Controlling biological processes using light has increased the accuracy and speed with which researchers can manipulate many biological processes. Optical control allows for an unprecedented ability to dissect function and holds the potential for enabling novel genetic therapies. However, optogenetic experiments require adequate light sources with spatial, temporal, or intensity control, often a bottleneck for researchers. Here we detail how to build a low-cost and versatile LED illumination system that is easily customizable for different available optogenetic tools. This system is configurable for manual or computer control with adjustable LED intensity. We provide an illustrated step-by-step guide for building the circuit, making it computer-controlled, and constructing the LEDs. To facilitate the assembly of this device, we also discuss some basic soldering techniques and explain the circuitry used to control the LEDs. Using our open-source user interface, users can automate precise timing and pulsing of light on a personal computer (PC) or an inexpensive tablet. This automation makes the system useful for experiments that use LEDs to control genes, signaling pathways, and other cellular activities that span large time scales. For this protocol, no prior expertise in electronics is required to build all the parts needed or to use the illumination system to perform optogenetic experiments.

Long-range projections link distant circuits in the brain, allowing efficient transfer of information between regions and synchronization of distributed patterns of neural activity. Understanding the functional roles of defined neuronal projection pathways requires temporally precise manipulation of their activity, and optogenetic tools appear to be an obvious choice for such experiments. However, we and others have previously shown that commonly-used inhibitory optogenetic tools have low efficacy and off-target effects when applied to presynaptic terminals. In my talk, I will present a new solution to this problem: a targeting-enhanced mosquito homologue of the vertebrate encephalopsin (eOPN3), which upon activation can effectively suppress synaptic transmission through the Gi/o signaling pathway. Brief illumination of presynaptic terminals expressing eOPN3 triggers a lasting suppression of synaptic output that recovers spontaneously within minutes in vitro and in vivo. The efficacy of eOPN3 in suppressing presynaptic release opens new avenues for functional interrogation of long-range neuronal circuits in vivo.

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.