Electrophysiology and fluorescent imaging are widely used to study neural activity in vivo. These methods, however, require invasive procedures and can only sample small populations of neurons, and whole-brain fluorescent imaging of activity indicators is only able to capture a snapshot of the last state of the brain prior to euthanasia. W propose LOCATER: a transformative system that will transcend the limitations of these previous techniques. Therein, we will capture single-cell activity traces in large populations of neurons using activity-reporting RNA barcodes, and then export these activity traces from the brain in exosomes for non- invasive capture via circulating blood. Exosomes are endosome-derived vesicles containing protein and RNA cargo that are secreted from neurons and that readily cross the blood-brain barrier. Hence, exosomes provide an ideal substrate for capturing nucleic acid cargo in neurons and exporting these cargoes into circulating blood. The use of LOCATER technology to capture activity-reporting single-cell RNA barcodes secreted by neuronal populations in circulating blood and quantify the abundance of individual barcode sequences using next generation sequencing (NGS) will facilitate scalable longitudinal monitoring of activity from small neuronal populations or the entire brain. To develop this system we will create an AAV-vector that uses an immediate early gene (IEG) promoter to drive transcription of an RNA barcode unique to each neuron under LOCATER surveillance. We will include bacteriophage RNA stem loops on each activity-reporting barcode to facilitate binding of RNA barcodes to bacteriophage coat protein-linked exosomal transmembrane proteins for exosomal localization of barcodes via normal endosomal protein trafficking. Epitope tags will also be added to the extracellular domains of exosomal transmembrane proteins to enable high-fidelity capture of barcode- containing exosomes from circulating blood. To monitor neural activity in vivo using LOCATER, we will express tagged exosomal membrane proteins and activity-reporting cellular RNA barcodes in sub-regions of the mouse brain and subject animals to stimulation to capture single cell neuronal activity profiles accompanying behavior. Beyond capturing activity traces from single neurons in the brain, the LOCATER system also makes it possible to map the spatial origins of activity-reporting cellular barcodes in the brain. Following longitudinal activiy monitoring, animals will be sacrificed, followed by sectioning of each brain and clearing of tissue using the CLARITY technique. We will subject the clarified tissue to FISH to identify multiplexed FISH barcodes included on each AAV-vector that specify a unique activity-reporting RNA barcode. In this manner, colorimetric readout from serial rounds of FISH will be used to identify the cellular origins of each activity-reporting barcode in the brain. The use of LOCATER to monitor single-cell activity states non-invasively in behaving animals and map these states back to their cellular origins in the brain will transform our understanding of behavior, sensation, and neurodevelopmental, psychiatric, and neurodegenerative disease.
We are developing LOCATER (Large-scale Observation of Cellular Activity Through Exosomal Reporters), a transformative technology that will enable whole-brain, single-neuron recording over multiple experiments in behaving animals. LOCATER works by engineering exosomes, extracellular vesicles secreted by neurons and glia that cross the blood-brain barrier, to report neural activity mapped to a 3-D neural circuit in the brain. Unlike conventional optical and electrophysiological recording methods, this novel non-invasive method requires only the collection of blood from behaving animals.
| Habib, Naomi; Li, Yinqing; Heidenreich, Matthias et al. (2016) Div-Seq: Single-nucleus RNA-Seq reveals dynamics of rare adult newborn neurons. Science 353:925-8 |
