understanding what drives foraging decisions in animals requires careful manipulation of the value of available options while monitoring animal choices. Value-based decision-making tasks, in combination with formal learning models, have provided both an experimental and theoretical framework to study foraging decisions in lab settings. While these approaches were successfully used in the past to understand what drives choices in mammals, very little work has been done on fruit flies. This is even though fruit flies have served as a model organism for many complex behavioural paradigms. To fill this gap we developed a single-animal, trial-based decision-making task, where freely walking flies experienced optogenetic sugar-receptor neuron stimulation. We controlled the value of available options by manipulating the probabilities of optogenetic stimulation. We show that flies integrate a reward history of chosen options and forget value of unchosen options. We further discover that flies assign higher values to rewards experienced early in the behavioural session, consistent with formal reinforcement learning models. Finally, we show that the probabilistic rewards affect walking trajectories of flies, suggesting that accumulated value is controlling the navigation vector of flies in a graded fashion. These findings establish the fruit fly as a model organism to explore the genetic and circuit basis of value-based decisions.

How does our brain rapidly determine if something is good or bad? How do we know our place within a social group? How do we know how to behave appropriately in dynamic environments with ever-changing conditions? The Tye Lab is interested in understanding how neural circuits important for driving positive and negative motivational valence (seeking pleasure or avoiding punishment) are anatomically, genetically and functionally arranged. We study the neural mechanisms that underlie a wide range of behaviours ranging from learned to innate, including social, feeding, reward-seeking and anxiety-related behaviours. We have also become interested in “social homeostasis” -- how our brains establish a preferred set-point for social contact, and how this maintains stability within a social group. How are these circuits interconnected with one another, and how are competing mechanisms orchestrated on a neural population level? We employ optogenetic, electrophysiological, electrochemical, pharmacological and imaging approaches to probe these circuits during behaviour.

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.

Olfactory navigation provides a tractable model for studying the circuit basis of sensori-motor transformations and goal-directed behaviour. Macroscopic organisms typically navigate in odor plumes that provide a noisy and uncertain signal about the location of an odor source. Work in many species has suggested that animals accomplish this task by combining temporal processing of dynamic odor information with an estimate of wind direction. Our lab has been using adult walking Drosophila to understand both the computational algorithms and the neural circuits that support navigation in a plume of attractive food odor. We developed a high-throughput paradigm to study behavioural responses to temporally-controlled odor and wind stimuli. Using this paradigm we found that flies respond to a food odor (apple cider vinegar) with two behaviours: during the odor they run upwind, while after odor loss they perform a local search. A simple computational model based one these two responses is sufficient to replicate many aspects of fly behaviour in a natural turbulent plume. In on-going work, we are seeking to identify the neural circuits and biophysical mechanisms that perform the computations delineated by our model. Using electrophysiology, we have identified mechanosensory neurons that compute wind direction from movements of the two antennae and central mechanosensory neurons that encode wind direction are are involved in generating a stable downwind orientation. Using optogenetic activation, we have traced olfactory circuits capable of evoking upwind orientation and offset search from the periphery, through the mushroom body and lateral horn, to the central complex. Finally, we have used optogenetic activation, in combination with molecular manipulation of specific synapses, to localize temporal computations performed on the odor signal to olfactory transduction and transmission at specific synapses. Our work illustrates how the tools available in fruit fly can be applied to dissect the mechanisms underlying a complex goal-directed behaviour.

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.